Antibacterial parenteral formulation and method thereof

Abstract

The present disclosure provides a formulation comprising a compound of formula (I) or a salt thereof together with a solubilizing agent, a hydrotropic agent, and a pH adjusting agent. The present disclosure also provides a lyophilized formulation and a reconstituted formulation. The present disclosure further provides a process for preparing the formulation and a method thereof. JPEG2025526229000072.jpg71170

Description

[Technical Field]

[0001] The present disclosure relates to the field of pharmaceutical formulations, and in particular to antibacterial formulations. More particularly, the present disclosure relates to formulations comprising the compound of formula (I): (S)-6-(5-(((2-(7-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-8-yl)ethyl)amino)methyl)-2-oxooxazolidin-3-yl)-2H-pyrazino[2,3-b][1,4]oxazin-3(4H)-one or a salt thereof, and processes for preparing the formulations. The present disclosure also relates to lyophilized injectable pharmaceutical formulations that exhibit improved solubility and storage stability.

[0002] [ka] [Background technology]

[0003] Globally, the ever-increasing challenges posed by the rising incidence and spread of antimicrobial resistance (AMR) have created the need to amplify efforts and prevent this widespread and major health threat. Recent pandemics have only reiterated the urgent need for thorough preparations and appropriate measures by the health sector to avert a major global catastrophe. It is essential to discover and develop novel, broad-spectrum antibiotics capable of tackling AMR and providing solutions to the most serious hospital-associated infections. Beyond hospital- and community-based health risks, the potential unexpected danger of bioterrorism is of particular concern, as it could result in a major public health emergency of severe scale, with panic-inducing, massive illness and mortality. Therefore, there is an urgent need for safe, potent, broad-spectrum antibacterial agents that are effective against all important and high-priority pathogens with multidrug-resistant backgrounds (WHO Antimicrobial Resistance. https: / / www.who.int / news-room / fact-sheets / detail / antimicrobial-resistance). In this regard, compounds of formula (I) can be identified as suitable for developing pharmaceutical formulations for antibiotic purposes.

[0004] The compound of formula (I) is a fully synthetic oxazolidinone-based broad-spectrum antibacterial agent, which is chemically designated as (S)-6-(5-(((2-(7-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-8-yl)ethyl)amino)methyl)-2-oxooxazolidin-3-yl)-2H-pyrazino[2,3-b][1,4]oxazin-3(4H)-one (WO2018225097). The broad-spectrum antibiotic compound of formula (I) is a novel chemical entity (NCE) with dual-target inhibition and potent activity against a broad spectrum of Gram-negative bacteria, including all major members of the order Enterobacteriaceae, as well as non-fermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii, and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) (WO2018225097). Extensive MIC studies using nearly 10,000 clinical isolates demonstrated a lack of cross-resistance to all currently used antibiotics, including fluoroquinolones.

[0005] However, the compounds of formula (I) need to be developed as parenteral antibiotics to treat a variety of bacterial infections and to have good solubility in pharmaceutically acceptable vehicles with a pH range of 3 to 8. Further formulations or drug products should be stable in formulation vehicles suitable for long-term storage.

[0006] Clearly, therefore, there remains a need to develop pharmaceutical injectable drug formulations of formula (I) that can be prepared and stored as ready-to-use medicaments and that meet the requirements for stability during longer periods of storage. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO20188225097 [Non-patent literature]

[0008] [Non-Patent Document 1] WHO antimicrobial resistance. https: / / www.who.int / news-room / fact-sheets / detail / antimicrobial-resistance [Non-patent document 2] Clinical and Laboratory Standards Institute CLSI. 2018. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th edition. CLSI standard M07 [Non-patent document 3] CLSI. 2021. Performance Standards for Antimicrobial Susceptibility Testing. 31st Edition. CLSI Supplement M100 [Non-patent document 4] USP <788> Chapter Particulate Matter in Injections Summary of the Invention [Means for solving the problem]

[0009] In one embodiment of the present disclosure, a) a compound of formula (I) or a salt thereof;

[0010] [ka]

[0011] Formulations are provided that include: b) a solubilizing agent; c) a hydrotropic agent; and d) a pH adjusting agent.

[0012] In another embodiment of the present disclosure, a) a compound of formula (I) or a salt thereof;

[0013] [ka]

[0014] A lyophilized formulation is provided that includes: b) a solubilizing agent; c) a hydrotropic agent; and d) a pH adjusting agent.

[0015] In one alternative aspect of the present disclosure, a reconstituted formulation is provided, comprising a lyophilized formulation as disclosed herein, together with a reconstitution agent and a diluent.

[0016] In yet another aspect of the present disclosure, a kit is provided comprising: a) a first compartment comprising a formulation as disclosed herein; b) a second compartment comprising a facilitating agent; and c) optionally accessories.

[0017] In a further aspect of the present disclosure, there is provided a process for preparing a formulation as disclosed herein, comprising the steps of: a) contacting a solubilizing agent with a compound of formula (I) or a salt thereof in the presence of a second solvent under stirring at a temperature in the range of 18°C to 40°C to obtain a first solution; b) adding a hydrotropic agent to the first solution under stirring to obtain a second solution; and c) mixing a pH adjusting agent with the second solution, followed by adding a vehicle to obtain the formulation.

[0018] In one or more embodiments of the present disclosure, there is provided a process for preparing a lyophilized formulation as disclosed herein, comprising: a) freezing a formulation as disclosed herein at a temperature in the range of 0° C. to −50° C., followed by drying at a pressure in the range of 10 μbar to 1 bar, to obtain the lyophilized formulation.

[0019] In a further aspect of the present disclosure, there is provided a process for preparing a reconstituted formulation as disclosed herein, comprising contacting a lyophilized formulation with a diluent, followed by adding a reconstitution agent.

[0020] In a further aspect of the present disclosure, there is provided a method of treating a bacterial infection, comprising administering to a subject in need thereof an effective amount of a formulation as disclosed herein.

[0021] In one or more embodiments of the present disclosure, there is provided a method of treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of a formulation as disclosed herein.

[0022] These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description and appended claims. This Summary is provided to introduce a selection of concepts in a simplified form. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 illustrates an XRD (X-ray diffraction) diffractogram of a compound of formula (I) formate salt, in accordance with the practice of the present disclosure. [Figure 2] FIG. 1 illustrates a DSC (differential scanning calorimetry) thermogram of a compound of Formula (I) formate salt, in accordance with the practice of the present disclosure. [Figure 3] 1 illustrates a pXRD (powder X-ray diffraction) diffractogram of the compound of formula (I) in accordance with the practice of the present disclosure. [Figure 4] FIG. 1 illustrates CFU / g of mouse thighs infected with Acinetobacter baumannii ATCC 17978 and treated subcutaneously with a formulation of the disclosure or meropenem (200 mg / kg) or polymyxin B (25 mg / kg) administered q8h according to the practice of the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0024] Those skilled in the art will recognize that the present disclosure is subject to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The disclosure further includes all such steps, features, compositions, and compounds individually or collectively referred to or described in this specification, and any and all combinations of any or more of such steps or features.

[0025] definition For convenience, before further description of the present disclosure, certain terms used in the specification and examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by one of ordinary skill in the art. The terms used herein have meanings that are recognized and known to those of ordinary skill in the art; however, for convenience and completeness, specific terms and their meanings are explained below.

[0026] The articles "a", "an" and "the" are used to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0027] The terms "comprise" and "comprising" are used in an inclusive and open sense, meaning that additional elements may be included. Throughout this specification, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to imply the inclusion of the specified element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps.

[0028] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0029] As used herein, the term "w / w" refers to the weight of a component relative to the total weight of the composition. As used herein, the term w / v refers to the weight of a component relative to the total volume of the composition. Furthermore, when water is used in the formulation, w / w and w / v are used interchangeably.

[0030] As used herein, the term "solubilizer" refers to a substance that aids in dissolving a substance that is otherwise poorly soluble in water. Solubilizers of the present disclosure include, but are not limited to, lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, and fumaric acid. The terms "solubilizer" and "dissolution agent" are used interchangeably. Solubilizers include, but are not limited to, lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or combinations thereof.

[0031] As used herein, the term "hydrotrope" refers to a chemical or substance added to an incompatible mixture of two or more different substances. Hydrotropes are added to increase the solubilizing capacity of the solvent in the mixture. Hydrotropes include, but are not limited to, niacinamide, sodium benzoate, sodium citrate, sodium acetate, or combinations thereof.

[0032] As used herein, the term "pH adjusting agent" refers to a substance added to a solution or mixture that can provide a desired pH. In this disclosure, pH adjusting agents include, but are not limited to, potassium hydroxide, sodium hydroxide, L-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof. The terms "pH adjusting agent" and "pH adjusting agent" are used interchangeably.

[0033] As used herein, the term "vehicle" refers to a substance or liquid used as a medium to disperse, suspend, or dissolve a material. In this disclosure, the term "vehicle" refers to water that is added to a formulation to make the formulation in an injectable form and for easy use.

[0034] As used herein, the term "diluent" refers to a substance used in a formulation to make the formulation easier to flow or pump. In this disclosure, diluents include, but are not limited to, dextrose, sorbitol, sodium bicarbonate, glucose, mannitol, sucrose, or sodium chloride.

[0035] The term "reconstituting agent" as used herein refers to a substance that is added to a substance and converts the dry substance into a fluid form. In the present disclosure, the reconstituting agent refers to a substance that is added to a lyophilized formulation, preferably to facilitate the use of the formulation as an injection formulation. The reconstituting agent of the present disclosure includes, but is not limited to, water. The lyophilized formulation is added with the reconstituting agent to obtain a reconstituted formulation.

[0036] The term "lyophilized formulation" as used herein refers to a dried formulation, wherein the prepared formulation is subjected to sequential processes such as heating and freezing to obtain a lyophilized formulation. The lyophilized formulation enhances the stability of the active ingredient in the formulation and provides long-term storage of the drug product. The lyophilized formulation is also referred to as a lyophilized cake, wherein the as-prepared formulation is subjected to a process selected from heating, freezing, drying, or a combination thereof, carried out in a sequential manner, to obtain a lyophilized formulation. The process used in obtaining a lyophilized formulation is also referred to as a freezing cycle.

[0037] As used herein, the term "therapeutic agent" refers to a substance or pharmaceutical material that can provide a therapeutic benefit, such as diagnosing, preventing, curing, mitigating, or treating a disease, disorder, condition, or infection.

[0038] Once a term is described, the same meaning applies to it throughout this disclosure.

[0039] As discussed in the Background Art, the compounds of formula (I) have the potential to treat a variety of bacterial infections. In particular, the compounds of formula (I) can treat a variety of clinical indications caused by both Gram-positive and Gram-negative bacterial species, such as complicated and uncomplicated urinary tract infections (cUTIs, e.g., pyelonephritis, cystitis), intra-abdominal infections (cIAIs), bloodstream infections, hospital-acquired bacterial pneumonia (HABP, hospital-acquired pneumonia) and ventilator-associated bacterial pneumonia (VABP), community-acquired bacterial pneumonia (CABP), secondary cystic fibrosis infections (CFIs), skin and soft tissue infections (SSTIs), endocarditis, meningitis, dysentery and diarrhea, typhoid, Clostridium difficile-associated colitis and diarrhea (CDAD), and Helicobacter pylori-associated peptic ulcers. It has the potential to treat bacterial sexually transmitted diseases caused by Chlamydia trachomatis, gonorrhea caused by Neisseria gonorrhoeae, and syphilis caused by Treponema pallidum, as well as bioterrorism-related bacterial diseases caused by anthrax (Bacillus anthracis), bubonic plague (Yersinia pestis), tularemia (Francisella tularensis), glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), and Q fever (Coxiella burnetii).

[0040] However, the solubility of the compound of Formula (I) or its salts is limited, which may limit its bioavailability. It should be noted that the compound of Formula (I) contains an amine group and can therefore readily form salts with acids, including hydrochlorides, formates, acetates, and the like. However, salt forms of the compound of Formula (I) have very limited solubility in water (2-4 mg / ml) and require further solubility improvement to 20-50 mg / ml strength in an appropriate pharmaceutical vehicle with a pH compatible with parenteral administration for human use. Therefore, the present disclosure provides formulations containing the compound of Formula (I) together with various ingredients, such as solubilizers, hydrotropes, and pH adjusters. Furthermore, the formulations must be stable for long periods of time, and the stability of the injectable form of the formulation must also be maintained. The formulations of the present disclosure use a combination of an acid for better solubility and a pH adjuster to balance the pH caused by the acid combination. Furthermore, the formulations of the present disclosure use a suitable hydrotropic agent to maintain maximum solubility of the compound of formula (I) in the formulation. Additionally, the present disclosure provides lyophilized formulations, reconstituted formulations, and processes for preparing the same.

[0041] In some embodiments of the present disclosure, a) a compound of formula (I) or a salt thereof;

[0042] [ka]

[0043] Formulations are provided that include: b) a solubilizing agent; c) a hydrotropic agent; and d) a pH adjusting agent.

[0044] In one embodiment of the present disclosure, there is provided a formulation as disclosed herein comprising a compound of formula (I) or a salt thereof in the weight range of 1% to 30% (w / w); a solubilizing agent in the weight range of 2% to 55% (w / w); a hydrotropic agent in the weight range of 1% to 30% (w / w); and a pH adjusting agent in the weight percentage range of 0.5% to 30% (w / w).

[0045] In another embodiment of the present disclosure, there is provided a formulation as disclosed herein comprising a compound of formula (I) or a salt thereof in the weight range of 2% to 20% (w / w); a solubilizing agent in the weight range of 5% to 35% (w / w); a hydrotropic agent in the weight range of 1% to 20% (w / w); and a pH adjusting agent in the weight percentage range of 0.5% to 20% (w / w).

[0046] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein, wherein the solubilizing agent is selected from lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or a combination thereof; the pH adjusting agent is selected from potassium hydroxide, sodium hydroxide, l-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof; the hydrotrope is selected from niacinamide, sodium benzoate, sodium citrate, or sodium acetate; and the salt of the compound of formula (I) is selected from formate, mesylate, esylate, besylate, tosylate, acetate, propionate, fumarate, maleate, tartrate, succinate, glycolate, glutamate, aspartate, hydrochloride, hydrobromide, or sulfate. In another embodiment of the present disclosure, the solubilizing agent is selected from lactic acid, ascorbic acid, acetic acid, or a combination thereof; the pH adjusting agent is selected from potassium hydroxide, l-arginine, or a combination thereof; and the hydrotrope is niacinamide or sodium benzoate.

[0047] In some embodiments of the present disclosure, there is provided a formulation as disclosed herein, wherein the solubilizing agent is a combination of lactic acid and ascorbic acid; the pH adjusting agent is l-arginine; the hydrotropic agent is niacinamide; and the salt of the compound of formula (I) is a formate salt.

[0048] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein that is stable at temperatures in the range of -70°C to 10°C.

[0049] In one embodiment of the present disclosure, there is provided a formulation as disclosed herein having a pH in the range of 2 to 6. In another embodiment of the present disclosure, the formulation has a pH in the range of 3 to 5.

[0050] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises a vehicle, wherein the vehicle is water.

[0051] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation is capable of killing or inhibiting the growth of a microbial organism, and the microbial organism is selected from a bacterium, a virus, a fungus, or a protozoan.

[0052] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation is an anti-infective against microbial organisms, particularly bacteria; the bacteria is selected from gram-negative bacteria, gram-positive bacteria, or a combination thereof.

[0053] In certain embodiments of the present disclosure, the microbial organism is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Citrobacter spp., Proteus spp., Serratia marcescens, Salmonella spp., Morganella morganii, Klebsiella oxytoca, Klebsiella aerogenes, and the like. aerogenes, Providencia spp., Neisseria gonorrhoeae, Mycoplasma spp., Campylobacter spp., Fusobacterium spp., Bacteriodes fragilis, Prevotella spp., Shigella spp., Helicobacter pylori, Ureaplasma spp., Burkholderia gladioli, Burkholderia multivorans, Pandorea apista, Burkholderia cepacia cepacia species, Burkholderia pseudomallei, Burkholderia mallei, Stenotrophomonas maltophilia, Achromobacter species, Ralstonia picketii, Legionella pneumophila, Clostridium difficile, Staphylococcus aureusaureus, Coagulase-negative Staphylococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

[0054] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein that inhibits an enzyme selected from bacterial gyrase, topoisomerase IV, or a combination thereof.

[0055] In certain embodiments of the present disclosure, there is provided a formulation as disclosed herein, further comprising an additive, a therapeutic agent, or a combination thereof.

[0056] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the excipient is selected from sorbitol, dextrose, monobasic sodium phosphate, dibasic sodium phosphate, sodium citrate, sodium bicarbonate, sodium chloride, or a combination thereof.

[0057] In some embodiments of the present disclosure, there is provided a formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; d) a pH adjusting agent; and e) a vehicle.

[0058] In some embodiments of the present disclosure, there is provided a formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; d) a pH adjusting agent; e) a vehicle; and f) an additive.

[0059] In some embodiments of the present disclosure, there is provided a formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; d) a pH adjusting agent; e) a vehicle; f) an additive; and g) a therapeutic agent.

[0060] In certain embodiments of the present disclosure, there is provided a formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent selected from lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or a combination thereof; c) a hydrotropic agent selected from niacinamide, sodium benzoate, sodium citrate, and sodium acetate; and d) a pH adjusting agent selected from potassium hydroxide, sodium hydroxide, l-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof.

[0061] In certain embodiments of the present disclosure, there is provided a formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent selected from lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or a combination thereof; c) a hydrotropic agent selected from niacinamide, sodium benzoate, sodium citrate, and sodium acetate; d) a pH adjusting agent selected from potassium hydroxide, sodium hydroxide, l-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof; and e) water as a vehicle.

[0062] In some embodiments of the present disclosure, a formulation is provided that includes: a) a compound of formula (I) formate salt; b) a solubilizer that is a combination of ascorbic acid and lactic acid; c) niacinamide; and d) l-arginine.

[0063] In one embodiment of the present disclosure, there is provided a lyophilized formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; and d) a pH adjusting agent.

[0064] In some embodiments of the present disclosure, a reconstituted formulation is provided, comprising a lyophilized formulation comprising a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; and d) a pH adjusting agent, together with a reconstituting agent and a diluent.

[0065] In one embodiment of the present disclosure, the reconstituted formulation as disclosed herein is provided, wherein the reconstitution agent is water; the diluent is selected from dextrose, sorbitol, sodium bicarbonate, glucose, mannitol, sucrose, or sodium chloride; and the diluent is present in the first solvent in a weight range of 0.9 to 25% (w / v). In another embodiment, the diluent is present in the first solvent in a weight range of 1 to 10% (w / v). In another embodiment, the reconstituted formulation as disclosed herein is provided, wherein the diluent is present in the first solvent in a weight range of 2 to 10% (w / v).

[0066] In certain embodiments of the present disclosure, there is provided a reconstituted formulation as disclosed herein, wherein the first solvent is water.

[0067] In some embodiments of the present disclosure, there is provided a reconstituted formulation comprising: i) a lyophilized formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent; c) a hydrotropic agent; d) a pH adjusting agent; ii) a reconstituting agent; and iii) a diluent.

[0068] In some embodiments of the present disclosure, there is provided a reconstituted formulation comprising: a) a compound of formula (I) or a salt thereof; b) a solubilizing agent selected from lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or a combination thereof; c) a hydrotropic agent selected from niacinamide, sodium benzoate, sodium citrate, and sodium acetate; d) a pH adjusting agent selected from potassium hydroxide, l-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof; e) water as a reconstituting agent; and f) a diluent selected from dextrose, sorbitol, sodium bicarbonate, glucose, mannitol, sucrose, or sodium chloride.

[0069] In some embodiments of the present disclosure, a reconstituted formulation is provided that includes: a) a compound of Formula (I) formate salt; b) a solubilizer that is a combination of ascorbic acid and lactic acid; c) niacinamide; d) l-arginine; e) water as a reconstituting agent; and f) dextrose as a diluent.

[0070] In certain embodiments of the present disclosure, a kit is provided that includes: a) a first compartment comprising a formulation as disclosed herein; b) a second compartment comprising a facilitating agent; and c) optionally accessories.

[0071] In certain embodiments of the present disclosure, a kit is provided that includes: a) a first compartment containing a formulation as disclosed herein; b) a second compartment containing water for injection; and c) optionally accessories selected from a diluent bag, tubing for infusion, needles, connectors, or combinations thereof.

[0072] In one embodiment of the present disclosure, there is provided a process for preparing a formulation, comprising: a) contacting a solubilizing agent with a compound of formula (I) or a salt thereof in the presence of a second solvent under stirring at a temperature ranging from 18°C to 40°C to obtain a first solution; b) adding a hydrotropic agent to the first solution under stirring to obtain a second solution; and c) mixing a pH adjusting agent with the second solution, followed by adding a vehicle to obtain the formulation.

[0073] In certain embodiments of the present disclosure, there is provided a process for preparing a formulation as disclosed herein, wherein the solubilizing agent is selected from ascorbic acid, lactic acid, acetic acid, succinic acid, or a combination thereof; and the two or more solubilizing agents are mixed prior to contacting with the compound of formula (I).

[0074] In certain embodiments of the present disclosure, there is provided a process for preparing a formulation as disclosed herein, wherein the first solution has a pH in the range of 2 to 3; and the second solution has a pH in the range of 2.5 to 4.

[0075] In certain embodiments of the present disclosure, there is provided a process for preparing a formulation as disclosed herein, wherein the formulation is filtered after adding the vehicle.

[0076] In certain embodiments of the present disclosure, there is provided a process for preparing a formulation as disclosed herein, wherein the second solvent is water; and the vehicle is water.

[0077] In an embodiment of the present disclosure, there is provided a process for preparing a lyophilized formulation, comprising: a) freezing a formulation as disclosed herein at a temperature in the range of 0° C. to −50° C., followed by drying at a pressure in the range of 10 μbar to 1 bar, to obtain a lyophilized formulation.

[0078] In one embodiment of the present disclosure, there is provided a process for preparing a lyophilized formulation as disclosed herein, wherein freezing is carried out for a time period ranging from 30 minutes to 30 hours; and drying is carried out for a time period ranging from 20 minutes to 150 hours. In another embodiment of the present disclosure, freezing is carried out for a time period ranging from 1 hour to 25 hours; and drying is carried out for a time period ranging from 1 hour to 140 hours. In another embodiment of the present disclosure, freezing is carried out for a time period ranging from 10 hours to 25 hours; and drying is carried out for a time period ranging from 50 hours to 130 hours.

[0079] In certain embodiments of the present disclosure, a process for preparing a reconstituted formulation is provided, comprising contacting a lyophilized formulation with a diluent, followed by adding a reconstitution agent.

[0080] In certain embodiments of the present disclosure, there is provided a process for preparing a reconstituted formulation as disclosed herein, which is carried out for a time period ranging from 60 seconds to 10 minutes.

[0081] In certain embodiments of the present disclosure, there is provided a process for preparing a reconstituted formulation as disclosed herein, wherein the reconstituted formulation is stable at a temperature in the range of 20° C. to 35° C. for a time period ranging from 20 hours to 30 hours.

[0082] In certain embodiments of the present disclosure, there is provided a process for preparing a reconstituted formulation as disclosed herein, wherein the reconstituted formulation has a pH in the range of 3 to 5; and an osmolality in the range of 300-500 mOs-mol / kg.

[0083] In certain embodiments of the present disclosure, there is provided the use of a formulation or a lyophilized formulation or a reconstituted formulation as disclosed herein.

[0084] In certain embodiments of the present disclosure, there is provided the use of a kit as disclosed herein.

[0085] In certain embodiments of the present disclosure, there is provided a use of a formulation or a lyophilized formulation or a reconstituted formulation as disclosed herein for the manufacture of a medicament.

[0086] In certain embodiments of the present disclosure, there is provided a method of treating a bacterial infection, comprising administering to a subject in need thereof an effective amount of a formulation as disclosed herein.

[0087] In certain embodiments of the present disclosure, there is provided a method of treating a bacterial infection, comprising administering to a subject in need thereof an effective amount of a lyophilized formulation as disclosed herein.

[0088] In certain embodiments of the present disclosure, a method of treating a bacterial infection is provided, comprising administering to a subject in need thereof an effective amount of a reconstituted formulation as disclosed herein.

[0089] In certain embodiments of the present disclosure, there is provided a method of treating a bacterial infection as disclosed herein, wherein the bacterial infection is caused by a bacterium, a virus, a fungus, or a protozoan.

[0090] In certain embodiments of the present disclosure, there is provided a method of treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of a formulation as disclosed herein.

[0091] In certain embodiments of the present disclosure, there is provided a method of treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of a lyophilized formulation as disclosed herein.

[0092] In certain embodiments of the present disclosure, a method of treating or preventing a disease or condition is provided, comprising administering to a subject in need thereof an effective amount of a reconstituted formulation as disclosed herein.

[0093] In certain embodiments of the present disclosure, methods are provided for treating or preventing a disease or condition as disclosed herein, wherein the disease or condition is mediated by a gram-positive, gram-negative bacterial species, or a combination thereof.

[0094] In certain embodiments of the present disclosure, the disease or condition is complicated and uncomplicated urinary tract infections (cUTIs, e.g., pyelonephritis, cystitis), intra-abdominal infections (cIAIs), bloodstream infections, hospital-acquired bacterial pneumonia (HABP, hospital-acquired pneumonia), ventilator-associated bacterial pneumonia (VABP), community-acquired bacterial pneumonia (CABP), secondary cystic fibrosis infections (CFIs), skin and soft tissue infections (SSTIs), endocarditis, meningitis, dysentery and diarrhea, typhoid, Clostridium difficile-associated colitis and diarrhea (CDAD), Helicobacter pylori-associated peptic ulcer, Chlamydia trachomatis, In some embodiments, methods are provided for treating or preventing a disease or condition as disclosed herein, wherein the disease or condition is selected from a sexually transmitted disease caused by Bacillus anthracis, gonorrhea caused by Neisseria gonorrhea, syphilis caused by Treponema pallidum, or a bioterrorism-associated bacterial disease caused by anthrax (Bacillus anthracis), bubonic plague (Yersinia pestis), tularemia (Francisella tularensis), glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), and Q fever (Coxiella burnetii).

[0095] Although the subject matter has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible, and therefore, the spirit and scope of the disclosure should not be limited to the description of the embodiments contained herein. [Example]

[0096] The following examples provide details about the synthesis, activity, and applications of the compounds and formulations of the present disclosure. It should be understood that the following are representative only, and that the disclosure is not limited by the details set forth in these examples. Abbreviation XRD - X-ray diffraction DMSO - Dimethyl Sulfoxide CFU - colony forming units MIC - Minimum Inhibitory Concentration pXRD - Powder X-ray Diffraction PPt - precipitate PBS - Phosphate Buffer Solution NMT - below WFI - Water for Injection mL - milliliter mg - milligram g - grams Qs - sufficient quantity °C - degrees Celsius Hrs - hours RH - Relative Humidity FD - Formulation Development API - Active Pharmaceutical Ingredients Ppt - Precipitate PVP K-12 - Polyvinylpyrrolidine HPβ-CD - Hydroxypropyl Beta Cyclodextrin NaOH - Sodium Hydroxide N - normality RT - room temperature USP - United States Pharmacopoeia Min - minutes mT - millitor R / H - Tilt / Hold IV - Intravenous w / w - mass / mass Sec - seconds mM - millimolar concentration

[0097] Methods and Materials For the purposes of this disclosure, the following materials were purchased and used without further purification.

[0098] [Table 1]

[0099] Powder X-ray diffraction analysis was carried out using a Bruker instrument, model D2 PHASER. Thermal analysis and differential scanning calorimetry were determined using a Perkin Elmer instrument, model DSC8000.

[0100] Example 1 Compounds of formula (I) The compound of formula (I) was prepared by the process defined in WO20188225097 and obtained as a crystalline formate salt (S)-6-(5-(((2-(7-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-8-yl)ethyl)amino)methyl)-2-oxooxazolidin-3-yl)-2H-pyrazino[2,3-b][1,4]oxazin-3(4H)-one, characterized as a single polymorph.

[0101] [ka]

[0102] The prepared formate salt of the compound of formula (I) was subjected to powder X-ray diffraction analysis, and a characteristic single polymorphic XRD diffractogram is shown in Figure 1. The distinct pattern of the diffractogram confirmed that a single polymorph was formed with crystalline characteristics. Figure 2 illustrates the differential scanning calorimetry (DSC) thermogram of the formate salt of the compound of formula (I). It can be inferred that the compound of formula (I) is crystalline in nature, as indicated by the high and sharp peaks in the thermogram.

[0103] Example 2 Antibacterial activity determination The compounds of formula (I) exhibit highly potent antibacterial activity against a variety of bacterial species, including Gram-negative and Gram-positive, aerobic and anaerobic bacteria, and biothreat bacterial pathogens. Bacterial susceptibility testing procedures for various bacterial species are described herein.

[0104] MIC Broth Microdilution Method For all bacterial strains except Neisseria gonorrhoeae, Helicobacter pylori, and anaerobes, the following MIC broth microdilution method was used:

[0105] Antimicrobials were prepared according to CLSI susceptibility testing standards (Clinical and Laboratory Standards Institute CLSI. 2018. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th Edition. CLSI Standard M07). Solutions of antimicrobials or combinations at 2x final concentrations were prepared in 50 mL tubes by dilution in CA-MHB (or buffered yeast extract alpha-ketoglutarate broth for Legionella pneumophila) and manually transferred into a 96-well master block. 50 μL of each well was then transferred from the master block into a 96-well plate. Microtiter plates were stored frozen at -80°C until the test date for up to 6 months and were not frozen / thawed more than once. Bacterial inocula were prepared to approximately 1 x 10 by diluting a 0.5 McFarland suspension in CA-MHB with TES 100-fold. 6 The antibacterial panel wells were thawed at room temperature, and then 50 μL of inoculum was added to each well to obtain approximately 5 × 10 CFU / mL. 5 A final density of CFU / mL and the desired test concentration of antibacterial agent was obtained. Test plates were incubated and visually read according to CLSI guidelines. The MIC value corresponded to the first well with no visible growth.

[0106] Agar dilution method for Neisseria gonorrhoeae / Helicobacter pylori The compound of formula (I) was dissolved in DMSO at 3200 μg / mL. Solutions at 100x the test range were prepared in DMSO by serial dilution, then diluted 100-fold in GC agar (for Neisseria gonorrhoeae) or Mueller-Hinton agar supplemented with 5.0% old (≥2-week-old) sheep blood (for H. pylori) and poured into dishes (final volume = 40 mL). MIC testing was performed by agar dilution according to CLSI susceptibility testing standards (CLSI. 2021. Performance Standards for Antimicrobial Susceptibility Testing. 31st Edition. CLSI Supplement M100). Bacterial inocula were prepared by the direct colony suspension method. Bacterial suspensions were adjusted to 0.5 McFarland in 0.9% NaCl. 200 μL of bacterial suspension was placed in wells of a 96-well plate, and 1 μL of each suspension (approximately 1 × 10 4 CFU) were deposited on the agar surface. The plates were incubated according to CLSI guidelines (CLSI. 2018. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th Edition. CLSI Standard M07).

[0107] Agar dilution method for antibacterial testing of anaerobic bacteria (Bacteroides spp., Fusobacterium spp., Prevotella spp., and Clostridium spp.) The compound of formula (I) or a salt thereof, preferably the formate salt of the compound of formula (I), was dissolved in DMSO at 3200 μg / mL. Solutions at 100x concentrations of the test range were prepared in DMSO by serial dilution, then diluted 100-fold with Brucella agar supplemented with 5 μg / mL hemin, 1 μg / mL vitamin K1, and 5.0% hemolyzed sheep blood medium and poured into dishes (final volume = 40 mL). The solutions were incubated at 35-37°C for 42-48 hours using a Bactron 600 anaerobic chamber containing an atmosphere of 5% CO2, 5% H2, and 90% N2, after which MIC values (CLSI. 2021. Performance Standards for Antimicrobial Susceptibility Testing. 31st Edition. CLSI Supplement M100) were determined.

[0108] The minimum inhibitory concentration (MIC) is the lowest concentration of an active ingredient or drug that facilitates the cessation of bacterial growth. 50 (Minimum inhibitory concentration that stops bacterial growth in 50% of the species tested) and MIC 90 The minimum inhibitory concentrations (minimum inhibitory concentrations that stop bacterial growth of 90% of the species tested) were determined and are tabulated below in Table 1. The formate salt of compound of formula (I) demonstrated potent antibacterial activity against Gram-positive, Gram-negative, anaerobic, and biothreat bacterial pathogens (Table 2).

[0109] [Table 2A]

[0110] [Table 2B]

[0111] [Table 3]

[0112] Example 3 Salt screening A salt screen of the compound of Formula (I) was evaluated to select suitable salts for the development of formulations with improved solubility in water. The solid-state properties of the compound of Formula (I) were studied using pXRD diffraction patterns. The free amine of the compound of Formula (I) was found to be amorphous in nature by pXRD diffractograms, as X-ray diffraction showed a halo diffraction pattern in the 2-40° (2-theta) range (Figure 3).

[0113] Counterion selection The free amine compound of formula (I) was found to be soluble only in a dichloromethane and methanol (1:1) mixture. Therefore, the same solvent system was used during salt screening experiments using a slow solvent evaporation technique. Acidic counterions were used to screen the salt formation tendency of the compound of formula (I) due to its weakly basic nature (pKa approximately 6.81). Theoretically, counterions with pKa values at least 2 units lower than the basic pKa value of the compound of formula (I) were considered for salt formation. Table 3 shows a list of selected counterions for salt screening.

[0114] [Table 4]

[0115] A common solvent method was optimized to allow for salt screening in which the drug and counterion were soluble. Qualitative solubility of the counterion was performed in water as the solvent, and a stoichiometric 1:1.125 ratio of drug and counterion was used to prepare the solutions during salt screening experiments.

[0116] The compound of formula (I) (100 mg) was dissolved in an appropriate amount of organic solvent system containing a 1:1 mixture of dichloromethane and methanol, and the required amount of counterion dissolved in methanol was added. The solution was then heated at 50-60°C for several minutes and allowed to equilibrate to room temperature to observe whether any salt formation occurred by crystallization or precipitation from the solution. The results of the salt screening are summarized in Table 4 below.

[0117] [Table 5]

[0118] The counterions that resulted in crystalline salts from the above experiments were further investigated in scale-up trials (200 mg scale). Selected crystalline salts were then evaluated for solubility in various aqueous media to investigate solubility enhancement of salts of the compound of formula (I).

[0119] The compound of formula (I) (200 mg) as the free amine was weighed into a round-bottom flask and dissolved in a sufficient amount of a mixture of dichloromethane and methanol (1:1) with the aid of sonication and mild heating in a water bath at approximately 40-50° C. The required amount of acidic counterion was then weighed (if solid) and dissolved in methanol or measured (if liquid counterion), then transferred to the above contents in the round-bottom flask and stirred overnight until a precipitated solid was observed, and the solid was collected after complete solvent evaporation.

[0120] Solubility of Selected Salts of Compounds of Formula (I) The as-prepared salts of the compound of formula (I) were evaluated for qualitative solubility in various aqueous media to select the best counterion based on enhanced solubility compared to the formate salt. 2 mg of the equivalent salt of the compound of formula (I) was weighed into a clean glass vial. 20 μL of the solvent listed in Table 5 below was added, sonicated, vortexed, and observed for dissolution. If not dissolved, additional increments of solvent were added as shown in Table 5. Sonication and vortexing were performed to dissolve the compound with each increment.

[0121] [Table 6]

[0122] Inferences from salt screening: To enhance the solubility of the compound of Formula (I), salt screening was performed to meet the high solubility requirements for the higher doses required for human administration. Various salts were screened using acidic counterions, including sulfonate, mesylate, succinate, tosylate, fumarate, formate, maleate, malate, oxalate, lactate, glucuronate, and HCl. Preliminary feasibility trials resulted in amorphous salts in the maleate, citrate, and oxalate trials. Even in the lactate and ascorbate salts, amorphous solids formed. However, from Table 5, it could be identified that only the formate salt of the compound of Formula (I) exhibited crystallinity and increased solubility.

[0123] The formate salt of the compound of formula (I) gave 10 mg / mL solutions in both 10% w / v aqueous ascorbic acid and 30% v / v aqueous lactic acid, while the HCl salt and malic acid gave clear solutions at 5 mg / mL in 30% aqueous lactic acid.

[0124] Determination of the saturation solubility of the formate salt of the compound of formula (I) Saturation solubility of the compound of formula (I) formate salt was performed at 25°C ± 2°C in various vehicles, acids, and additives as listed in Table 6A.

[0125] Water for injection (WFI) and various solvents (5 mL or 5.0 g) were placed in 10 mL clear glass vials. An appropriate amount of the formate salt of Formula (I) was added to each vial, which was then closed with a stopper. These glass vials were attached to an orbital shaking water bath. Shaking was carried out for 72 hours, with the temperature maintained at approximately 25±2°C. The resulting test samples were then evaluated for drug concentration determination in each relevant solvent at 24, 48, and 72 hours.

[0126] [Table 7]

[0127] The compound of formula (I) formate exhibited very limited solubility in water and different aqueous solvents, but showed maximum solubility in individual acids such as ascorbic acid, acetic acid, and lactic acid. The combination of acids with hydroxypropyl β-cyclodextrin (HPβ-CD) and polyvinylpyrrolidone (PVP) K-12 showed enhanced solubility of the compound of formula (I) formate. It could be observed that the use of a combination of ascorbic acid, lactic acid, and glacial acetic acid with HPβCD and PVP K-12 resulted in maximum solubility of the compound of formula (I) formate.

[0128] pH solution stability study pH solution stability studies were performed at 25°C ± 2°C in the following buffers listed in Table 6B (50 mM acetate buffer for pH 3.0, pH 4.0, pH 5.0 and 50 mM phosphate buffer for pH 6.0).

[0129] To determine the pH solution stability of the compound of formula (I) formate salt at different pH ranges, each buffer solution was prepared by dissolving the buffer salt in WFI. A weighed amount of the compound of formula (I) formate salt was added to each buffer system and dissolved. The volume was adjusted with WFI. Five mL samples were filled into 5 mL USP Type I vials. The vials were stoppered and sealed. These vials were kept at 25°C for the stability study. A blank buffer / placebo sample was also placed alongside the active sample. The samples were evaluated for profile, pH, assay, and related substances at 24, 48, and 72 hours.

[0130] [Table 8]

[0131] [Table 9]

[0132] [Table 10]

[0133] At pH 3.0, 4.0, and 5.0, the formulation was found to be a clear, pale yellow solution; however, at pH 6.0, it was an off-white suspension. There was droplets in the assay after 24 hours at 25°C across all pH ranges. The droplets ranged from 0.4% to 3.5%. This indicated that the formate salt of Formula (I) had solution stability at pH 3.0 to 5.0 for up to 24 hours. Related substance (RS) data showed an increase in impurity levels after 48 hours at 25°C. The increase in RS was significant at pH 6.0 compared to pH 3.0, 4.0, and 5.0. Therefore, the target pH range for formulation development is between pH 3.0 and 5.0.

[0134] Example 4 suitability study The drug herein is referred to as the active ingredient, which is a compound of Formula (I) or a salt thereof. Compatibility studies of the drug with formulation components were conducted to identify suitable formulation components for achieving an acceptable impurity profile. To achieve this, compatibility and stability studies were conducted on various components in solutions / suspensions such as buffers, solvents, and surfactant mixtures at 25°C / 60% RH and 40°C / 75% RH for one month. All these batches were submitted for stability studies in mixtures at 25°C / 60% RH and 40°C / 75% RH for one month.

[0135] Table 7 below provides details of the container closure systems used in this disclosure.

[0136] [Table 11]

[0137] Formulations for compatibility studies Various formulations F1 to F5 were prepared using the ingredients as set out in Table 8 below. Additionally, stability and solubility studies were performed on each formulation, with the results tabulated in Tables 9, 10 and 11.

[0138] [Table 12]

[0139] [Table 13]

[0140] [Table 14]

[0141] [Table 15]

[0142] In the drug-ingredient compatibility study, significant changes in appearance, assay, and related substances (RS) were observed under both conditions, i.e., 25°C ± 2°C / 60 ± 5% RH and 40°C ± 2°C / 75 ± 5% RH, as illustrated in Tables 9, 10, and 11. Changes in appearance (solution / suspension color) were observed primarily due to ascorbic acid discoloration under both conditions, as placebo (formulation without the compound of Formula (I)) samples containing ascorbic acid also showed discoloration. Droplets in the assay were more prevalent for batches placed as formulations compared to assays of batches placed similarly for the individual ingredients, with the exception of the batch with L-arginine (F4).

[0143] The impurity profile in the batches with the individual components (F1, F2, F3 and F4) was found to be lower compared to the batch manufactured as formulation (F5).

[0144] Solubility enhancement of the compound of formula (I) or its salt The compound of formula (I) formate salt has limited solubility in water and had to be further improved to achieve the target formulation strength, which was achieved by using a combination of acids such as ascorbic acid, lactic acid, and glacial acetic acid.

[0145] The compound of formula (I) formate was unstable at higher pH ranges, i.e., in the neutral to alkaline pH range (pH 5.0 to 10.0), and was very stable at acid pH (<5). Therefore, the desired target pH range for formulations containing the compound of formula (I) formate is between pH 3.0 and pH 5.0. The compound of formula (I) formate is very unstable in the liquid state (solution / suspension); therefore, the drug formulation was prepared as a lyophilized drug formulation to achieve a stable product.

[0146] To improve the solubility of the compound of formula (I) formate salt using a combination of acids, different prototype formulations were investigated. The compound of formula (I) formate salt was dissolved by vortexing and mixing at 35°C to 40°C. All observations were recorded once the solution temperature reached 25°C to 30°C. Among the solubilizing agents investigated, the compound of formula (I) formate salt was most soluble in acidic conditions and in combinations of acids such as ascorbic acid, lactic acid, and acetic acid. Furthermore, the addition of solubilizing agents such as hydroxypropyl β-CD and polyvinylpyrrolidine K-12 enhanced the solubility to over 25 mg / mL.

[0147] [Table 16A]

[0148] [Table 16B]

[0149] Evaluation of the use and formulation of L-arginine as a pH adjuster Formulations were evaluated using l-arginine as a pH adjuster to maintain the formulations between pH 3.0 and 4.0. Two different batches (SF19000421 & SF19000435), each 10 mL, of the compound of Formula (I) formate salt were prepared. The formulations prepared for evaluation are shown in Table 13. Formulation 35A contained l-arginine in addition to the ingredients of Formulation 21A.

[0150] [Table 17]

[0151] [Table 18]

[0152] Table 14 illustrates the solution stability initially and 18-20 hours after preparation of the formulation. It can be seen that L-arginine exhibits a stable pH, and the appearance of the solution remains clear for up to 18 hours. Therefore, L-arginine has been found to be suitable for use as a pH adjuster for formulations of the compound of formula (I).

[0153] Evaluation of pH modifiers in combination with hydrotropes. Formulations with pH adjusters (pH modifiers) and hydrotropes (with and without) were evaluated with formulation pHs between 3.5 and 4.0. Table 15 provides formulations for solution stability at varying pH ranges of the compound of Formula (I) formate salt, and their corresponding stability studies are shown in Table 16.

[0154] [Table 19]

[0155] [Table 20]

[0156] Among the pH adjusters and solubilizers evaluated, the combination of l-arginine and niacinamide showed better stability (solution appearance) for up to 3 to 4 hours and up to 20 hours (formulation 465B). Therefore, l-arginine and niacinamide were used as the pH adjuster and hydrotrope, respectively. It was further observed that the pH adjuster should be incorporated in a specific weight percent range, i.e., in the range of 0.5% to 30%, and if incorporated outside of this range, it resulted in precipitation of the formulation.

[0157] Estimation of the concentration of acid (solubilizer) to solubilize the compound of formula (I) formate salt. Formulations prepared using different acid concentrations were evaluated to obtain the desired concentration of solubilization of the compound of formula (I) formate salt as shown in Table 17, and the observations are tabulated below in Table 18.

[0158] [Table 21]

[0159] [Table 22]

[0160] The concentrations of glacial acetic acid and lactic acid had a significant effect on the solubilization of the compound of formula (I). Formulations 470B and 470G had the indicated solubility of the compound of formula (I) formate at a concentration of 25 mg / mL. In the remaining formulations in Table 17, the compound of formula (I) formate could not be completely solubilized. Formulation 470A was examined as a control sample. Therefore, to solubilize the compound of formula (I) formate at different concentrations, a minimum concentration of acid was required, as shown in Table 19.

[0161] [Table 23]

[0162] Based on the solubility improvements observed with organic acids, formulations were further evaluated using a combination of ascorbic acid and lactic acid to increase solubility and reduce overall acid strength, with a desired pH range between 4.00 and 4.50.

[0163] Evaluation of the solubility of the compound of formula (I) formate (without acetic acid) Formulations containing the compound of formula (I) formate salt were prepared with varying concentrations of lactic acid as a solubilizing agent and without acetic acid, as shown in Table 20. The as-prepared formulations were observed for their appearance, pH, and were recorded in Table 21.

[0164] [Table 24]

[0165] [Table 25]

[0166] From Table 21 above, it can be seen that the compound of formula (I) was solubilized at a concentration of 20 mg / mL using a combination of ascorbic acid and lactic acid, and the pH of the solution was found to be in the range of pH 4.5 to 5.3. Therefore, it was speculated that acetic acid could optionally be used as a solubilizing agent.

[0167] Example 5 Evaluation of freeze-drying cycles for the compound of formula (I) formate salt The lyophilization cycle for the formulations of the present disclosure was evaluated based on the lyophilization cycle of a formulation having two acid combinations, i.e., ascorbic acid and lactic acid. The formulations were prepared using batch sizes of 100 ml each, and are shown in Table 22.

[0168] [Table 26]

[0169] The formulations in Table 22 (Table 26) were prepared as described herein. Ascorbic acid (solubilizer) was dissolved in water for injection (approximately 70% of the batch size, 25°C-30°C). Lactic acid (solubilizer) was then added and dissolved by stirring for 5 minutes to obtain a solution. The solution was heated to 35°C-40°C. The formate salt of Formula (I) was added to the solution and dissolved by stirring for 2 to 3 hours. The solution was then allowed to cool to room temperature under continuous stirring. Niacinamide (hydrotrope) was added to the first solution and dissolved by stirring for 10 minutes to obtain a second solution. L-arginine was added to the above solution and dissolved by stirring for 10 minutes to obtain the formulation. Mannitol (depending on the batch) was then added and dissolved by stirring for 10 minutes. The solution volume was brought up to the maximum batch size with water for injection (25°-30° C.) (vehicle) and mixed for 15 minutes.

[0170] The solution was filtered through a 0.2μ PES filter (47 mm) using a vacuum filtration assembly. 25.0 mL of the filtered solution was filled into 50 mL clear USP Type I Tubular vials, which were half-stoppered and loaded for lyophilization. The lyophilization cycle was performed as illustrated in Table 23 below.

[0171] [Table 27]

[0172] After lyophilization, the vacuum was discontinued, the vials were stoppered, removed from the lyophilizer, and sealed. The observed moisture content in both batches was 5.52% w / w for 529A and 7.65% w / w for 529B, and the observed moisture content for the lyophilized samples is shown in Table 24.

[0173] [Table 28]

[0174] The above batches of lyophilization cycles provided acceptable cake appearance and reconstitution times and were further used to generate prototype stability batches. Based on moisture content, secondary drying was required to be performed at a higher temperature, i.e., 25-30°C. Therefore, further lyophilization cycle optimization was performed for prototype batch development.

[0175] Predilution studies for the compound of formula (I) formate salt as an injection sample The lyophilized formulations as prepared above were evaluated for dilution studies, and 5% glucose (GS, diluent) solution, sterile water for injection were used for the dilution studies. The batches of formulations used were B.No. 529A and 529B.

[0176] The lyophilized formulation was reconstituted with sterile water for injection (reconstituting agent), and the reconstituted formulation was further diluted with different diluents having a 1:5 ratio of compound of Formula (I) formate salt and diluent. The reconstituted and diluted samples were evaluated for appearance, pH, and osmolality as shown in Tables 25 and 26.

[0177] [Table 29]

[0178] [Table 30]

[0179] The reconstituted solution was a clear, pale yellow solution that was stable at room temperature for up to 6 hours. The product diluted with 5% glucose solution exhibited an osmolality between 380 and 410 mOsmol / Kg, an acceptable range for low-volume parenterals. However, samples diluted with sterile water for injection exhibited a low osmolality solution (140 to 170 mOsmol / Kg).

[0180] Example 6 Prototype stability batch of formulation for injection Prototype formulation batches for stability studies were prepared as detailed in Table 27 below, each with a batch size of 800 mL, and lyophilized formulations were prepared as illustrated in Table 28. Stability studies were performed on the lyophilized formulations, and the results were recorded for each batch as shown in Tables 29 through 32.

[0181] [Table 31]

[0182] [Table 32]

[0183] [Table 33]

[0184] [Table 34A]

[0185] [Table 35]

[0186] [Table 36A]

[0187] [Table 36B]

[0188] All batches of formulations of the formate salt of formula (I) compound showed cake meltback / collapse after 6 months at 40°C ± 2°C / 75% RH ± 5% RH, and at 25°C ± 2°C / 60% RH ± 5% RH, a slightly collapsed cake structure with an appearance change was observed in all batches compared to the initial state. In all batches, the lyophilized cake after 6 months at 40°C ± 2°C / 75% RH ± 5% RH and 25°C ± 2°C / 60% RH ± 5% RH showed difficulty in reconstitution, resulting in a yellow to orange suspension.

[0189] The stability of the lyophilized formulations (all batches) after 6 months at 2°-8°C showed a cake structure similar to that of the initial cake, and there were no difficulties in reconstituting the lyophilized formulations. All batches of formulation showed no change in pH of the formulations stored at all stability conditions. A decrease in assay and an increase in impurities were observed for formulations stored at 40°C ± 2°C / 75% RH ± 5% RH and 25°C ± 2°C / 60% RH ± 5% RH, while no significant differences were observed in the assay and impurity profiles for formulation batches stored at 2°-8°C. There was no significant effect of mannitol on the stability of the drug product.

[0190] Therefore, lyophilized formulations of compound of formula (I) formate salt for injection were evaluated at different pH conditions, i.e., pH 3.50, 4.00 & 4.50, and with storage conditions of 2°-8°C selected for the pH range study. The developed formulations were further slightly modified for process optimization.

[0191] Process optimization study of formulation for injection Although the compound of formula (I) formate salt was not solubilized at room temperature even after stirring for 3 hours, heating up to 40-50°C solubilized the compound of formula (I) in the formulation with the vehicle. This resulted in a significant effect of temperature and stirring time on the related substances of the formulation in unfiltered and filtered samples, especially in the sample prepared at 50°C with 3 hours of stirring. Therefore, the process was further optimized to solubilize the compound of formula (I) formate salt at lower temperatures.

[0192] Effect of the initial volume of WFI on the solubilization of the compound of formula (I) formate salt Formulations of the present disclosure were prepared by varying the processing temperature of the preparation of the formulations to study their effect on the solubilization of the compound of formula (I) formate salt, each using a batch size of 50 ml.

[0193] [Table 37]

[0194] Ascorbic acid and lactic acid were dissolved in 20% of the total batch size of water for injection (25°C-30°C). Then, the compound of formula (I) formate salt was added to the above solution and continued stirring at the respective temperature conditions as shown in Table 33 (Table 37). Niacinamide was added to the above solution at room temperature (RT) and dissolved by stirring. L-arginine was added to the above solution and dissolved by stirring. The volume was made up to 50 mL with WFI and continued stirring at room temperature. Samples were kept at room temperature for physical observation.

[0195] [Table 38]

[0196] From Table 34 above, it can be seen that the compound of formula (I) formate salt can be solubilized at 25°C (room temperature, RT) and 45°C using a smaller amount of WFI at an earlier stage of the manufacturing process, i.e., 20% of the total batch size.

[0197] Solutions produced using this process remained stable at room temperature (for 5 to 18 hours), demonstrating the ability to solubilize the compound of formula (I) formate salt in a given formulation of vehicle as exemplified herein while eliminating a heating step in the manufacturing process.

[0198] Differential scanning calorimetry (DSC) thermogram analysis was performed to determine the glass transition temperature. No glass transition temperature was observed in the DSC thermogram. In both thermograms, an exothermic peak was observed at -20°C to -25°C; this was due to water crystallization in the sample, and the endothermic peak at 0°C indicated the ice melting phenomenon.

[0199] During cooling to -70°C (second time), a peak was observed at -32°C to -36°C. However, this peak may be due to recrystallization of the frozen matrix. Using DSC analysis, it was difficult to detect the glass transition temperature of the formulation, as the formulation was composed of an acid and a different salt, likely with a much lower glass transition temperature. Further investigation was required to identify the glass transition temperature or collapse temperature.

[0200] Freeze-drying cycle development for pharmaceuticals Based on the freeze-dried formulation developed in this invention, further optimization was carried out. To form larger ice crystals, an annealing step was incorporated into the freezing stage. The preparation process is as follows: solubilizing the compound of formula (I) formate salt at room temperature, and further developing the freeze-drying cycle for higher solid content.

[0201] [Table 39]

[0202] The formulations shown in Table 35 (Table 39) were prepared by the process defined herein. Ascorbic acid (solubilizer) was dissolved in water for injection (second solvent, approximately 20% of the batch size, 25°C-30°C), followed by the addition of lactic acid (solubilizer) under stirring for 5 minutes. The compound of Formula (I) as the formate salt was then added to the solution and dissolved by stirring at room temperature for 3 hours to obtain a first solution. Niacinamide (hydrotrope) was then added to the first solution and dissolved for 10 minutes to obtain a second solution, followed by the addition of L-arginine (pH adjuster) under stirring for 10 minutes to obtain the formulation. The formulation volume was brought up to the maximum batch size using WFI (25°C-30°C) and mixed for 15 minutes. The solution was filtered through a 0.2μ PES filter (47 mm) using a vacuum filtration assembly. A 300 mL batch size of each formulation was prepared.

[0203] Table 36 below shows an exemplary process for the lyophilization cycle of the compound of Formula (I) formate salt. The formulations were subjected to sequential freezing at temperatures ranging from -50°C to 0°C, preferably from -45°C to 0°C, as shown in Table 36 below. After freezing, the formulations were subjected to additional freezing and drying to obtain lyophilized samples. Drying was carried out at pressures ranging from 10 μbar to 1 bar. After lyophilization, the vacuum was interrupted with nitrogen gas. The vials were stoppered, removed from the lyophilizer, and sealed. The lyophilized formulations were analyzed for their appearance and characteristics, as shown in Table 37.

[0204] [Table 40]

[0205] [Table 41]

[0206] The annealing step during freezing helped improve the cake structure and provided an intact cake. The lyophilized cake was easily reconstituted with water for injection within 2 minutes to form a clear, pale yellow solution. No change in pH / shift in pH of the reconstituted solution was observed upon lyophilization. This lyophilization cycle was slightly modified at the end of the primary drying step to target a 96-hour lyophilization cycle, which provided similar lyophilized cake structure, reconstitution time, and higher solids content in the lyophilized cake. The same lyophilization cycle was adopted for further development.

[0207] Diluent compatibility studies of formulations Diluent compatibility studies were conducted using 5% dextrose injection as strengths of 1.0 mg / mL, 3.33 mg / mL, and 10.0 mg / mL after dilution.

[0208] [Table 42]

[0209] The lyophilized formulations shown in Table 38 were prepared according to the process exemplified below. Water for injection (WFI) was purged with nitrogen gas for 1 hour. 20% of the total batch size of nitrogen-purged water for injection (RT) was collected in a glass beaker. To this, a weighed amount of ascorbic acid was added and kept under stirring at room temperature for 5 minutes using a magnetic stirrer. The solution appeared clear and was a light pale yellow solution. A weighed amount of lactic acid was added to the above solution under stirring at room temperature. The solution appeared clear and was still a light pale yellow solution. A weighed amount of the formate salt of Formula (I) was added to the above solution and kept under stirring at room temperature for 120 minutes. The solution now became translucent and was a dark yellow solution with some fine particulate matter. 40% of the total batch size of nitrogen-purged WFI was added to the above solution and kept under stirring at room temperature for 10 minutes. A translucent yellow solution with some fine particulate matter was observed. A weighed amount of niacinamide was added to the above and kept under stirring at room temperature for 15 minutes. A translucent dark yellow solution with some fine particulate matter was observed. A weighed amount of L-arginine was added to the above and kept under stirring at room temperature for 15 minutes. The solution became a translucent dark yellow solution with some fine particulate matter. Nitrogen-purged WFI (RT) was used to make the volume of the process solution up to the maximum required batch size and stirred for 15 minutes at room temperature. (Appearance: (externally) clear to translucent dark yellow solution with some fine particulate matter). The solution was filtered through a 0.2μ PES (polyethersulfone) membrane filter. The appearance (after filtration) was a clear yellow solution. The filtered solution was stored at 5°±3°C for approximately 18 hours until further use.

[0210] The filtered bulk solution was thawed from 5°±3°C to room temperature for 1 hour. The solution appeared to be a clear, dark yellow solution and free of any visible particles. The details of the diluent and IV set are shown in Table 39 below. Table 40 details the dilution stability and IV set suitability of the formate salt of Formula (I) for injection using different concentrations, and Table 41 shows the chemical analysis and particulate matter of the formulation.

[0211] [Table 43]

[0212] [Table 44]

[0213] [Table 45]

[0214] [Table 46]

[0215] [Table 47]

[0216] Formulations (bulk solutions) containing the compound of formula (I) formate salt for injection were diluted with 5% glucose injection at different concentrations. The diluted solutions were stored at room temperature (20-30°C) along with the IV set. These diluted solutions were evaluated for various physicochemical parameters, such as pH, osmolality, and assay of the compound of formula (I) formate salt, related substances, and particulate matter.

[0217] The diluted solutions were found to be clear, slightly pale yellow solutions with no visible particles and remained stable at room temperature (20°C to 30°C) for up to 8 hours. The diluted solutions exhibited a pH of 4.50 ± 0.20 and remained stable for up to 8 hours. The diluted solutions were isotonic in nature at the 1.25 mg / mL concentration (270 to 330 mOsmol / kg) as shown in Table 41 and remained stable for up to 24 hours. The remaining concentration diluted solutions (2.50, 3.50, 4.0, and 5.0 mg / mL) were slightly hyperosmolar solutions (370 to 460 mOsmol / kg); however, solutions in this osmolality range can be administered as large-volume parenteral solutions. The diluted solutions remained stable for up to 8 hours, and no significant changes were observed in the assay of the compound of Formula (I) formate salt. There was an increase in the impurity levels observed from the initial (0 hour) to the 8 hour holding period; however, the impurity levels were within the specified limits for the bulk solution of the formulation of the formate salt of Formula (I) for injection (total impurities: NMT 4%, Table 42). Particulate matter in the diluted solution was within the limits specified in USP <788> This is within the limits described for large-volume parenterals in the Particulate Matter in Injections section. The initial (0 hour) particulate matter (Table 43) for placebo was not within the limits. This may be due to contamination that occurred during sample handling / analysis.

[0218] Based on in-use stability studies of the compound of formula (I) formulation for injection (as a formate bulk solution), it is recommended that the bulk solution of the compound of formula (I) formate for injection should be further diluted with 5% glucose injection (free-flex bags) and used within 6 to 8 hours of dilution with the proposed IV set when stored at room temperature (20°-30°C) and protected from light. The following concentrations of the compound of formula (I) formate formulation in 5% glucose injection diluted solution are recommended for administration: 1.25 mg / mL, 2.50 mg / mL, 3.50 mg / mL, 4.0 mg / mL, and 5.0 mg / mL.

[0219] Each placebo solution of different strengths should also be used within 6 hours of its dilution with the proposed IV set when stored at room temperature (20°-30°C) and protected from light.

[0220] Formulation stability studies A lead batch for a stability study for a formulation of the formate salt of compound of Formula (I) for injection having a batch size of 500 mg / vial and 1600 mL was prepared using the ingredients set forth in Table 44.

[0221] [Table 48]

[0222] The formulations were prepared according to the process described herein. Ascorbic acid was dissolved in water for injection (20% of the batch size, 25°C-30°C) and stirred for 30 minutes. Lactic acid was then added under stirring for 15 minutes at room temperature, and the formate salt of Formula (I) was added under stirring for 150 minutes at room temperature to obtain a first solution. Niacinamide was added to the first solution and dissolved under stirring for 30 minutes at room temperature to obtain a second solution. An additional 40% (total batch size) of WFI was added to the second solution under stirring for 45 minutes, and L-arginine (a pH adjuster) was added under stirring for 30 minutes at room temperature to obtain the formulation. The volume of the formulation solution was adjusted to the batch size by adding WFI. The solution was then filtered through a 0.2μ PES filter (47 mm, Lot No. 071715407, Sartorius) using a vacuum filtration assembly to obtain the formulation. 25.7 mL of the filtered solution was filled into 50 mL clear USP Type I Tubular vials, the vials were half-stoppered and loaded for lyophilization. The lyophilization cycle used for the formulations was as follows:

[0223] [Table 49]

[0224] After lyophilization, the vacuum was broken with nitrogen gas, and the vials were stoppered, removed from the lyophilizer, and sealed. The formulation was then an off-white to pale yellow dry cake with a dark yellow color at the bottom of the cake. There was dry, collapsed / melt-back cake at the corners of the vial. The lyophilized cake was easily reconstituted with water for injection within 3 minutes to form a clear, yellow solution. No change in pH / shift in pH of the reconstituted solution was observed upon lyophilization, and the results are shown in Table 46.

[0225] [Table 50]

[0226] As-prepared batches were placed on stability studies for up to 12 months at 25°C ± 2°C / 60% RH ± 5% RH and 2°C to 8°C. Placebo batches (batch size: 400 mL) for each formulation were also manufactured and processed identically to the active batches.

[0227] [Table 51A]

[0228] [Table 51B]

[0229] From Table 47 above, it can be inferred that the color of the lyophilized cake was observed to change from yellow to reddish after 2 months at 25°C ± 2°C / 60% RH ± 5% RH and at 2°C-8°C. A change in the pH of the formulation was observed at both stability conditions. A decrease in assay and an increase in impurities were observed at 25°C ± 2°C / 60% RH ± 5% RH. No significant differences were observed in the assay and impurity profiles at 2°C-8°C. Based on the observation of the lyophilized cake of the 500 mg / vial formulation of the compound of Formula (I) formate salt for injection, it was further evaluated at 250 mg / vial to improve the lyophilized cake structure to make it more refined and pharmaceutically acceptable.

[0230] Formulation of Compound of Formula (I) Formate Salt for Injection 250 mg / vial for Lyophilization Cycle Validation and Stability Studies

[0231] [Table 52]

[0232] The formulations shown in Table 48 (Table 52) were prepared by the process described below. Ascorbic acid was dissolved in water for injection (20% of the batch size, 25°C to 30°C) and stirred for 30 minutes to dissolve. Subsequently, lactic acid was added under stirring for 15 minutes at room temperature, and the formate salt of Formula (I) was added under stirring for 150 minutes at room temperature to obtain a first solution. Niacinamide was added to the first solution and dissolved under stirring for 30 minutes at room temperature to obtain a second solution. An additional 40% (total batch size) of WFI was added to the second solution under stirring for 45 minutes, and L-arginine (a pH adjuster) was added under stirring for 30 minutes at room temperature. WFI was added to bring the volume of the solution to the batch size. The solution was then filtered through a 0.2μ PES filter (47 mm, Lot No. 071715407, Sartorius) using a vacuum filtration assembly to obtain the formulation. 25.7 mL of the filtered solution was filled into 50 mL clear USP Type I Tubular vials, the vials were half-stoppered, and loaded for lyophilization. The lyophilization cycle used for the formulations is as provided below in Table 49.

[0233] [Table 53]

[0234] The formate salt of formula (I) took a longer time to solubilize, i.e., about 3 hours, and some fine dust like particles were found to be visible. The unfiltered bulk solution was translucent yellow, but after filtration through a 0.2 μ filter, it was found to be a clear yellow solution. There was no significant change in the assay results of the unfiltered and filtered bulk solutions.

[0235] After lyophilization, the vacuum was interrupted with nitrogen gas, and the vial was stoppered, removed from the lyophilizer, and sealed. The dry, intact cake was off-white to pale yellow with a dark yellow color at the bottom of the cake. No shrinkage or meltback of the lyophilized cake was observed. A yellow ring was observed at the bottom of the vial, which may be due to phase separation of the drug substance during lyophilization. The cake was reconstituted using 11.5 mL of WFI, and the resulting solution was a clear, yellow solution. For reconstitution, 11.5 mL of WFI was added to the vial, which was then allowed to stand at room temperature for 5 minutes to ensure adequate wetting of the solid, followed by vigorously shaking for 2-3 minutes. The top, intact cake was easily solubilized with the addition of WFI without any shaking. The dried, yellow layer at the bottom required vigorous shaking to solubilize.

[0236] [Table 54]

[0237] Inference for the compound of formula (I) formate lyophilized formulation at 250 mg / vial strength Lyophilization of the compound of formula (I) formate lyophilized formulation at 250 mg / vial strength resulted in the formation of a pharmaceutically acceptable lyophilized cake structure. The batches were subjected to stability studies at 25°C ± 2°C / 60% RH ± 5% RH and at 2°C to 8°C for up to 24 months. A placebo batch of the formulation was also manufactured and processed identically to the active batch. Compatibility studies of the manufacturing process and its process components were conducted, and the results obtained were satisfactory for all process components. Therefore, it can be concluded that the compound of formula (I) formate formulation for injection is compatible with components such as stainless steel 316L, USP Type 1 glass, 0.2μ PES filters, 0.2μ PVDF filters, PharmaPure AL242029 tubing, and Sani Tech 50® tubing for up to 24 hours.

[0238] It can be appreciated that a reconstituted formulation of the compound of formula (I) formate salt for injection having 250 mg / vial, when stored at room temperature, should be used within 24 hours of its reconstitution.

[0239] The reconstituted solution of compound of formula (I) formate salt for injection 250 mg / mL should be further diluted with 5% dextrose injection IP and used within 10 hours after dilution when stored at room temperature. Concentrations of compound of formula (I) formate salt such as 1.0 mg / mL and 3.33 mg / mL when diluted with 5% dextrose injection IP are recommended for administration.

[0240] Temperature range study and freeze-thaw study Two different batches of a lyophilized formulation of the formate salt of Formula (I) and a placebo were prepared to evaluate freeze-thaw and temperature excursion stability. For the freeze-thaw cycling stability study, the exposure conditions were -20°C for 24 hours, followed by 25°C ± 2°C / 60% ± 5% RH for 24 hours. This exposure was considered to be one completed freeze-thaw cycle. The formulation together with the secondary pack was exposed to a total of three freeze-thaw cycles. For the lyophilized formulation, i.e., the formate salt of Formula (I) for IV injection together with the secondary pack, was exposed to each storage condition and each duration. The exposure conditions were -20°C for 24 and 48 hours, and 40°C ± 2°C / 75% ± 5% RH for 24 and 48 hours.

[0241] Formulations of the compound of Formula (I) formate drug product for injection 250 mg / vial have been found to be stable for up to 24 hours, even when the formulations are exposed to different temperature conditions from -20°C to 40°C ± 2°C.

[0242] The compound of formula (I) formate salt for injection 250 mg / vial was also found to be stable for up to 144 hours (3 cycles of freeze-thaw study duration) even when the formulation was exposed to different temperature conditions, i.e., -20°C ± 2°C to 25°C ± 2°C.

[0243] photostability studies Lyophilized formulations of the formate salt of the compound of formula (I) were prepared as described herein. The formulations were packaged in various packages and exposed to light to evaluate their photostability.

[0244] Lyophilized formulations (250 mg / vial) in 50 mL clear USP Type 1 glass vials were kept intact for exposure to light until the required overall illumination was achieved. In another example, lyophilized formulations in 50 mL clear USP Type 1 glass vials were kept in a second cardboard box and exposed to a light source until the required overall illumination was achieved. Similarly, formulations were packaged in aluminum foil and cardboard boxes and exposed to illumination. For comparison purposes, control samples and placebos were also exposed to illumination. Table 51A (Table 55) shows details of the packaging materials used. Similarly, only the active ingredient, i.e., the formate salt of the compound of Formula (I), was subjected to a photostability study, and details of the packaging materials are shown in Table 51B (Table 56).

[0245] [Table 55]

[0246] [Table 56]

[0247] The photostability of formulations containing the compound of formula (I) formate salt for injection and the compound of formula (I) formate salt alone was evaluated according to ICH-Q1B guidelines.

[0248] The formulation of the formate salt of the compound of Formula (I) for injection, 250 mg / vial, was found to be insensitive to light, with the remaining components of the formulation providing light protection by forming a solid cake structure above the API layer at the bottom of the lyophilized cake.

[0249] The photostability study showed that the compound of formula (I) as a formate salt was found to be sensitive to light, as discoloration was observed. Therefore, the compound of formula (I) as a formate salt should be protected from light during the manufacturing process of the formulation. However, the formulation was found to be non-photosensitive. Therefore, the formulation of the present disclosure is photo-stable and enhances the stability of the compound of formula (I) as a formate salt.

[0250] Vmax research Volume Maximization Study (Vmax) The objective of the constant pressure study was to determine the capacity of sterilizing grade filters and membrane-based prefilters and to provide sizing for sterilizing grade filters and membrane-based prefilters for a given unit operation.

[0251] Trials were performed manually using a Virus Vmax chamber with a filter connected to the chamber with the aid of a Luer-lock connection. Prior to operation, the filter was wetted / conditioned with water for injection and buffer. Furthermore, prior to the process, the apparatus was properly vented to avoid any air entrapment. Cumulative volume measurements as a function of time were recorded during the experiment and, accordingly, used as a measure of filter performance (at constant pressure). By plotting t / V (time / volume) as a function of time (t), a slope line was constructed to calculate the filter's capacity (Vmax) and initial volumetric flow rate (Ji) based on a stepwise pore clogging model. The following equation was used to calculate the minimum filtration area (Amin) required for a batch volume (VB) at time (tB):

[0252]

number

[0253] In the above equation, V = VB = process volume (L)

[0254] Filters evaluated: Sartoguard 1.2μ / 0.2μ PES nominal filter as pre-filter; followed by Sartopore 0.2μ PES absolute filter as sterilizing filter as shown in Table 51C.

[0255] [Table 57]

[0256] The compound of Formula (I) formate formulation for injection 250 mg / vial was found to be easily filterable. No filter clogging or flow rate decline was observed. The results (Table 51D) showed no adsorption of the compound of Formula (I) formate drug product formulation onto the filter. There was no significant change in related substances.

[0257] [Table 58]

[0258] Observations on the formulations of the present disclosure From the above examples relating to the development of formulations of the compound of formula (I) formate salt, it can be seen that the drug product, i.e., the compound of formula (I) formate salt, was not stable in solution for a longer sustained period of time and therefore, it was necessary to prepare the formulation and lyophilize the formulation for improved stability.

[0259] Furthermore, based on 6-month stability data of prototype formulation batches, formulations of the formate salt of Formula (I) for injection were found to be stable at 2°C to 8°C. As the pH decreased from pH 4.50 to pH 3.50, the impurity profile of the formulation also decreased at higher temperatures, i.e., 40°C ± 2°C / 75% RH ± 5% RH and 25°C ± 2°C / 60% RH ± 5% RH. Additionally, formulations with l-arginine strengths of 500 mg / vial and 250 mg / vial were introduced for long-term stability studies. The formulations also contain a combination of two acids, i.e., ascorbic acid and lactic acid. Furthermore, l-arginine was added to reduce the acid concentration of the formulation. Reducing the strength from 500 mg / vial to 250 mg / vial improved the refinement of the lyophilized cake structure with pharmaceutically acceptable properties, and lyophilized cakes were produced using a 3-4 day lyophilization cycle time period as disclosed herein.

[0260] For use in injection, the lyophilized formulation was reconstituted with a reconstituting agent and a diluent. The reconstituting agent was water for injection, and 5% dextrose (diluent) was used for dilution. The reconstituted formulation was found to be stable and compatible at room temperature for up to 10 hours after dilution. If dilution is required, diluted formulations of 1.0 mg / mL and 3.33 mg / mL can be used. Product strength was targeted at 250 mg / vial with a formulation containing 20 mg / mL of the compound of Formula (I) formate drug product. Accordingly, formulations of the present disclosure are provided in Table 52 below.

[0261] [Table 59]

[0262] A kit comprising the formulation. The formulations of the present disclosure can be used in the form of a kit, wherein the kit comprises a first compartment and a second compartment. The first compartment contains the as-prepared formulation. In another example, the kit contains a reconstituted formulation or a lyophilized formulation. The second compartment contains water. The kit optionally comprises accessories selected from a diluent bag, tubing for injection, a needle, or a connector.

[0263] Methods for Treating Bacterial Infections Dose-ranging study of formulations in a neutropenic murine model of thigh infection caused by A. baumannii ATCC 17978 The objective of this study was to investigate the efficacy of formulations containing the compound of Formula (I) formate salt as disclosed herein over a dose range of 10 mg / kg to 120 mg / kg administered by subcutaneous (SC) injection every eight hours (q8h) using a 26-hour neutropenic murine double-thigh infection model infected with A. baumannii ATCC 17978. The well-established antibiotics meropenem and polymyxin B were used as comparative controls.

[0264] Mice were infected intramuscularly with A. baumannii ATCC 17978 2 hours before treatment with the compound of formula (I) formate salt administered subcutaneously (SC) at 10 mg / kg, 30 mg / kg, 60 mg / kg, 90 mg / kg, and 120 mg / kg q8h, and with the comparators meropenem (200 mg / kg SC q8h) and polymyxin B (25 mg / kg SC q8h). Animals were euthanized 2 hours post-infection (pre-treatment controls) or 26 hours post-infection (vehicle controls and all treatment groups). Data were obtained from all animals at early time points.

[0265] Lyophilized vials of the compound of formula (I) formate salt as a formulation were reconstituted with SWFI, and the reconstituted solution was diluted with SWFI to the required dosage strength for administration via subcutaneous route. Similarly, lyophilized placebo vials were reconstituted with SWFI and diluted to the required strength using SWFI for administration to control animals.

[0266] When a SC dose range of 10 mg / kg to 120 mg / kg q8h was evaluated, the compound of formula (I) formate salt was effective in reducing A. baumannii ATCC 17978 bacterial load at 10 mg / kg and 30 mg / kg q8h, and demonstrated either bacteriostatic or bactericidal effects against A. baumannii ATCC 17978 at a dose of 60 mg / kg q8h, with logarithmic kill being achieved at higher doses of 90 mg / kg and 120 mg / kg q8h in the neutropenic mouse thigh model.

[0267] Compound (I) formate was more effective against this A. baumannii strain than polymyxin B (25 mg / kg q8h administered SC) and subcutaneous meropenem (200 mg / kg q8h) at doses ≥90 mg / kg q8h. Figure 4 shows the CFU / g of femurs in mice infected with Acinetobacter baumannii ATCC 17978 and treated subcutaneously with [compound (I) formate]], meropenem (200 mg / kg), or polymyxin B (25 mg / kg) administered q8h (every 8 hours). To demonstrate the efficacy of compound (I) formate when administered subcutaneously in vivo, a neutropenic murine double-thigh model of A. baumannii ATCC 17978 was used. When a SC dose range of 10 mg / kg to 120 mg / kg q8h was evaluated across three replicate studies, the compound of formula (I) formate salt was effective in reducing A. baumannii ATCC 17978 bacterial load at 10 mg / kg and 30 mg / kg q8h, causing log kill at ≥ 60 mg / kg q8h in a general dose-dependent manner.

[0268] Thus, formulations containing the compound of Formula (I) formate salt demonstrated either bacteriostatic or killing effects against A. baumannii ATCC 17978 at a dose of 60 mg / kg q8h, with logarithmic killing achieved at higher doses of 90 mg / kg and 120 mg / kg q8h in the neutropenic mouse thigh model. Formulations of the present disclosure were more effective against this A. baumannii strain than polymyxin B (25 mg / kg q8h administered SC) and subcutaneous meropenem (200 mg / kg q8h) at doses ≥ 90 mg / kg q8h.

[0269] Advantages of the present disclosure The present disclosure provides a formulation comprising a compound of Formula (I) formate salt together with a solubilizing agent, a pH adjusting agent, and a hydrotropic agent. The formulation of the present disclosure is stable for a long period of time and is stable in a pH range of 2 to 6. The formulation of the present disclosure is stable at temperatures ranging from -25°C to 10°C. Moreover, the formulation of the present disclosure can be lyophilized, and stability can be improved. The present disclosure also provides a reconstituted formulation that makes the formulation in an injectable form. The present disclosure further provides a process for preparing the formulation that is easy to adapt and suitable for large-scale production. The present disclosure also provides a formulation that can treat infectious diseases caused by microbial organisms. The present disclosure also provides methods of treating or preventing diseases or conditions mediated by microbial organisms, including complicated and uncomplicated urinary tract infections (cUTIs, e.g., pyelonephritis, cystitis), intra-abdominal infections (cIAIs), bloodstream infections, hospital-acquired bacterial pneumonia (HABP, hospital-acquired pneumonia), ventilator-associated bacterial pneumonia (VABP), community-acquired bacterial pneumonia (CABP), secondary cystic fibrosis infections (CFIs), skin and soft tissue infections (SSTIs), endocarditis, meningitis, dysentery and diarrhea, typhoid, Clostridium difficile-associated colitis and diarrhea (CDAD). , Helicobacter pylori-associated peptic ulcer disease, sexually transmitted diseases caused by Chlamydia trachomatis, gonorrhea caused by Neisseria gonorrhea, syphilis caused by Treponema pallidum, or bioterrorism-related bacterial diseases caused by anthrax (Bacillus anthracis), bubonic plague (Yersinia pestis), tularemia (Francisella tularensis), glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), and Q fever (Coxiella burnetii).

Claims

1. a. A compound of formula (I) or a salt thereof; 【Chemical 1】 b.Solubilizers; c. hydrotropic agents; and d. pH adjuster A formulation comprising:

2. 2. The formulation of claim 1, comprising a compound of formula (I) or a salt thereof in the weight range of 1% to 30% (w / w); a solubilizing agent in the weight range of 5% to 55% (w / w); a hydrotropic agent in the weight range of 1% to 30% (w / w); and a pH adjusting agent in the weight range of 0.5% to 30% (w / w).

3. 2. The formulation of claim 1, wherein the solubilizing agent is selected from lactic acid, ascorbic acid, acetic acid, propionic acid, succinic acid, gluconic acid, benzoic acid, tartaric acid, glutaric acid, malic acid, fumaric acid, or a combination thereof; the pH adjusting agent is selected from potassium hydroxide, sodium hydroxide, l-arginine, histidine, glycine, sodium bicarbonate, or a combination thereof; the hydrotrope is selected from niacinamide, sodium benzoate, sodium citrate, sodium acetate, or a combination thereof; and the salt of the compound of formula (I) is selected from formate, mesylate, esylate, besylate, tosylate, acetate, propionate, fumarate, maleate, tartrate, succinate, glycolate, glutamate, aspartate, hydrochloride, hydrobromide, or sulfate.

4. 10. The formulation of claim 1, which is stable at temperatures ranging from -70°C to 10°C.

5. 10. The formulation of claim 1 having a pH in the range of 2 to 6.

6. 10. The formulation of claim 1, comprising a vehicle, wherein the vehicle is water.

7. 10. The formulation of claim 1, wherein the formulation is capable of killing or inhibiting the growth of a microbial organism, the microbial organism being selected from a bacterium, a virus, a fungus, and a protozoan.

8. 10. The formulation of claim 1, which is an anti-infective agent against microbial organisms.

9. The microbial organisms may be Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Citrobacter spp., Proteus spp., Serratia marcescens, Salmonella spp., Morganella morganii, Klebsiella oxytoca, Klebsiella aerogenes, Providencia spp., Neisseria gonorrhoeae, Mycoplasma spp., Campylobacter sp., Fusobacter spp., Bacteroides fragilis, Prevotella spp., Shigella spp., Helicobacter pylori, Ureaplasma spp., Burkholderia gladioli, Burkholderia multivorans, 9. The formulation of claim 7 or 8, wherein the bacterium is selected from Pandoraea apista, Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia mallei, Stenotrophomonas maltophilia, Achromobacter spp., Ralstonia picketii, Legionella pneumophila, Clostridium difficile, Staphylococcus aureus, coagulase-negative Staphylococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

10. 10. The formulation of claim 1, which inhibits an enzyme selected from bacterial gyrase, topoisomerase IV, or a combination thereof.

11. 10. The formulation of claim 1, further comprising an additive, a therapeutic agent, or a combination thereof.

12. 12. The formulation of claim 11, wherein the excipient is selected from sorbitol, dextrose, monobasic sodium phosphate, dibasic sodium phosphate, sodium citrate, sodium bicarbonate, sodium chloride, or combinations thereof.

13. A lyophilized formulation comprising the formulation of claim 1.

14. 14. A reconstituted formulation comprising the lyophilized formulation of claim 13 or the formulation of claim 1 together with a reconstitution agent and a diluent.

15. 15. The reconstituted formulation of claim 14, wherein the reconstitution agent is water; the diluent is selected from dextrose, sorbitol, sodium bicarbonate, glucose, mannitol, sucrose, or sodium chloride; and the diluent is in the mass range of 0.9 to 25% (w / v) in the first solvent.

16. 16. The formulation of claim 15, wherein the first solvent is water.

17. a. a first compartment containing the formulation of claim 1 or 13 or 14 b. a second compartment containing an accelerator; and c. Optional accessories Kit including:

18. 18. The kit of claim 17, wherein the facilitator is selected from water for injection; and the accessories are selected from a diluent bag, tubing for injection, needles, and connectors.

19. 10. A method for preparing the formulation of claim 1, comprising: a. contacting a solubilizing agent with a compound of formula (I) or a salt thereof in the presence of a second solvent under stirring at a temperature in the range of 18°C to 40°C to obtain a first solution; b. adding a hydrotrope to the first solution under stirring to obtain a second solution; and c. mixing a pH adjuster into the second solution, optionally followed by adding a vehicle to obtain the formulation A method comprising:

20. 20. The method of claim 19, wherein the two or more solubilizing agents are mixed prior to contacting with the compound of formula (I) or a salt thereof.

21. 20. The method of claim 19, wherein the first solution has a pH in the range of 2 to 3; and the second solution has a pH in the range of 2.5 to 4.

22. 20. The method of claim 19, wherein the formulation is filtered after adding the vehicle.

23. 20. The method of claim 19, wherein the second solvent is water; and the vehicle is water.

24. 20. A method for preparing a lyophilized formulation as defined in claim 19, comprising the steps of: a) freezing the formulation of claim 1 at a temperature in the range of 0 to -50°C, followed by drying at a pressure in the range of 10 μbar to 1 bar to obtain a lyophilized formulation.

25. 25. The method of claim 24, wherein freezing is carried out for a time period ranging from 30 minutes to 40 hours; and drying is carried out for a time period ranging from 20 minutes to 150 hours.

26. 15. A method for preparing the reconstituted formulation of claim 14, comprising contacting the lyophilized formulation of claim 13 with a diluent, followed by adding a reconstitution agent.

27. 27. The method of claim 26, carried out for a period of time ranging from 60 seconds to 10 minutes.

28. 27. The method of claim 26, wherein the reconstituted formulation is stable at a temperature in the range of 20°C to 35°C for a time period ranging from 20 hours to 30 hours.

29. 27. The method of claim 26, wherein the reconstituted formulation has a pH in the range of 3 to 5; and an osmolality in the range of 300-500 mOs-mol / kg.

30. Use of a formulation according to claim 1 or claim 13 or claim 14.

31. Use of the kit according to claim 17.

32. 15. A method of treating a microbial infection, comprising administering to a subject in need thereof an effective amount of the formulation of claim 1 or claim 13 or claim 14.

33. 33. The method of claim 32, wherein the microbial infection is caused by a bacterium, a virus, a fungus, or a protozoan.

34. 15. A method of treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of the formulation of claim 1 or claim 13 or claim 14.

35. 35. The method of claim 34, wherein the disease or condition is mediated by a gram-positive, gram-negative bacterial species, or a combination thereof.

36. The disease or condition may be complicated and uncomplicated urinary tract infections (cUTI, e.g., pyelonephritis, cystitis), intra-abdominal infections (cIAI), bloodstream infections, hospital-acquired bacterial pneumonia (HABP, hospital-acquired pneumonia), ventilator-associated bacterial pneumonia (VABP), community-acquired bacterial pneumonia (CABP), secondary cystic fibrosis infections (CFI), skin and soft tissue infections (SSTI), endocarditis, meningitis, dysentery and diarrhea, typhoid, Clostridium difficile-associated colitis and diarrhea (CDAD), Helicobacter pylori-associated peptic ulcer, chlamydia trachomatis, and urinary tract infections (URI).

35. The method of claim 34, wherein the bacterial infection is selected from a sexually transmitted disease caused by S. lachomatis, gonorrhea caused by Neisseria gonorrhea, syphilis caused by Treponema pallidum, or a bioterrorism-associated bacterial disease caused by anthrax (Bacillus anthracis), bubonic plague (Yersinia pestis), tularemia (Francisella tularensis), glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), or Q fever (Coxiella burnetti).

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