Pharmaceutical development

Abstract

To provide a pharmaceutical product in the form of a storage stable lyophilisate or a pharmaceutical formulation in the form of a sterile solution for parenteral administration.SOLUTION: Provided are a lyophilisate and a solution, both consisting essentially of a polymyxin selected from polymyxin E, polymyxin B, or a pharmaceutically acceptable derivative thereof, and zidovudine or a pharmaceutically acceptable derivative thereof, and the solution further includes an aqueous carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical preparations in the form of a storage-stable lyophilisate and a sterile solution for parenteral administration, both of which contain polymyxin and zidovudine, or pharmaceutically acceptable derivatives thereof, and which may be useful in the treatment of Gram-negative bacterial infections, such as infections caused by Enterobacteriaceae or Enterobacter species. [Background technology]

[0002] The emergence of multidrug-resistant Gram-negative bacteria that cause hospital-acquired infections is becoming a global problem. In fact, in the United States, more than 70% of bacteria that cause hospital-acquired infections are resistant to at least one of the major antimicrobial agents typically used to treat infections (Nature Reviews, Drug Discovery, 1, 895-910(2002)).

[0003] These limited treatment options have led to the increasing clinical use of polymyxin antibiotics, colistin and polymyxin B, both of which were developed more than 50 years ago. Unlike many other antimicrobial agents, these compounds retain activity against many multidrug-resistant pathogens. This is likely due to their limited clinical use. For example, parenteral formulations of colistin were approved in the 1960s but quickly abandoned due to the availability of more suitable treatment options. Nevertheless, polymyxins are now considered critically important antimicrobial agents and are attracting widespread interest and research.

[0004] Polymyxins are a group of naturally occurring, multicomponent, cyclic polypeptide antibiotics produced by certain strains of the spore-forming soil bacterium Paenibacillus polymyxa (Bacillus polymyxa var. colistinus). Five major, chemically distinct members of this group are recognized, designated polymyxins A, B, C, D, and E, of which B and E are commercially available and approved in the United States and Europe. Polymyxin B is approved for topical use, while polymyxin E, commonly referred to as colistin, is approved for oral, parenteral, and inhaled use. Two forms of colistin are in clinical use: colistin sulfate for oral administration and its prodrug, colistimethate sodium (CMS), for parenteral and inhaled administration. Li et al. (Lancet Infect Dis. 2006 Sep;6(9):589-601) describe colistin as "a re-emerging antibiotic for multidrug-resistant Gram-negative infections."

[0005] However, resistance to polymyxins is on the rise, and growing evidence suggests that monotherapy may result in the emergence of resistance to polymyxins. A potential solution to this problem is polymyxin combination therapy, which has been demonstrated to improve bacterial kill and prevent the emergence of resistance. Hu et al. (Hu et al., Antimicrob Agents Chemother. 2018 Dec 21;63(1)) describe how azidothymidine, in combination with colistin, exhibits synergistic activity against antibiotic-resistant Enterobacteriaceae.

[0006] Azidothymine (AZT), also known as zidovudine, is a nucleoside analogue reverse transcriptase inhibitor and a type of antiretroviral drug approved in the United States and Europe for the treatment of HIV / AIDS infection. In addition to its antiretroviral activity against HIV, the antibacterial effects of zidovudine have been demonstrated in experimental models of Gram-negative bacterial infection both in vitro and in vivo (Herman et al., Antimicrob Agents Chemother. 1992 May;36(5):1081-1085).

[0007] The synergistic effect between polymyxin and zidovudine was first discovered by the present inventors and is described in International Patent Application PCT / GB2014 / 050878, published as WO2014 / 147405A1. Further experiments on the ratio of zidovudine to colistin were also conducted by the present inventors and are described in International Patent Application PCT / GB2016 / 053901, published as WO2017 / 098274A1.

[0008] However, the art does not disclose or teach methods for preparing pharmaceutical formulations or medicaments for successful parenteral administration of a therapeutically effective amount of polymyxin in combination with a therapeutically effective amount of zidovudine. Each of these compounds is commercially available only as a monotherapy product, and WO2014 / 147405A1 and WO2017 / 098274A1 disclose the combination itself, along with the possibility of preparing pharmaceutical compositions containing the combination. However, neither of these prior applications discloses a feasible method for preparing a medicament containing the combination that can be used to parenterally administer therapeutically effective amounts of both compounds to a subject in need of treatment. These prior disclosures focus on the synergistic effect between the two active substances in an in vitro environment. There is also no disclosure of a suitably storage-stable form of this combination that would allow for the effective and reliable preparation of an injectable solution.

[0009] Therefore, commercial exploitation of the combination of polymyxin (e.g., colistin) and zidovudine requires the development of a formulation that is acceptable in terms of storage stability and ease of preparation. However, because of the synergistic relationship between the two compounds, formulating an acceptable drug product or pharmaceutical formulation is not straightforward and presents challenges not encountered when formulating the individual compounds. In particular, it is not possible to predict how this relationship will affect the solubility of each compound in organic or other solvents and / or whether the product will be storage stable.

[0010] It is an object of the present invention to provide a pharmaceutically useful formulation of polymyxin and zidovudine, in particular a formulation comprising a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, and zidovudine or a pharmaceutically acceptable derivative thereof as active ingredients. Summary of the Invention

[0011] In a first aspect, the present invention provides a pharmaceutical product in the form of a lyophilizate, the lyophilizate consisting essentially of a therapeutically effective amount of a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof, and optionally one or more buffering agents. The lyophilizate is shelf-stable and can be stored in vials or cartridges for reconstitution with an aqueous carrier (e.g., water or an aqueous buffer solution) to produce a sterile solution for parenteral administration.

[0012] Thus, in a second aspect, the present invention provides a pharmaceutical formulation in the form of a sterile solution for parenteral administration, the solution consisting essentially of a therapeutically effective amount of a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof, an aqueous carrier, and optionally one or more buffering agents.

[0013] Advantages of the first and second aspects include the preparation of a shelf-stable form of the combination that allows for the efficient and reliable preparation of a pharmaceutically useful formulation, i.e., a sterile solution for parenteral administration, which is also shelf-stable, as described in more detail below.

[0014] In a third aspect, the present invention provides a medicament or pharmaceutical formulation as defined herein for use in the treatment of a Gram-negative bacterial infection. Preferably, the Gram-negative bacterial infection is caused by a bacterium selected from the genera Enterobacteriaceae, Enterobacter, Pseudomonas and Acinetobacter, such as Enterobacteriaceae or Enterobacter. In various embodiments, the infection is caused by a (multi-)drug-resistant strain of bacteria.

[0015] In a fourth aspect, the present invention provides a method for treating a Gram-negative bacterial infection, comprising administering a medicament or pharmaceutical formulation as defined herein to a subject in need of treatment. Preferably, the Gram-negative bacterial infection is caused by a bacterium selected from the genera Enterobacteriaceae, Enterobacter, Pseudomonas and Acinetobacter, such as Enterobacteriaceae or Enterobacter. In various embodiments, the infection is caused by a (multi-)drug-resistant strain of bacteria.

[0016] In a fifth aspect, the present invention provides a sealed vial containing a medicament or pharmaceutical formulation as defined herein.

[0017] In a sixth aspect, the present invention provides a method for producing a pharmaceutical product in the form of a shelf-stable lyophilizate, the method comprising mixing a therapeutically effective amount of a polymyxin selected from polymyxin E, polymyxin B, or a pharmaceutically acceptable derivative thereof, a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof, and an aqueous carrier to form a solution, sterile filtering the solution, filling the filtered sterile solution into one or more vials, and lyophilizing the filled vials. Each individual vial has a fill volume of about 10 mL to about 20 mL. A buffer may be present.

[0018] In various embodiments of the invention, the polymyxin is polymyxin E or a pharmaceutically acceptable derivative thereof. For example, the polymyxin E or a pharmaceutically acceptable derivative thereof can be selected from the group consisting of colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

[0019] In various embodiments of the invention, the therapeutically effective amount of polymyxin is from about 0.5 million international units (MIU) to about 14 million international units (MIU). Preferably, the amount of polymyxin is from about 1 MIU to about 12 MIU, and more preferably, the amount of polymyxin is from about 2 MIU to about 9 MIU. The units used to dose polymyxin are discussed in more detail below.

[0020] In various embodiments of the present invention, the therapeutically effective amount of zidovudine is about 50 mg to about 1500 mg. Preferably, the amount of zidovudine is about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0021] In various embodiments of the invention, the medicament or pharmaceutical formulation comprises a greater amount of polymyxin compared to zidovudine by weight, for example, the weight ratio of polymyxin to zidovudine can be from about 8:1 to about 11:10, e.g., about 2:1.

[0022] In various embodiments of the methods of the present invention, the polymyxin is mixed with the aqueous carrier prior to mixing with the zidovudine.

[0023] These aspects and embodiments are set out in the accompanying independent and dependent claims. It will be understood that features of the dependent claims may be combined with each other and with features of the independent claims in combinations other than those explicitly set out in the claims. Further, the methodology described herein is not limited to the specific embodiments described below, but includes and contemplates any suitable combination of the features described herein. [Brief explanation of the drawings]

[0024] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0025] [Figure 1] 1 shows an overlay of CMS peak patterns from the stability study obtained in Example 1A. From bottom to top of the graph, the patterns are for the CMS drug substance, the sample at TO, the sample after 24 hours at 5°C, and the sample after 24 hours at 25°C. DETAILED DESCRIPTION OF THE INVENTION

[0026] Aspects and features of certain examples and embodiments are discussed and described herein. Some aspects and features of certain examples and embodiments may be conventionally implemented and will not be discussed or described in detail for the sake of brevity. Accordingly, it will be understood that aspects and features of the apparatus and methods discussed herein that are not described in detail may be implemented in accordance with conventional techniques for implementing such aspects and features.

[0027] As described herein, the present invention relates to a pharmaceutical product of polymyxin and zidovudine that exhibits storage stability both at controlled room temperature and under accelerated conditions. Surprisingly, this pharmaceutical product is a lyophilizate consisting essentially of the two active ingredients and, optionally, one or more buffer(s). In a preferred embodiment, the lyophilizate consists of the two active ingredients. In other words, the lyophilizate does not contain any additives, such as solubilizers or stabilizers, that significantly affect the stability of the lyophilizate or the solubility of the active ingredients. This is a surprising solution to the problem of developing a stable pharmaceutical product for the synergistic combination of colistin and zidovudine. Because the lyophilization process typically depends on the additives selected, lyophilizates almost always contain one or more additives, such as polyol-based stabilizers, to stabilize the active ingredients contained therein. Thus, the inventors were surprised to discover that the combination of polymyxin and zidovudine does not require any stabilizing or solubilizing additives to prepare a storage-stable lyophilizate, and that the stable lyophilizate is further suitable for reconstitution with an aqueous carrier (e.g., water) to prepare a sterile solution for parenteral administration. The solution may be administered after reconstitution or may be combined with an infusion solution and then administered. The inventors have surprisingly found that infusion solutions containing the sterile solution of the present invention are stable under both ambient and storage conditions, e.g., at 2-8°C.

[0028] The present inventors were also surprised to discover that aqueous carriers for pharmaceutical formulations of polymyxin and zidovudine are useful. Although zidovudine is poorly soluble in water, the presence of polymyxin, particularly CMS, was found to enhance the solubility of zidovudine, allowing the use of aqueous solvent systems.

[0029] definition As used herein, the phrases "consists essentially of" or "consisting essentially of" mean that a product or formulation contains, in addition to the required ingredients listed, other ingredients, such as trace amounts of impurities, that do not materially affect the essential characteristics of the composition. It is noteworthy that the phrases "consists essentially of" or "consisting essentially of" exclude additives that have a significant effect on the stability of the lyophilizate and / or the solubility of the active agent in an aqueous carrier. The present invention is based on the discovery that polymyxin and zidovudine can be lyophilized to stable lyophilizates without the need for any added stabilizers and further formulated in aqueous solvent systems suitable for parenteral administration without the need for solubilizing agents.

[0030] With the exception of optional buffering agents, the term "excipient" is used herein to refer to "substances other than a pharmacologically active drug or prodrug that are included in the manufacturing process or contained in the finished pharmaceutical dosage form," as defined by the International Pharmaceutical Excipients Council. Thus, the term excipient can include, but is not limited to, bulking agents such as sugars, amino acids, polymers, complexing agents, solubilizing agents such as surfactants and cosolvents, tonicity agents, additional antimicrobial agents, and collapse temperature modifiers.

[0031] As used herein, the phrases "consists of" or "consisting of" mean that the product or formulation contains only the essential ingredients listed.

[0032] As used herein, the term "shelf-stable" means that a product or formulation is stable when stored under specified conditions, such as 5°C ± 3°C or room temperature (25°C), for a specified period of time, such as 6 to 30 hours. Stability can be measured by methods known in the art and can include analyzing the formulation after storage for changes in the amount of zidovudine or polymyxin or impurity levels. These changes can be measured using techniques known in the art. For example, such techniques include high-performance liquid chromatography (HPLC) and variations thereof, such as reverse-phase HPLC (RP-HPLC).

[0033] HPLC typically uses pressure to force a mobile phase through a column containing a stationary phase, and those skilled in the art would understand how to perform HPLC and RP-HPLC to measure the stability of a formulation. HPLC can be used to assess stability, for example, by analyzing the impurity profile generated. For example, one measure is the peak area % of an impurity detected by HPLC, or the total peak area % of all impurities detected by HPLC. These measurements can be compared to measurements of the formulation before storage or to measurements of a formulation standard.

[0034] In various embodiments of the invention, the formulations are storage stable in that there is no significant change in the levels of zidovudine, polymyxin and / or impurities over a period of time, where significant means statistical significance as typically determined in the art.

[0035] Suitable stability assays are also described in the Examples. These include a hybrid RP-HPLC method for simultaneously quantifying the purity and quantity of zidovudine and CMS before and after storage for 6, 12, 24, or 30 hours at, for example, 5°C ± 3°C or room temperature (approximately 25°C). This hybrid method is an improvement over the RP-HPLC method for CMS described in Bai et al., "A simple HPLC method for separation of colistimethate sodium and colistin sulfate," J Chromatograph Separat Techniq 2011:2(1). The method by Bai et al. (which is incorporated herein by reference) can be performed to quantify CMS and then modified to quantify zidovudine. The modifications include decreasing the gradient from 3% / min to 1.5% / min and increasing the detection wavelength from 214 nm to 265 nm to improve resolution and separate impurities from the main zidovudine peak. Chromatographic conditions are described in Example 1.

[0036] The stability of CMS and zidovudine can also be assessed according to European Pharmacopoeia (Ph.Eur.) methods, which are incorporated herein by reference, including the RP-HPLC method for related substances of CMS-Na according to European Pharmacopoeia 9.5 (07 / 2017:0319); the method for determination of free colistin according to European Pharmacopoeia 9.5 (07 / 2017:0319); and the RP-HPLC method for zidovudine according to Monograph European Pharmacopoeia 9.5 (01 / 2017:1059).

[0037] Other suitable methods for analyzing colistimethate sodium and identifying the various components of CMS include the HPLC method described in WO 2014 / 195405 A1 (Xellia Pharmaceuticals APS) and the aqueous HPLC method described in Li et al. Antimicro. Agents. Chemo (2003) 47, 4. Analytical methods for determining the potency of colistimethate sodium (CMS) in synergistic antimicrobial combinations are disclosed in co-pending application GB1910777.0, which is incorporated herein by reference. For combinations of CMS and zidovudine, the potency of CMS in the combination can be determined using Pseudomonas aeruginosa in an agar diffusion assay.

[0038] The term "lyophilisate" is used herein to refer to a material produced by a lyophilisation or freeze-drying process. There is no limitation on the specific conditions used for freeze-drying, and it is believed that one of ordinary skill in the art would be able to readily determine suitable conditions for obtaining a lyophilisate according to the present invention.

[0039] The term "sterile" is used herein in the medical context to mean aseptic or free from bacteria or other living microorganisms. The pharmaceutical formulations according to the present invention are sterile solutions in the sense that they can be administered parenterally to a subject in need of treatment.

[0040] The term "pharmaceutically acceptable derivative" is used herein to refer to a pharmaceutically acceptable prodrug, salt, or ester of a particular compound. Pharmaceutically acceptable salts of the compounds of the present invention include suitable acid addition or base salts thereof. A comprehensive list of suitable pharmaceutical salts can be found in Berge et al., J Pharm Sci, 66, 1-19 (1977). Salts are formed with strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid, or hydrohalogen acids; with strong organic carboxylic acids, such as unsubstituted or substituted (e.g., by halogens) alkanecarboxylic acids having 1 to 4 carbon atoms; with saturated or unsaturated dicarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, or tetraphthalic acid; with hydroxycarboxylic acids, for example, ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid; with amino acids, for example, aspartic acid or glutamic acid; with benzoic acid; or with organic sulfonic acids, for example, unsubstituted or substituted (e.g., by halogens) (C1-C4) alkyl or aryl sulfonic acids, for example, methanesulfonic acid or p-toluenesulfonic acid.

[0041] Esters are formed using organic acids or alcohols / hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids such as alkanecarboxylic acids having 1 to 12 carbon atoms, unsubstituted or substituted (e.g., by halogen), such as acetic acid; saturated or unsaturated dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, or terephthalic acid; hydroxycarboxylic acids such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid; amino acids such as aspartic acid or glutamic acid; benzoic acid; or organic sulfonic acids, such as unsubstituted or substituted (e.g., by halogen) (C1-C4) alkyl or aryl sulfonic acids, such as methanesulfonic acid or p-toluenesulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and aluminum hydroxide. Alcohols include alkane-alcohols having 1 to 12 carbon atoms, which may be unsubstituted or substituted (e.g., by halogen).

[0042] The present invention also includes solvated forms of the compounds.

[0043] The term "therapeutically effective amount" is used herein in reference to an amount useful in a medical context. The specific amount of each active agent to be incorporated into a formulation can be determined based on the active agent and the intended end use of the formulation. Therapeutically effective amounts of polymyxin E, polymyxin B, and their pharmaceutical derivatives are typically expressed in the art in units "IU," "IU," "MIU," or "MIU." These acronyms stand for "International Unit" or "Million International Units." The same nomenclature is used herein.

[0044] For polymyxin B and its pharmaceutically acceptable derivatives, such as polymyxin B sulfate, it is generally accepted in the art that 1 mg is equivalent to approximately 10,000 IU, which means that 5 MIU is considered to be equal to 500 mg (10,000 x 500 = 5,000,000).

[0045] For polymyxin E and its pharmaceutically acceptable derivatives, such as colistin sulfate, colistin methanesulfonate, and colistin methanesulfonate sodium, the meaning of 1 MIU depends on the compound used and its potency. For example, colistin base has a potency of approximately 30,000 IU / mg, while colistin sulfate has a potency of approximately 20,500 IU / mg, and CMS has a potency of approximately 12,500 IU / mg. This means that 5 MIU of colistin sulfate, with a potency of 120,500 IU / mg, corresponds to an amount of approximately 244 mg, and 5 MIU of CMS, with a potency of 2,500 IU / mg, corresponds to an amount of 400 mg.

[0046] The potency of polymyxin E or its derivatives is determined according to the European Pharmacopoeia Microbiological Assay for Antibiotics: European Pharmacopoeia 2.7.2. This assay, incorporated herein by reference, is used to determine antibiotic activity by comparing the inhibition of growth of susceptible microorganisms produced by three doses of a reference substance and three doses of a test antibiotic. It is well recognized and understood in the art that one skilled in the art can easily convert the IU or MIU units herein to the amount of antibiotic used in mg. As mentioned above, the potency of CMS in synergistic combination with zidovudine can be determined by the method disclosed in co-pending application GB1910777.0.

[0047] It is also known that dosage labeling for colistin-based products differs between the United States and Europe. In the United States, CMS is labeled and administered according to milligrams of colistin reference activity (CBA). CBA is calculated based on international units, and manufacturers often report both CBA and IU or MIU values. In Europe, CMS is labeled and administered according to international units of CMS. The European SmPC for colomycin includes the following approximate CMS conversion table, which is used herein:

[0048] [Table 1]

[0049] Combination Products The pharmaceutical products and pharmaceutical formulations of the present invention are therapeutic combination products of polymyxin and zidovudine, where polymyxin is selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, and zidovudine is the compound itself or a pharmaceutically acceptable derivative thereof.

[0050] Polymyxin E, polymyxin B, and zidovudine are well known in the art and, for the sake of brevity, will not be described in detail herein. Polymyxin E, also known as colistin, along with its pharmaceutically acceptable derivatives, such as colistin sulfate, colistin methanesulfonate, and colistin methanesulfonate sodium, as well as polymyxin B or its pharmaceutically acceptable derivatives (e.g., polymyxin B sulfate) can be obtained from commercial sources or by known methods. CMS can be produced, for example, by chemical modification of colistin, a mixture of antibiotics. In particular, the primary amino group of colistin is converted to a methylsulfonate group (Barnette et al., Brit. J. Pharmacol. (1964), 23, 552). Commercial sources of polymyxin active substance include Sigma-Aldrich, Livzon Pharmaceutical Group (China), and Xellia Pharmaceuticals ApS (Denmark).

[0051] Zidovudine can be obtained by known methods or from commercial sources such as Sigma-Aldrich, Hetero Labs Ltd. (India), or EDQM (European Directorate for the Quality of Medicines & Healthcare).

[0052] Pharmaceutically acceptable derivatives of polymyxin E and polymyxin B are known in the art and include the known prodrugs colistin methanesulfonate and colistin methanesulfonate sodium, as well as pharmaceutically acceptable salts of polymyxin E and polymyxin B, such as colistin sulfate and polymyxin B sulfate.

[0053] The medicaments and formulations of the present invention contain a therapeutically effective amount of each of polymyxin and zidovudine, the term "therapeutically effective amount" being defined above.

[0054] In various embodiments, the medicament or pharmaceutical formulation is utilized for therapeutic purposes, particularly for the treatment of gram-negative bacterial infections, which may be caused by drug-resistant strains of bacteria and / or may be caused by bacteria selected from the genera Enterobacteriaceae, Enterobacter, Pseudomonas, or Acinetobacter, for example. This use entails parenteral administration of the reconstituted lyophilizate by injection in a dose reflecting the prescribed treatment.

[0055] In various embodiments, the therapeutically effective amount of zidovudine is about 50 mg to about 1500 mg. Preferably, the amount of zidovudine is about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to 500 mg. It will be understood that these weight ranges can be combined with the therapeutically effective amounts of polymyxin described below. It will also be understood that these weight ranges can be combined with other features of the invention described herein, such as the weight ratio of polymyxin to zidovudine.

[0056] For example, when administered as an injection volume of about 20 mL, the above amount of zidovudine corresponds to a concentration range of about 2.5 mg / mL to about 75 mg / mL, preferably about 5 mg / mL to about 50 mg / mL, more preferably about 7.5 mg / mL to about 40 mg / mL, and most preferably about 7.5 mg / mL to about 25 mg / mL. Other injection volumes are, of course, possible, and one skilled in the art can readily convert the required therapeutically effective amount of zidovudine in mg units to a mg / mL concentration.

[0057] As explained above, amounts of polymyxin antibiotics are conventionally reported in international units or million international units. In various embodiments, a therapeutically effective amount of polymyxin or a pharmaceutically acceptable derivative thereof is from about 5000 IU to about 14 MIU. Preferably, it is from about 5000 IU to about 12 MIU, and more preferably, it is from about 5000 IU to about 9 MIU.

[0058] When the polymyxin is polymyxin B or a pharmaceutically acceptable derivative thereof (e.g., polymyxin B sulfate), the therapeutically effective amount of polymyxin in the pharmaceutical product or pharmaceutical formulation of the present invention is about 5000 IU to about 5 MIU, preferably about 5000 IU to about 4 MIU, and more preferably about 5000 IU to about 1 MIU.

[0059] When the polymyxin is polymyxin E or a pharmaceutically acceptable derivative thereof (e.g., colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium), the therapeutically effective amount of polymyxin in the medicament or pharmaceutical formulation of the present invention is about 0.5 MIU to about 14 MIU, preferably about 1 MIU to about 12 MIU, more preferably about 2 MIU to about 9 MIU, and most preferably about 2 MIU to about 5 MIU.

[0060] In the case of a CMS in which the polymyxin has a potency of 12,500 IU / mg, these values ​​correspond to an amount of about 40 mg to about 1,120 mg, preferably about 80 mg to about 960 mg, more preferably about 160 mg to about 720 mg, and most preferably about 160 mg to about 400 mg.

[0061] In various embodiments of the invention, the medicament or formulation contains polymyxin B or a pharmaceutically acceptable derivative thereof in an amount of about 5000 IU to about 5 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to 800 mg, and most preferably about 150 mg to about 500 mg.

[0062] In various embodiments of the invention, the medicament or formulation contains polymyxin B or a pharmaceutically acceptable derivative thereof in an amount of about 5000 IU to about 4 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0063] In various embodiments of the invention, the medicament or formulation contains polymyxin B or a pharmaceutically acceptable derivative thereof in an amount of about 5000 IU to about 1 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0064] In various embodiments of the invention, the pharmaceutical preparation comprises polymyxin E or a pharmaceutically acceptable derivative thereof in an amount of about 0.5 MIU to about 14 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0065] In various embodiments of the invention, the pharmaceutical preparation comprises polymyxin E or a pharmaceutically acceptable derivative thereof in an amount of about 1 MIU to about 12 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0066] In various embodiments of the invention, the medicament or formulation comprises polymyxin E or a pharmaceutically acceptable derivative thereof in an amount of about 2 MIU to about 9 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0067] In various embodiments of the invention, the medicament or formulation comprises polymyxin E or a pharmaceutically acceptable derivative thereof in an amount of about 2 MIU to about 5 MIU and zidovudine or a pharmaceutically acceptable derivative thereof in an amount of about 50 mg to about 1500 mg. Preferably, zidovudine is present in an amount of about 100 mg to about 1000 mg, more preferably about 150 mg to about 800 mg, and most preferably about 150 mg to about 500 mg.

[0068] The examples of the present application demonstrate how preferred embodiments contain polymyxin in a weight amount greater than the amount of zidovudine. Specifically, if the amount of polymyxin in IU or MIU is converted to X mg by weight and zidovudine is contained in Y mg by weight, then X>Y. In more preferred embodiments, the weight ratio of polymyxin to zidovudine can be defined. Suitable weight ratios of polymyxin to zidovudine are disclosed in WO 2017 / 098274 A1, and these weight ratios are incorporated herein by reference. However, a more preferred weight ratio is about 8:1 to about 11:10 (polymyxin:zidovudine).

[0069] Thus, in various embodiments of the present invention, the weight ratio of polymyxin to zidovudine is about 8:1 to about 11:10, preferably about 6:1 to about 9:7, more preferably about 5:1 to about 6:4, and most preferably about 3:1 to about 2:1.

[0070] As noted above, in preferred embodiments, the medicaments and pharmaceutical formulations of the present invention do not contain any additives other than any buffering agent(s). The term "consisting essentially of" is used herein to define the medicaments and pharmaceutical formulations such that other undefined additives are excluded. Such additives include those well known in the art and approved by regulatory agencies for parenteral administration. Examples include bulking agents, solubilizers, tonicity agents or adjusters, additional antimicrobial agents, and collapse temperature adjusters.

[0071] Bulking agents, as the name suggests, provide bulk to the lyophilisate and provide sufficient structure to the cake. They are also referred to herein as stabilizers and can be sugars such as mannitol, lactose, sucrose, trehalose, sorbitol, glucose and raffinose, amino acids such as arginine, glycine and histidine, or polymers such as dextran and polyethylene glycol. Mannitol and glycine are the most commonly used bulking agents, followed by glucose, sucrose, lactose, trehalose and dextran.

[0072] Solubilizers are added when the drug has low solubility or when the drug is crystalline and remains amorphous or difficult to crystallize after lyophilization. They can be complexing agents such as EDTA or cyclodextrin, surfactants such as polysorbates (e.g., polysorbate 80), or cosolvents such as tert-butyl alcohol, isopropyl alcohol, dichloromethane, ethanol, acetone, or glycerol. Surfactants are typically added to low-dose products to minimize losses due to surface adsorption. Cosolvents can be used to increase the rate of primary drying by increasing the rate of sublimation, improve product stability, shorten reconstitution times by improving drug wettability and solubility, and even improve sterility assurance of sample solutions.

[0073] Isotonicity agents or adjusters are added to parenteral formulations to make them isotonic with human plasma. Examples include sodium chloride, sucrose, mannitol, and glucose, with glucose being the most commonly used. Additional antimicrobial agents can be added if the product is intended for multiple uses and / or to prevent microbial growth during storage. Examples include benzyl alcohol, phenol, m-cresol, methylparaben, ethylparaben, and mixtures thereof.

[0074] Freeze-drying amorphous materials requires that the primary drying temperature be kept below the collapse temperature of the formulation. However, some amorphous excipients have very low collapse temperatures, which significantly lengthens the primary drying period. In such cases, the use of collapse temperature modifiers can increase the overall collapse temperature, shortening the primary drying cycle without compromising product quality. Examples include dextran, hydroxyethyl starch, Ficoll®, and gelatin.

[0075] Optionally, one or more buffering agents may be included if a particular pH is desired and can be any acid or salt combination, such as acetate, tartrate, or citrate, that is pharmaceutically acceptable and capable of maintaining the solution within a particular pH range. The buffering agent can be selected from the group consisting of citric acid, sodium citrate, potassium citrate, tartaric acid, sodium phosphate, sodium acetate, potassium acetate, and mixtures thereof.

[0076] In various embodiments, the medicament or pharmaceutical formulation does not include a bulking agent. In various embodiments, the medicament or pharmaceutical formulation does not include a bulking agent selected from sugars, amino acids, and polymers.

[0077] The pharmaceutical preparation of the present invention is suitable for parenteral administration. As used herein, parenteral administration refers to direct injection into the body, bypassing the skin and mucous membranes. In various embodiments, parenteral administration refers to intradermal, intraperitoneal, intramuscular, subcutaneous and intravenous administration. Preferably, the pharmaceutical preparation of the present invention is suitable for intravenous administration. Therefore, due to the presence of an aqueous carrier, the pharmaceutical preparation of the present invention is in the form of a solution, particularly a sterile solution.

[0078] In various embodiments, the pharmaceutical formulation of the present invention is a single-vial injectable concentrate, which is a sterile solution in a single vial for dilution with an infusion fluid. In other embodiments, the pharmaceutical formulation is a double-vial injectable concentrate that must be mixed with a diluent before it can be further diluted with an infusion fluid, or a diluted injectable concentrate, which is a double-vial injectable concentrate mixed with a diluent for further dilution with an infusion fluid. Alternatively, the pharmaceutical formulation is a final diluent for infusion, which is a single-vial injectable concentrate for administration or a diluted injectable concentrate, combined with an infusion fluid.

[0079] Therefore, the aqueous carrier contained in the pharmaceutical formulation of the present invention can refer to the liquid used to prepare a single-vial injectable concentrate, diluent, and / or infusion solution. An infusion solution is typically a sterile isotonic solution used to dilute an injectable concentrate for administration to a patient. Based on this, an aqueous carrier can be defined as a pharmaceutically acceptable liquid containing water as the primary solvent. In various embodiments, the aqueous carrier is composed of at least about 75% water by weight. In various embodiments, the aqueous carrier is composed of at least about 80% water by weight, preferably at least about 85% water by weight, more preferably at least about 90% water by weight, and most preferably at least about 95% water by weight.

[0080] In various embodiments, the aqueous carrier may further be selected from the group consisting of water, saline, and sugar solutions such as glucose or dextrose solutions. For example, the aqueous carrier may be a 0.9% by weight saline (sodium chloride) solution, a 5% by weight glucose solution, or a 5% by weight dextrose solution. However, the invention is not limited to these specific examples, and other pharmaceutically acceptable aqueous carriers are known in the art and can be used herein.

[0081] In various embodiments, the aqueous carrier is completely free of organic solvents.

[0082] Medical Use The present invention further provides medicaments and pharmaceutical formulations as defined herein for use in the treatment of a Gram-negative bacterial infection. The present invention also provides a method of treating a Gram-negative bacterial infection comprising administering to a subject in need thereof a medicament or pharmaceutical formulation as defined herein.

[0083] In particular, the medicaments or pharmaceutical formulations can be used to kill vegetative and / or clinically latent bacteria associated with Gram-negative bacterial infections. Thus, reference herein to treating a bacterial infection includes killing vegetative and / or clinically latent bacteria associated with such infections. Preferably, the products and formulations of the present invention are used to kill clinically latent bacteria associated with Gram-negative bacterial infections.

[0084] As used herein, "kill" means loss of viability as assessed by lack of metabolic activity.

[0085] As used herein, "clinically latent bacteria" refers to bacteria that are metabolically active but have a growth rate below the threshold for manifestation of infection. The threshold for manifestation of infection refers to a growth rate threshold below which there are no symptoms of infection in the host. The metabolic activity of clinically latent bacteria can be measured by several methods known to those skilled in the art, such as by measuring mRNA levels in the bacteria or by measuring the uridine incorporation rate of the microorganism. In this regard, compared to bacteria under logarithmic growth conditions (in vitro or in vivo), clinically latent bacteria exhibit a lower, but still significant, metabolic activity. (i) mRNA levels (e.g., 0.0001 to 50%, e.g., 1 to 30%, 5 to 25%, or 10 to 20% of the level of mRNA); and / or (ii) uridine (e.g., [ 3 H]uridine) incorporation levels (e.g., [ 3 0.0005 to 50%, e.g., 1 to 40%, 15 to 35%, or 20 to 30% of the level of [H]uridine incorporation) It has.

[0086] Clinically latent bacteria typically have many distinguishing characteristics. For example, they may be viable but non-culturable; that is, the microorganisms are usually undetectable by standard culture techniques, but are detectable and quantifiable by techniques such as broth dilution counting, microscopy, or molecular techniques such as polymerase chain reaction. Furthermore, clinically latent bacteria are phenotypically resistant and therefore susceptible (in log phase) to the bacteriostatic effects of conventional antimicrobial agents (i.e., bacteria for which the minimum inhibitory concentration (MIC) of a conventional antimicrobial agent remains substantially unchanged), but are significantly reduced in susceptibility to drug-induced killing (e.g., bacteria for which the ratio of minimum microbicide concentration (e.g., minimum bactericidal concentration, MBC) to MIC is 10 or greater for any given conventional antimicrobial agent).

[0087] As used herein, the term "bacteria" (and derivatives thereof, such as "bacterial infection") includes, but is not limited to, reference to the following classifications and specific types of organisms (or infections by organisms):

[0088] Gram-negative cocci, e.g. Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava, and Neisseria weaveri; Enterobacteriaceae, e.g., Escherichia coli, Enterobacter spp. (e.g., Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter agglomerans and Enterobacter cloacae), Citrobacter spp. (such as Citrobacter freundii and Citrobacter divernis), Hafnia spp. (e.g., Hafnia alvei), Erwinia spp. (e.g., Erwinia persicinus), Morganella morganii, Salmonella spp. (Salmonella enterica and Salmonella typhi), Shigella spp. (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Klebsiella (e.g., Klebs. pneumoniae, Klebs. oxytoca, Klebs. ornitrichica),ornitholytica, Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs. granulomatis (Calymmatobacterium granulomatis and Klebs. rhinoscleromatis), Proteus (e.g., Proteus mirabilis, Proteus rettgeri and Proteus vulgaris), Providencia (e.g., Providencia alcalifaciens), alcalifaciens, Providencia rettgeri, and Providencia stuartii), Serratia (e.g., Serratia marcescens and Serratia liquifaciens) and Yersinia (e.g., Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis); Helicobacter (e.g., Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fenella). fennellii); Acinetobacter species (e.g., Acinetobacter baumanii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter ruwofii, Acinetobacter spp. ...lwoffi and Acinetobacter radioresistens; Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas maltophilia, Stenotrophomonas maltophilia), Pseudomonas alcaligenes, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas oryzihabitans, Pseudomonas spp. oryzihabitans, Pseudomonas pertocinogena, Pseudomonas pseudalcaligenes, Pseudomonas putida, and Ps. stutzeri; Bacteriodes fragilis; Haemophilus species (e.g., Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus) parahaemolyticus); Actinobacillus spp. (e.g., Actinobacillus actinomycetemcomitans, Actinobacillus equuli, Actinobacillus hominis, Actinobacillus lignieresi)Actinobacillus lignieresii, Actinobacillus suis, and Actinobacillus ureae; Brucella (e.g., Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis); Campylobacter (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus); Vibrio (e.g., Vibrio cholerae and Vibrio parahaemolyticus) parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus and Vibrio vulnificus; Spirochetaceae, for example, Borrelia (e.g., Borrelia recurrentis, Borrelia burgdorferi, Borrelia afzelii, Borrelia andersonii). andersonii, Borrelia bissettii, Borrelia garinii, Borrelia japonica, Borrelia lusitaniae, Borrelia tanukitanukii, Borrelia turdi, Borrelia valaisiana, Borrelia caucasica, Borrelia crocidurae, Borrelia duttoni, Borrelia graingeri, Borrelia hermsii, Borrelia hispanica, Borrelia latyschewii, Borrelia mazzottii, Borrelia parkeri, Borrelia persica, Borrelia turikatae turicatae and Borrelia venezuelensis) and Treponema spp. (Treponema pallidum ssp. pallidum, Treponema pallidum ssp. endemicum, Treponema pallidum ssp. pertenue and Treponema carateum); Pasteurella spp. (e.g., Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septicaseptica, Pasteurella pneumotropica, and Pasteurella stomatis; Rickettsia (e.g., Ricksettsii or Coxiella burnetii); Legionella (e.g., Legionella anisa, Legionella birminghamensis, Legionella bozemanii, Legionella cincinnatiensis, Legionella dumoffii, Legionella feeleii, Legionella gormanii, Legionella hakelia, hackeliae, Legionella israelensis, Legionella jordanis, Legionella lansingensis, Legionella longbeachae, Legionella maceachernii, Legionella micdadei, Legionella oakridgensis, Legionella pneumophila, Legionella sainthelensi, Legionella tucsonensis, and Legionella wadhwasi wadsworthii); Moraxella catarrhalis; Stenotrophomonas maltophilia; Burkholderia cepaciacepacia; Burkholderia mallei and Burkholderia pseudomallei; Francisella tularensis; Cardnerella spp. (e.g., Gardneralla vaginalis and Gardneralla mobiluncus); Streptobacillus moniliformis; Flavobacteriaceae, e.g., Capnocytophaga spp. (e.g., Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa ... granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea, and Capnocytophaga sputigena; Bartonella spp. (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana, and Bartonella vinsonii arupensis); Leptospira spp. (e.g., Leptospira biflexa) biflexa), Leptospira borgpetersenii, Leptospira inadai, Leptospira interrogansinterrogans), Leptospira kirschneri, Leptospira noguchi Leptospira noguchii, Leptospira santarosai, and Leptospira weilii; Spirillium (e.g., Spirillum minus); Bacteroides (e.g., Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides merudae) merdae, Bacteroides ovatus, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchinicus, Bacteroides stercoris, Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus and Bacteroides vulgatus);Species of the genus Prevotella (e.g., Prevotella bivia, Prevotella buccae, Prevotella corporis, Prevotella dentalis (Mitsuokella dentalis), Prevotella denticola, Prevotella disiens, Prevotella enoeca, Prevotella heparinolytica, Prevotella intermedia, Prevotella loeschii, Prevotella melaninogenica, Prevotella nigrescens) nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulora, Prevotella tannerae, Prevotella venoralis and Prevotella zoogleoformans);Porphyromonas spp. (e.g., Porphyromonas asaccharolytica, Porphyromonas cangingivalis, Porphyromonas canoris, Porphyromonas cansulci, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas crevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas levii and Porphyromonas macacae);Fusobacterium species (e.g., Fusobacterium gonadiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrogenes, Fusobacterium necrophorum necrophorum, Fusobacterium necrophorum fundiliforme, Fusobacterium nucleatum nucleatum, Fusobacterium nucleatum fusiforme, Fusobacterium nucleatum polymorphum) polymorphum, Fusobacterium nucleatum vincentii, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium ulcerans, and Fusobacterium varium; Chlamydia (e.g., Chlamydia trachomatis); Chlamydophila (e.g., Chlamydophila abortus, Chlamydia psittaci, Chlamydophila pneumoniae, Chlamydophila pneumoniae), and Chlamydophila psittaci)(Chlamydia psittaci));The genus Leuconostoc (e.g., Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc pseudomesenteroides); and the genus Gemella (e.g., Gemella bergeri, Gemella haemolysans, Gemella morbillorum, and Gemella sanguinis);

[0089] Specific bacteria that can be treated with the products or formulations of the present invention include enterobacteria such as Escherichia coli, Klebsiella (e.g., Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris); Enterobacter, Pseudomonas, and Acinetobacter. Preferably, the bacterium is an enterobacterium such as Escherichia coli, Klebsiella (e.g., Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g., Proteus mirabilis, Proteus rettgeri and Proteus vulgaris), or an Enterobacter species. Most preferably, the bacterial infection being treated is an infection caused by one or more of Escherichia coli, Klebs. pneumoniae or Enterobacter species. In all embodiments, the combination therapy is synergistic compared to administration of the combination components taken alone.

[0090] The medicaments and pharmaceutical formulations of the present invention are particularly useful in the treatment of (multiple)drug-resistant ((M)DR) bacteria. For Enterobacteriaceae, drug resistance is most often mediated by carbapenemase-resistant strains and "extended-spectrum β-lactamase" (ESBL) strains, such as New Delhi metallo-β-lactamase-1 (NDM-1)-resistant Klebsiella pneumoniae. It should be noted that while the claimed combinations can first be shown to be functional in the treatment of (M)DR strains, they can also be used to treat non-resistant strains. This is particularly beneficial for the presently claimed combinations, as the primary therapy for Enterobacteriaceae is expensive, patent-protected antimicrobial drugs. Replacing such "prescription" drugs with "generic" antibiotic combinations would be beneficial from a therapeutic standpoint, as well as from a financial / economic standpoint, at a time when governments are seeking to reduce healthcare costs.

[0091] Specific diseases that can be treated using the medicament or pharmaceutical formulation of the present invention include abscesses, bone and joint infections, burns, cystitis, empyema, endocarditis, typhoid fever, epididymitis, eye infections, furuncles, genital infections, inguinal granulomas, infected burns, infections after dental surgery, prosthetic device-related infections, intra-abdominal abscesses, liver abscesses, mastoiditis, nervous system infections, osteomyelitis, orchitis, pancreatitis, pelvic peritonitis, peritonitis, peritonitis associated with appendicitis, pleural effusions, post-operative wound infections, prostatitis, pyelonephritis, pyoderma (e.g., impetigo), salmonellosis, sialadenitis, septic arthritis, typhoid fever, and wound infections; or infections caused by Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, These include infections caused by Proteus oxytoca, Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris.

[0092] It will be clear that references herein to "treatment" extend to prophylaxis as well as the treatment of existing diseases or conditions.

[0093] The pharmaceutical agent or pharmaceutical preparation of the present invention can be provided in a unit dosage form, for example, as described in "Remington: The Science and Practice of Pharmacy," Lippincott Williams and Wilkins, 21 st Edition, (2005). In a preferred embodiment, the pharmaceutical preparation is prepared according to the process of the present invention as described herein.

[0094] Manufacturing method In various embodiments of the present invention, the pharmaceutical product of the present invention is prepared according to the method of the present invention, which comprises mixing a therapeutically effective amount of polymyxin, as defined above, a therapeutically effective amount of zidovudine, as defined above, and an aqueous carrier, as defined above, to form a solution. The solution is sterile filtered and transferred to one or more vials, and the filled vials are lyophilized. The fill volume of each vial is about 10 mL to about 20 mL.

[0095] The addition of the components (i.e., polymyxin, zidovudine, and aqueous carrier) can be accomplished by methods known in the art. For example, one or more components can be added to each other and then placed in a common container for mixing, the components can be added to a common container in a specific order, or the components can be added to a common container simultaneously. In various embodiments, the aqueous carrier can be added to a container followed by the polymyxin and zidovudine. In various embodiments, the polymyxin and zidovudine are added simultaneously. In other embodiments, the polymyxin is added first, followed by the zidovudine.

[0096] The ingredients may be mixed by methods known in the art. For example, the ingredients may be mixed by simple mixing, or may be mixed continuously, periodically, or a combination thereof using a mixing device. Mixing devices include, but are not limited to, magnetic stirrers, shakers, paddle mixers, homogenizers, and any combination thereof. The addition and mixing of one or more ingredients may be performed under controlled conditions. For example, the addition and mixing of the ingredients may be performed under conditions such as nitrogen or a specific humidity, or the addition and mixing may be performed at a specific temperature. In certain embodiments, the addition and mixing may be performed at a temperature of about 5°C to about 40°C. Furthermore, the addition and mixing may be performed under controlled light, such as yellow light, or under protection from direct exposure to light.

[0097] After the solution is prepared, it is sterilized by sterilization or aseptic filtration (e.g., filtration through a 0.2 μm disposable sterile membrane filter or other suitable filter known in the art). The sterile solution is filled into one or more pre-sterilized vials, aseptically stoppered, and then lyophilized.

[0098] Lyophilization is a freeze-drying process well characterized in the art. It is a process in which water is frozen and typically removed from a sample in two steps: first, sublimation in a step called "primary drying," and second, desorption in a step called "secondary drying." Most lyophilized formulations contain additives to improve the functional properties and stability of the lyophilized product. However, the present invention does not involve the use of such additives. The lyophilization process performed as part of the process of the present invention is not particularly limited and is believed to be readily determinable by one of skill in the art. Once lyophilized, the vials can be stored at a temperature of about 2°C to about 10°C.

[0099] The present disclosure will now be illustrated with reference to the following non-limiting examples. [Example]

[0100] Example 1: Solubility Test The objective of Example 1 was to provide a combination formulation containing CMS or colistin methanesulfonate sodium and zidovudine. CMS is an inactive prodrug that is enzymatically hydrolyzed to colistin sulfate, a salt of the antimicrobial agent polymyxin E, after parenteral administration. The antiretroviral agent zidovudine is also a prodrug that must be triphosphorylated to be effective and acts as an antibiotic resistance buster (ARB). Colistin sulfate and zidovudine are two registered drugs, and they should be combined into a single antibiotic combination. However, this has never been done before, and the possibility of solubility mismatches between the two active substances cannot be ruled out. Therefore, the inventors conducted extensive solubility and stability studies to identify common, compatible solvents.

[0101] A) Solubility and stability in aqueous solvent systems To identify compatible solvents for both agents, CMS, zidovudine, and their combination were screened in aqueous solvent systems. A binary buffer system consisting of acetate and phosphate (5 mM acetate and 5 mM phosphate) was used to cover a pH range of approximately 4.5 to 7.5. After complete dissolution of the solids, the pH was adjusted to the desired value using hydrochloric acid.

[0102] The target dose was 400 mg CMS and 200 mg zidovudine per vial, and the materials were dissolved in a volume of appropriate solvent suitable for lyophilization: 10 mL to 20 mL. The target concentrations were 40 mg / mL CMS (400 mg / 10 mL) and 20 mg / mL zidovudine (200 mg / 10 mL). If the solids did not dissolve at the target concentrations, additional buffer was added to achieve concentrations of 20 mg / mL CMS and 10 mg / mL zidovudine.

[0103] The stability of the active substance in solution was tested when samples were stored for 24 hours at 2-8°C and 25°C, as shown in Table 1. Samples were analyzed before and after storage by the hybrid RP-HPLC method described below.

[0104] Table 1: Stability storage conditions in buffered aqueous systems [Table 2]

[0105] Materials and Methods The materials used in Example 1A are listed in Table 2 and were obtained from commercial sources.

[0106] [Table 3]

[0107] A hybrid RP-HPLC method was used to simultaneously quantify the purity and quantity of zidovudine and CMS. This hybrid method was an improvement of the RP-HPLC method for CMS described by Bai et al. (Bai et al., "A simple HPLC method for separation of colistimethate sodium and colistin sulfate," J Chromatograph Separation Techniq 2011:2(1)). This method was performed to quantify CMS according to Bai et al. (the method is incorporated herein by reference) and then improved to quantify zidovudine. The changes were to decrease the gradient from 3% / min to 1.5% / min and increase the detection wavelength from 214 nm to 265 nm to improve resolution and separate impurities from the main zidovudine peak. The following chromatographic conditions were used:

[0108] [Table 4]

[0109] Mobile phase A was prepared by adding 0.5 mL of TFA to 1000 mL of purified water. Mobile phase B was prepared by adding 0.5 mL of TFA to 1000 mL of ACN. The sample and drug substance were dissolved in mobile phase A at 2 mg / mL for CMS and 1 mg / mL for zidovudine.

[0110] Results and Conclusions Both active agents were dissolved in aqueous solvent systems at all pH values. All solutions containing CMS had a pH between 7.1 and 7.8, indicating that CMS also exhibits strong buffering capacity at neutral to slightly basic pH values. Zidovudine did not affect the pH of the solutions.

[0111] During stability screening, no degradation products of zidovudine were observed. Zidovudine appears to be stable in aqueous solution regardless of pH (4.5-7.5). CMS was most stable at pH 7.5, as evidenced by a peak pattern comparable to that of CMS bulk material after 24 hours at 25°C (see Figure 1).

[0112] Therefore, the aqueous solvent system with a fill volume of 20 mL is a promising candidate for pharmaceutical formulation of CMS and zidovudine.

[0113] B) Solubility and stability in organic solvent systems Following the results of Example 1A, further screening was performed in an organic solvent system. C, Z, and C+Z were screened by varying the concentration of the organic solvent tert-butanol (TBA). The target dose was set at 240 mg of CMS and 200 mg of zidovudine per vial. The material was dissolved in a volume appropriate to justify the use of an organic solvent-based system (reducing the fill volume per vial by a factor of four, resulting in a fill volume of ∼5 mL for the organic solvent-based system). Samples were tested for stability of the API in solution when stored at 2-8°C and 25°C for 24 hours, and the samples were analyzed before and after storage by the hybrid RP-HPLC method described above. Chromatographic conditions were the same as those described above for Example 1A.

[0114] material The following materials were used (Table 3), all of which were obtained from commercial sources. [Table 5]

[0115] Results and Conclusions In the solubility test, the TBA concentration was varied in four steps from 20% (by weight) to 60% (by weight). The target concentrations were 48 mg / mL CMS (240 mg / 5 mL) and 40 mg / mL zidovudine (200 mg / 5 mL).

[0116] CMS was found to dissolve at all TBA concentrations in 5 mL. The solid dissolved within minutes. Zidovudine did not dissolve in 5 mL of 20% TBA (solids were allowed to dissolve under stirring for 30 minutes), but did dissolve in 5 mL of 30%, 50%, and 60% TBA. Similar results were observed for mixtures of CMS and zidovudine as for zidovudine alone (all solids completely dissolved in 5 mL of 30%, 50%, and 60% TBA).

[0117] In the stability study, C+Z solutions containing 30%, 50%, and 60% TBA were prepared and stored at room temperature and 5°C for 24 hours, respectively, and then monitored. No zidovudine degradation products were observed at either 25°C or 5°C during the stability screening of the mixed C+Z solution. With 30% TBA, slight precipitation was observed with the naked eye in the vials at 5°C. However, the peak areas of zidovudine and CMS remained unchanged by hybrid RP-HPLC. Most likely, zidovudine precipitated due to its relatively low solubility in 30% TBA at low temperatures. The amount of precipitated zidovudine was too small to be quantified by RP-HPLC.

[0118] Unfortunately, the peak pattern from CMS in the mixed C+Z DP dissolved in a TBA-based solvent system showed differences from the CMS standard dissolved in water. The reason for the different peak pattern of CMS dissolved in a TBA-based solvent system was thought to be the formation / presence of different methanesulfonic acid derivatives in TBA than in water, but this was not clear and could not be verified by further peak evaluation.

[0119] Example 2: Freeze drying with organic solvent system To determine whether the organic solvent system is suitable for the production of lyophilized forms of C+Z, lyophilization studies were performed on the C+Z combination in a TBA / water mixture. The formulations listed in Table 4 were lyophilized.

[0120] [Table 6]

[0121] The formulations were mixed and filled into washed and heat-sterilized 20R glass vials. The filled vials were stoppered with autoclaved and dried lyophilization stoppers, sealed in lyophilization bags, and placed in the lyophilizer. Samples were lyophilized using the appropriate lyophilization cycle. Ten vials were prepared for each variant. Chamber pressure was monitored and recorded via online data acquisition to detect the end of sublimation. Samples were then analyzed by degradation profile immediately after lyophilization (T0).

[0122] material Table 5 lists the materials used in Example 2, all of which were obtained from commercial sources.

[0123] [Table 7]

[0124] method Measurement of free colistin: Measurement of free colistin was performed according to the European Pharmacopoeia 9.5 (07 / 2017:0319).

[0125] Preparation of hydrazine sulfate solution: 1.0 g of hydrazine sulfate was dissolved in purified water, diluted with the same solvent to 100.0 mL, and allowed to stand for 4 to 6 hours.

[0126] Preparation of hexamethylenetetramine solution: 2.5 g of hexamethylenetetramine was dissolved in 25.0 mL of purified water and placed in a 100 mL glass stoppered volumetric flask.

[0127] Preparation of the primary milky suspension: 25.0 mL of the hydrazine sulfate solution was added to the hexamethylenetetramine solution in the measuring flask, mixed, and left to stand for 24 hours.

[0128] Preparation of milky white standard: 15.0 mL of the primary milky white suspension was diluted to 1000.0 mL with purified water. This suspension was freshly prepared and stored for up to 24 hours.

[0129] Preparation of reference suspension: For calibration of the turbidimeter, reference suspensions were prepared according to Table 6.

[0130] [Table 8]

[0131] Assay for free colistin according to European Pharmacopoeia 9.5: One lyophilizate containing 240 mg of CMS was dissolved in 10 mL of purified water. CMS BDS was analyzed by dissolving 80 mg of CMS BDS in 3 mL of purified water. After complete dissolution, 0.1 mL of 100 g / mL tungstosilicic acid was added to 3 mL of test sample (reconstituted lyophilizate or dissolved BDS). The turbidity of the sample was recorded after 10 to 20 seconds. The solution should not be significantly more turbid than the Milky White II standard.

[0132] Freeze-drying procedure: The vials were washed with purified water. The vials were then dried, and the heat-treated stoppers were autoclaved and then dried. The formulated bulk solution was compounded according to Table 4 above. CMS and zidovudine were weighed into a beaker. A TBA / water mixture was added. The solution was stirred at ambient temperature until complete dissolution was observed. Vials were filled with 5 g of the formulated bulk solution via pipette. The stoppers were manually placed in the lyophilization position. The product vials were placed on a stainless steel rack, placed in a lyophilization bag, sealed, and placed in the lyophilizer. Pressure control was achieved via a pressure gauge using a dosing valve and a vacuum valve. The freeze-drying cycle described in Table 7 was performed.

[0133] [Table 9]

[0134] After the lyophilization cycle was completed, the lyophilization chamber was evacuated to 750 mbar with nitrogen and the vials were closed by folding the lyo-shelves together. After evacuating to atmospheric pressure, the vials were removed, capped, and stored at 5°C.

[0135] result The lyophilisates of the test lyophilisations in organic solvent systems were analysed for free colistin. Table 8 shows a summary of the results obtained.

[0136] [Table 10]

[0137] Both lyophilisates (50% TBA and 60% TBA) contained significant amounts of free colistin, meaning that neither was a stable product for commercial use.

[0138] conclusion The levels of free colistin in organic solvent-based lyophilisates were unacceptable, therefore the TBA solvent system is not suitable for combination products.

[0139] CMS is known to those skilled in the art to be stable against hydrolysis because it forms micelles in aqueous solutions of 3.5 mM (approximately 5.7 mg / mL) or higher (Wallace SJ, Li J, Nation RL, Prankerd RJ, Velkov T, Boyd BJ. Colistin and its prodrug colistin methansulfonate, self-assembly behavior of colistin and its prodrug colistin methansulfonate: implications for solution stability and solubilization, J Phy. Chem B, 2010; 114, p. 23. Chem B, 2010; 114, pp. 4836-4840). Below this concentration, rapid hydrolysis of CMS to colistin has been observed (Wallace SJ, Li J, Rayner CR, Coulthard K, Nation RL. Wallace SJ, Li J, Rayner CR, Coulthard K, Nation RL. Stability of Colistin Methanesulfonate in Pharmaceutical Products and Solutions for Administration to Patients. Antimicrob. Agents Chemother, 2008, pp. 3047-3051).

[0140] Without wishing to be bound by any theory, the inventors believe that the organic solvent TBA included in the lyophilization solution shifts the critical micelle concentration (CMC) of CMS to a higher concentration, resulting in hydrolytic instability of CMS in the organic lyophilization solution at a working concentration of 48 mg / mL. Based on the data obtained for the 50% TBA / water and 60% TBA / water mixtures, it is not possible to lyophilize drug-containing CMS using organic solvent-based lyophilization solutions.

[0141] Example 3: Freeze drying using an aqueous solvent system In Example 3, the feasibility of freeze-drying a mixed pharmaceutical product using an aqueous-based solvent system was investigated. Formulations were compounded and filled into cleaned and heat-sterilized 30R glass vials. The filled vials were stoppered with autoclaved and dried lyophilization stoppers, sealed in lyophilization bags, and placed in the lyophilizer. Samples were lyophilized using the same lyophilization cycle as in Example 2. Ten vials were prepared for each variant. Chamber pressure was monitored and recorded via online data acquisition to detect the end of sublimation. Immediately after lyophilization (T0), samples were analyzed for refolding rate and behavior; European Pharmacopoeia degradation profiles for CMS and zidovudine; and residual moisture content by the conventional Karl Fischer oven method.

[0142] material Table 9 lists the materials used in Example 3. All materials were obtained from commercial sources.

[0143] [Table 11]

[0144] method RP-HPLC of related substances of CMS-Na: The RP-HPLC method for related substances of CMS-Na was performed according to the European Pharmacopoeia 9.5 (07 / 2017:0319). The following chromatographic conditions were used:

[0145] [Table 12]

[0146] Preparation of 0.05M phosphate buffer pH 6.5: 7.8 g of sodium dihydrogen phosphate dihydrate was dissolved in approximately 900 mL of purified water. The pH was adjusted to 6.5 with 15% aqueous sodium hydroxide. Finally, the solution was filled to 1000 mL with purified water and filtered through a 0.22 μm membrane filter.

[0147] Preparation of mobile phase A: 25 mL of acetonitrile was added to 475 mL of 0.05 M phosphate buffer pH 6.5.

[0148] Preparation of mobile phase B: 250 mL of acetonitrile was added to 250 mL of 0.05 M phosphate buffer pH 6.5.

[0149] Preparation of sample and reference solutions: The sample and standard solutions were prepared by dissolving 24 mg of CMS-Na in 1 mL of purified water. Immediately after dissolution, the solution was diluted to 2 mg / mL with methanol.

[0150] Measurement of free colistin: Measurement of free colistin was performed according to European Pharmacopoeia 9.5 (07 / 2017:0319). Hydrazine sulfate solution, hexamethylenetetramine solution, primary milky suspension, milky standard, and reference suspension were prepared according to the methods described in Example 2. The assay for free colistin was also prepared according to Example 2.

[0151] RP-HPLC method for zidovudine: RP-HPLC chromatography for zidovudine quantity and purity was performed according to Monograph European Pharmacopoeia 9.5 (01 / 2017:1059). The following chromatographic conditions were used for the analysis:

[0152] [Table 13]

[0153] Preparation of mobile phase A: Mobile phase A was prepared by dissolving 2 g of ammonium acetate in 800 mL of purified water, and the pH was adjusted to 6.8 with acetic acid. The solution was made up to 1 L with purified water.

[0154] Preparation of dilutions: A diluent was prepared by mixing 76 mL of mobile phase A with 4 mL of ACN and 20 mL of methanol.

[0155] Preparation of sample and reference solutions: The sample solution was prepared by dissolving 1 mg of zidovudine in 1 mL of diluent. Reference solution A: 2 mg of impurity C and 2 mg of impurity B were dissolved in 50 mL of diluent. 1 mL of this solution was diluted with 19 mL of diluent. System compatibility solution: 5 mg of zidovudine for SST (containing impurities A, G, and H) was dissolved in 5 mL of reference solution A.

[0156] Karl Fischer titration: 50–150 mg of the corresponding lyophilizate was weighed into a glass vial and sealed with a crimp cap. The sample was transferred to the oven of a Karl Fischer coulometer (756 / 774, Metrohm) heated to 100 °C. The septum of the cap was pierced with a syringe needle, and the generated water vapor was transferred directly to the titration chamber of the Karl Fischer coulometer via dry nitrogen. Two vials per variant were used, with two samples per vial analyzed. An empty glass vial was used for blank correction.

[0157] Restore behavior: The dissolution behavior of the lyophilisate was monitored by adding 10 mL / 20 mL of purified water. The reconstitution process was monitored for dissolution time and behaviour.

[0158] Freeze-drying procedure: The vials were washed with purified water. Then, the vials were dried and heat treated. The stoppers were autoclaved and then dried. The formulation bulk solution was formulated according to Table 10. CMS and zidovudine were weighed into a beaker. Water was added. The solution was stirred at room temperature until complete dissolution was observed.

[0159] [Table 14]

[0160] Vials were filled with 20 mL of formulation bulk liquid using a pipette. Stoppers were manually placed in the freeze-drying position. The product vials were placed on a stainless steel rack, sealed in a freeze-drying bag, and placed in the freeze-dryer. After pressure control, the same freeze-drying cycle as in Example 2 was performed, except that primary drying was terminated after sublimation was complete. After the freeze-drying cycle was completed, the freeze-drying chamber was evacuated to 750 mbar with nitrogen and the vials were closed. After evacuating to atmospheric pressure, the vials were removed, capped, and stored at 5°C.

[0161] result Residual moisture Table 11 gives an overview of the residual moisture content of the lyophilisates.

[0162] [Table 15]

[0163] The water samples showed very low moisture content of less than 1.0%.

[0164] Restoration behavior A reconstitution volume of 20 mL was required to obtain a clear solution.

[0165] Analysis of lyophilisates using the European Pharmacopoeia method (i) Zidovudine related substances The lyophilisates of the test lyophilisates in aqueous lyophilisate solutions were analysed using the method for related substances of zidovudine. The results obtained are shown below.

[0166] [Table 16]

[0167] The purity of the lyophilized zidovudine is comparable to the reference standard, indicating that zidovudine is stable during the lyophilization process.

[0168] (ii) CMS-related substances and compositions The lyophilisates of the test lyophilisation in aqueous lyophilisate were analysed using methods for related substances and composition of CMS-Na. The peak pattern remained unchanged during lyophilisation.

[0169] (iii) Free colistin in the lyophilisate The lyophilisates of the test lyophilisations in aqueous systems were analysed for free colistin and the table gives a summary of the results obtained.

[0170] [Table 17]

[0171] Table 13 shows how the lyophilisates contained small amounts of free colistin, but these amounts were only slightly above the Milky White II standard and significantly reduced compared to the organic solvent system of Example 2. This demonstrates that the aqueous solvent system is an improvement over the organic solvent system and provides a useful formulation of CMS and zidovudine.

[0172] conclusion Surprisingly, we found that a combination of CMS and zidovudine could be prepared using an aqueous solvent system, which provided improved free colistin levels compared to organic solvent systems. All quality attributes of the resulting lyophilizates from the aqueous solvent system were satisfactory, and the free colistin assay showed significant improvement over the organic solvent system.

[0173] Example 4: Stability test of lyophilized product To determine the storage stability of the lyophilisate, the following protocol was carried out on the lyophilisate of Example 3.

[0174] [Table 18]

[0175] Testing at each time point, indicated by A, included solubility, visible particles, Z identification, Z assay, CMS identification, and CMS assay. At each time point, indicated by B, the following properties were evaluated in addition to those indicated for time point A: pH, and sterility. After 3 months at 25°C ± 2°C and 60% ± 5% RH, the lyophilizate was readily soluble in water with a reconstitution time of 29 seconds and contained no visible particles. HPLC and assay results are shown in Tables 15-16.

[0176] [Table 19]

[0177] [Table 20]

[0178] Based on stability data, the drug product is expected to remain within specifications for at least 3 months at controlled room temperature (25°C) and accelerated conditions (40°C).

[0179] Example 5: Infusion stability test Example 5 investigates the stability of the lyophilized product after reconstitution and dilution with infusion solutions. Two infusion solutions (0.9% NaCl and 5% glucose), two storage conditions (+2 to +8°C and +20 to +25°C), and four time points (0 hours (T0), 6 hours (T1), 24 hours (T2), and 30 hours (T3)) were tested. To prepare the infusion solutions, the lyophilized product from Example 2 was first reconstituted with 20 mL of sterile water. The solution was then added to each infusion solution. Details are as follows:

[0180] Stability was monitored using the microbial assay method disclosed in co-pending application GB1910777.0. A randomized block design was used for assay design, with triplicate treatments of test samples and standards in each of six petri dishes. Data evaluation was performed using European Pharmacopoeia 5.3 (Statistical Analysis of Results of Biological Assays and Tests) using the parallel line model. Generally, the CMS activity in the test solution used in the assay should be the same as that in the standard solution. The microorganism used in the assay was Pseudomonas aeruginosa ATCC 27853. The standard solution was colistimethate sodium CRS.

[0181] The expected activity of CMS in the resulting solution was 3,075,000 IU in 100 mL = 30,750 IU / mL. Since the activity of CMS in solution T3 used in the microbial assay should be 12,300 IU / mL, the infusion solution was diluted. For this dilution, a pH 6.0 buffer solution prepared from monopotassium dihydrogen phosphate, sodium hydroxide, and sterile water was used. To ensure the same solvent for all solutions (T3, T2, and T1), further dilution steps were performed using the same mixture of water, infusion solution, and pH 6.0 buffer solution (hereafter referred to as "serial dilutions"). Details are as follows:

[0182] Preparation of stock solutions and reference / standard solutions To prepare the standard stock solution (SL-S), the entire contents of one vial of colistimethate sodium was completely dissolved in 2.0 mL of sterile water. The certified activity in IU / vial was 285,000 IU / vial, meaning that this solution contained approximately 142,500 IU / mL of CMS. The standard stock solution was prepared fresh for each time point.

[0183] Standard solution S0 was prepared by mixing 0.9 mL of SL-S with 3.6 mL of infusion solution (either 0.9% NaCl or 5% glucose). The expected activity of S0 was approximately 28,500 IU / mL CMS. Standard solution S3 was prepared by diluting 2.2 mL of S0 solution to 5.0 mL with buffer solution pH 6.0. The resulting solution S3 contained approximately 12,540 IU / mL CMS. Standard solution S2 was prepared by mixing 2.0 mL of S3 solution with 2.0 mL of serial dilution solution, yielding an expected activity of 6,270 IU / mL CMS. Standard solution S1 was prepared by mixing 2.0 mL of solution S2 with 2.0 mL of serial dilution solution, yielding an expected activity of 3,135 IU / mL CMS.

[0184] Preparation of infusion solutions Test solution TO was prepared by adding 20 mL of sterile water to four vials containing the lyophilized combination. The combination consisted of CMS and zidovudine. After dissolving the contents, a reconstituted solution was obtained with an expected activity of 153,750 IU / mL CMS.

[0185] 20 mL of reconstituted solution was removed from each vial and added to either 80 mL of sterile NaCl infusion or 80 mL of sterile 5% glucose infusion. The expected activity of the resulting solution, TO, was approximately 30,750 IU / mL CMS.

[0186] Immediately after preparation of the infusion solution, approximately 10 mL was removed from each vial and used to prepare the four test solutions described below. The remaining portions of the infusion solution were stored at either 2-8°C or 20-25°C. Approximately 10 mL was removed from the storage solution 6 hours, 24 hours, and 30 hours after preparation of the infusion solution and used to prepare the test solutions described below.

[0187] Preparation of test solutions for combination products 4.0 mL of each of the four TO solutions (or solutions sampled after 6, 24, or 30 hours) was diluted to 10.0 mL with buffer pH 6.0. The expected activity of the resulting solution, T3, was approximately 12,300 IU / mL CMS.

[0188] Solution T2 was prepared by mixing 3.0 mL of T3 solution with 3.0 mL of serial dilution solution to yield a solution with an expected activity of approximately 6,150 IU / mL. Solution T1 was prepared by mixing 2.0 mL of T2 solution with 2.0 mL of serial dilution solution to yield a solution with an expected activity of approximately 3,075 IU / mL.

[0189] Preparation of inoculum of test organisms 100 μL of P. aeruginosa ATCC 27853 glycerol stock solution was inoculated into 5 mL of Caso broth and incubated overnight at 30–35°C for 18–24 hours. Microbial counts of the overnight cultures were determined using the pour plate method. Serial 10-fold dilutions were prepared from the overnight cultures, and 10-fold dilutions were analyzed from these dilutions. -6 and 10 -7 100 μL of each was taken and placed in a sterile Petri dish. The molten, cooled agar was poured into the Petri dish and mixed. After the agar solidified, the plates were inverted and incubated at 30-35°C for 24-72 hours, during which time the overnight cultures were stored at 2-8°C. After incubation, the microbial count was measured, and the overnight cultures showed a microbial count of approximately 1 x 10 7 An inoculum of CFU / mL was prepared.

[0190] Agar plate preparation Two time points (0 and 6 h, and 24 and 30 h), two infusion solutions (0.9% NaCl and glucose for each test point and solution, respectively), and two incubation temperatures (2-8°C and 20-25°C for each test point and infusion solution) were tested per day. Six plates (φ14.5 cm) were required for each condition, resulting in 48 plates per day (96 plates for the entire study).

[0191] The medium Caso-agar was prepared according to methods known in the art. Flasks containing solid cultures were liquefied and thermostated at 45-55°C. An inoculum of P. aeruginosa (approximately 1 x 10 7 Two mL of the liquified inoculated medium (CFU / mL) was pipetted into each flask of warmed medium and mixed thoroughly. To prepare the agar plates, 57 mL of the liquefied and inoculated medium was measured and transferred to each of 48 Petri dishes. All plates were placed precisely level at room temperature for at least 1 hour to allow the agar to solidify. Seven 6 mm diameter cavities were made in each of the 48 agar plates using a sterile biopsy punch, and the plates were stored at 2–8°C until the next day.

[0192] procedure For each time point (including 0 h), 50 μL of sample solutions T1, T2, and T3 and 50 μL of standard solutions S1, S2, and S3 were pipetted into the cavity of each of 24 agar plates. 50 μL of buffer solution was pipetted into the central cavity of each plate. For pre-spreading, the plates were precisely leveled at room temperature for 3 h. All agar plates were then incubated at 30°C–35°C for 18 h. After incubation, the zones of inhibition were measured to the nearest 0.1 mm and evaluated according to the European Pharmacopoeia 5.3.

[0193] result Calculations of the microbial activity of the samples were based on statistical analysis of the results of the bioassays described in European Pharmacopoeia 5.3 using the parallel line model and randomized block design of the assays. These results are presented to three significant figures in Tables 17-20 below.

[0194] [Table 21]

[0195] [Table 22]

[0196] [Table 23]

[0197] [Table 24]

[0198] These results show that all solutions, whether refrigerated or at room temperature, were stable for up to 6 hours. The refrigerated solutions were also stable for up to 30 hours. The room temperature samples were stable for up to 24 hours. This stability is comparable to that of CMS alone, which means that the infusion solution should come with instructions stating that it should be stored at room temperature for no more than 6 hours and refrigerated for no more than 24 hours.

[0199] Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in chemistry or related fields are intended to be encompassed by the appended claims.

[0200] Abbreviation ACN: acetonitrile ARB: Antibiotic Resistance Breaker API: Active Pharmaceutical Ingredient BDS: Active Pharmaceutical Ingredients CMC: Critical micelle concentration CMS: colistimethate sodium C: CMS / Colistimethate sodium DP: Pharmaceuticals DS: Pharmaceutical substance EMA: European Medicines Agency HPLC: High-performance liquid chromatography HS-GC: Headspace gas chromatograph HPLC: High-performance liquid chromatography LC-MS: Liquid chromatography-mass spectrometry RP-HPLC: reversed-phase high-performance liquid chromatography RT: retention time SD: standard deviation TBA: tert-butanol TFA: Trifluoroacetic acid USP: United States Pharmacopoeia Z: Zidovudine

Claims

1. 1. A pharmaceutical product in the form of a storage-stable lyophilisate, said lyophilisate consisting essentially of a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, zidovudine or a pharmaceutically acceptable derivative thereof, and optionally one or more buffering agents.

2. 2. The pharmaceutical product of claim 1, wherein the lyophilisate consists of polymyxin and zidovudine.

3. 1. A pharmaceutical formulation in the form of a sterile solution for parenteral administration, said solution consisting essentially of a therapeutically effective amount of a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof, an aqueous carrier, and optionally one or more buffering agents.

4. 4. The pharmaceutical product of claim 1 or claim 2, or the pharmaceutical formulation of claim 3, wherein the polymyxin is polymyxin E or a pharmaceutically acceptable derivative thereof, preferably the polymyxin E or a pharmaceutically acceptable derivative thereof is selected from the group consisting of colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

5. 5. The medicament or pharmaceutical formulation according to any one of claims 1 to 4, wherein the therapeutically effective amount of the polymyxin or pharmaceutically acceptable derivative thereof is from about 0.5 M.I.U. to about 14 M.I.U., preferably from about 1 M.I.U. to about 12 M.I.U., more preferably from about 2 M.I.U. to 9 M.I.U.

6. The pharmaceutical product or pharmaceutical formulation according to any one of claims 1 to 5, wherein the therapeutically effective amount of zidovudine is from about 50 mg to about 1500 mg, preferably from about 100 mg to about 1000 mg, more preferably from about 150 mg to about 800 mg.

7. 7. A pharmaceutical product or pharmaceutical formulation according to any one of claims 1 to 6, wherein the polymyxin is present in an amount, by weight, that is greater than the amount of zidovudine.

8. 8. The pharmaceutical product or pharmaceutical formulation of claim 7, wherein the weight ratio of polymyxin to zidovudine is from about 8:1 to about 11:10, preferably about 2:

1.

9. A pharmaceutical product or pharmaceutical formulation according to any one of claims 1 to 8 for use in the treatment of a gram-negative bacterial infection.

10. 10. The medicament or pharmaceutical formulation for use according to claim 9, wherein the Gram-negative bacterial infection is caused by Enterobacteriaceae, Enterobacter, Pseudomonas or Acinetobacter, preferably Enterobacteriaceae or Enterobacter.

11. 11. The medicament or pharmaceutical formulation for use according to claim 9 or 10, wherein the infection is caused by a (multi-)drug resistant strain of bacteria.

12. A method for treating a gram-negative bacterial infection, comprising administering the pharmaceutical or pharmaceutical formulation according to any one of claims 1 to 8 to a subject in need of treatment.

13. 13. The method of claim 12, wherein the bacterial infection is caused by Enterobacteriaceae, Enterobacter, Pseudomonas or Acinetobacter, preferably Enterobacteriaceae or Enterobacter.

14. 14. The method according to claim 12 or claim 13, wherein the infection is caused by a (multi-)drug resistant strain of bacteria.

15. A sealed vial containing a pharmaceutical product or pharmaceutical formulation according to any one of claims 1 to 8.

16. A method for producing a pharmaceutical product in the form of a storage-stable lyophilizate, the method comprising mixing a therapeutically effective amount of a polymyxin selected from polymyxin E, polymyxin B or a pharmaceutically acceptable derivative thereof, a therapeutically effective amount of zidovudine or a pharmaceutically acceptable derivative thereof, and an aqueous carrier to form a sterile solution, sterile filtering the solution, filling the filtered solution into vials, and lyophilizing the filled vials.

17. 17. The method of claim 16, wherein the polymyxin is polymyxin E or a pharmaceutically acceptable derivative thereof, preferably wherein the polymyxin E or a pharmaceutically acceptable derivative thereof is selected from the group consisting of colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

18. The method of claim 16 or 17, wherein the therapeutically effective amount of the polymyxin or pharmaceutically acceptable derivative thereof is from about 0.5 M.I.U. to about 14 M.I.U., preferably from about 1 M.I.U. to about 12 M.I.U., and more preferably from about 2 M.I.U. to 9 M.I.U.

19. 19. The method of any one of claims 16 to 18, wherein the fill volume per vial is from about 10 mL to about 20 mL.

20. The method of any one of claims 16 to 19, wherein the therapeutically effective amount of zidovudine is from about 50 mg to about 1500 mg, preferably from about 100 mg to about 1000 mg, more preferably from about 150 mg to about 800 mg.

21. 21. The method of any one of claims 16 to 20, wherein the polymyxin is mixed with the aqueous carrier prior to the addition of the zidovudine.

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