Antibacterial effect of halogenated fluorescein against colistin-resistant Gram-negative bacteria

Abstract

The present invention relates to a method for treating Gram-negative bacteria that are resistant to colistin (MIC > 50 mg / mL), an antibacterial compound, excluding those of the genera Burkholderia, Proteus, and Serratia, comprising contacting the bacteria with an aqueous pharmaceutical composition containing a rose bengal (RB) compound of formula I, dissolved or dispersed at a concentration of about 0.01 to about 15 mg / mL, and irradiating the contacted bacteria with light of a wavelength of about 500 nm to about 600 nm for a period of about 1 to about 10 minutes, at a concentration of about 16 to about 160 J / cm². 2 The aim is to create a method that involves obtaining a certain amount of light to treat and kill irradiated bacteria. [Formula 1] JPEG2026511692000028.jpg5864 (where X, R 1 , R 2 (and M+ are defined herein)

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 455,501, filed on 29 March 2023, entitled "ANTI-BACTERIAL EFFECT OF HALOGENATED FLUORESCEINS AGAINST COLISTIN-RESISTANT GRAM-NEGATIVE BACTERIA," the entire contents of which are incorporated herein by reference. explanation [Background technology]

[0002] Multidrug-resistant (MDR) Gram-negative bacteria have a significant impact on morbidity and mortality [1-4]. Colistin (polymyxin E), an antibiotic first discovered nearly 60 years ago, is effective against most multidrug-resistant Gram-negative bacteria [5, 6]. Except in patients with cystic fibrosis [7-10], colistin has not been widely used since the early 1980s due to its nephrotoxicity. However, colistin has been reintroduced into clinical practice as a last-line drug to treat severe hospital-acquired infections caused by multidrug-resistant Gram-negative bacteria

[11] . Numerous studies have shown a rapid increase in colistin resistance among Enterobacteriaceae [12–14]. Clinical isolates of some of these bacteria, including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Salmonella spp., and Klebsiella spp., have acquired resistance to colistin [15–20]. Therefore, colistin resistance is considered a serious public health problem due to the lack of alternative treatment options. Interestingly, other Pseudomonadota (Proteobacteria) species, such as Serratia spp., Proteus spp., and Burkholderia spp., exhibit spontaneous resistance to colistin

[21] . Colistin-resistant Gram-negative bacteria are often cross-resistant to other antibacterial agents [22, 23]. For example, meropenem, a broad-spectrum carbapenem, is effective at low concentrations (0.03–6.25□g / mL) against drug-susceptible Klebsiella pneumoniae, Pseudomonas aeruginosa, and A. baumannii, but colistin-resistant strains of Pseudomonas aeruginosa, A. baumannii, and Klebsiella pneumoniae show reduced susceptibility to meropenem and other antibiotics [24, 25].

[0003] Colistin-resistant Klebsiella pneumoniae strains are difficult to treat with the currently available drug arsenal [26, 27]. Much research has been dedicated to optimizing the use of currently available drugs or identifying any combination thereof, but none of the pipeline drugs are directed toward resolving drug-resistant bacteria associated with colistin resistance [28-33]. Therefore, there is a need for drugs effective against colistin-resistant strains or methods to prevent the acquisition of colistin resistance during treatment. Singer et al.'s U.S. Patents 8,530,675, 9,273,022, and 9,422,260 describe and claim the synthesis of highly purified rose bengal, as well as similarly purified compounds containing different halogen substituents and different numbers of those halogen substituents, and their lactone derivatives. The highly purified rose bengal used in the above patents was a clinical-stage formulated rose bengal (HB-RBf) composition containing 10% rose bengal w / v in 0.9 percent aqueous sodium chloride (NaCl), provided for research use by Provectus Biopharmaceuticals, Inc., Knoxville, TN) under the name PV-10®. Rose bengal compounds are collectively referred to herein as "halogenated xanthenes," and more specifically as "halogenated fluorescein."

[0004] RB dyes have been clinically investigated for the treatment of melanoma and other solid tumors, particularly when injected directly into cancerous lesions

[36] and PCT / US22 / 05407. The photodynamic antibacterial properties of RB have been reported sporadically

[37] and PCT / US22 / 054076. Dees et al., U.S. Patent No. 8,974,363, instructs the use of topical formulations of RB at 10–100 μM (i.e., 10 μg / mL–100 μg / mL) in combination with green light irradiation in the 500–600 nm wavelength band against Gram-positive and Gram-negative antibiotic-resistant bacteria, but provides no details regarding the bacterial strains used, the light source, or the intensity or duration of the irradiation.

[0005] The inventors screened tissue library molecules containing FDA-approved and unapproved antibacterial agents against two species of Burkholderia, one species of Proteus, one species of Serratia, colistin-resistant Pseudomonas aeruginosa, and wild-type Pseudomonas aeruginosa (reference bacterial strain). The inventors revealed that rose bengal (RB; 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) exhibited strong bactericidal activity (0.05~6.25□g / mL) against colistin-resistant Gram-negative strains under fluorescent light (see Table 1 and

[35] below). However, RB did not show clear antibacterial activity against wild-type other Gram-negative bacteria (Escherichia coli, A. baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) (MIC < 12.5□g / mL). Based on the antibacterial properties of RB described above, the inventors decided to investigate the effectiveness of RB against Gram-negative bacteria that have acquired colistin resistance. To verify the bactericidal effect of RB against colistin-resistant strains, the inventors prepared moderate to high colistin-resistant strains of Escherichia coli, A. baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia species, Salmonella enterica Typhimurium, and Serratia species

[34] . In this specification, the inventors report the susceptibility of pharmaceutical-grade RB [high-purity RB formulation (HP-RBf)] to these colistin-resistant Gram-negative bacteria. [Overview of the project]

[0006] This invention aims to treat and kill Gram-negative bacteria that are resistant to the antibacterial compound colistin (MIC ≥ 50 mg / mL) by irradiating them with light at wavelengths of approximately 500 nm to 600 nm for a period of approximately 1 to 10 minutes, in combination with a rose bengal derivative. In one embodiment, the present invention aims to treat and kill Gram-negative bacteria, excluding those of the genera Burkholderia, Proteus, and Serratia, that are resistant to the antibacterial compound colistin (MIC ≥ 50 mg / mL), by irradiating them with light at wavelengths of approximately 500 nm to 600 nm for a period of approximately 1 to 10 minutes, in combination with a rose bengal derivative. In one aspect, Gram-negative colistin-resistant bacteria, and in certain embodiments, those other than the genus Burkholderia, Proteus, and Serratia, are contacted with an aqueous pharmaceutical composition containing a rose bengal (RB) compound of Formula I in which the bacteria are dissolved or dispersed at a concentration of about 0.01 to about 15 mg / mL, and the contacted bacteria are irradiated with light having a wavelength of about 500 nm to about 600 nm for a period of about 1 to about 10 minutes to obtain a light dose of about 16 to about 160 J / cm 2 are treated in a method comprising the steps. The RB compound of Formula I is shown below:

[0007]

Chemical formula

[0008] Structural formulas of exemplary aromatic ring substituents are shown below:

Chemical formula

Chemical formula

[0009] In a preferred embodiment, the method is performed to inhibit postoperative infection by Gram-negative colistin-resistant bacteria in the area where surgery is performed. The intended aqueous pharmaceutical composition containing the RB compound of Formula I can be applied to the surface of the surgical area by spraying, applying, or any other convenient method, as well as to the intra- and extra-intra [Brief explanation of the drawing]

[0010] In drawings forming part of this disclosure [Figure 1A-1F] Figures 1A, 1B, 1C, 1D, 1E, and 1F are graphs showing the time-to-death dynamics of HP-RBf (10% RB in physiological saline) against colistin-resistant [colistin®] Gram-negative bacteria under fluorescent lighting (17W, 63.8cm2, 0-40J / cm2). MIC values ​​were multiplied by 5: Escherichia coli ATCC35218 colistin®: MIC 400 mg / mL (colistin), Pseudomonas aeruginosa ATCC27853 colistin®: MIC > 400 mg / mL (colistin), Klebsiella pneumoniae ATCC 19606 colistin®: MIC 100 mg / mL (colistin), and A. baumannii ATCC BAA-1800 colistin®: MIC 400 mg / mL (colistin). [Modes for carrying out the invention]

[0011] The present invention relates to a method for treating Gram-negative bacteria that are resistant to the antibacterial compound colistin (MIC ≧ 50 mg / mL), and in one embodiment, Gram-negative bacteria that are resistant to the antibacterial compound colistin excluding the genera Burkholderia, Proteus, and Serratia, the method comprising contacting the bacteria with an aqueous pharmaceutical composition containing a rose bengal (RB) compound of the following formula I dissolved or dispersed at a concentration of about 0.01 to about 15 mg / mL, and irradiating the contacted bacteria with light having a wavelength of about 500 nm to about 600 nm for a period of about 1 to about 10 minutes to obtain a light dose of about 16 to about 160 J / cm 2 to treat and kill the irradiated bacteria.

[0012] The contemplated RB compounds have the following structural formula (Formula I) wherein X is O (oxygen) or N (nitrogen), and "n" is zero or 1.

Chemical formula

[0013] For simplicity, aromatic esters or aromatic amides are collectively referred to as aromatic derivatives. Therefore, these derivatives are formed from preferably monosubstituted alcohols or amines having a single 5- or 6-membered aromatic ring or a 5,6- or 6,6-condensed aromatic ring system, each containing 0, 1, or 2 heterocyclic atoms that are independently nitrogen, oxygen, or sulfur. Examples of such aromatic alcohol ester moieties are shown and named below, where O is an oxygen atom, the line-O indicates that the ring-oxygen may originate from any available carbon in the ring, and the O-line intersecting the wavy line indicates that the depicted alkoxy group is part of another molecule, an esterified RB molecule.

[0014] [ka] During the ceremony, [ka] These provide esters or monosubstituted amines, respectively. [ka] That is the case.

[0015] Rose bengal (RB) is a preferred RB compound, and its disodium salt, rose bengal disodium (RBD), is the most preferred RB compound. These compounds are used herein by reference as a group of RB compounds. The chemical name of rose bengal is 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein. The preferred form, rose bengal disodium (RBD), has the following structural formula: [ka] It has. Certain details of this preferred embodiment of the composition to be contemplated are described in U.S. Patents 5,998,597, 6,331,286, 6,493,570, and 8,974,363, whose disclosures are incorporated herein by reference in their entirety. The aforementioned patents describe the use of RBD for killing cancer cells.

[0016] In one embodiment, mammalian cells, such as human cells, and bacteria infected with Gram-negative colistin-resistant bacteria are brought into contact with an RB compound that is taken up by the infected cells. In another embodiment, Gram-negative colistin-resistant bacteria are present on the surface to be disinfected. Light can be administered together with the intended RB-containing pharmaceutical composition containing the intended RB compound, in which the intended RB compound is present, at a concentration of about 0.01 to about 15 mg / mL, preferably about 0.2 to about 3.1 mg / mL, dissolved or dispersed in a pharmaceutically acceptable diluent, and brought into contact with the infected area. Light can also be administered immediately after application of the pharmaceutical composition for contact with bacteria or mammalian cells containing bacteria, preferably within about 2 to about 5 minutes. Bacteria can be treated while present within infected mammalian cells or on surfaces such as examination tables or operating tables. Although superficially similar to previous treatments, the concentration of the RB compound is lower than previously used, and the light intensity is thought to be about one-tenth or less of that of the previous method. In addition, this treatment is targeted at Gram-negative bacteria other than those of the genera Burkholderia, Proteus, and Serratia that are also resistant to colistin treatment.

[0017] In this specification, colistin resistance, in its various grammatical forms, is used to mean that the MIC is approximately equal to or greater than (≧) a colistin concentration of approximately 50 mg / mL. In most cases, the MIC is ≧ approximately 100 mg / mL, and often ≧ approximately 400 mg / mL. As a result, fewer antibacterial RB compounds can be used, and lower-cost lighting can be used, which makes the process more viable. The infected area treated with RB is irradiated for approximately 1 to 10 minutes, more preferably 2 to 5 minutes. Such irradiation is approximately 16 to 160 J / cm². 2 This results in a light intensity of approximately 32 to 80 J / cm², more preferably about 32 to 80 J / cm². 2 It provides a certain amount of light.

[0018] Pharmaceutical halogenated xanthene composition The intended liquid compositions can be formulated for oral, parenteral, or topical administration. Exemplary halogenated xanthene (fluorescein) compositions are illustrated by reference to the use of rose bengal disodium, a preferred halogenated xanthene compound. Considering parenteral or topical treatment compositions first, the previously mentioned PV-10® composition is an example of a parenterally administered pharmaceutical composition containing a particularly preferred RB compound, rose bengal disodium. The delivery of the halogenated fluorescein component of the intended composition is most favorable when the composition has a pH value close to physiological pH (i.e., approximately pH 7), particularly when the pH value is above approximately 4, thereby ensuring that the halogenated fluorescein remains in a dibasic form within the composition. Therefore, in preferred embodiments, the pH value of the composition is about 5 to about 9, more preferably about 6 to about 7.5, and most preferably about pH 6.5 to about pH 7.4. At these pH values, the halogenated fluorescein typically remains in a dibasic form rather than the lactone that forms at lower pH values.

[0019] RB compounds such as rose bengal are dibasic, with pKa values ​​of 2.52 and 1.81. The determination of the pKa values ​​of several intended fluorescein halides can be found in Batsitela et al., Spectrochim Acta Part A 79(5):889-897 (Sept. 2011). A hydrophilic vehicle is a preferred medium for pharmaceuticals to maximize the distribution preference of halogenated fluorescein components, particularly RB compounds in acid and / or salt forms, into tissues. Therefore, in a preferred embodiment, the vehicle contains a minimum amount of non-hydrophilic components that may interfere with such distribution. Accordingly, a preferred formulation of the composition contains a particularly preferred RB, or RB disodium, in a hydrophilic, preferably water-containing, vehicle. When administered parenterally by means other than suppositories, the RB compound-containing pharmaceutical composition preferably contains a water-soluble electrolyte comprising at least one cation selected from the group consisting of sodium, potassium, calcium, and magnesium, and at least one anion selected from the group consisting of chloride, phosphoric acid, and nitrate. The electrolyte is preferably in concentrations of about 0.1% (w / v) and about 2% (w / v).

[0020] Alternatively, the electrolyte is present at a level sufficient to yield a molal osmotic concentration exceeding approximately 100 mOsm / kg (milliosmoles per kilogram of water) up to approximately 600 mOsm / kg. More preferably, the molal osmotic concentration of the pharmaceutical composition exceeds 250 mOsm / kg, and most preferably is approximately 300-500 mOsm / kg. The electrolyte is preferably sodium chloride. The electrolyte is present at a concentration of about 0.5 to about 1.5%, more preferably about 0.8 to about 1.2%, and most preferably about 0.9%, similar to that present in physiological saline. The aqueous medium (diluent) of the composition is preferably water only, meeting the standards for use in injection. Up to about 20 volume percent of the diluent may be one or more C1-C6 monohydric or polyhydric alcohols such as methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, glycerol, ethylene glycol, propylene glycol, 1,2-butanediol, 2,3-butanediol, erythritol, treitol, trimethylolpropane, and sorbitol. More preferably, the alcohol is present in the intended composition at less than about 10 volume percent, and more preferably less than about 5 volume percent, of the diluent.

[0021] The terms “physiologically acceptable salt” and “pharmaceutically acceptable salt” refer to any non-toxic cations commonly used in the pharmaceutical industry, such as alkali metals, alkaline earth metals, and ammonium salts, including sodium, potassium, lithium, calcium, magnesium, barium, and protamine zinc salts, which can be prepared by methods known in the art, in their various grammatical forms. The intended cations result in water-soluble RB salts. Preferably, the salts are either sodium, potassium, and calcium in monobasic or dibasic salt forms. Readers are directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 for a list of commonly used physiologically (or pharmaceutically) acceptable acids and bases that form physiologically / pharmaceutically acceptable salts with pharmaceutical compounds. The pH value of a RB-containing pharmaceutical composition can be adjusted or modified by any suitable means known to those skilled in the art. The composition can be buffered, or the pH value can be adjusted by adding acids, bases, etc. Since RB or its physiologically acceptable salts are weak acids, depending on their concentrations and / or electrolyte concentrations, the pH value of the composition may not require buffers and / or pH modifiers. However, it is particularly preferable that the composition does not contain buffers, allowing it to adapt to the biological environment once administered. It is preferable for the pharmaceutical composition to contain no preservatives at all, as many preservatives may adversely interfere with the pharmaceutical composition or its formulation, or may form complexes with, otherwise interact with, or interfere with the delivery of, the active ingredient of the RB compound-containing composition. As long as a preservative is used, imidourea is a preferred preservative because it does not interact with RB compounds in the pharmaceutical composition or during administration.

[0022] The intended liquid pharmaceutical composition may also be adapted for oral administration to the mammalian subject being treated. In a preferred embodiment, the RB compound is dissolved or dispersed in an aqueous diluent when administered to the mammalian subject, as described above. The aqueous diluent is more preferably free of isotonic agents, except for sugars and / or buffering agents present as flavoring agents. Up to about 20 volume percent of the diluent, one or more C1-C6 monohydric or polyhydric alcohols may be used, as described above. More preferably, the alcohol is present in the intended composition at less than about 10 volume percent, and more preferably less than about 5 volume percent, of the diluent. Topically applied liquid compositions are also being considered. One such liquid composition undergoing clinical trials for the treatment of psoriasis is a developmental drug called PH-10® by Provectus Biopharamceuticals, Inc. of Knoxville, TN. This drug contains at least one builder present at a level sufficient to give the drug a viscosity of 10–1000 cps, along with sodium chloride as an electrolyte present at a concentration of 0.9% w / v, sufficient to give the drug a molar osmotic pressure concentration of 100 mOsm / kg–500 mOsm / kg, and RBD present at a concentration of 0.001–0.01% w / v dissolved or dispersed in an aqueous diluent. This drug is described and claimed, for example, in U.S. Patent No. 8,974,363.

[0023] Treatment method The proposed treatment method involves contacting Gram-negative bacteria that are resistant to the antibacterial compound colistin (MIC ≥ 50 mg / mL). In one embodiment, with the exception of Burkholderia, Proteus, and Serratia species, the bacterial cells are treated and killed by irradiating them with light having a wavelength of about 500 nm to about 600 nm for a period of about 1 to about 10 minutes, along with a composition containing an anti-Gram-negative amount of the RB compound. The preferred wavelength for irradiation is about 500 to about 575 nm, more preferably about 510 to about 550 nm. The proposed RB compound-containing composition is considered to provide an effective treatment if the MIC, as determined herein, is about 10 mg / mL or less. In one embodiment, Gram-negative bacterial cells are present on or within (e.g., in an infection) the target mammal. For example, the target mammal may have a dermatological Gram-negative bacterial infection, such as those of the genera Klebsiella, Escherichia, or Pseudomonas, or as treatment for an existing infection and for the prevention of subsequent Gram-negative bacteria, especially in the case of local treatment of open or surgical wounds, where the infecting bacteria are colistin-resistant.

[0024] In another embodiment, surfaces such as examination tables, operating room floors and / or walls and / or equipment can be cleaned and irradiated with the composition intended as described above to disinfect or prevent the growth of one or more Gram-negative bacteria as described above. Cleaning and irradiation can be performed concurrently with surgery or other procedures for mammalian subjects to aid disinfection or to improve the overall cleanliness of the mammalian subject and treatment area. The mammals to be treated may include humans, apes such as chimpanzees or gorillas, primates such as crab-eating macaques or macaques, laboratory animals such as rats, mice or rabbits, companion animals such as dogs, cats or horses, or food animals such as cows or bulls, sheep, lambs, pigs, goats or llamas. Each intended composition is typically administered repeatedly until the bacterial disease (infection) being treated has subsided to a desirable level, such as being undetectable. Therefore, administration to the mammalian subject requiring the treatment can be done multiple times a day, daily, weekly, monthly, or over a period of several months to several years, as directed by the treating physician. Gram-negative bactericidal effective doses of RB are administered to the mammalian target requiring them and can be formulated using conventional liquids, gels, creams, or other forms. In most cases, RB is administered with irradiation using a light source that preferably includes emission wavelengths of about 500 to about 600 nm, more preferably about 510 to about 550 nm. Exemplary sources of light sources, their intensity, and their duration are described below.

[0025] result Pharmaceutical-grade rose bengal preparations (HP-RBf) effectively inhibit the growth of colistin-resistant Gram-negative bacteria. The antibacterial activity of rose bengal (RB) has been reported in commercial-grade rose bengal (80–95% pigment content) containing known impurities (e.g., transhalogenated substances) generated by historical manufacturing processes

[36] . Therefore, commercial-grade RB lacks pharmaceutically appropriateness. The RB used in this study had a purity of >99.5% and was synthesized and purified under current Good Manufacturing Practices. The high-purity RB was initially formulated in physiological saline (10% RB in physiological saline; HP-RBf), and then further diluted with physiological saline to the desired concentration.

[0026] The minimum inhibitory concentration (MIC, μg / mL) is approximately 23.0 kJ / cm³ over a 24-hour period. 2 ) and LED lights (approximately 29.0 kJ / cm²) 2 It was obtained by the broth dilution method under irradiation conditions. For example, it was obtained by diluting approximately 0.96 (approximately 1) kJ / cm over 60 minutes (min). 2 Alternatively, approximately 16.0 J / cm² per minute. 2 It will be 17W, 63.8cm 2For studies conducted using fluorescent lamps with exposure times ranging from zero to 30 minutes, the amount of light corresponds to approximately 0 to 480 J / cm². 2 The irradiation described in

[35] for about 1 to 10 minutes is approximately 16 to 160 J / cm². 2 This is the result. HP-RBf effectively inhibited the growth of colistin-resistant Gram-negative bacteria at MIC levels of 0.05–12.5 μg / mL. The bactericidal activity of HP-RBf against colistin-resistant strains observed in Table 1 below did not differ significantly depending on the light source, and the MIC values ​​were equal or very similar for fluorescent and LED lamps

[35] .

[0027] [Table 1]

[0028] HP-RBf showed bactericidal activity against colistin-resistant Escherichia coli (ATCC 35218, >400 g / mL against colistin) with an MIC value of 0.05 g / mL (Table 2 below, Registration 2). Meropenem susceptibility to E. coli was significantly lower when it had acquired colistin resistance (Registration 1 vs 2). E. coli serotype O157 (wild type) is a major foodborne pathogen that has shown susceptibility to colistin, meropenem, and amikacin (Registration 3). The inventors were able to isolate its colistin-resistant mutant with an MIC level of 100 μg / mL (Registration 4).

[0029] [Table 2] JPEG2026511692000013.jpg182161 JPEG2026511692000014.jpg229163

[0030] Wild-type Escherichia coli serotype O157 strain showed very low susceptibility to HP-RBf (Registration 3), but HP-RBf effectively killed its colistin-resistant variant (MIC: 3.13□g / mL) (Registration 3 vs. 4). Drug-susceptible and multidrug-resistant variants of Acinetobacter baumannii (ATCC19606, Registration 5) and A. baumannii (ATCC BAA1800, Registration 7) showed resistance to HP-RBf. On the other hand, their colistin-resistant variants were susceptible to HP-RBf, and the MIC value of HP-RBf was 0.05 μg / mL for these two strains (Registrations 6 and 8). Two wild-type Klebsiella pneumoniae strains (ATCC 19606 and NR48569) were resistant to HP-RBf (MIC > 100 μg / mL) (Registrations 9 and 11), but their colistin-resistant mutants showed increased susceptibility to HP-RBf, with MIC values ​​of 6.3 and 12.5 μg / mL, respectively (Registrations 10 and 12). Furthermore, the inventors created colistin-intermediate (MIC 50 μg / mL) and high (MIC > 400 μg / mL) resistant Pseudomonas aeruginosa mutants (ATCC 27853) (Registered Nos. 14 and 15). The HP-RBf susceptibility of Pseudomonas strains depended on their degree of colistin resistance, with MIC values ​​of 0.05 and 6.3 μg / mL for higher and intermediate colistin-resistant strains, respectively. Similar to the Pseudomonas aeruginosa (ATCC 27853) studied in Table 1, Pseudomonas aeruginosa MRSN 1356 (NR51521) and Pseudomonas aeruginosa MRSN 1380 (NR51522) strains showed low levels of intrinsic susceptibility to HP-RBf under irradiation conditions (Registered Nos. 16 and 18)

[37] . These colistin-resistant strains showed increased HP-RBf susceptibility (Registered Nos. 17 and 19).

[0031] Some Gram-negative bacteria, such as species of the genus Burkholderia, have been reported to be inherently resistant to colistin, with MIC values ​​often >200 mg / mL (see above). Wild-type Burkholderia multivorans CGD1 was found to be colistin resistant at an MIC level of 200 mg / mL (Table 1, Table 2, registration 20)

[21] . Colistin-resistant mutants of B. multivolance CGD1 were highly susceptible to HP-RBf (Registration 21). Similarly, two other Burkholderia species (B. thailandensis E264 and B. cepacia genomovar III, LMG16656) were examined, and their wild types also showed high colistin resistance (Registrations 22 and 24)

[38] . Their colistin-resistant mutants had 8-fold and 16-fold increased susceptibility to HP-RBf (Registrations 23 and 25). Colistin effectively killed most Salmonella species with MIC values ​​of 0.8–1.6 mg / mL (Registrations 26 and 28). HP-RBf was able to inhibit the growth of wild-type Salmonella tiphimuria at an MIC value of 12.5 mg / mL (Registration 26). A 15-fold colistin-resistant mutant of Salmonella tiphimuria showed increased susceptibility to HP-RBf (Registration 27). Colistin-acquiring mutants of other Salmonella species (S. enterica pelsylvenia, Serovar tiphimuria) also showed increased susceptibility to HP-RBf (Registration 29). Proteus and Serratia species are other bacteria that exhibit colistin resistance (Registrations 30 and 32). HP-RBf effectively killed these wild-type strains at low concentrations (Registrations 30 and 32). As observed in all other registrations in Table 2, their colistin-acquiring mutants showed increased susceptibility to HP-RBf (Registrations 31 and 32).

[0032] HP-RBf has the characteristic of rapidly killing colistin-resistant Gram-negative bacteria. The inventors have reported the photodynamic inhibition of growth of Gram-positive bacteria by HP-RBf, and have shown that it inhibits the growth of Gram-positive bacterial cultures (1.3 to approximately 4.6 × 10⁻¹⁰) including drug-resistant strains. 8 The addition of HP-RBf in CFU / ml was able to cause a reduction of less than 6 log of bacterial cells in less than 2 minutes (min)

[35] . Similarly, selected colistin resistance (colistin R ) Mutant strain (Escherichia coli ATCC35218 colistin) R Pseudomonas aeruginosa ATCC27853 colistinR Klebsiella pneumoniae ATCC 19606 colistin R , and A. Baumani ATCC BAA-1800 colistin R A time-course study on ) was conducted over 24 minutes (40 J / cm²). 2 The tests were conducted under fluorescent lighting at a concentration five times that of MIC (HP-RBf). A graphical explanation of the results is shown in Figure 1. The reference molecules used were amikacin (10 mg / mL) and meropenem (10 mg / mL). HP-RBf was 1.3 to approximately 1.9 × 10⁻⁶ in all colistin-resistant strains. 8 Colony-forming units (CFU) were reduced by 6 log in 2 minutes. CFU was measured in culture medium treated with HP-RBf (0.025 or 31.5 mg / mL) at concentrations of 1 × 10⁻¹⁴. 3 Dilutions were not counted (Figures 1A-1D). In the same study, amikacin and meropenem were found in E. coli ATCC35218 colistin, respectively. R Approximately 3.5% (30 min) and 0% (30 min) of the strains were reduced (Figures 1E and 1D). These results demonstrate that HP-RBf has the property of rapidly killing colistin-resistant Gram-negative bacteria under irradiation conditions

[35] .

[0033] Consideration The inventors evaluated the antibacterial activity of pharmaceutical-grade RB formulation products (HP-RBf) under irradiation conditions. The inventors recently reported a comprehensive evaluation of HP-RBf against Gram-positive bacteria and Mycobacterium spp., examining over 45 bacterial strains. HP-RBf demonstrated rapid bactericidal activity against Gram-positive bacteria (MIC 0.3–3.1 mg / mL, less than 2 minutes)

[35] . HP-RBf exhibits strong affinity for the Gram-positive peptidoglycan layer, inducing photodynamic activation and generating reactive oxygen species. Under irradiation conditions, HP-RBf shows moderate bactericidal activity against Mycobacterial spp. with MIC values ​​of 12.5–25.0 mg / mL (12 hours). The inventors hypothesize that a thick cell wall containing mycolic acid reduces the cellular uptake of HP-RBf, considering the MIC values ​​for mycobacterial species, which are higher than the MIC values ​​for Gram-positive bacteria. In previous studies, the inventors concluded that common hospital-acquired Gram-negative bacteria are not susceptible to HP-RBf, and that their MIC values ​​are 25 mg / mL or >50 mg / mL

[35] .

[0034] The inventors expanded their antibacterial screening against Gram-negative bacteria and found that HP-RBf is effective against certain Proteobacteria (Pseudomonadota) species, including Burkholderia, some Salmonella, Proteus, and Serratia species

[21] . The MIC values ​​of HP-RBf are 0.8–12.5 mg / mL compared to their wild types. One characteristic of their drug susceptibility is that they are inherently highly resistant to the action of colistin. HP-RBf killed colistin-resistant mutants of Gram-negative bacteria (Escherichia coli, A. baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) at low concentrations. Colistin-enhanced strains of Burkholderia, Salmonella, Proteus, and Serratia species also showed increased susceptibility to HP-RBf. These data clearly suggest that the acquisition of colistin resistance in Gram-negative bacteria alters the structure of the outer membrane, increasing its affinity for HP-RBf.

[0035] It is an established fact that identified colistin-resistant Gram-negative bacteria are involved in structural changes of lipopolysaccharide (LPS) in which colistin interacts with the loaded electrolipid A of LPS [39-41]. Therefore, increasing the positively charged LPS component (e.g., cationic forms of 4-amino-L-arabinose, phosphoethanolamine, and galactosamine) in colistin-resistant strains could enhance the affinity of loaded HP-RBf. The inventors are in the process of further elucidating the mechanism of action of HP-RBF against colistin-resistant Gram-negative bacteria. The rapid antibacterial photodynamic activity of HP-RBF offers several advantages, including 1) reducing the frequency of drug-resistant strain development and 2) enhancing the safety profile of disinfection and sterilization applications.

[0036] In summary, the inventors' research reported herein suggests that HP-RBf is a drug candidate for treating intrinsic and acquired colistin-resistant Gram-negative bacterial infections. Colistin-resistant Gram-negative bacteria are involved in membrane structure modifications that lead to resistance to other important antibacterial agents used for Gram-negative bacterial infections [40, 41]. Interestingly, these structural changes make HP-RBf more susceptible to colistin-resistant Gram-negative bacteria. The inventors previously reported that HP-RBf, at a concentration of 200 mM (203.4 mg / mL) under 1 hour (h) of fluorescent light, does not alter the integrity of human skin tissue

[35] . Therefore, the selectivity and therapeutic index of HP-RBf for topical application are very high. The inventors' toxicological studies have shown that HP-RBf, at therapeutic concentrations, has no systemic toxicological effects, potential mutagenicity, or effects on female reproduction and development [42, 43]. In summary, HP-RBf is an attractive drug candidate as a rapid bactericidal agent applicable to skin, oral, and surgical wound infections. HP-RBf has the potential to function as a broad-spectrum antibacterial agent for Gram-positive bacteria (previous studies) and Gram-negative bacteria (this study) in combination with anti-Gram-negative agents.

[0037] Materials and methods Overview / Chemicals and Reagents All chemicals and antibiotics were purchased from commercial sources, including Sigma-Aldrich, and used without further purification unless otherwise noted. High-purity rose bengal was synthesized by a team at Provectus Biopharmaceuticals, Inc. (USA). All bacterial culture media used in the growth inhibitory activity tests were purchased from Fisher Scientific. Fluorescent lamp [17W, 63.8cm²] 2 , Sunblaster Holdings, ULC (Langly BC, CA)] and LED (9.5W, 28.3cm 2 I purchased the Philips product from Amazon.com. I purchased the Lessazurin (Alamar Blue) from Sigma-Aldrich.

[0038] bacterial strain Escherichia coli (ATCC 35218), Escherichia coli serotype O157 (TW07793), A. baumannii (ATCC 19606), A. baumannii (ATCC BAA 1800), Klebsiella pneumoniae (ATCC 19606), Klebsiella pneumoniae (CRE) CHS67 (NR48569), Klebsiella pneumoniae VA360 (NR48977), Pseudomonas aeruginosa (ATCC 27853), Pseudomonas aeruginosa MRSN 1356 (NR51521), Pseudomonas aeruginosa MRSN 1380 (NR51522), Salmonella tiphimurium (ATCC BAA All bacteria studied in this project, including Salmonella tiphimuria (NR4333), Burkholderia cepacia (UCB717), and Streptococcus pneumoniae (ATCC6301), were purchased or obtained from the American Type Culture Collection (ATCC) or BEI Resources (NIAID). Colistin-resistant strains were prepared by the following method. A preparation of pharmaceutical-grade rose bengal in physiological saline (HP-RBf). Rose bengal disodium salt preparation (10%) in physiological saline (HP-RBf) was provided by Provectus Biopharmaceuticals, Inc. (Knoxville, TN, USA). HP-RBf (>99.5%) was synthesized according to Provectus' proprietary process for synthesizing and utilizing the RB molecule as a viable active pharmaceutical ingredient for commercial use. Detailed procedures are described previously in

[35] , as well as in U.S. Patents 8,530,675, 9,273,022, and 9,422,260 by Singer et al., as previously mentioned.

[0039] Logarithmic-phase bacterial culture All liquid bacterial cultures were performed in Erlenmeyer flasks equipped with air filters. Single colonies of bacterial strains were grown under the recommended conditions proposed by ATCC. The culture flasks were incubated for 24 hours (h) in a 37°C shaking incubator with a shaking rate of 200 rpm until the midpoint of the logarithmic phase (optical density -0.5). Optical density was monitored at 600 nm using a 96-well microplate reader. Determination of the minimum inhibitory concentration (MIC) All assays were performed according to guidelines established by the Clinical & Laboratory Standards Institute (CLSI; Wayne, PA, USA). Minimum inhibitory concentration (MIC) was determined by broth-diluted microplate Alamer Blue assay or by OD measurement. All commercially available compounds were stored in DMSO or physiological saline (1 mg / 100 μL concentration). A 10% rose bengal disodium salt preparation (HP-RBf) in physiological saline was diluted with physiological saline to form a 1 mg / 100 μL storage solution. Aliquots of the storage solution were stored at 4°C for the duration of the experiment.

[0040] Each compound from the storage solution was placed in the first well of a sterile 96-well plate, and serial dilutions were performed with culture broth (total volume 10 μL). A logarithmic-phase bacterial suspension (190 μL) was placed in each well (total volume 200 μL), treated with serial dilutions of antibacterial agents under aerobic conditions, and incubated at 37°C for 24 hours. MIC research on HR-RBf under fluorescent lamp (17W, 63.8cm) 2 (Sunblaster Holdings) and LED (9.5W, 28.3cm) 2 The procedure was performed under Philips standards. OD was measured using a UV-Vis spectrophotometer. Resazurin (20 μL) was incubated at 37°C for 2 hours on a shaking incubator. Refer to the National Committee for Clinical Laboratory Standards [(NCCLS); Methods (pink = growth, blue = no visible growth)]. OD measurements were performed for all studies before performing colorimetric assays. Absorbance measurements were performed at 570 nm and 600 nm using a Biotek Synergy XT, 96-well plate reader.

[0041] Creation of drug-resistant Gram-negative bacterial strains Colistin-resistant mutants of the tested bacterial strains were prepared using the same procedure. (1 × 10⁶ of 100 μL of bacterial culture) 7 A 55cm agar plate containing colistin [minimum bactericidal concentration (MBC)] (CFU / mL) 2 Colonies were plated with colistin-containing agar plates, collected, and suspended in PBS buffer (approximately 1 × 10⁻⁶). 7 A 100 □ L bacterial suspension (CFU / mL) was plated on an agar plate containing the antibiotic (1.5 × MBC). This process was repeated until the cells achieved >10 times the MIC level of the wild type, gradually increasing the antibiotic concentration (2.0 ×, 2.5 ×, 3.0 ×, 3.5 ×, 4.0 ×, 5.0 ×, 7.0 ×, 8.0 ×, 9.0 ×, 10.0 ×, 20 ×, and 50 × MBC). Isolated resistant cells were confirmed by MIC assays using the generated resistant strains. Time-death kinetics assay Time-retention assays for antimicrobial agents were performed based on CLSI guidelines with some modifications. Multiple time points were observed in the time-retention assays for HP-RBf and reference molecules. Bacterial cultures grown in broth were subjected to 1 × 10⁶ sampling with a storage dilution of HP-RBf prepared at 5 times the MIC value. 8 ~5.0×10 9 The solution was diluted to a concentration between CFU / mL.

[0042] Equivolutes of the specified bacteria in a 96-well plate were inoculated into the test compound. Microtiter plates were incubated under fluorescent light (conditions summarized in the table above) at 37°C for durations of 1 to 30 minutes. Aliquots of the culture medium were taken from each well and serially diluted. The diluted cultures were incubated at 37°C and the CFU / mL was counted. Bactericidal activity was defined as a reduction of less than 3 log of colony-forming units. [Outside 1] JPEG2026511692000015.jpg231170[Outside 2] JPEG2026511692000016.jpg223170[Outside 3] JPEG2026511692000017.jpg237170[Outside 4] JPEG2026511692000018.jpg247170[Outside 5] JPEG2026511692000019.jpg248170[Outside 6] JPEG2026511692000020.jpg244170[Outside 7] JPEG2026511692000021.jpg80170

Claims

1. A method for treating Gram-negative bacteria other than those of the genera Burkholderia, Proteus, and Serratia, which are also resistant to the antibacterial compound colistin, a) The step of contacting the Gram-negative bacteria with an aqueous pharmaceutical composition containing a rose bengal (RB) compound of the following formula I, which is dissolved or dispersed at a concentration of about 0.01 to about 15 mg / mL, and b) Irradiate the bacteria that have come into contact with the light with a wavelength of approximately 500 nm to approximately 600 nm for a period of approximately 1 to approximately 10 minutes, at a rate of approximately 16 to approximately 160 J / cm². 2 Steps to obtain bacteria irradiated with a certain amount of light. A method that includes this. 【Chemistry 1】 (In the formula, if X is oxygen, then n is zero, and R 2 It does not exist, and if X is nitrogen, then n is 1, R 2 X is oxygen or nitrogen, and n is zero or 1, If X is oxygen, then R 1 Hydrogen (H) is a pharmaceutically acceptable cation M. + , C 1 -C 4 Selected from the group consisting of alkyl and aromatic rings as defined below, When X is nitrogen, R 1 and R 2 are the same or different and are selected from the group consisting of hydrogen, C 1 -C 4 alkyl, or together with the amide nitrogen atom form a 5- or 6-membered ring and the aromatic rings defined hereinafter, The aromatic ring is a monoring containing 5 or 6 members, or a 5,6- or 6,6-condensed aromatic ring system, and the aromatic ring or ring system independently contains 0, 1, or 2 heterocyclic atoms which are nitrogen, oxygen, or sulfur.

2. The method according to claim 1, wherein the RB compound is rose bengal disodium.

3. The method according to claim 1, wherein the Gram-negative colistin-resistant bacterium is one or more of Escherichia coli (E. coli), A. baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica.

4. The method according to claim 1, wherein the Gram-negative colistin-resistant bacteria are present inside or on mammalian cells at the time of contact.

5. The method according to claim 1, wherein the Gram-negative colistin-resistant bacteria are present on or within a surgical wound or other wound in a mammal.

6. The method according to claim 1, wherein the Gram-negative colistin-resistant bacteria are present on one or more of the examination table, operating room floor, walls, and equipment.

7. The Gram-negative colistin-resistant bacteria were irradiated for a period of approximately 2 to 5 minutes at an intensity of approximately 32 to 80 J / cm². 2 The method according to claim 1, for obtaining the amount of light.

8. The aforementioned aromatic ring substituent is 【Chemistry 2】 (In the formula, 【Transformation 3】 Each provides an ester or a monosubstituted amine. 【Chemistry 4】 (is) The method according to claim 1, wherein one or more types are selected from a group consisting of one or more types.

9. A pharmaceutical composition for treating Gram-negative bacteria other than those of the genera Burkholderia, Proteus, and Serratia, which are also resistant to the antibacterial compound colistin, comprising the following rose bengal (RB) compound of formula I. 【Transformation 5】 (In the formula, if X is oxygen, then n is zero, and R 2 It does not exist, and if X is nitrogen, then n is 1, R 2 X is oxygen or nitrogen, and n is zero or 1, If X is oxygen, then R 1 Hydrogen (H) is a pharmaceutically acceptable cation M. + , C 1 -C 4 Selected from the group consisting of alkyl and aromatic rings as defined below, If X is nitrogen, then R 1 and R 2 are the same or different, hydrogen, C 1 -C 4 Selected from the group consisting of alkyl groups, or together with an amide nitrogen atom to form a 5- or 6-membered ring and an aromatic ring as defined below, The aromatic ring is a monoring containing 5 or 6 members, or a 5,6- or 6,6-condensed aromatic ring system, and the aromatic ring or ring system independently contains 0, 1, or 2 heterocyclic atoms which are nitrogen, oxygen, or sulfur. A pharmaceutical composition containing the following:

10. The composition according to claim 9, wherein the RB compound is rose bengal disodium.

11. The composition according to claim 9, wherein the RB compound has a pH value of 6.5 to 7.

4.

12. The composition according to claim 9, wherein the RB compound has a pKa value of 2.52 or 1.

81.

13. The composition according to claim 9, further comprising a water-soluble electrolyte containing at least one cation selected from the group consisting of sodium, potassium, calcium, and magnesium, and at least one anion selected from the group consisting of chloride, phosphoric acid, and nitric acid.

14. The composition according to claim 13, wherein the water-soluble electrolyte is sodium chloride.

15. The composition according to claim 13, wherein a water-soluble electrolyte is present in the composition at a concentration of 0.1 mass / volume percent to 2 mass / volume percent.

16. The composition according to claim 13, wherein a water-soluble electrolyte is present in the composition at a concentration sufficient to yield a molal osmotic pressure concentration of 300 milliosmoles per kilogram of water to 500 milliosmoles per kilogram of water.

17. C present in the diluent at a concentration of less than 5 volume percent 1 -C 6 The composition according to claim 9, further comprising a diluent containing a monohydric or polyhydric alcohol.

18. The aqueous composition according to claim 9, provided in the form of a liquid, gel, or cream.

19. A fungicide for treating Gram-negative bacteria other than those of the genera Burkholderia, Proteus, and Serratia, which are also resistant to the antibacterial compound colistin, comprising the following rose bengal (RB) compound of formula I. 【Transformation 6】 (In the formula, if X is oxygen, then n is zero, and R 2 It does not exist, and if X is nitrogen, then n is 1, R 2 X is oxygen or nitrogen, and n is zero or 1, If X is oxygen, then R 1 Hydrogen (H) is a pharmaceutically acceptable cation M. + , C 1 -C 4 Selected from the group consisting of alkyl and aromatic rings as defined below, If X is nitrogen, then R 1 and R 2 are the same or different, hydrogen, C 1 -C 4 Selected from the group consisting of alkyl groups, or together with an amide nitrogen atom to form a 5- or 6-membered ring and an aromatic ring as defined below, The aromatic ring is a monoring containing 5 or 6 members, or a 5,6- or 6,6-condensed aromatic ring system, and the aromatic ring or ring system independently contains 0, 1, or 2 heterocyclic atoms which are nitrogen, oxygen, or sulfur. It contains, The aforementioned disinfectant is designed to be used in combination with a fluorescent light source having a wavelength of approximately 500 nm to approximately 600 nm. The aforementioned light source is applied to Gram-negative bacteria for a period of approximately 1 to 10 minutes, resulting in a radiation level of approximately 16 to 160 J / cm². 2 It brings about a certain amount of light. Disinfectant.

20. The disinfectant according to claim 19, which is applied to at least one of the surfaces of a surgical area, surgical instruments, the surgeon's hand, the internal parts of the patient's body, and the external parts of the surgical patient's body.

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