Antibacterial effect of halogenated fluorescein on colistin-resistant gram-negative bacteria

Abstract

The present invention contemplates a method for treating gram-negative bacteria other than the genus Burkholderia, Proteus and Serratia, said gram-negative bacteria also exhibiting drug resistance (MIC > = 50 mg / mL) against the bacterial compound colistin, said gram-negative bacteria also exhibiting drug resistance (MIC > = 50 mg / mL) against the bacterial compound colistin, such as colistin, colistin, colistin, colistin, colistin, colistin, colistin, colistin, colistin, colistin, colistin, colistin and colistin. The method comprises contacting bacteria with an aqueous pharmaceutical composition containing a rose-bengal (RB) compound of the following formula I dissolved or dispersed therein at a concentration of from about 0.01 to about 15 mg / mL, and irradiating those contacted bacteria with light having a wavelength of from about 500 nm to about 600 nm for a period of from about 1 to about 10 minutes to provide a light dose of from about 16 to about 160 J / cm2, treating and killing the irradiated bacteria, wherein X, R1, R2 and M + are defined herein.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 455,501, filed March 29, 2023, entitled “Antibacterial Action of Halogenated Fluorescein Against Colistin-Resistant Gram-Negative Bacteria,” the entire contents of which are incorporated herein by reference.

[0003] describe Background Technology

[0004] 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 MDR Gram-negative bacteria [5,6]. Except for patients with cystic fibrosis [7–10], it has not been widely used since the early 1980s due to its nephrotoxicity. However, it has been reintroduced into clinical practice as a last-line treatment for serious hospital-acquired infections caused by MDR Gram-negative bacteria

[11] .

[0005] Numerous studies have shown a rapid increase in the prevalence of colistin resistance in Enterobacteriaceae [12-14]. Several clinical isolates of these bacteria, including *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, *Escherichia coli*, *Salmonella* species, and *Klebsiella* species, 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.

[0006] Of particular concern is that other Pseudomonadota (Proteobacteria) species, such as Serratia spp., Proteus spp., and Burkholderia spp., exhibit natural resistance to colistin

[21] . Colistin-resistant Gram-negative bacteria also typically exhibit cross-resistance to other antibacterial agents [22,23]. For example, meropenem, an extended-spectrum carbapenem, is effective against drug-sensitive Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii at low concentrations (0.03–6.25 g / mL), while colistin-resistant strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae have reduced susceptibility to meropenem and other antibiotics [24,25].

[0007] Colistin-resistant Klebsiella pneumoniae strains are difficult to treat with the currently available drug library [26,27]. Although many studies have been dedicated to optimizing the use of currently available agents or identifying any combinations thereof, no pipeline drug is available to address drug-resistant bacteria associated with colistin resistance [28-33]. Therefore, there is a need for drugs that are effective against colistin-resistant strains or methods to prevent the acquisition of colistin resistance during treatment.

[0008] U.S. Patents 8,530,675, 9,273,022, and 9,422,260 by Singer et al. describe and claim protection for highly purified rose bengal, as well as similarly purified compounds containing different halogen substituents and different numbers of those halogen substituents, and the synthesis of their lactone forms. The highly purified rose bengal used in the aforementioned patents is a clinically formulated rose bengal (HB-RBf) composition containing 10% rose bengal w / v dissolved in a 0.9% sodium chloride (NaCl) aqueous solution, produced by Provectus Biopharmaceuticals, Inc., Knoxville, TN, under the name... Provided for research purposes. The rose-red compounds are collectively referred to herein as "halogenated fluorescein" and more specifically as "halogenated fluorescein".

[0009] RB dyes have been clinically investigated for the treatment of melanoma and other solid cancers (

[36] ; and PCT / US22 / 05407), particularly when injected directly into cancerous lesions. The photodynamic antibacterial properties of RBs have been reported sporadically (

[37] and PCT / US22 / 054076).

[0010] U.S. Patent No. 8,974,363 to Dees et al. teaches the use of a 10-100 μM (i.e., 10 μg / mL to 100 μg / mL) topical RB formulation in combination with green light irradiation at a wavelength of 500-600 nm to combat Gram-positive and Gram-negative antibiotic-resistant bacteria, without details regarding the bacterial strains used, the light source, its intensity, or the duration of irradiation.

[0011] We screened an internal library of molecules, including FDA-approved and unapproved antibacterial agents, against two Burkholderia species, one Proteus species, one Serratia species, colistin-resistant Pseudomonas aeruginosa, and wild-type Pseudomonas aeruginosa (reference bacterial strain). We determined that rose rubrum (RB; 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) showed strong bactericidal activity (0.05–6.25 g / mL) against colistin-resistant Gram-negative strains under fluorescence (see Table 1 below and

[35] ). However, RB did not show significant antibacterial activity against wild-type of the other Gram-negative bacteria examined (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) (MIC <12.5 g / mL).

[0012] The antibacterial properties of RB discussed above led us to investigate the efficacy of RB against colistin-resistant Gram-negative bacteria. To verify the bactericidal effect of RB against colistin-resistant strains, we prepared moderate to highly colistin-resistant strains of Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia species, Salmonella enterica Typhimurium, and Serratia species

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

[0013] Invention Summary

[0014] The present invention considers the combined use of a rose red derivative with irradiation of Gram-negative bacteria with light of a wavelength of about 500 nm to about 600 nm for a period of about 1 to about 10 minutes to treat and kill irradiated bacteria that also exhibit resistance to the antibacterial compound colistin (MIC ≥ 50 mg / mL).

[0015] In one embodiment, the invention considers the combined use of a rose red derivative with irradiation of Gram-negative bacteria, excluding Burkholderia, Proteus, and Serratia, with light of a wavelength of about 500 nm to about 600 nm for a period of about 1 to 10 minutes, to treat and kill the irradiated bacteria, which also exhibit resistance to the antibacterial compound colistin (MIC ≥ 50 mg / mL).

[0016] In one aspect, Gram-negative colistin-resistant bacteria, and in one embodiment, Gram-negative colistin-resistant bacteria other than Burkholderia spp., Proteus spp., and Serratia spp., are treated in a method comprising the following steps: contacting the bacteria with an aqueous pharmaceutical composition containing a rose red (RB) compound of formula I dissolved or dispersed therein 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 to provide about 16 to about 160 J / cm³. 2 The light dose. The RB compound of formula I is shown below:

[0017]

[0018] Where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and R 2 It does not exist, but when X is nitrogen, n is 1 and R 2 It exists. When X is oxygen, R 1 M is selected from hydrogen (H) as a pharmaceutically acceptable cation. + The group consisting of C1-C4 alkyl groups and substituents containing aromatic rings as defined below. When X is nitrogen, R 1 and R 2 The substituents may be the same or different, and are selected from the group consisting of hydrogen, C1-C4 alkyl groups, or 5- or 6-membered rings formed together with an amide nitrogen atom, and aromatic ring substituents. The aromatic ring substituents are monocyclic rings containing 5 or 6 members, or 5,6- or 6,6-fused aromatic ring systems, and the aromatic ring or ring system substituents may contain 0, 1, or 2 heterocyclic atoms that are independently nitrogen, oxygen, or sulfur.

[0019] Exemplary aromatic ring substituents are described below:

[0020]

[0021] in for Esters or monosubstituted amines are provided, respectively.

[0022] In a preferred embodiment, the method is performed in the area where surgery is performed to inhibit postoperative infection by Gram-negative colistin-resistant bacteria. The aqueous pharmaceutical composition of the RB compound of formula I is intended to be applied to the surface of the surgical area by spraying, wiping, or any other conventional method, such as to internal and external areas of the surgical patient's body, as well as to surgical instruments, the surgeon's hands, the hands of other persons involved in the surgery, and various instruments present in the surgical area. If necessary, one or more lamps emitting light of a wavelength from about 500 nm to about 600 nm in the aforementioned amounts can be used to illuminate the entire surgical area or a smaller area. Brief description of the attached diagram

[0024] In the accompanying drawings that form part of this disclosure

[0025] Divided as Figure 1 The six figures: A, 1B, 1C, 1D, 1E, and 1F. Figure 1 This indicates that under a fluorescent lamp (17W, 63.8cm) 2 0-40J / cm 2 Under these conditions, HP-RBf (10% RB saline solution) showed resistance to colistin [colistin] R [Graph of time-kill kinetics of Gram-negative bacteria. 5 times the MIC concentration: Escherichia coli ATCC35218 colistin] R MIC 400 mg / mL (colistin); Colistin of Pseudomonas aeruginosa ATCC27853 R MIC > 400 mg / mL (colistin); Klebsiella pneumoniae ATCC 19606 colistin R MIC 100 mg / mL (colistin); and Acinetobacter baumannii ATCC BAA-1800 colistin. R MIC 400mg / mL (colistin).

[0026] Detailed description of the preferred implementation scheme

[0027] This invention relates to a method for treating Gram-negative bacteria that also exhibit resistance to the antibacterial compound colistin (MIC ≥ 50 mg / mL), and in one embodiment, the Gram-negative bacteria, other than Burkholderia spp., Proteus spp., and Serratia spp., also exhibit resistance to the antibacterial compound colistin. The method comprises contacting the bacteria with an aqueous pharmaceutical composition containing a rose red (RB) compound of formula I dissolved or dispersed therein 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 to provide about 16 to about 160 J / cm³.2 The light dose is used to treat and kill irradiated bacteria.

[0028] The RB compound under consideration has the following structural formula (Formula I), where X is O (oxygen) or N (nitrogen), and "n" is 0 or 1.

[0029]

[0030] When X is oxygen, n is 0 and does not exist, therefore the RB compound is: a) rose red, where -XR 1 b) A pharmaceutically acceptable salt of -OH, wherein -XR 1 -OM + M + c) C1-C4 alkyl esters, or d) aromatic esters as defined below, are pharmaceutically acceptable cations.

[0031] Alternatively, when X is a nitrogen atom, n is 1 and R 2 With R 1 They exist together. Therefore, R 1 and R 2 They can be the same or different, and C(O)NR 1 R 2 It is an amide, and its nitrogen atom is a) unsubstituted [-X-(R-1) ..."-1'"-1'"-1'"-1'"-1"-1"-1"-1"-2"-12"""" """""""""" 1 R 2 ), and R 1 and R 2 Both are hydrogen (H)], b) substituted with one or two C1-C4 alkyl groups or forming a 5- or 6-membered ring with the nitrogen atom of the amide group, or c) aromatic amides, preferably monosubstituted, wherein R 1 It is hydrogen, and R 2 For aromatic substituents discussed below.

[0032] For ease of description, aromatic esters or aromatic amides are collectively referred to as aromatic derivatives. Thus, these derivatives are formed from alcohols or amines (preferably monosubstituted) and have a single 5- or 6-membered aromatic ring or a 5,6- or 6,6-fused aromatic ring system containing 0, 1, or 2 heterocyclic atoms that are independently nitrogen, oxygen, or sulfur.

[0033] Illustrative examples of such aromatic alcohol ester moieties are shown and named below, where O is an oxygen atom, and the line-O indicates that the ring-oxygen can come from any available carbon in the ring, and the O-line crossed by the wavy line indicates that the depicted alkoxy group is part of another molecule (an esterified RB molecule).

[0034]

[0035]

[0036] in for Esters or monosubstituted amines are provided, respectively.

[0037] Rose red (RB) is a preferred RB compound, and its disodium salt, disodium rose red (RBD), is the most preferred RB compound. These compounds are used illustratively in this document as part of the RB compound group.

[0038] The chemical name of rose red is 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein. The preferred form, disodium rose red (RBD), has the following structural formula:

[0039]

[0040] Certain details of this preferred embodiment of the considered composition are described in U.S. Patents 5,998,597, 6,331,286, 6,493,570, and 8,974,363, the disclosures of which are incorporated herein by reference in their entirety. The aforementioned patents describe the use of RBD in killing cancer cells.

[0041] On one hand, mammalian cells (such as human cells) infected with Gram-negative colistin-resistant bacteria come into contact with the bacteria via RB compounds taken up by the infected cells. On the other hand, Gram-negative colistin-resistant bacteria are present on the surface to be disinfected.

[0042] Light can be applied in conjunction with a pharmaceutical composition containing RB (in which the RB compound is present), the pharmaceutical composition being applied to the infected area at a concentration of about 0.01 to about 15 mg / mL, and preferably about 0.2 to about 3.1 mg / mL, dissolved or dispersed in a pharmaceutically acceptable diluent. Light can also be applied shortly after the pharmaceutical composition has been applied to contact bacteria or mammalian cells containing bacteria, preferably within about 2 to about 5 minutes. Bacteria can be treated within infected mammalian cells or when present on surfaces such as examination tables or operating tables.

[0043] Although it appears similar to previous treatments, it is believed that the concentration of the RB compound is lower than previously used, and the light intensity is approximately one-tenth or less of that of previous methods. Furthermore, this treatment targets Gram-negative bacteria other than Burkholderia, Proteus, and Serratia, which are also resistant to colistin treatment.

[0044] Colistin resistance (in its various grammatical forms) is used herein to refer to a colistin concentration with a MIC approximately equal to or greater than (≥) approximately 50 mg / mL. In most cases, the MIC is ≥ approximately 100 mg / mL, and typically ≥ approximately 400 mg / mL. Therefore, fewer antibacterial RB compounds can be used, and less expensive light can be employed, making this method more feasible.

[0045] The infected area treated with RB is irradiated for approximately 1 to 10 minutes, more preferably for approximately 2 to 5 minutes. This irradiation provides approximately 16 to 160 J / cm². 2 The light dose, and more preferably, about 32 to about 80 J / cm². 2 Light dose.

[0046] Pharmaceutical halogenated saxon composition

[0047] The liquid compositions under consideration can be formulated for oral, parenteral, or topical administration. Illustrative xanthones (fluorescein) compositions are illustrated by reference to the preferred xanthones compound, disodium rose red.

[0048] First, let's discuss parenteral or topical therapeutic compositions, as mentioned earlier. The composition describes a pharmaceutical composition for parenteral administration containing a particularly preferred RB compound, disodium rose rubes. Delivery of the halogenated fluorescein component of the composition is most advantageous when the composition has a pH close to physiological pH (i.e., approximately pH 7), and especially when the pH is greater than approximately 4, thus ensuring that the halogenated fluorescein remains in a binary form within the composition.

[0049] Therefore, in a preferred embodiment, the pH of the composition is from about 5 to about 9, more preferably from about 6 to about 7.5, and most preferably from about pH 6.5 to about pH 7.4. At these pH values, the halogenated fluorescein generally remains in a binary form, rather than as a lactone formed at low pH values.

[0050] RB compounds such as rose red are binary, with pKa values ​​of 2.52 and 1.81. The pKa values ​​of several considered halogenated fluoresceins can be determined in Batsitela et al., Spectrochim Acta Part A 79(5):889-897 (September 2011).

[0051] Hydrophilic carriers are a preferred medium for pharmaceuticals to maximize the distribution of halogenated fluorescein components to tissues, particularly for RB compounds in acidic and / or salt form. Therefore, in preferred embodiments, the medium contains minimal non-hydrophilic components that might interfere with such distribution. Thus, preferred formulations contain RB or disodium RB, particularly preferably in a hydrophilic medium, and more preferably in an aqueous medium.

[0052] When administered parenterally, in addition to being in suppositories, pharmaceutical compositions containing RB compounds preferably include 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, phosphate, and nitrate. The concentration of the electrolyte is preferably about 0.1% (w / v) and about 2% (w / v).

[0053] Alternatively, the electrolyte is present at a level sufficient to provide an isotonic molar concentration of more than about 100 mOsm / kg (milliosmol / kg water) up to about 600 mOsm / kg. More preferably, the isotonic molar concentration of the pharmaceutical composition is greater than 250 mOsm / kg, and most preferably about 300-500 mOsm / kg.

[0054] The electrolyte is preferably sodium chloride. The electrolyte is preferably present at a concentration of about 0.5% to about 1.5%, and even more preferably at a concentration of about 0.8% to about 1.2%, and most preferably at a concentration of about 0.9%, as is present in physiological saline.

[0055] The aqueous medium (diluent) of the composition is preferably only water that meets the standards for injection. Up to about 20% by volume 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, threitol, trimethylolpropane, sorbitol, etc. More preferably, the alcohol is present in the considered composition at less than about 10% by volume of the diluent, and more preferably less than about 5% by volume.

[0056] The terms “physiologically acceptable salt” and “pharmaceutically acceptable salt” in their various grammatical forms refer to any non-toxic cation, such as alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry, including sodium, potassium, lithium, calcium, magnesium, barium, and protamine zinc salts, which can be prepared by methods known in the art. The cations considered provide water-soluble RB salts. Preferably, the salts are sodium, potassium, and calcium in the form of monobasic or dibasic salts. For a list of commonly used physiologically (or pharmaceutically) acceptable acids and bases that form physiologically / pharmaceutically acceptable salts with pharmaceutical compounds, the reader may refer to Berge, J. Pharm. Sci. 1977 68(1):1-19.

[0057] The pH of a pharmaceutical composition containing RB can be adjusted or modified in any suitable manner known to those skilled in the art. The composition can be buffered, or the pH can be adjusted by adding acids, bases, etc. Since RB or its physiologically acceptable salts are weak acids, the pH of the composition may not require buffers and / or pH adjusters, depending on their concentrations and / or electrolyte concentrations. However, it is particularly preferred that the composition is buffer-free, allowing it to conform to the biological environment upon administration.

[0058] It is also preferred that the pharmaceutical composition does not contain any preservatives, as many preservatives may harmfully interfere with the pharmaceutical composition or its formulation, or may complex with or otherwise interact with or interfere with the delivery of the active component of the composition containing the RB compound. Regarding the use of preservatives, imidolidinyl urea is a preferred preservative because it does not interact with the RB compound in the pharmaceutical composition or at the time of administration.

[0059] The liquid pharmaceutical compositions under consideration are also suitable for oral administration to mammalian subjects to be treated. In a preferred aspect, when administered to mammalian subjects, the RB compounds, as previously discussed, are dissolved or dispersed in an aqueous diluent. More preferably, the aqueous diluent is free of tonicity agents, except for those sugars and / or buffers present as flavoring agents.

[0060] The diluent may be one or more C1-C6 monohydric or polyhydric alcohols as previously discussed, up to about 20% by volume. More preferably, the alcohol is present in the composition under consideration in an amount of less than about 10% by volume of the diluent, and more preferably less than about 5% by volume.

[0061] Topical liquid compositions were also considered. One such liquid composition currently undergoing clinical trials for the treatment of psoriasis is called [name of composition]. The development of a pharmaceutical product. This pharmaceutical product contains RBD present at a concentration of 0.001 to 0.01% w / v dissolved or dispersed in an aqueous diluent, and at least one builder (present at a level sufficient to provide the pharmaceutical product with a viscosity of 10-1000 cps), and sodium chloride as an electrolyte (present at a concentration of 0.9% w / v, sufficient to provide the pharmaceutical product with an isotonic molar concentration of 100 mOsm / kg to 500 mOsm / kg). This pharmaceutical product is described and claimed, for example, in U.S. Patent No. 8,974,363.

[0062] processing method

[0063] The considered treatment methods include contact with Gram-negative bacteria that exhibit resistance to the antibacterial compound colistin (MIC ≥ 50 mg / mL). In one embodiment, in addition to Burkholderia spp., Proteus spp., and Serratia spp., these 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 using a composition containing an RB compound that is effective against Gram-negative bacteria. Preferred wavelengths for this irradiation are about 500 to about 575 nm, more preferably about 510 to about 550 nm. As discussed herein, the considered RB compound-containing composition is considered to provide effective treatment if the determined MIC is about 10 mg / mL or lower.

[0064] In one implementation, Gram-negative bacterial cells are present on or in the test mammal (e.g., in an infection). Illustratively, the test mammal may have a dermatological Gram-negative bacterial infection, such as Klebsiella, Escherichia, or Pseudomonas infection, or particularly in the case of local treatment of open or surgical wounds, as a treatment for current infection and as a prophylaxis for subsequent Gram-negative bacteria in which the infecting bacteria are also colistin-resistant.

[0065] In another embodiment, surfaces in the surgical area, such as examination tables, floors, and / or walls and / or equipment, may be washed and irradiated with the compositions considered above to disinfect or prevent the growth of one or more Gram-negative bacteria discussed above. Washing and irradiation may be performed concurrently with surgery or other treatments on mammalian subjects to help disinfect or improve the overall cleanliness of the mammalian subjects and treatment areas.

[0066] The test mammals for treatment can be primates such as humans, apes such as chimpanzees or gorillas, monkeys such as cynomolgus monkeys or macaques, laboratory animals such as rats, mice or rabbits, companion animals such as dogs, cats, horses, or food animals such as cows or steers, sheep, lambs, pigs, goats, llamas, etc.

[0067] Each of the considered compositions is typically administered repeatedly until the treated bacterial disease (infection) is reduced to a desired level, such as becoming undetectable. Therefore, administration to mammalian subjects in need can occur multiple times within a day, daily, weekly, monthly, or over a period of months to years, as directed by the treating physician.

[0068] A Gram-negative bactericidal effective amount of RB is administered to mammalian subjects in need, and it can be formulated using common liquid, gel, cream, or other forms. In most cases, RB is administered in conjunction with irradiation, preferably using a light source with an emission wavelength of about 500 to about 600 nm, and more preferably about 510 to about 550 nm. Illustrative sources for the light source, their intensity, and their duration are discussed below.

[0069] result

[0070] Pharmaceutical-grade rose rube formulation (HP-RBf) effectively inhibits the growth of colistin-resistant Gram-negative bacteria.

[0071] Antibacterial activity of rose red (RB) has been reported in commercial grade rose red (80-95% dye content), which contains known impurities (e.g., transhalogenated substances) resulting from historical preparation methods

[36] . Therefore, commercial grade RB lacks drug relevance.

[0072] 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 prepared in brine (a 10% brine solution of RB; HP-RBf) and further diluted with brine to form the desired concentration.

[0073] The fluorescence (approximately 23.0 kJ / cm²) was obtained by broth dilution method. 2 ) and LED lights (approximately 29.0 kJ / cm²) 2 Under irradiation conditions, the lowest inhibitory concentration (MIC, μg / mL) was obtained over a 24-hour period. Illustratively, this translates to approximately 0.96 (approximately 1) KJ / cm³ over 60 minutes (min). 2 Or approximately 16.0 J / cm³ within one minute. 2 For irradiation times of 0 to 30 minutes, using 17W, 63.8cm... 2The light intensity for studies conducted using fluorescent lamps will be approximately 0-480 J / cm². 2 The irradiation of about 1 to about 10 minutes discussed in

[35] will reach about 16 to about 160 J / cm. 2 .

[0074] HP-RBf effectively inhibited the growth of colistin-resistant Gram-negative bacteria, with MIC levels ranging from 0.05 to 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; for fluorescence and LED light, the MIC values ​​were equal or very close

[35] .

[0075] Table 1

[0076] The discovery of bactericidal molecules targeting colistin-resistant bacterial strains.

[0077]

[0078]

[0079] All studies were repeated three times. MIC values ​​were determined by OD and colorimetric assays using either Risazurin or Malachite green.

[0080] b bacteria were purchased from ATCC or obtained from BEIResources.

[0081] C Colistin-resistant strains were prepared in the Kurosu laboratory according to the procedure described below.

[0082] The light source used in d is 17W and 63.8cm. 2 Fluorescent lamp or 9.5W, 28.3cm 2 LED lights. After 24 hours of treatment (compared to 23.0 KJ / cm² for fluorescent lamps). 2 Or, for LED lights, it is 29.0 KJ / cm². 2 Then determine the MIC.

[0083] HP-RBf exhibited bactericidal activity against colistin-resistant Escherichia coli (ATCC 35218, colistin >400 g / ml), with a MIC of 0.05 g / ml (Table 2, item 2 below). Meropenem showed significantly lower susceptibility to E. coli when it acquired colistin resistance (item 1 vs. item 2). E. coli serotype O157 (wild type) is a major foodborne pathogen that showed susceptibility to colistin, meropenem, and amikacin (item 3). We were able to isolate its colistin-resistant mutant with a MIC level of 100 μg / ml (item 4).

[0084] Table 2. Sensitivity of HP-RBf to colistin-resistant Gram-negative bacterial strains (a)

[0085]

[0086]

[0087]

[0088] All studies were repeated three times. MIC values ​​were determined by OD and colorimetric assays using risazurin or malachite green.

[0089] b bacteria were purchased from ATCC or obtained from BEI Resources.

[0090] C Colistin-resistant strains were prepared in the Kurosu laboratory according to the procedure described below.

[0091] The light source used in d is 17W and 63.8cm. 2 Fluorescent lamp or 9.5W, 28.3cm 2 LED lights. After 24 hours of treatment (compared to 23.0 KJ / cm² for fluorescent lamps). 2 Or, for LED lights, it is 29.0 KJ / cm². 2 The MIC was then determined. The MIC values ​​obtained under fluorescent and LED lights were the same.

[0092] Wild-type *Escherichia coli* serotype O157 showed very low susceptibility to HP-RBf (entry 3), but HP-RBf effectively killed its colistin-resistant mutant (MIC: 3.13 g / mL) (entry 3 vs. entry 4). Drug-sensitive and multidrug-resistant mutants of *Acinetobacter baumannii* (ATCC 19606, entry 5) and *Acinetobacter baumannii* (ATCC BAA 1800, entry 7) showed resistance to HP-RBf. On the other hand, their colistin-resistant mutants were sensitive to HP-RBf; the MIC of HP-RBf against these two strains (entries 6 and 8) was 0.05 μg / mL. Two wild-type Klebsiella pneumoniae strains (ATCC 19606 and NR48569) were resistant to HP-RBf (MIC>100 μg / mL) (Entries 9 and 11), while their colistin-resistant mutants increased susceptibility to HP-RBf; MIC values ​​were 6.3 and 12.5 μg / mL, respectively (Entries 10 and 12).

[0093] In addition, we have generated moderate (MIC 50 μg / mL) and high (MIC > 400 μg / mL) colistin-resistant P. aeruginosa mutants from P. aeruginosa (ATCC 27853) (Entries 14 and 15). HP-RBf susceptibility of P. aeruginosa strains depends on the degree of their colistin resistance; the MIC values ​​for highly resistant and moderately resistant strains are 0.05 and 6.3 μg / mL, respectively. Similar to the P. aeruginosa (ATCC 27853) studied in Table 1, P. aeruginosa MRSN 1356 (NR51521) and P. aeruginosa MRSN 1380 (NR51522) strains showed low levels of inherent susceptibility to HP-RBf under irradiation conditions (Entries 16 and 18)

[37] . Their colistin-resistant strains increased HP-RBf susceptibility (Entries 17 and 19).

[0094] It has been reported that some Gram-negative bacteria, such as species of Burkholderia, are inherently resistant to colistin, with MIC values ​​typically >200 mg / mL (see above). Wild-type Burkholderia polyphaga CGD1 is recognized to be resistant to colistin, with an MIC level of 200 mg / mL (Table 1, entry 20 in Table 2)

[21] .

[0095] Colistin-resistant mutant strains of Burkholderia polyphaga CGD1 were highly susceptible to HP-RBf (entry 21). Similarly, two other Burkholderia species (Burkholderia thaiensis E264 and Burkholderia cepacia genomic type III, LMG 16656) were examined; these wild types also showed high colistin resistance (entries 22 and 24)

[38] . Their acquired colistin-resistant mutants showed 8-fold and 16-fold increased susceptibility to HP-RBf (entries 23 and 25). Colistin effectively kills most Salmonella species, with MIC values ​​of 0.8–1.6 mg / mL (entries 26 and 28).

[0096] HP-RBf inhibited the growth of wild-type *Enterobacter typhimurium*, with a MIC of 12.5 mg / mL (entry 26). A 15-fold higher colistin-resistant mutant of *Enterobacter typhimurium* enhanced its HP-RBf susceptibility (entry 27). An acquired colistin mutant of another *Salmonella* species (*Enterobacter tinea*, typhimurium serotype) also enhanced its HP-RBf susceptibility (entry 29). *Proteus* and *Serratia* species are other bacteria exhibiting resistance to colistin (entries 30 and 32). HP-RBf effectively killed these wild-type strains at low concentrations (entries 30 and 32). As observed in all other entries in Table 2, their acquired colistin mutants enhanced HP-RBf susceptibility (entries 31 and 32).

[0097] HP-RBf exhibits rapid killing properties against colistin-resistant Gram-negative bacteria.

[0098] We have reported the photodynamic growth inhibition of HP-RBf on Gram-positive bacteria; in bacterial cultures of Gram-positive bacteria (including drug-resistant strains) (1.3 to approximately 4.6 × 10⁻⁶). 8 Adding HP-RBf to CFU / ml can cause a bacterial cell reduction of more than 6 logs in less than 2 minutes

[35] . Similarly, for selected colistin-resistant (colistin) R Mutant strain (Escherichia coli ATCC35218 colistin) R Pseudomonas aeruginosa ATCC27853 colistin R Klebsiella pneumoniae ATCC19606 colistin R Acinetobacter baumannii ATCC BAA-1800 colistin R The time-course study was conducted under fluorescent light for 24 minutes (40 J / cm²) at a concentration 5 times the MIC (HP-RBf). 2 The graphical representations of those results are shown... Figure 1 middle.

[0099] The reference molecules used were amikacin (10 mg / mL) and meropenem (10 mg / mL). HP-RBf reduced 1.3 × 10⁻⁶ cells / mL within 2 minutes. 8 From approximately 1.9 × 10 8 (Colony forming units: CFU) all colistin-resistant strains showed a 6-log reduction; for media treated with HP-RBf (0.025 or 31.5 mg / mL) at a concentration of 1 × 10⁻⁶. 3 (Dilution), no CFU count ( Figure 1 (A-1D). In the same study, amikacin and meropenem reduced colistin levels in E. coli ATCC35218. R The strains were approximately 3.5% (30 minutes) and 0% (30 minutes) ( Figure 1 E and 1D). These results demonstrate that HP-RBf has a rapid killing effect on colistin-resistant Gram-negative bacteria under irradiation conditions

[35] .

[0100] discuss

[0101] We evaluated the antibacterial activity of pharmaceutical-grade RB formulations (HP-RBf) under irradiation conditions. We recently reported a comprehensive evaluation of HP-RBf against Gram-positive bacteria and Mycobacterium spp.; more than 45 bacterial strains were tested, and HP-RBf showed rapid bactericidal activity against Gram-positive bacteria (MIC 0.3–3.1 mg / mL, less than 2 minutes)

[35] . HP-RBf has a strong affinity for Gram-positive peptidoglycan layers and induces photodynamic activation, generating reactive oxygen species. HP-RBf showed moderate bactericidal activity against Mycobacterium spp., with MIC values ​​of 12.5–25.0 mg / mL (12 hours) under irradiation conditions.

[0102] Since the MIC values ​​for mycobacterial species are higher than those for Gram-positive bacteria, we believe that thick cell walls containing mycolic acid reduce cellular uptake of HP-RBf. In previous studies, we concluded that common hospital Gram-negative bacteria are not sensitive to HP-RBf; their MIC values ​​are 25 mg / mL or greater than >50 mg / mL

[35] .

[0103] We have expanded our antibacterial screening to include Gram-negative bacteria and have recognized that HP-RBf exhibits susceptibility to some Proteobacteria (Pseudomonas) species such as Burkholderia, some Salmonella, Proteus, and Serratia

[21] . The MIC values ​​of HP-RBf against their wild-types range from 0.8 to 12.5 mg / mL. One characteristic of their drug susceptibility is their inherently high resistance to the action of colistin.

[0104] HP-RBf kills colostin-resistant mutants of Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) at low concentrations. Enhanced colostin-resistant strains of Burkholderia, Salmonella, Proteus, and Serratia species also show increased HP-RBf susceptibility. These data clearly indicate that the acquisition of colostin resistance in Gram-negative bacteria alters the outer membrane structure, increasing the affinity of HP-RBf.

[0105] It is established that the identified colistin-resistant Gram-negative bacteria involve changes in the lipopolysaccharide (LPS) structure, in which colistin interacts with the negatively charged lipid A of LPS [39-41]. Therefore, an increase in the positively charged LPS components (e.g., cationic forms of 4-amino-L-arabinose, phosphoethanolamine, and galactosamine) in colistin-resistant strains can enhance the affinity for negatively charged HP-RBf.

[0106] We are further determining the mechanism of action of HP-RBF against colistin-resistant Gram-negative bacteria. The rapid antibacterial photodynamic activity of HP-RBf has several advantages, including: 1) reducing the frequency of resistant strain emergence, and 2) increasing the safety profile for disinfection and sterilization applications.

[0107] In summary, the research reported here demonstrates that HP-RBf is a potential drug candidate for treating infections caused by both endogenous and acquired colistin-resistant Gram-negative bacteria. Colistin-resistant Gram-negative bacteria involve membrane structural alterations that lead to resistance to other important antibacterial agents used to treat Gram-negative bacterial infections [40,41]. Of interest, these structural changes make HP-RBf more susceptible to colistin-resistant Gram-negative bacteria.

[0108] We previously reported that HP-RBf at a concentration of 200 mM (203.4 mg / mL) did not alter the integrity of human skin tissue under fluorescent light for 1 hour (h)

[35] . Therefore, HP-RBf for topical application has a very high selectivity and therapeutic index. Our toxicology studies showed that HP-RBf had no systemic toxicological effects, mutagenic potential, or effects on female reproduction and development at therapeutic concentrations [42,43].

[0109] In summary, HP-RBf is an attractive drug candidate as a rapid bactericidal agent applicable to skin, oral, and surgical wound infections. HP-RBf in combination with anti-Gram-negative drugs has the potential to act as a broad-spectrum antibacterial agent against both Gram-positive bacteria (previous studies) and Gram-negative bacteria (this study).

[0110] Materials and methods

[0111] General Chemicals and Reagents

[0112] Unless otherwise stated, all chemicals and antibiotics were purchased from commercial sources, including Sigma-Aldrich, and used without further purification. High-purity rose red was synthesized by the team at Provectus Biopharmaceuticals, Inc. (USA). All bacterial culture media used for growth inhibition activity assays were purchased from Fisher Scientific. Fluorescent lamp [17W, 63.8cm] 2 , Sunblaster Holdings, ULC (Langly BC, CA)] and LED (9.5W, 28.3cm 2 Purchased from Amazon.com (Philips). Alamar Blue (Almar Blue) purchased from Sigma-Aldrich.

[0113] bacterial strains

[0114] All bacteria studied in this project were purchased or obtained from the American Type Culture Collection (ATCC) or BEI Resources (NIAID), including *Escherichia coli* (ATCC 35218), *Escherichia coli* serotype O157 (TW07793), *Acinetobacter baumannii* (ATCC 19606), *Acinetobacter baumannii* (ATCC BAA1800), *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), and *Salmonella typhimurium* (ATCC BAA). 2721), *Salmonella enterica* serotype *Typhimurium* (NR4333), *Burkholderia cepacia* (UCB717), and *Streptococcus pneumoniae* (ATCC 6301). Colistin-resistant strains were generated using the following method.

[0115] Preparation of pharmaceutical grade rose red (HP-RBf).

[0116] The saline solution (HP-RBf) of the disodium rosenbergii preparation (10%) was supplied by Provectus Biopharmaceuticals, Inc. (Knoxville, TN, USA). HP-RBf (>99.5%) was synthesized according to Provectus’ proprietary method of synthesizing and utilizing the RB molecule as a viable pharmaceutical substance for commercial use. Detailed steps were previously described in

[35] and in U.S. Patent Nos. 8,530,675, 9,273,022 and 9,422,260 to Singer et al., mentioned above.

[0117] Logarithmic phase bacterial culture

[0118] All liquid bacterial cultures were performed in Erlenmeyer flasks equipped with air filters. Single bacterial strains were grown under the recommended conditions suggested by the ATCC. Culture flasks were incubated in a shaking incubator at 37°C with shaking at 200 rpm for 24 h, reaching mid-log (optical density -0.5). Optical density was monitored at 600 nm using a 96-well microplate reader.

[0119] Determination of the minimum inhibitory concentration (MIC)

[0120] All assays were performed according to guidelines established by the Clinical and Laboratory Standards Institute (CLSI; Wayne, PA, USA). Minimum inhibitory concentrations (MICs) were determined by broth dilution microplate alpha blue assay or by OD measurement. All commercial compounds were stored in DMSO or saline (1 mg / 100 μL concentration). A 10% saline solution (HP-RBf) of disodium rose rubrum was diluted with saline to prepare a 1 mg / 100 L stock solution. Aliquots of the stock solution were stored at 4°C for the duration of the assay.

[0121] Each compound from the stock solution was placed in the first well of a sterile 96-well plate and serially diluted 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 serially diluted antibacterial agent under aerobic conditions, and incubated at 37°C for 24 hours.

[0122] In a fluorescent lamp (17W, 63.8cm) 2 , Sunblaster Holdings) and LED (9.5W, 28.3cm 2 MIC studies of HR-RBf were performed using a UV-Vis spectrophotometer. OD was measured using a resazurin (20 μL) incubated at 37°C for 2 hours on a shaking incubator. See the National Committee for Clinical Laboratory Standards (NCCLS) [(NCCLS); Methods (pink = growth, blue = no visible growth)]. OD measurements were performed for all studies prior to colorimetric assays. Absorbance was measured at 570 nm and 600 nm using a Biotek SynergyXT 96-well plate reader.

[0123] Generation of drug-resistant Gram-negative bacterial strains

[0124] Colistin-resistant mutants of the test bacterial strains were generated using the same procedure. Bacterial cultures (100 μL, 1 × 10⁻⁶) were then cultured. 7 CFU / mL was plated on agar plates (55 cm²) containing colistin [minimum bactericidal concentration (MBC)]. 2 Collect colonies grown on agar plates containing colistin and suspend them in PBS buffer (approximately 1 × 10⁻⁶). 7The bacterial suspension was prepared in CFU / mL and 100 μL of the suspension was plated on agar plates containing antibiotic (1.5 × MBC). This process was repeated until the cells achieved a MIC level >10 times higher than the wild type; the antibiotic concentration was increased incrementally (2.0x, 2.5x, 3.0x, 3.5x, 4.0x, 5.0x, 7.0x, 8.0x, 9.0x, 10.0x, 20x, and 50x MBC). The isolated resistant cells were confirmed by MIC determination using the generated resistant strains.

[0125] Time-kill kinetics determination

[0126] Time-kill kinetics of antimicrobial agents were performed based on the CLSI guidelines (with slight modifications). Multiple time points were analyzed for both HP-RBf and the reference molecule. Bacterial cultures grown in broth were diluted to 1 × 10⁻⁶ with stock dilution of HP-RBf. 8 Up to 5.0×10 9 The concentration was CFU / mL, and the diluent was prepared at 5 times the MIC value.

[0127] Inoculate the test compound with an equal volume of the designated bacteria placed in a 96-well plate. Incubate the microtiter plate under fluorescent light at 37°C for a duration of 1–30 minutes (conditions summarized in the table above). Remove an aliquot of the culture from each well and perform serial dilutions. Incubate the diluted cultures at 37°C and count the CFU / mL. Bactericidal activity is defined as a reduction of colony-forming units greater than 3 logarithmic times.

[0128] References

[0129] 1. Boucher, HW, Talbot, GH, Benjamin, DK, Bradley, J., Guidos, RJ, Jones, RN, Murray, BE, Bonomo, RA, Gilbert, D. New drugs for Gram-negativebacilli. Clin. Infect. Dis. 56, 1685-1693 (2013).

[0130] 2. Bassetti, M., Peghin, M., Vena, A., Giacobbe, DRTreatment of infections due to MDR Gram-negative bacteria. Front. Med. (Lausanne). 16, 74 (2019).

[0131] 3.Bassetti,M.,Vena,A.,Giacobbe,D.R.,Castaldo,N.Management ofinfections caused by multidrug-resistant Gram-negative pathogens:Recentadvances and future directions.Arch.Med.Res.52,817-827(2021).

[0132] 4.Oliveira,J.,Reygaert,W.C.Gram negative bacteria.2022Oct 8.In:StatPearls[Internet].Treasure Island(FL):StatPearls Publishing;2022Jan.PMID:30855801.

[0133] 5.Loho,T.,Dharmayanti,A.Colistin:an antibiotic and its role inmultiresistant Gram-negative infections.Acta Med.Indones.47,157-68(2015).

[0134] 6.Gharaibeh,M.H.,Shatnawi,S.Q.An overview of colistin resistance,mobilized colistin resistance genes dissemination,global responses,and thealternatives to colistin:A review.Vet.World.12,1735-1746(2019).

[0135] 7.Pike,M.,Saltiel,E.Colistin-and polymyxin-induced nephrotoxicity:focus on literature utilizing the RIFLE classification scheme of acute kidneyinjury.J.Pharm.Pract.27,554-561(2014).

[0136] 8.Ordooei,Javan.A.,Shokouhi,S.,Sahraei,Z.A review on colistin nephrotoxicity.Eur.J.Clin.Pharmacol.71,801-810(2015).

[0137] 9.Jafari,F.,Elyasi,S.Prevention of colistin induced nephrotoxicity:areview of preclinical and clinical data.Expert Rev.Clin.Pharmacol.14,1113-1131(2021).

[0138] 10.Eljaaly,K.,Bidell,M.R.,Gandhi,R.G.,Alshehri,S.,Enani,M.A.,Al-Jedai,A.,Lee,T.C.Colistin nephrotoxicity:Meta-analysis of randomizedcontrolled trials.Open Forum Infect.Dis.8(2021).

[0139] 11.Biswas,S.,Brunel,J.M.,Dubus,J.C.,Reynaud-Gaubert,M.,Rolain,J.M.Colistin:an update on the antibiotic of the 21st century.ExpertRev.Anti.Infect.Ther.10,917-934(2021).

[0140] 12.Qadi M,Alhato S,Khayyat R,Elmanama AA.Colistin Resistance amongEnterobacteriaceae Isolated from Clinical Samples in Gaza Strip.Can J InfectDis Med Microbiol.2021 Apr 20;2021:6634684.

[0141] 13.Aghapour,Z.,Gholizadeh,P.,Ganbarov,K.,Bialvaei,A.Z.,Mahmood,S.S.,Tanomand,A.,Yousefi,M.,Asgharzadeh,M.,Yousefi,B.,Kafil,H.S.Molecularmechanisms related to colistin resistance in Enterobacteriaceae.Infect.Drug.Resist.24,965-975(2019).

[0142] 14.Zafer,M.M.,El-Mahallawy,H.A.,Abdulhak,A.,Amin,M.A.,Al-Agamy,M.H.,Radwan,H.H.Emergence of colistin resistance in multidrug-resistant Klebsiellapneumoniae and Escherichia coli strains isolated from cancer patients.Ann.Clin.Microbiol.Antimicrob.18,40(2019).

[0143] 15.Azimi,L.,Lari,A.R.Colistin-resistant Pseudomonas aeruginosaclinical strains with defective biofilm formation.GMS Hyg.Infect.Control.10(2019).

[0144] 16.Bialvaei,A.Z.,Kafil H.S.Colistin,mechanisms and prevalence ofresistance.Curr.Med.Res.Opin.31,707-721(2015).

[0145] 17.Qureshi,Z.A.,Hittle,L.E.,O’Hara,J.A.,Rivera,J.I.,Syed,A.,Shields,R.K.,Pasculle,A.W.,Ernst,R.K.,Doi,Y.Colistin-resistant Acinetobacterbaumannii:beyond carbapenem resistance.Clin.Infect.Dis.60,1295-1303(2015).

[0146] 18.Humphrey,M.,Larrouy-Maumus,G.J.,Furniss,R.C.D.,Mavridou,D.A.I.,Sabnis,A.,Edwards,A.M.Colistin resistance in Escherichia coli confersprotection of the cytoplasmic but not outer membrane from the polymyxinantibiotic.Microbiology(Reading)167,001104(2021).

[0147] 19.Lima,T.,Domingues,S.,Da Silva,G.J.Plasmid-mediated colistinresistance in Salmonella enterica:A review.Microorganisms 19,55(2019).

[0148] 20.Janssen,A.B.,Doorduijn,D.J.,Mills,G.,Rogers,M.R.C.,Bonten,M.J.M.,Rooijakkers,S.H.M.,Willems,R.J.L.,Bengoechea,J.A.,van Schaik,W.Evolution ofcolistin resistance in the Klebsiella pneumoniae complex follows multipleevolutionary trajectories with variable effects on fitness and virulence characteristics.Antimicrob.Agents Chemother.16,e01958-20(2020).

[0149] 21.Gogry,F.A.,Siddiqui,M.T.,Sultan,I.,Haq,Q.M.R.Current update onintrinsic and acquired colistin resistance mechanisms in bacteria.Front.Med.(Lausanne)12,677720(2021).

[0150] 22.Napier,B.A.,Burd,E.M.,Satola,S.W.,Cagle,S.M.,Ray,S.M.,McGann,P.,Pohl,J.,Lesho,E.P.,Weiss,D.S.Clinical use of colistin induces cross-resistance to host antimicrobials in Acinetobacter baumannii.mBio.21,e00021-13(2013).

[0151] 23.Dobias,J.,Poirel,L.,Nordmann,P.Cross-resistance to human cationicantimicrobial peptides and to polymyxins mediated by the plasmid-encoded MCR-1?Clin.Microbiol.Infect.23,676.e1-676.e5(2017).

[0152] 24.Hirsch,H.A.,McCarthy,C.G.,Finland,M.Polymyxin B and colistin;activity,resistance,and cross-resistance in vitro.Proc.Soc.Exp.Biol.Med.103,338-342(1960).

[0153] 25.Livermore,D.M.,Mushtaq,S.,Warner,M.Selectivity of ertapenem forPseudomonas aeruginosa mutants cross-resistant to other carbapenems.J.Antimicrob.Chemother.55,306-311(2005).

[0154] 26.Petrosillo,N.,Taglietti,F.,Granata,G.Treatment options forcolistin resistant Klebsiella pneumoniae:Present and future.J.Clin.Med.28,934(2019).

[0155] 27.Jana,B.,Cain,A.K.,Doerrler,W.T.,Boinett,C.J.,Fookes,M.C.,Parkhill,J.,Guardabassi,L.The secondary resistome of multidrug-resistant Klebsiellapneumoniae.Sci.Rep.15,42483(2017).

[0156] 28.Boucher,H.W.,Talbot,G.H.,Benjamin,D.K.,Bradley,J.,Guidos,R.J.,Jones,R.N.,Murray,B.E.,Bonomo,R.A.,Gilbert,D.New drugs for Gram-negativebacilli.Clin.Infect.Dis.56,1685-1693(2013).

[0157] 29.Meyer,A.L.Prospects and challenges of developing new agents fortough Gram-negatives.Curr.Opini.Pharmacol.5,490-494(2005).

[0158] 30.El-Gawad El-Sayed Ahmed,M.A.,Zhong,L-L.,Shen,C.,Yang,Y.,Doi,Y.,Tian,G-B.Colistin and its role in the Era of antibiotic resistance:anextended review(2000-2019).Emerg.Microbes.Infect.9,868-885(2020).

[0159] 31.Aris,P.,Robatjazi,S.,Nikkhahi,F.,Marashi,S.M.A.Molecularmechanisms and prevalence of colistin resistance of Klebsiella pneumoniae inthe Middle East region:A review over the last 5 years.J.Glob.Antimicrob.Resist.22,625-630(2020).

[0160] 32.Zhang,Q.,Chen,S.,Liu,X.,Lin,W.,Zhu,K.Equisetin restores colistinsensitivity against multi-drug resistant Gram-negative bacteria.Antibiotics(Basel)18,1263(2021).

[0161] 33.Elio,C.,PhD;Erica,R.,Marcello,M.Infections due to antibiotic-resistant Gram-negative bacteria in pediatrics:Possible managementstrategies.Pediatric Infect.Dis.J.41,e283-e285(2022).

[0162] 34.Lelovic,N.,Mitachi,K.,Yang,J.,Lemieux,M.R.,Ji,Y.,Kurosu,M.Application of Mycobacterium smegmatis as a surrogate to evaluate drugleads against Mycobacterium tuberculosis.J.Antibiotics 73,780-789(2020).

[0163] 35.Kurosu,M.,Mitachi,K.,Yang,J.,Pershing,E.V.,Horowitz,B.D.,Wachter,E.A.,Lacey,J.W.3 rd .,Ji Y.,Rodrigues,D.J.Anti-bacterial activity ofpharmaceutical-grade rose bengal:An application of a synthetic dye in anti-bacterial therapies.Molecules 27,322(2022).

[0164] 36.Patel,S.P.,Carter,B.W.Percutaneous hepatic injection of rosebengal disodium(PV-10)in metastatic uveal melanoma.J.Clin.Oncol.38,3143(2020).

[0165] 37.Nakonieczna,J.,Wolnikowska,K.Rose bengal-mediatedphotoinactivation of multidrug resistant Pseudomonas aeruginosa is enhancedin the presence of antimicrobial peptides.Front.Microbiol.9,1949(2018).

[0166] 38.Zhou,K.,Cattoir,V.,Xiao,Y.Intrinsic colistin resistance.LancetInfect.Dis.16,1227-1228(2016).

[0167] 39.Li,X.,Gu,Y.,Dong,H.,Wang,W.,Dong,C.Trapped lipopolysaccharide andLptD intermediates reveal lipopolysaccharide translocation steps across theEscherichia coli outer membrane.Sci.Rep.7,11883(2015).

[0168] 40.Sabnis,A.,Hagart,K.L., A.,Becce,M.,Evans,L.E.,Furniss,R.C.D.,Mavridou,D.A.,Murphy,R.,Stevens,M.M.,Davies,J.C.,Larrouy-Maumus,G.J.,Clarke,T.B.,Edwards,A.M.Colistin kills bacteria by targetinglipopolysaccharide in the cytoplasmic membrane.Elife 6,e65836(2021).

[0169] 41.Freinkman,E.,Chng,S-S.,Kahne,D.The complex that insertslipopolysaccharide into the bacterial outer membrane forms a two-proteinplug-and-barrel.PNAS 108,2486-2491(2010).

[0170] 42.Wachter,E.,Dees,C.,Harkins,J.,Scott,T.,Petersen,M.,Rush,R.E.,Cada,A.Topical rose bengal:pre-clinical evaluation of pharmacokinetics andsafety.Lasers Surg.Med.32,101-10(2003).

[0171] 43.Lee,Y.C.,Park,C.K.,Kim,M.S.,Kim,J.H.In vitro study for stainingand toxicity of rose bengal on cultured bovine corneal endothelialcells.Cornea 15,376-385(1996).

Claims

1. A method for treating Gram-negative bacteria other than Burkholderia, Proteus, and Serratia, said Gram-negative bacteria also exhibiting resistance to the antibacterial compound colistin, said method comprising the following steps: a) Contact the Gram-negative bacteria with an aqueous pharmaceutical composition containing a rose red (RB) compound of formula I dissolved or dispersed therein at a concentration of about 0.01 to about 15 mg / mL; and b) Irradiate the contacted bacteria with light of a wavelength of approximately 500 nm to approximately 600 nm for a period of approximately 1 to approximately 10 minutes, providing the irradiated bacteria with approximately 16 to approximately 160 J / cm³. 2 light dose, Where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and R 2 It does not exist, and when X is nitrogen, n is 1 and R 2 exist; When X is oxygen, R 1 M is selected from hydrogen (H) as a pharmaceutically acceptable cation. + The group consisting of C1-C4 alkyl groups and aromatic rings as defined below; When X is nitrogen, R 1 and R 2 The same or different, and selected from the group consisting of hydrogen, C1-C4 alkyl, or 5- or 6-membered rings formed together with amide nitrogen atoms, and aromatic rings as defined below; The aromatic ring is a 5- or 6-membered monocyclic ring, or a 5,6- or 6,6-fused aromatic ring system, wherein the aromatic ring or ring system contains 0, 1 or 2 heterocyclic atoms that are independently nitrogen, oxygen or sulfur.

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

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

4. The method of claim 1, wherein, upon contact, the Gram-negative colistin-resistant bacteria are present within or on mammalian cells.

5. The method of claim 1, wherein the Gram-negative colistin-resistant bacteria are present on or in surgical or other wounds of a mammalian subject.

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

7. The method of claim 1, wherein the Gram-negative colistin-resistant bacteria are irradiated for a period of about 2 to about 5 minutes to provide about 32 to about 80 J / cm³. 2 Light dose.

8. The method of claim 1, wherein the aromatic ring substituent is selected from one or more of the group consisting of: in for Esters or monosubstituted amines are provided, respectively.

9. A pharmaceutical composition for treating Gram-negative bacteria other than Burkholderia, Proteus, and Serratia, which also exhibit resistance to the antibacterial compound colistin, the pharmaceutical composition comprising a rose red (RB) compound of formula I. Where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and R 2 It does not exist, and when X is nitrogen, n is 1 and R 2 exist; When X is oxygen, R 1 M is selected from hydrogen (H) as a pharmaceutically acceptable cation. + The group consisting of C1-C4 alkyl groups and aromatic rings as defined below; When X is nitrogen, R 1 and R 2 The same or different, and selected from the group consisting of hydrogen, C1-C4 alkyl, or 5- or 6-membered rings formed together with amide nitrogen atoms, and aromatic rings as defined below; The aromatic ring is a 5- or 6-membered monocyclic ring, or a 5,6- or 6,6-fused aromatic ring system, wherein the aromatic ring or ring system contains 0, 1 or 2 heterocyclic atoms that are independently nitrogen, oxygen or sulfur.

10. The composition of claim 9, wherein the RB compound is disodium rose red.

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

4.

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

81.

13. The composition of claim 9, wherein the composition further comprises 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, phosphate and nitrate.

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

15. The composition of claim 13, wherein the water-soluble electrolyte is present in the composition at a concentration of 0.1 wt% to 2 wt%.

16. The composition of claim 13, wherein the water-soluble electrolyte is present in the composition at a concentration sufficient to provide an isotonic molar concentration of 300 mIsmol / kg water to 500 mIsmol / kg water.

17. The composition of claim 9, wherein the composition further comprises a diluent comprising a C1-C6 monohydric alcohol or polyhydric alcohol present in the diluent at a concentration of less than 5% by volume.

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

19. A bactericide for treating Gram-negative bacteria other than Burkholderia, Proteus, and Serratia, which also exhibit resistance to the antibacterial compound colistin, said bactericide comprising a rose red (RB) compound of formula I. Where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and R 2 It does not exist, and when X is nitrogen, n is 1 and R 2 exist; When X is oxygen, R 1 M is selected from hydrogen (H) as a pharmaceutically acceptable cation. + The group consisting of C1-C4 alkyl groups and aromatic rings as defined below; When X is nitrogen, R 1 and R 2 The same or different, and selected from the group consisting of hydrogen, C1-C4 alkyl, or 5- or 6-membered rings formed together with amide nitrogen atoms, and aromatic rings as defined below; The aromatic ring is a 5- or 6-membered monocyclic ring, or a 5,6- or 6,6-fused aromatic ring system, wherein the aromatic ring or ring system contains 0, 1, or 2 heterocyclic atoms that are independently nitrogen, oxygen, or sulfur. The bactericide is designed for use in conjunction with a fluorescent light source having a wavelength of about 500 nm to about 600 nm. The light source is applied to the Gram-negative bacteria for a period of approximately 1 to 10 minutes to provide approximately 16 to 160 J / cm². 2 Light dose.

20. The bactericide of claim 19, wherein the bactericide is applied to at least one of the surface of the surgical area, surgical instruments, the surgeon's hand, and internal and external regions of the patient's body.

Images