Description

BACKGROUND ART

The increasing emergence of multidrug- resistant (MDR) Gram-positive bacteria is one of the major public health threats [1-3] . Particularly, MDR strains of Staphylococcus , Enterococcus , and Streptococcus spp. have a significant impact on morbidity and mortality [4] . The increasing resistance rates of these pathogens against the critically important antibacterial agents (e.g., β-lactams, macrolides, aminoglycoside, fluoroquinolones, glycopeptide, oxazolidinones , cyclic peptide, and depsipeptides ) are of great concern [5] . To date, very few new chemical entities have been examined in late-stage clinical studies for the treatment of infections caused by MDR bacterial pathogens [6] .

Naturally occurring and synthetic dyes have been applied as antibacterial or antiprotozoal agents [7] . For example, methylene blue and clofazimine are still considered to be important orphan drugs [8,9] .

Commercial -grade Rose Bengal (RB)

Rose bengal (RB) is a bright rose-red xanthene derivative compound that was first synthesized in the 19 ^th century as a wool dye and subsequently used as a food dye in Japan (food red no. 105) [28] . More particularly, RB is a derivative of the xanthene compound fluorescein. Compared to fluorescein, RB has two types of additional halogens: four chloride and four iodide substituents .

The use of RB for the visual diagnosis of human ocular surface damage (via ocular instillation) was first described in 1914 [29] . RB was later introduced as an intravenously- administered, relatively rapid diagnostic aid to evaluate the functional capacity of a human liver after a single 100 mg dose [30] . In 1971, ¹³¹ I RB (Robengatope®, rose bengal sodium ¹³¹ I injection USP) was approved by the U.S. Food and Drug Administration (FDA) for use as a diagnostic aid in determining liver function [31,32] .

In 2009, Robengatope' s manufacturer Bracco Diagnostics Inc. formally withdrew the RB product from the U.S. market because of the emergence of newer methods of liver imaging, such as computed tomography. In 1974, Barnes-Hind Pharmaceuticals Inc. (Barnes-Hind) introduced a medical device product of 1% RB in an aqueous solution for the disclosure of corneal injury, the diagnosis of keratitis, keratoconjunctivitis, and sicca, and the detection of foreign bodies in the eye [33] . In 1981, Barnes-Hind introduced ophthalmic strips of the same concentration for the same indications. Although both products were accepted by the U.S. Food and Drug Administration (FDA) for marketing, neither the solution and strip devices nor their respective claims were approved because their introductions predated formal FDA review and approval .

Commercial-grade RB, with marketed dye contents that can vary between 80% and 95% RB, including gross contaminants and substance-related impurities, is manufactured using an historical process developed by Gnehm in the 1880s. It is thought that RB used in diagnostic applications is a commercial-grade RB that contains some impurities [34] . The United States Pharmacopeia (USP) previously listed RB as an analytical standard. RB was removed from the USP in 2019. Thus, commercial- grade RB lacks relevance in the context from modern diagnostic and therapeutic settings. Therefore, it poses significant regulatory challenges to validate RB for application to the treatment of human diseases .

Rose Bengal (RB) dye ( 4 , 5, 6, 7-tetrachloro- 2 ', 4 ', 5 ', 7 ' -tetraiodofluorescein) has been clinically investigated for the treatment of melanoma and the other solid cancers [10-14] . Photodynamic antibacterial properties of RB have been sporadically reported [15-27 and 70] . For example, Dees et al. [75] teaches use of RB at 1-10 μM in conjunction with green light irradiation in the 500-600 nm wavelength band against Gram-positive and Gram-negative antibiotic-resistant bacteria without specifics as to the light source, its intensity or duration of irradiation. That disclosure did report use of RB with light at 100 J/cm2 of 532 nm light at an intensity of 200 mW/cm2 for treating tumors (Table 3) .

Here, we report the antibacterial activity of a pure form of RB and its scope and limitations as an antibacterial agent for the treatment of MDR Gram-positive bacterial infections in the presence and absence of light. BRIEF SUMMARY OF THE INVENTION

The present invention contemplates the combined use of a rose bengal derivative in conj unction with irradiation of bacteria with light of the wavelength about 500 nm to about 600 nm for a time period of about 1 to about 10 minutes to treat and kill the irradiated bacteria . In one aspect , Gram-positive bacteria are treated in a method that includes the steps of contacting the bacteria with an aqueous pharmaceutical composition containing a rose bengal (RB ) compound of Formula I , below, dissolved or dispersed therein at a concentration of about 0 . 2 to about 3 . 1 μg/mL ; and irradiating those contacted bacteria with light of the wavelength about 500 nm to about 600 nm for a time period of about 1 to about 10 minutes to provide a light dose of about 0 . 7 to about 7 . 2 J/cm ² . The RB compound of

Formula I is shown below : wherein X is oxygen or nitrogen, "n" is zero or 1 such that when X is oxygen, n is zero and R2 is absent , whereas when X is nitrogen, n is 1 and R ² is present . When X is oxygen, R ¹ is selected from the group consisting of hydrogen (H) , M ⁺ that is a pharmaceutically acceptable cation, C ₁ -C ₄ alkyl, and an aromatic ring-containing substituent as defined herein after. When X is nitrogen, 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 amido nitrogen atom form a 5- or 6-membered ring, and an aromatic ring substituent. The aromatic ring substituent is a single ring containing 5- or 6-members, or a 5, 6- or 6, 6-fused aromatic ring system, and the aromatic ring or ring system substituent can contain 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur .

Structural formulas of exemplary aromatic ring substituents are set out below: ester or a monosubstituted amine, respectively.

Illustrative treated Gram-positive bacteria include one or more of drug-susceptible and drug-resistant S. aureus, S. epidermis , E. faecalis and E. f aecium, as well as one or more of Bacillus subtilis , Bacillus cereus, and Streptococcus salivarius . Gram-positive bacteria are present within or on mammalian cells when contacted or present as a biofilm. Preferably, the bacteria are irradiated for a time period of about 2 to about 5 minutes to provide a light dose of about 1.4 to about 3.6 J/cm2.

A similar method is also contemplated for treating Gram-negative bacteria that is one or more of Burkholderia Salmonella, and Proteus . Here, an above RB compound is present dissolved or dispersed in an aqueous pharmaceutical composition at a concentration of about 2 to about 15 M.

BRIEF DESCRIPTION OF THE FIGURES

In the drawings forming a part of this disclosure,

Fig. 1, in six parts as Figs. 1A, IB, 1C, ID, IE, and IF, is Table 1 that shows minimal inhibitory concentrations (MICs) of the growth of Gram-positive and Gram-negative bacteria contacted with varying concentrations of rose bengal disodium RBD) dissolved in an aqueous solution of 0.9 N sodium chloride, when illuminated with a fluorescent light, an LED light, sun light (through a window- pane) and in the dark. All studies were done in triplicate. The MIC values were determined via OD and colorimetric assays using risazurin or malachite green. Bacteria were purchased from ATCC or acquired from BEI Resources (BEI Resources, American Type Culture Collection, Manassas, VA, USA) . A 17W, 63.8cm ² fluorescent light was used. The MIC was determined after 24 hours (h) of treatment (23.0 KJ/cm ² ) . A 9.5W, 28.3cm ² LED light was used. The MIC was determined after 24h of treatment (29.0 KJ/cm ² ) . The studies were performed in the BSL-2 lab on the 5th floor, College of Pharmacy, University of Tennessee Health Science Center (UTHSC) . The 96- well plates were placed on the east side of the laboratory and exposed to sunlight filtered through an architectural window. The studies were terminated after 9h (8AM-5PM, sunny, 34 °C (outside) , 27 °C (inside) ) . The studies were performed in the dark room. The 96-well plates were covered with aluminum foil. The MIC values were determined after 24h. "g" = MIC was not determined.

Fig. 2 shows the MIC and minimum bactericidal concentrations (MBCs) of eleven bacterial strains contacted with RBD by agar dilution and broth dilution illuminated by fluorescent light. All studies were done in triplicate. The MIC values were determined via counting the colony-forming units (CFUs) . Bacteria were purchased from ATCC or acquired from BEI Resources, as above. A 17W, 63.8cm ² fluorescent light was used. The MIC was determined after 24 hours (h) of treatment (23.0 KJ/cm ² ) . MBC : minimum bactericidal concentrations ( μg/mL) . "Sun" studies carried out as in Fig. 1. "Dark" studies were performed in the dark room. RB-agar prepared in the 24-well plates and 35 mm culture dishes were covered with aluminum foil, or in the dark (by agar dilution) .

Fig. 3, in three panels as Figs. 3A, 3B and 3C, in a time-kill kinetics study showing cell viability (log ₁₀ CFU/mL) versus time in minutes using PV-10®, a formulated rose bengal (HB-RBf) composition containing 10% rose bengal w/v in 0.9 percent aqueous sodium chloride (NaCl) available from Provectus Biopharmaceuticals, Inc., of Knoxville, TN) , HB-RBf at 5.0 mg/mL, HB-RBf at 1.6 mg/mL, ciprofloxacin at 18 mg/mL and linezolid at 10 mg/mL in Fig. 3A; twice the MIC concentration of HB- RBf was used against S. aureus BAA-44™ (3.2 mg/mL) , S. aureus 71080 (VRS8) (0.8 mg/mL) and E. faecium NR-32065 (1.6 mg/mL) in the time-kill kinetics study against drug-resistant Gram-positive bacteria in Fig. 3B; and Fig. 3C provides graphs illustrating time-kill kinetics of RB and of anti-Gram-negative antibiotics against B. cepacia (UCB707) ;

Fig. 4, in three panels as Fig. 4A, Fig. 4B and Fig. 4G, illustrates a comparison of noted concentrations of linezolid and HB-RBf, with the HB- RBf treatment being under fluorescent light or in the dark, in the treatment of biofilms due to each of three Gram-positive bacteria; S. aureus 6508™ in Fig. 4A, S. aureus 71080 (VRS8) in Fig. 4B, and E. Faecium NR-32065 in Fig. 4G;

Fig. 5 is a graph showing the time in hours (h) for cell confluence at four concentrations of HB-RBf (zero μM, 100 μM, 200 μM and 300 μM) when irradiated under fluorescent light;

Fig. 6, in two panels as Fig. 6A (upper group) and Fig. 6B (lower group) , are photomicrographs of multi-layered human epithelial keratinocytes (HEKa) upon treatment with HB-RBf (i.e. "PV-10", see definition above) under fluorescent light in which the cells were grown for 25 days using the Nunc™ Cell Culture Insert System (CLS-AN-047W) [67] , with the cells being grown in the absence (control) , 10 μM, 100 μM and 200 μM RB under the fluorescent light for one hour (17 W, 63.8 cm2, 0.96KJ/cm2) , recovered and then stained with hematoxylin and eosin, and in which the photomicrographs of Fig . 6A are a top view at X10 , and those of Fig . 6B are a side view at x40 .

Some Gram-positive bacterial strains are referred to herein as being "resistant" or "drug resistant" or other grammatical variants of resistance . The "resistance" meant here is of the bacteria to treatment with one or more antibacterial pharmaceutical products that are usually deemed to be bactericidal at known concentrations and bacterial cell densities to that type of bacteria . There are thus , both "drug-susceptible" and "drug- resistant" strains of bacteria such as Staphyl ococcus aureus ( S . aureus) , Staphyl ococcus epidermidi s (S . epidermi s) , Enterococcus faecali s ( E. faecali s) and Enterococcus faeci um (E. f aeci um) . I llustrative drugs to which several common bacteria strains have become resistant include vancomycin, methicillin and gentamicin .

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

One embodiment of the present invention contemplates the medicinal use in the presence of light having a wavelength of about 500 to about 600 nm of rose bengal (RB ) or a derivative thereof (RB compound) ( shown hereinafter ) as a bactericide against Gram-positive bacteria . In one aspect , mammalian cells such as those of humans , which are infected by the Gram-positive bacteria, and the bacteria are contacted by RB compound which is taken-up by the infected cells . The light can be administered along with a contemplated RB-containing pharmaceutical composition in which a contemplated RB compound is present contacting the infected mammalian cells at a concentration of about 0 . 2 to about 3.1 μg/mL dissolved or dispersed in a pharmaceutically acceptable diluent. The light can also be administered shortly after application of the pharmaceutical composition to contact the bacteria or mammalian cells containing the bacteria, e.g., within about 2 to about 5 minutes. The bacteria can be treated within infected mammalian cells or when present as a biofilm.

Although seemingly similar to prior treatments, it is believed that the concentrations of the RB compound are less than those used previously, and that the light amount is about one tenth or less than that of a prior method. As a consequence, less of the anti-bacterial RB compound can be used, and lower-priced lighting so a to make home use more practicable.

The infected mammalian cells treated with RB are irradiated for a time period of about 1 to about 10 minutes, and more preferably about 2 to about 5 minutes. Such irradiation provides a light dose of about 0.7 to about 7.2 J/cm2, and more preferably provide a light dose of about 1.4 to about 3.6 J/cm^.

Exemplary Gram-positive bacteria for treatment and killing include one or more of drug- susceptible and drug-resistant S. aureus, S. epidermis , E. faecalis and E. f aecium, as are one or more Bacillus subtilis , Bacillus cereus, and Streptococcus salivarius .

A contemplated RB compound has the structural formula (Formula I) below, where X is 0 (oxygen) or N (nitrogen) , and "n" is zero or one.

When X is oxygen, n is zero and absent so that the RB compound is a) rose bengal where -X-R ¹ is -O-H, b) is a pharmaceutically acceptable salt of RB where X-R ¹ is -O- M ⁺ and where M ⁺ is a pharmaceutically acceptable cation, c) a C ₁ -C ₄ alkyl ester, or is d) an aromatic ester as defined below.

Alternatively, when X is a nitrogen atom, n is 1 and R2 is present along with R^ . As such, R ¹ and R^ can be the same or different and -C (O) -NR ¹ R ² is an amide whose nitrogen atom is a) unsubstituted [-X- (R ¹ R ² ) and both R^ and R2 are hydrogen (H) ] , is b) substituted with one or two C ₁ -C ₄ alkyl groups or together with the amido nitrogen atom form a 5- or 6-membered ring, or is c) an aromatic amide that is preferably monosubstituted in that R^ is hydrogen and R^ is the aromatic substituent discussed below.

For ease of description, an aromatic ester or aromatic amide are collectively referred to as an aromatic derivative. As such, those derivatives are formed from an alcohol or amine, preferably monosubstituted, having a single 5- or 6-membered aromatic ring, or a 5, 6- or 6, 6-fused aromatic ring system that contains 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur.

Illustrative examples of such aromatic alcohol ester portions are shown and named below, where 0 is an oxygen atom and line-0 indicates the ring-oxygen can be from any available carbon of the ring and the 0-line crossed by a wavy line indicates that the depicted alkoxy group is a portion of another molecule, the esterified RB molecule. ester or a monosubstituted amine, respectively.

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 illustratively herein for the group of RB compounds.

The chemical name for rose bengal is 4 , 5, 6, 7-tetrachloro-2' , 4 ' , 5 ' , 7 ' -tetraiodo - fluorescein. A preferred form, rose bengal disodium (RBD) , has the following structural formula:

Certain details of this preferred embodiment for a contemplated composition are described in U.S. Patents No. 5,998,597, No. 6,331,286, No. 6,493,570, and No. 8,974,363, whose disclosures are incorporated by reference herein in their entireties. The above patents describe the use of RBD to kill cancer cells.

A similar method to that described above is contemplated a in an embodiment for treatment of Gram-negative bacteria that are one or more of Burkholderia Salmonella, and Proteus . In this embodiment, a rose bengal (RB) compound of Formula I, described previously, is dissolved or dispersed in an aqueous pharmaceutical composition at a concentration of about 2 to about 15 μM.

Pharmaceutical Compositions

Rose Bengal (RB)

Liquid Compositions

A contemplated liquid composition can be formulated for oral administration, parenteral administration or topical administration.

Turning first to a parenteral composition, the previously noted PV-10® composition is illustrative of a parenterally-administrable pharmaceutical composition containing the particularly preferred RB compound, rose bengal disodium. Delivery of the halogenated fluorescein component of a contemplated composition is most favorable when the composition has a pH value close to physiologic pH (i.e., approximately pH 7) , and especially when the pH value is greater than about 4, thereby assuring that a halogenated fluorescein remains in dibasic form in the composition. Thus, in a preferred embodiment, the pH value of the composition is about 5 to about 9, and 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 fluoresceins typically remain in dibasic form, rather than the lactone that forms at low pH values.

An RB compound such as rose bengal is dibasic, having pK _a values of 2.52 and 1.81. pK _a value determinations for several contemplated halogenated fluorescein s can be found in Batsitela et al., Spectrochim Acta Part A 79 (5) : 889-897 (Sept. 2011) .

A hydrophilic vehicle is one preferred medium for the medicament to maximize preference for partitioning of the halogenated fluorescein component into tissue, particularly for RB compounds in the acid and/or salt form. Accordingly, in a preferred embodiment, the vehicle contains a minimum of non-hydrophilic components that might interfere with such partitioning. Accordingly, a preferred formulation of the composition contains RB, or RB disodium that is particularly preferred, in a hydrophilic, preferably water-containing vehicle.

When administered parenterally, other than in a suppository, RB compound-containing pharmaceutical composition preferably includes 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 electrolyte is preferably at a concentration of about 0.1% (w/v) and about 2% (w/v) .

Alternately, the electrolyte is present at a level sufficient to provide an osmolality of greater than approximately 100 mOsm/kg up to about 600 mOsm/kg. More preferably, the osmolality of the medicament composition is greater than 250 mOsm/kg, and most preferably approximately 300-500 mOsm/kg.

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 approximately 0.9% as is present in physiological saline .

The aqueous medium (diluent) of the composition is preferably only water that meets the criteria for use in injection. Up to about 20 percent by volume of the diluent can be one or more C ₁ -C ₆ mono-or polyhydric alcohols such as methanol, ethanol, propanol, isopropanol, butanol, sec- butanol, glycerol, ethylene glycol, propylene glycol, 1 , 2-butanediol , 2 , 3-butanediol , erytritol, threitol, trimethylolpropane, sorbitol and the like. More preferably, an alcohol is present in a contemplated composition at less than about 10 percent by volume of the diluent, and more preferably at less than about 5 percent by volume. The terms "physiologically acceptable salt" and "pharmaceutically acceptable salt" in their various grammatical forms refer to any non- toxic cation such as an alkali metal , alkaline earth metal , and ammonium salt commonly used in the pharmaceutical industry, including the sodium, potassium, lithium, calcium, magnesium, barium, and protamine zinc salts , which can be prepared by methods known in the art . A contemplated cation provides a water-soluble RB salt . Preferably, the salts are sodium, potassium, and calcium in either the mono or dibasic salt form . The reader is directed to Berge , J. Pharm . Sci . 1977 68 ( 1 ) : 1- 19 for lists of commonly used physiologically ( or pharmaceutically) acceptable acids and bases that form physiologically/pharmaceutically acceptable salts with pharmaceutical compounds .

The pH value of the RB-containing pharmaceutical composition can be regulated or adj usted by any suitable means known to those of skill in the art . The composition can be buf fered or the pH value adj usted by addition of acid or base or the like . As RB, or physiologically acceptable salts thereof , are weak acids , depending upon its concentration and/or electrolyte concentration, the pH value of the composition may not require the use of a buf fer and/or pH value-modi fying agent . It is especially preferred, however, that the composition be free of buf fer, allowing it to conform to the biological environment once administered .

It is also preferred that the pharmaceutical composition not include any preservatives , many of which can deleteriously interfere with the pharmaceutical composition or formulation thereof , or may complex or otherwise interact with or interfere with the delivery of the RB compound-containing composition active component . To the extent that a preservative is used, imidurea is a preferred preservative as it does not interact with RB compounds , either in the pharmaceutical composition or upon administration .

A contemplated liquid pharmaceutical composition can also be adapted for oral administration to the mammalian subj ect to be treated . In a preferred aspect , the RB compound, as previously discussed, is dissolved or dispersed in an aqueous diluent when administered to a mammalian subj ect . It is more preferred that the aqueous diluent be free of tonicity agents except for those sugars and/or buf fering agents present as flavorants .

Up to about 20 percent by volume of the diluent can be one or more C ₁ -C ₆ mono-or polyhydric alcohols as was previously discussed . More preferably, an alcohol is present in a contemplated composition at less than about 10 percent by volume of the diluent , and more preferably at less than about 5 percent by volume .

A topically-applied liquid composition is also contemplated . One such liquid composition that is undergoing clinical trials for treatment of psoriasis is a developmental medicament called PH- 10® by Provectus Biopharamceuticals , Inc . of Knoxville , TN . This medicament contains RBD present at a concentration of 0 . 001 to 0 . 01 % w/v dissolved or dispersed in an aqueous diluent , along with at least one builder present at a level suf ficient to provide a viscosity of 10- 1000 cps to the medicament, sodium chloride as an electrolyte present at a concentration of 0.9% w/v of at a level sufficient to provide an osmolality of 100 mOsm to 500 mOsm/kg to the medicament. This medicament is described and claimed in, e.g., U.S. Patent No. 8, 974,363.

Solid Compositions

It is further contemplated that the RB compound such as RB itself or disodium RB, be administered in a solid pharmaceutical composition for oral administration that is enterically-coated to pass through the stomach and release the RB compound relatively close to the site of the bacterial infection so that there will be a lesser amount of wasted RB compound bound to tissues dorsal to the site of the infection and less likely visible tissue staining. The RB compound is typically dissolved in or dispersed in or on a solid diluent matrix .

There are several factors at play in the dissolution of an orally administered solid pharmaceutical product in a mammalian body. Among those factors are residence time of the medicament at different locations along the GI tract, particle size, solubility of the individual components of the medicament in the bodily fluids likely to be encountered from mouth to anus, the order in which various coating layers, when present, are applied to the medicament, as well as the pH value at which a particular coating layer is soluble.

For example, the highly acidic gastric environment (pH 1.5-2 in the fasted state; pH 3-6 in the fed state) rises rapidly to about pH 6 in the duodenum and increases along the small intestine to pH 7 . 4 at the terminal ileum . The pH value in the cecum drops j ust below pH 6 and again rises in the colon reaching pH 6 . 7 at the rectum [Hua, Front Pharmacol ll : Article 524 (April 2020 ) ] . Observation of solutions of disodium RB mixed into a water solution having the acidic pH value of the human stomach revealed rapid clouding of the admixture and clumping of the previously soluble disodium RB, presumably into the lactone form .

Gastric transit can range from 0 to 2 hours in the fasted state and can be prolonged up to 6 hours in the fed state . In general , the transit time in the small intestine is considered relatively constant at around 3 to 4 hours , but can range from 2 to 6 hours in healthy individuals . Colonic transit times can be highly variable , with ranges from 6 to 70 hours reported [Hua, Front Pharmacol ll : Article 524 (April 2020 ) ] .

One approach useful for predictable release of a medicament to a particular location in the GI tract relies upon pH-speci fic coatings and matrices that dissolve or disintegrate at preselected GI tract pH values such as those noted previously . Particularly preferred illustratively, for release in or near the colon, neutral or slightly alkaline pH values are utili zed to release the drug in the distal part of the small intestine or in the colon .

The table below shows some examples of pH- dependent polymer coatings that have been used for the purpose of colonic targeting ( local treatment ) either alone or in combination, including some methacrylic resins ( commercially available from Evonik Industries, AG, Essen, Germany as Eudragit®) , and hydroxypropyl methylcellulose (HPMC; available from DuPont, Wilmington, DE as Methocel™; and Ashland, Inc., as Benecel™, Wilmington, DE) derivatives. In addition to triggering release at a specific pH value range, the enteric coating can protect the incorporated RB compound active agent against the harsh GI tract environment (e.g., gastric juice, bile acid, and microbial degradation) and can create an extended and delayed drug release profile to enhance therapeutic efficiency.

The Table below lists several commercially available enteric coating polymers and the "published pH release" value from their manufacturer. The "published pH release" values are not absolute for all compositions or environments, and pH values for dissolution or disintegration stated herein are based on those published values. For the local treatment of colonic infections, colon-targeted drug delivery systems have been actively pursued because conventional non- targeted therapy can have undesirable side-effects and low efficacy due to the systemic absorption of drug before reaching the target site. Liu et al., Eur. J. Pharm. Biopharm. 74:311-315 (2010) , adopted dual coating approach by using the alkaline aqueous solution of Eudragit® S with buffering agents for inner layer and the organic solution of Eudragit® S for outer layer, accelerating the drug dissolution at pH values greater than 7. Subsequently, Varum et al., Eur. J. Pharm. Biopharm. 84:573-577 (2013) , evaluated in vivo performance of this dual coated system in humans, demonstrating more consistent disintegration of dual coated tablets mainly in the lower intestinal tract.

Hashem et al., Br. J. Pharm. Res. 3:420- 434 (2013) , developed microspheres combining time- and pH-dependent systems for colonic delivery of prednisolone. By using a combination of Eudragit® S and ethyl cellulose, they achieved greater colonic drug delivery while preventing premature drug release in the upper intestine.

Eudracol® is another example of a multi- unit technology providing targeted drug delivery to the colon, with delayed and uniform drug release. This system is based on coating the pellet with Eudragit® RL/RS and Eudragit® FS 30D, providing colon-specific drug release in a pH-and time- dependent manner [Patel, Expert Opin. Drug Deliv. 8:1247-1258 (2011) ] . One composition that targets the small intestine comprises a diluent matrix of sugar/ sucrose beads coated with particulate rose bengal (RB ) that is coated with one or a plurality of layers of a (meth) acrylate copolymer that is composed of about 60 to about 95% by weight free radical polymeri zed C ₁ -C ₄ -alkyl esters of acrylic or methacrylic acid and about 5 to about 40% by weight (meth) acrylate monomers with an acidic group in the alkyl radical .

Particularly suitable (meth) acrylate copolymers include about 10 to about 30% by weight methyl methacrylate , about 50 to about 70% by weight methyl acrylate and about 5 to about 15% by weight methacrylic acid (Eudragit® FS type ) . Similarly suitable , are (meth) acrylate copolymers of about 20 to about 40% by weight methacrylic acid and about 80 to about 60% by weight methyl methacrylate (Eudragit® S type ) . The word " (meth) acrylate" is used to mean that either or both of acrylate and methacrylate monomers can be used .

These coating polymers permit little i f any RB compound release prior to the particles leaving the stomach . The pH value of the fluid within the duodenum typically is about 6 and rises to about 7 . 4 toward the ileum . Thus , i f the bacterial infection is closer to the stomach, a coating polymer with a greater amount of free carboxylic acid groups is utili zed, whereas i f the tumor is further toward the ileum, a polymer with a lesser of amount of acid groups can be utili zed .

A usual tablet or lozenge can be prepared by admixture of lactose ( 20% ) and active ingredient ( 80% ; RB compound) mixed in a high-speed mixer ( DIOSNA type PI O , Osnabruck, Germany) . An aqueous solution containing the excipient polyvinyl- pyrrolidone ( PVP ) such as povidone ( Sigma-Aldrich International GmbH, Buchs , CH) is added in small amounts until a homogeneous composition is obtained . The moist powder mixture is screened . Tablets are subsequently made therefrom as is well-known, and dried .

The resulting tablets or lozenges are thereafter preferably coated with a protective polymer film, often using fluidi zed bed equipment . Film- forming polymers are normally mixed with plastici zers and release agents by a suitable process . The film formers can in this case be in the form of a solution or suspension . The excipients for the film formation can likewise be dissolved or suspended . Organic or aqueous solvents or dispersants can be used . Stabili zers can be used in addition to stabili ze the dispersion ( for example : Tween® 80 or other suitable emulsi fiers or stabili zers ) .

Examples of release agents are glycerol monostearate or other suitable fatty acid derivatives , silicic acid derivatives or talc . Examples of plastici zers are propylene glycol , phthalates , polyethylene glycols , sebacates or citrates , and other substances mentioned above and in the literature .

Another preferred type of medicament is a water-soluble capsule or blister containing a plurality of particles of an RB compound such as rose bengal disodium or rose bengal lactone that are covered with one or more layers of polymeric resin that release the RB compound quickly upon dissolution or disintegration of the capsule in water or body fluid. Capsules are typically made of gelatin and are often referred to as gelcaps. Gelatin is an animal product. Vegetarian capsules are often made of hydroxypropyl methyl cellulose (HPMC) .

In some embodiments, the RB compound is directly layered with one or more coats of the polymer to form particles that are generally spherical in shape. Such particles are often referred to as beads. In a preferred aspect, particles (beads) are sized so as that about 90 percent by weight pass through a 20 mesh sieve (opening = 850 m) screen and about 90 percent by weight are retained on an 80 mesh sieve (opening = 180 pm) screen.

Exemplary pH value-sensitive coating polymeric resins are discussed above. Exemplary pH value-insensitive coating polymeric resins are also discussed above. The pH value-sensitivity of coating polymeric resins is to be understood in terms of physiologically present pH values along the GI tract such as those discussed previously.

In other embodiments, small pellets such as sugar/starch seeds, non-pareils or prills, which are small, generally spherically-shaped cores, are coated with one or a plurality of layers of the RB compound and one or more layers of polymeric coating. Illustrative sugar/starch cores are sugar spheres NF that pass through an about 40 mesh sieve (425 mm opening) screen to an about 50 mesh sieve (300 mm opening) screen, that contain not less than 62.5 percent and not more than 91.5 percent sucrose, calculated on the dry basis, the remainder consisting primarily of starch. (USP NF 1995 2313) .

In an illustrative example, a 100 kilogram (kg) quantity of disodium rose bengal, a 7.1 kg quantity of cross-linked carboxymethyl cellulose (preferably croscarmellose sodium NF) , and an 11.9 kg quantity of starch NF, are each divided in half, and the three constituents are blended together to form two identical batches. Each of the batches is milled through an 80 mesh screen using a mill such as a Fitzpatrick Mill. The two milled batches are then blended to form a mixture, which is tested for composition in accordance with accepted quality assurance testing methods that are well-known by those skilled in the art.

The disodium rose bengal mixture is subsequently divided into three equal parts, with a first part remaining whole, and second and third parts each divided into lots of 50 percent, 30 percent and 20 percent. A 25.6 kg quantity of 40-50 mesh sugar/starch seeds (e.g., sugar spheres NF) is placed in a stainless steel coating pan. An 80 liter (L) quantity of 5 percent povidone/iso- propanol (IPA) solution is prepared for spraying onto the particles.

The coating pan is started with the sugar spheres, onto which is sprayed an application (approximately 0.173 kg per application) of the povidone/IPA solution, and onto which is sifted an application (approximately 0.32 kg) of the disodium rose bengal mixture from the first part (that part that remained whole) . Sifting is done using a standard sifter. The spraying and sifting steps are continued until the first part of the mixture has been applied to the sugar spheres to form a batch of partially coated spheres .

The partially coated spheres are then divided into two equal lots , each lot being placed in a coating pan . Separately for each of the two lots , spraying of the povidone/ IPA solution and si fting of the disodium rose bengal mixture as divided into the 50 percent lots continues until the 50 percent lots have been applied to the spheres . Following application of the 50 percent lots , the spheres can be screened using a 25 mesh screen i f necessary .

The spraying of the povidone/ IPA solution and si fting of the disodium rose bengal mixture as divided into the 30 percent lots commences and continues until the 30 percent lots have been applied to the spheres . The coated spheres can be rescreened using a 25 mesh screen .

Spraying of the povidone/ IPA solution and si fting of the disodium rose bengal mixture continues using the mixture as divided into the 20 percent lots until the 20 percent lots have been applied to the spheres . At this point in the process , the entire quantity of the disodium rose bengal mixture has been applied to the spheres , and about 50 kg of the 5 percent povidone/ IPA solution has been applied to the spheres .

A 7 . 5 percent povidone/ IPA solution is prepared and applied to the spheres as a sealant . The sealed spheres are tumble dried for about one hour, weighed, and placed in an oven at about 122 °F ( 50 °C ) for 24 hours . After drying, the spheres are screened through a 20 mesh screen and a 38 mesh screen to form the immediate ( quick or fast as compared to delayed) release particles .

The above-discussed RB compound-containing spheres or their capsule ( or blister ) can also be coated with a pH value-sensitive enteric coating polymer as discussed previously so that once released in the GI tract , the spheres do not provide their active ingredient , RB compound, to their surroundings unless the pH value is that of a desired GI tract location .

Another way to control the location of RB compound release is to further coat the spheres (RB- coated particles ) discussed above , with a dissolution-controlling coat of polymeric resin applied to the surface of the spheres such that the release of the RB compound from the spheres is controlled and released over a 6- 10 hour period . The materials used for this purpose can be , but are not limited to , ethylcellulose , hydroxypropylmethyl- cellulose , hydroxypropylcellulose , methylcellulose , hydroxyethylcellulose , nitrocellulose , carboxymethyl-cellulose , as well as copolymers of ethacrylic acid and methacrylic acid (Eudragit®) , or any other acrylic acid derivative ( Carbopol®, etc . ) can be used .

In addition, an enteric coating material can also be employed, either singularly, or in combination to the above non-pH-sensitive coatings . These materials include , but are not limited to , hydroxypropylmethylcellulose phthalate and the phthalate esters of all the cellulose ethers . In addition, phthalate esters of the acrylic acid derivatives (Eudragit®) , or cellulose acetate phthalate . These coating materials can be employed in coating the surfaces in an amount of about 1 . 0% (W/W) to about 25% (W/W) . Preferably, these coating materials are present at about 8 . 0 to about 12 . 0 percent (W/W) .

Excipients

Excipients customary in pharmacy can be employed in a manner known per se in the production of the drug form . These excipients can be present in the core or in the coating agent .

Polymers

Polymeric materials used as adhesives in helping to adhere an RB compound to a sugar prill or sphere is deemed to be an excipient where coating layers of an RB compound are employed . I llustrative of such polymers are polyvinyl pyrrolidone and polyvinyl alcohol as are other water-soluble , pharmaceutically acceptable film- forming polymers such as hydroxypropyl cellulose .

Dryers (non-stick agents )

Dryers have the following properties : they have large speci fic surface areas , are chemically inert , are free- flowing and comprise fine particles . Because of these properties , they reduce the tack of polymers containing polar comonomers as functional groups . Examples of dryers are : alumina, magnesium oxide , kaolin, talc, fumed silica, barium sulphate and cellulose .

Dis integrants

Disintegrants are added to oral solid dosage forms to aid in their disaggregation . Disintegrant are formulated to cause a rapid break- up of solids dosage forms on contacting moisture . Disintegration is typically viewed as the first step in the dissolution process. Illustrative disintegrants include sodium croscarmellose, an internally cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone ( crospovidone ) and sodium starch glycolate.

Release Agents

Examples of release agents are: esters of fatty acids or fatty amides, aliphatic, long-chain carboxylic acids, fatty alcohols and their esters, montan waxes or paraffin waxes and metal soaps; particular mention should be made of glycerol monostearate, stearyl alcohol, glycerol behenic acid ester, cetyl alcohol, palmitic acid, carnauba wax, beeswax, and the like. The usual proportionate amounts are in the range from 0.05% by weight to 5, preferably 0.1 to 3, % by weight based on the copolymer .

Other excipients customary in pharmacy Mention should be made here of, for example, stabilizers, colorants, antioxidants, wetting agents, pigments, gloss agents. They are typically used as processing aids and are intended to ensure a reliable and reproducible production process and good long-term storage stability. Further excipients customary in pharmacy may be present in amounts from 0.001% by weight to 10% by weight, preferably 0.1 to 10% by weight, based on the polymer coating.

Plasticizers

Substances suitable as plasticizers ordinarily have a molecular weight between 100 and 20,000 and comprise one or more hydrophilic groups in the molecule, e.g., hydroxyl, ester or amino groups. Citrates, phthalates, sebacates, castor oil are suitable . Examples of further suitable plastici zers are alkyl citrates , glycerol esters , alkyl phthalates , alkyl sebacates , sucrose esters , sorbitan esters , dibutyl sebacate and polyethylene glycols 4000 to 20 000 . Preferred plastici zers are tributyl citrate , triethyl citrate , acetyl triethyl citrate , dibutyl sebacate and diethyl sebacate . The amounts used are between 1 and 35 , preferably 2 to 10 , % by weight , based on the (meth) acrylate copolymer .

Optimizing Systemic Bioavail ability

Although the solid pharmaceutical compositions described herein have been discussed in the context of optimi zing direct delivery to bacterially diseased tissue in the GI tract , these approaches for controlling site of delivery and kinetics of delivery are equally applicable to optimi zing bioavailability for systemic uptake based on controlling site of delivery and kinetics .

As used herein, "administration" is used to mean the beginning of a treatment regimen . Thus , swallowing a liquid, tablet or other per os dosage form is the beginning of a treatment regimen, as are the time at which an IV flow is begun or a topical composition is applied .

The Method

A contemplated treatment method comprises contacting Gram-positive bacterial cells with a composition containing an anti-Gram-positive bacterial amount of a RB compound while those bacterial cells are irradiated with light that includes wavelengths of about 500 to about 575 nm, and more preferably at about 510 to about 550 nm .

In one embodiment , the Gram-positive bacterial cells are present on or in ( e . g . , infecting) a subj ect mammal . I llustratively, a subj ect mammal can have a dermatological Gram- positive bacterial infection such as that of Streptococcus pyogenes , or particularly in the case of a topical treatment of an open wound, as a treatment for a present infection and as a preventative from subsequent Gram-positive bacterial infection .

A treated subj ect mammal can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque , a laboratory animal such as a rat , mouse or rabbit , a companion animal such as a dog, cat , horse , or a food animal such as a cow or steer, sheep, lamb, pig, goat , llama or the like .

Each contemplated composition administration is typically repeated until the treated bacterial disease ( infection) is diminished to a desired extent , such as not being detectable . Thus , the administration to a mammalian subj ect in need can occur a plurality of times within one day, daily, weekly, monthly or over a period of several months to several years as directed by the treating physician .

A Gram-positive bactericidal ef fective amount of RB is administered to the mammalian subj ect in need and can be formulated using usual liquid, gel , solid or other formats . In most instances , the RB is administered with irradiation preferably using a light source having a lambda-max ( _max ) of about 500 to about 600 nm, and more preferably at about 510 to about 550 nm.

Illustrative sources for the light source, its intensity and its duration are discussed hereinafter .

Results

Therapeutic grade of rose bengal (RB)

Provectus Biopharmaceuticals, Inc. of Knoxville, TN, (Provectus) used commercial-grade RB and sought a purification process to prepare a pure form of RB as a drug substance. However, several challenges in the purification of RB were found due to commercial-grade reactions occurring during the dye manufacturing process, and the other by-products which lost one or more of the iodide substituents.

It was concluded that commercial-grade RB is not capable of efficiently yielding a pharmaceutical-grade material in sufficient quantity to support clinical development and registration by the FDA and other global drug regulatory agencies. A novel multi-step approach for synthesizing and manufacturing RB was established by Provectus

[35,36] . A key element of that work was the ability to eliminate the conditions that lead to the formation of the key historical impurities.

Those synthesis and purification methods have been applied to a good manufacturing practice (GMP) , producing RB with a pharmaceutical grade. This RB, PV-10®, is sterile 10% solution of rose bengal disodium (RBD) in 0.9% aqueous saline available from Provectus that is manufactured under the guidelines of The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) , and designed to apply as an injectable pharmaceutical. The pharmaceutical grade RBD is also present in a differently-formulated topical medication [36-39] referred to as PH-10®. Both preparations utilize rose bengal disodium (RBD) , whose structural formula was shown previously, as the active ingredient. Because many soluble salts of rose bengal can be utilized in addition to the disodium salt, "RB" is used herein as the designation for any pharmaceutically acceptable salt of rose bengal, whereas the designation "RBD" refers specifically to rose bengal disodium.

Antibacterial activity of RB

The antibacterial activity of RB via photodynamic approaches has been studied in several research groups [15-27] . Applications of photodynamic therapy of RB are not limited to skin infections, including cellulitis, erysipelas, impetigo, folliculitis, and furuncles and carbuncles. RB that was immobilized on polymer- supports were successfully applied for eradication of bacteria on material surfaces and in water [17,18] . However, spectrum of activity, rate of killing, and biofilm eradication activity of RB have been reported sporadically.

We have reinvestigated the bactericidal activity of pharmaceutical-grade of RB against a battery of Gram-positive and -negative aerobic bacteria including Mycobacterium spp . and a Saccharomyces sp . under different light sources (fluorescence, LED, and sun light) and in a dark condition. Fluorescent light used was 17W (1, 647 lumens, 63.8 cm ² ) and LED was a 9.5W (800 lumens, 28.3 cm ² ) , specifics about which are discussed hereinafter. In the studies under sunlight through an architectural window, the 96-well plates were placed on the east side of the building (the BSL-2 lab on the 5 ^th floor, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA) and the growth inhibition studies were performed between 8 AM and 5 PM (on June 18 ^th , 2021) .

A more preferred light source is a narrow band LED light such as the Explux PAR38 120 W equivalent flood light, which produces 870 Lumens at approximately 540 nm. The Explux green LED lamp produces 870 Lumens from a 4.7" diameter surface (110 cm ² ) . t the surface of the lamp, this equates to approximately 7.9 Lumens/cm ² . 1 Lumen is 1/683 Watts and is equal to 1/683 J/s. Thus, 7.9 Lumens/cm ² is 0.012 J//cm ² -s. Using a 5-minute exposure from such a lamp provides a light dose of about 3.5 J/cm^.

Minimum inhibitory concentrations (MICs, in μg/mL ) obtained via broth dilution and agar dilution methods [24 hours (h) under fluorescent (23.0 KJ/cm ² ) and LED light (29.0 KJ/cm ² ) conditions, and 9h under sunlight] are summarized in Table 1 and Table 2 that are provided as Fig. 1 and Fig 2, respectively. In these studies, a series of FDA- approved drugs (meropenem, colistin, amikacin, linezolid, rifampicin, ciprofloxacin, azithromycin, isoniazid, capreomycin, and 5-f luorouracil ) and preclinical antibacterial agents ( tunicamycin, APPB) were included as positive- and negative-controls of each MIC test [40-44] . RB used in Table 1 and 2 studies is a diluted form of PV-10®, discussed previously, that was provided by Provectus Biopharmaceuticals Inc. (Knoxville, TN, USA) [35, 36] .

The RB provided as PV-10® effectively killed a wide range of Gram-positive bacteria with the MIC level of 0.20-3.1 μg/mL under illumination conditions (entries 1-23 in Table 1) . The bactericidal activity of RB observed in entries 1-23 (Table 1) was not noticeably different depending on the light sources; the MIC values were equal or very close for fluorescent and LED lights.

RB killed Gram-positive Bacillus spp . at 0.39-0.78 μg/mL concentrations (entries 1-3) . A MIC standard strain of Staphylococcus aureus displayed less susceptibility to RB; it required 1.6 μg/mL of RB to kill >99% of bacteria (entry 4) .

RB' s bactericidal activity was examined against a panel of 7 methicillin-resistant S. aureus (MRSA) with different SCCmec types (entries 5-11) of Table 1 [45] . All MRSA strains tested in Table 1 were killed by RB at 0.78-3.1 μg/mL concentrations under the fluorescent or LED light. RB was further examined against 4 vancomycin-resistant S. aureus strains (entries 12-16) ; all vancomycin-resistant strains were killed at below 1.0μg/mL concentrations under either lighting condition.

Staphylococcus epidermidis is an anaerobic bacterium, but grows well under aerobic conditions. It was effectively killed by RB under an aerobic condition (entry 16) . Drug-susceptible and drug-resistant Enterococcus faecalls including vancomycin-resistant strains were killed at a concentration range of 0.39-0.78 μg/mL of RB (entries 17-21) . Streptococcus salivarius was susceptible to RB (entry 23) , whereas Streptococcus pneumoniae showed resistance to RB (entry 24) . The basis for that resistance is presently unknown.

Under the dark condition, RB showed antibacterial activity against all Gram-positive bacteria listed in entries 1-23 (Table 1) at 25.0- 100 μg/mL concentrations. Under the dark condition RB is known to display antibacterial activity at high concentrations. Our data support the idea that RB has one or more unknown mechanisms to inhibit the growth of bacteria other than via the excitation mechanism of triplet oxygen, generating cytotoxic reactive oxygen species.

Although excellent antibacterial activities were observed against Gram-positive bacteria, Gram-negative bacteria examined: three E. coli, two Pseudomonas aeruginosa, two Klebsiella pneumoniae, and two Aci netobacter baumannii strains, showed resistance to RB . The MIC levels were 50 or >100 μg/mL against these Gram-negative bacteria (entries 31-39) . An anaerobic Gram-negative bacterium, Bacteroides fragilis tolerated the RB treatment at 100 μg/mL or higher concentrations.

Although efficacy of RB against a large group of Gram-negative bacteria has not thoroughly been investigated, a photodynamic approach using RB was studied to inhibit growth of Salmonella and Burkholderia spp . Our data summarized in entries 41-42 (Table 1) suggest that RB is effective in killing Burkholderia, Salmonella, and Proteus spp. at 3.13-12.5 μg/mL concentrations under fluorescent or LED lights as shown in entries 43-47.

All Gram-negative bacteria assayed in Table 1 were not susceptible to RB under the dark condition; the MICs were determined to be >100μg/mL (entries 31-47) . Antibacterial activity of RB against 5 Mycobacterium spp . was examined (entries 25-29) ; the MIC values of RB under the illuminated conditions were 12.5-25.0 μg/mL (entries 25-29) , which are 15-60-fold higher than those for the Gram- positive bacteria (entries 1-23) . Interestingly, under the dark condition, these Mycobacteria were killed at equal or similar MIC to those observed under illumination conditions.

We examined the MIC of RB against a limited number of bacteria under sunlight (filtered through glass window) . The MIC values for eight Gram-positive bacteria and three Gram-negative bacteria displayed good agreement with those obtained under fluorescent and LED lights (entries 1-4, 16, 21-23, 43 and 47) . RB inhibited growth of Saccharomyces cerevisiae at the same MIC level under the three different light sources and at much higher concentrations under the dark condition (entry 30) .

We observed that under the illumination conditions the MIC values of RB determined by the agar dilution method are lower than those determined by the broth dilution method (Table 1) . Selected examples of difference in the MIC values determined by the two methods are summarized in Table 2.

The growth of Gram-positive bacteria such as B. subtilis, B. cereus, and S. aureus were inhibited at 0.01-0.10 μg/mL concentrations (entries 1-4 in Table 2) , which are 7-70-fold less than the MIC values determined by the broth dilution method. E. coli (35218™) and B. cepacia (UCB717) strains were also far more susceptible to RB under fluorescent light in the agar dilution method than those in the broth dilution method (entries 5 and 6) . Similarly, M. smegmatis (ATCC607™) was killed at lower concentration on the drug-containing agar plates (or wells) than in those in broth (entry 7) [46] .

Under the dark condition, the MICs of RB determined via the agar dilution method displayed good agreement with the values measured in the broth dilution method (entries 1-7) . Minimum bactericidal concentrations (MBCs) of RB against the selected bacteria are also summarized in Table 2. RB has a cytostatic effect against the fungus S. cerevisiae sp . ; it displayed a 50% growth inhibition at 1.6 pg/mL, but required 400 pg/mL (MBC) to kill >99% of the yeast under the fluorescent light. S. cerevisiae was not killed by 500 pg/mL of RB under the dark condition (entry 11) .

Time-kill kinetics of RB

Photodynamic growth inhibitions of RB against a several bacteria have been previously studied; Sabbahi et al. reported that under a visible light exposure about 80% of a S. aureus strain lost its viability in 10 minutes (min) with 19.5 pg/mL of RB (a light fluence dose of 30 J/cm ² ) [26] . Considering the MIC values determined under the fluorescent light (24h, 23.0 KJ/cm ² , Table 1) , we performed the time-kill kinetics assays of RB with one drug-susceptible strain of S. aureus 6538™ and three drug-resistant Gram-positive bacterial strains (S. aureus BAA-44, S. aureus 71080 (VRS8) , and E. faecium NR-32065) , and one Gram-negative bacterium (B. cepacia UCB717) . Our preliminary studies suggested that RB can kill both Gram-positive and -negative bacteria with a 6-log reduction within 2 hours of contact.

Thus, the time-course experiments on these bacteria were conducted under the fluorescent light for 2h (0-1,130 J/cm ² ) at concentrations of about 2 to about 8-fold the MIC (RB) . Reference molecules used were linezolid (10μg/mL ) and ciprofloxacin (10μg/mL ) for the Gram-positive bacteria and amikacin (10μg/mL ) and meropenem (10μg/mL ) for the Gram- negative bacterium. Rose bengal reduced 4.6 x 10 ⁸ (colony forming units: CFUs) of S. aureus 6538™ by a log reduction of 6 in 1 minute. No CFU were counted after 2 minutes at 1.6 and 5.0 μg/mL RB concentrations (Fig. 3A) .

Similarly, RB killed S. aureus BAA-44, S. aureus 71080 (VRS8) , and E. faecium NR-32065 within 2 minutes at a concentration twice that of the RB MIC (Fig. 3B) . B. cepacia UCB717 (2.9 x 10 ⁸ CFU) was killed in concentration- and time-dependent manners by the treatment of RB (Fig. 3C) at 4-times the MIC concentration, and provided >5 log reduction of the bacteria in 5 minutes. RB required 40 minutes to reduce a number of bacteria with a 5-log reduction at 2-times the MIC concentration.

Anti -biofilm activity of Therapeutic grade RB

The fast-killing antibacterial character of RB observed in the previous section encouraged us to evaluate antibiofilm efficacy of RB in Gram- positive bacteria. The data summarized above indicate that RB possesses a significant drug affinity or permeability onto (or into) Gram- positive bacteria. Antimicrobial and antifungal photodynamic therapy have been studied with the photosensitizers under biofilm conditions; however, a limited number of bacterial biofilms have been examined with RB [15] .

Recently, antibiofilm activity of RB against cariogenic oral bacteria, harboring on the tooth surface, was demonstrated under blue light LED (Hirose et al. 2021) [20] . Here, we examined efficacy of RB against biofilms of a drug- susceptible S. aureus 6508™, and drug-resistant S. aureus 71080 (VRS8) and E. faecium NR-32065 under the f lorescent light and dark conditions. Linezolid is not an effective drug in eradicating biofilms of Gram-positive bacteria, but has a beneficial effect in prevention of biofilm formations [47] .

We applied linezolid as a positive control at very high concentration of 600 μg/mL (>100xMIC for the planktonic cells) in our biofilm assays. We have confirmed that all strains tested here form strong biofilms on the polystyrene well plates. Under the fluorescent light, RB could eradicate the biofilms of S. aureus 6508 with a 7-log reduction at 30.0 μg/mL (38xMIC) concentration, which demonstrated the same level of efficacy as observed for linezolid (at 600μg/mL ) (Fig. 4A) .

RB showed a biofilm eradication activity in a dose-dependent manner. As seen in Fig. 4A, at 60.0 μg/mL ( 77xMIC) concentration, over a 5-log reduction was achieved. No CFU were counted at 100μg/mL . Although requiring much higher concentrations , RB showed a biofilm eradication activity under the dark condition. At 50.0μg/mL (2xMIC under dark) concentration, RB significantly reduced the number of viable bacteria. At 500 μg/mL (20xMIC) concentration, only about 110 to about 150 CFU/mL were observed. No viable bacteria were appeared at 1, 000 μg/mL concentration.

These trends could be observed in the biofilms of the drug-resistant S. aureus 71080 (VRS8) and E. f aecium NR-32065 with much lower RB concentrations (Figs. 4B and 4C) . Under the fluorescent light, Fig. 4B shows an over 6-log reduction of viable bacteria was observed at 10.0 μg/mL concentration. No CFU were counted at 30.0 μg/mL or higher concentrations.

There is some debate as to whether S. aureus colonies can be considered as air-exposed biofilms [48] . Nonetheless, our studies have shown that the colonies of S. aureus grown on agar plate (at 37 °C for 2 days) are not possible to reduce in a few hours using the FDA- approved antibiotics.

The data of Table 3, below, summarize the effect of RB on air-exposed biofilms of a MRSA, S aureus BAA-44™, under fluorescent light (17W, 1h, 0.57 KJ/cm ² ) . Spraying RB (5.0 μg/mL or 10μg/mL solution, 250 L (twice) ) and fluorescent light exposure (for 1 h) eradicated significant viable bacteria in the biofilms with a 4-log reduction (determined at a dilution of 5.8 xlO ⁹ ) .

The studies summarized in Table 3 indicate that RB can readily permeate biofilm matrix and diffuse across Gram-positive bacterial cell walls. These observations strongly support that RB has the potential to treat serious bacterial skin infections .

Antibacterial mechanisms of RB

Antibacterial photodynamic therapy of RB has been reported in several articles. Permeation of RB through bacterial cell walls and binding to cell membranes followed by production of reactive oxygen species are likely bactericidal mechanisms in illumination conditions [49,50] . Although relatively high concentrations are required, RB kills a majority of Gram-positive bacteria including Mycobacterium spp . in dark conditions. It also effectively eradicates biofilms of Gram-positive bacteria (above) . Antibacterial activity of RB in dark conditions remains far from completely understood [51,52] .

RB kills Mycobacterial spp. at 12.5-25.0 mg/mL in illumination conditions, and at 25.0-50.0 mg/mL in dark conditions (Table 1) . In both conditions, RB kills five Mycobacterial spp. at a slower rate than that of Gram-positive bacteria. RB seems to have low-permeability of mycobacterial cell walls. Due to rapid bactericidal effect of RB against Gram-positive bacteria even in the dark condition, generation of RB-resistant mutants of Gram-positive bacteria is an extremely difficult task .

We successfully generated RB-resistant mutants of M. smegmatis, which had the MIC value of 200 mg/mL [46] . The RB-resistant strain was susceptible to most of TB drugs [amikacin, capreomycin, rifampicin, aminouridyl phenoxypiperidinylbenzyl butanamide (APPB) , and ethionamide] (Table 4, below) .

However, it showed a cross-resistance to

Isoniazid (INH) . INH is a prodrug that requires oxidative activation by the enzyme KatG, which belongs to catalase-peroxidase families. KatG oxidizes INH to form an electrophilic species, an isonicotinoyl radical molecule, which reacts with the NADH-dependent enoyl-ACP (acyl carrier protein) reductase, an enzyme involved in the biosynthesis of mycolic acids of mycobacteria (Scheme 1, below) [53,54] .

Scheme 1

The RB-resistant M. smegmatis strain acquired medium-INH resistance but did not show resistance to ethionamide (ETH) . The major mechanism of INH resistance is mutation in katG, while ETH is activated by the monooxygenase EthA [55] . Our observations may imply that the RB' s antibacterial mechanisms share one or more INH metabolic enzymes to form bactericidal species.

To elucidate a potential mechanism of action, we performed a whole-genome sequencing analysis of a RB-resistant M. smegmatis ATCC607 strain using the next-generation of DNA sequencing technologies [56] . We identified that one insertion mutation occurred in anti-sigma E factor gene (rseA: evidenced TG:104 vs. T:0) and aquaporin family protein gene (evidenced GCACCCT:71 vs. G:0) , respectively .

Consequently, these insertion mutations caused the reading frame changes in the corresponding proteins and generated the truncated proteins compared to its parental strain. It has been reported that RseA functions as a specific anti-sigma E factor in Mycobacterium tuberculosis and that the sigma E factor (SigE) enables the mycobacterial organisms to tolerate a variety of stress responses [57,58] . Thus, the expression of a non-functional RseA in the RB-resistant mutant may affect the activity of SigE, increasing the bacterial tolerance to RB . On the other hand, the aquaporin family proteins exist in various organisms and play a critical role in bidirectional flux of water and uncharged solutes cross cell membranes.

It was reported that a null mutation of the Streptococcal aquaporin homolog increased the intracellular H2O2 retention, indicating that aquaporin mediates transporting H2O2 in Streptococcal spp . [59] . Therefore, we hypothesize that RB may inhibit the aquaporin function, leading to accumulation of H2O2 within the bacterial cells. Interestingly, a single nucleotide deletion causing the frameshift mutation was observed in molybdopterin-dependent oxidoreductase (evidenced T:74 vs. TC:0) of the RB-resistant strain.

The oxidoreductase systems can form superoxide by reduction of molecular oxygen or NO by reduction of inorganic nitrate. RB may serve as a single-electron acceptor in the redox of the oxidoreductases that will produce the radical anion (RB'-) or RB triplet state, undergoing electron- transfer reaction with oxygen. As such, we propose the involvement of molybdopterin-dependent oxidoreductase in the generation of reactive oxygen or nitrogen spp. through the excitation of RB under dark conditions ( Scheme 2 , below) . The oxidoreductase in Scheme 2 is molybdopterin- dependent oxidoreductase xanthine dehydrogenase family protein . The " • OH + OH-" in Scheme 2 is a reactive oxygen species (ROS ) or a reactive nitrogen species .

Scheme 2

The ka tG gene was intact in the RB- resistant strain . Thus , it remains di f ficult to speculate a mechanism that confers the cross- resistance with INH . However, we observed mutations in several transcriptional regulators of the RB- resistant strain that may af fect the expression level of KatG, suppressing the INH-activation . It generates hydroxy radicals ( reactive oxygen species ) through the Fenton reaction of H ₂ O ₂ . Requirement of relatively high concentration of RB to display bactericidal activity against Gram-positive bacteria including Mycobacterial spp . may imply that af finity of RB with catalases is moderate . Similarly, a cytotoxicity mechanism of RB in mammalian cells may be explained . As shown in Scheme 3 , below . Scheme 3

Cytotoxicity of RB under lights

The cytotoxicity of RB has been extensively evaluated in Provectus' oncology drug development program, where it was investigated via intralesional administration for the treatment of melanoma and hepatic tumors [37,38] . The results of Provectus' toxicology studies showed that RB does not have systemic toxicological effects, mutagenic potential, and female reproductive and development effects at the therapeutic concentrations [60] . These data are described in Provectus' U.S. patents (Eagle et al. 2019) [61] . Besides the toxicity studies in systemic applications, cytotoxicity of pharmaceutical-grade RB against mammalian cells in illumination conditions have not been discussed.

RB has been used for over 50 years to diagnose eye and liver disorders. It is often useful as a stain in diagnosing certain medical issues, such as conjunctival and lid disorders (vide supra) . In these applications, 0.1-2.0% RB has been used .

RB in concentrations below 2.0% are considered to be safe under natural and artificial lights [62] . The cytotoxicity level of RB against healthy cells under illumination conditions should be clarified for photodynamic antibacterial chemotherapy; however, these data are not publicly available. We chose two healthy cell lines, Vero (the kidney of an African green monkey) cells and skin (human epidermal keratinocytes (HEKa) cells to determine in vitro cytotoxicity of RB under the fluorescent light.

We have generated large data sets of cytotoxicity of antibacterial and anticancer agents against Vero cells, which allow us to compare the toxicity level of new molecules [63, 64] . The cytotoxicity against HEKa cells provides useful toxicology information for the development of safe topically-applied antibacterial agents [65, 66] .

In these studies, HEKa cells were differentiated to a stratified squamous epithelium via air-liquid interface; this type of epithelium can be applied as a physiological tissue to study epidermal necrolysis by the treatment of RB . In a 24h study under the dark, RB showed the IC50 value of 300 μM (292 pg/mL) against Vero cells.

Under the fluorescent light condition, RB displayed cytotoxicity against Vero cells in a time- and -concentration dependent manner. Fig. 5 summarizes the time effect of the confluent Vero cell from the treatment of RB (0-300 μM) . Nearly 50% of confluency was lost by the treatment of 100 μM (97.4 pg/mL) concentration of RB in 4h; however, the monolayer was intact for Ih exposure of 100 μM of RB . About 18% of confluence was lost in Ih at 200 μM, whereas over 80% of confluence was lost in 4h. At 300 μM concentration, complete loss of the cell viability was observed within 2h. Thus, it was concluded that under the fluorescent light Vero cells are tolerated at 100- 200 μM for Ih. Time-kill kinetic studies summarized previously indicated that RB kills Gram-positive bacteria in 1-2 min and Gram-negative bacteria in 5 min, respectively. The selectivity index (SI) , a ratio that measures the window between cytotoxicity and antibacterial activity, was determined to be >62.5 (for Gram-positives) and >7.9 (for Gram- negatives) for an Ih treatment time.

These favorable toxicity profiles of RB were further supported by the cytotoxicity studies with HEKa cells (vide supra) . RB was localized in the stratum layers. RB did not cause necrosis of the stratum corneum cells at 10 and 100 μM for Ih exposure. Some necrosis was observed on the surface tissue when the concentration increased to 200 μM (Fig. 6) . Therapeutic concentrations of RB are likely to be between 5-10 μM. Thus, these in vitro cytotoxicity tests imply that skin infections can be treated with RB without causing cytotoxicity of host cells under illumination conditions.

Conclusions

Provectus has established a manufacturing and purification process for pharmaceutical-grade RB that fulfills both cGMP and ICH requirements. We have evaluated the antibacterial activity and cytotoxicity of a pharmaceutical-grade RB formulated product (PV-10®) as an exemplary RB compound in illuminating and dark conditions. The comprehensive MIC data for RB via saline-diluted PV-10® summarized here indicate that RB is very effective in killing most Gram-positive bacteria (MIC 0 . 39-3μg./1mL ) , except Streptococcus pneumoniae sp .

S . pneumonia is one of very few Gram- positive bacteria that is susceptible to colistin (polymixin E ) , an anti-Gram-negative drug . We have studied the relationship between RB' s bactericidal ef fect and colistin-resistance in both Gram-positive and -negative bacteria . The resistance mechanism of S . pneumoniae against RB will be reported elsewhere .

RB kills Mycobacterial spp . with the MIC values of 12 . 5-25 . 0 μg/mL under the illumination conditions . It is speculated that the mycolic acid- containing thick cell walls reduce the cellular uptake of the charged RB, increasing its MIC values against Mycobacterial spp . much higher than those of Gram-positive bacteria .

We confirm that RB is an excellent agent to eradicate biofilms of Gram-positive bacteria, including drug-resistant strains . Under fluorescent and dark conditions , RB signi ficantly reduced a number of viable cells of the drug-resistant strains of S . aureus and E. faeci um in a concentration- dependent manner . RB displayed a signi ficant bactericidal ef fect on the air-exposed biofilms of a MRSA strain under fluorescent light exposure within Ih . These studies indicate that RB has the potential antibacterial agent to kill drug-resistant bacteria in any growth phases .

RB is an anionic photosensiti zer with high singlet oxygen quantum yield . This is a primary mode of action of antibacterial activity in illumination conditions . We could success fully generate RB-resistant mutants of M. smegma ti s under the dark condition . It showed a cross-resistance to first-line TB drug, INH . We performed whole genome analyses of the generated resistant mutant . It revealed the unique mutations that may confer mechanisms of RB' s bactericidal activity . Several redox systems , transcriptional factors , and aquaporin may be responsible for RB' s resistance mechanisms . Based on these data, it is speculated that RB can be shi fted to an excited state by the enzymes associated with the oxidoreductases , forming reactive oxygen or nitrogen spp . under the dark conditions . Certain cooperative mechanisms may exist in bactericidal activity of RB under dark conditions .

To summari ze , we demonstrated that a pharmaceutical-grade formulation of RB, PV- 10®, has an appropriate selectivity index for the treatment of Gram-positive bacterial infections under illumination conditions . Rapid bactericidal ef fect of RB that is ef fective against bacterial biofilms is an unusual drug characteristic and promises the advancement of RB as an antiseptic for clinical applications .

Materials and Methods

Lights

The LED light source used in these studies was a Phillips 9 . 5 Watt soft white , 800 Lumens , 2700-3000 Kelvin, Model number B07CFRCGKC ; part No . 479576 . The light output from this bulb is said to be comparable to that emitted by a 60 W similar bulb .

The fluorescent light source was a Sun Blaster™, 18 inch, T5 horticultural lighting mercury fluorescent lamp, 17 W, 6400 Kelvin, part No.

090035 .

Antibiotics

All antibiotics were purchased from commercial sources [amikacin disulfate salt (Sigma Aldrich, A1774-1G) , capreomycin sulfate (Sigma Aldrich, C4142-1G) , ciprofloxacin hydrochloride monohydrate (TCI, C2227) , ethionamide (TCI, E0695) , isoniazid (Sigma Aldrich, I3377-5G) , linezolid (Chem-Impex, 29723) , meropenem trihydrate (Ark Pharm, AK161987) , rifampicin (Sigma Aldrich, R3501- 1G) ] and used without further purification unless otherwise noted. APPB ( aminouridylphenoxypiperi- dinylbenzyl butanamide) was synthesized according to the reported procedure. [ (a) Mitachi et al., ACS Omega 2018, 3:1726-1739; and (b) Mitachi et al., MethodsX 2019, 6:2305-2321.]

Formulation of pharmacological grade rose bengal in saline (PV-10 ^® ) .

Rose bengal disodium salt was synthesized according to Provectus' proprietary procedure. The detailed procedure was as described in U.S. Patents No. 8,530, 675, No 9,273,022 and No. 9,422,260.

Acquisition of bacteria

The drug susceptible bacteria and yeasts used in this program were purchased from ATCC (The American Type Culture Collection, Manassas, VA) . The drug-resistant strains were acquired from BEI Resources (NIAID) . Log phase bacterial culture

All liquid bacterial culturing was performed with a conical flask with an air filter. A single colony of a bacterial strain was grown according to the conditions recommended by ATCC. Seed cultures and larger cultures of bacteria were obtained using media recommended by ATCC. M. smegmatis (ATCC 607) was cultured on a 0.5% Tween® 80 Middlebrook 7H10 nutrient agar (0.4% glycerol) [46] . The culture flasks were incubated for 3-4 days for M. smegmatis (ATCC 607) , and for 10-12 days for M. tuberculosis H37RV in a shaking incubator at 37 °C with a shaking speed of 200 rpm and cultured to mid-log phase (optical density - 0.5) . The optical density was monitored at 600 nm using a 96 well microplate reader.

MIC assays

All testing follows the guidelines set by the Clinical & Laboratory Standards Institute (CLSI) [68] . Minimum inhibitory concentrations (MICs) were determined by broth dilution microplate alamar blue assay or by CD measurement. All compounds were stored in DMSO or saline (1 mg/100 μL concentration) . This concentration was used as the stock solution for all MIC studies. Each compound from stock solution was placed in the first well of a sterile 96 well plate and a serial dilution was conducted with the culturing broth (total volume of 10 μL) . The bacterial suspension at log phase (190μL ) was added to each well (total volume of 200 μL ) , and was incubated for 24 h at 37 °C 20 μL of resazurin (0.02%) was added to each well and incubated for 4 h, (National Committee for Clinical Laboratory Standards (NCCLS ) method (pink = growth, blue = no visible growth) ) . The CD measurements were performed for all experiments prior to performing colorimetric assays . The absorbance of each well was also measured at 570 and 600 nm via UV-Vis .

Minimal bactericidal concentration (MBC) assays

A single colony of a speci fic bacterium ( grown on an agar plate ) was inoculated into the culture broth . A bacterial culture was grown overnight ( about 18 hours ) , then diluted in growth- supporting broth to a concentration between 1 x 10 ⁵ and 1 x 10 ⁹ CFU/mL . Based on the MIC values , agar plates containing a drug (MIC, 2x - 20x MIC ) were prepared . A series of drug-containing agar plates were inoculated with equal volumes of the speci fic bacterium . The agar plates were incubated at the appropriate temperature and duration . CFU/mL were counted . The MBC values were determined by reduction of >99 . 9% of bacteria .

Time-kill kinetic assays

Time-kill kinetics assay for antimicrobial agents was performed using CLS I guidelines with a minor modi fication . The multiple time points in time-kill kinetics assays for RB and the reference molecules were performed . The bacterial culture grown in the broth was diluted to a concentration between 1 x 10 ⁸ and 5 . 0 x 10 ⁹ CFU/mL .

A stock dilution of the antimicrobial test substance was prepared at approximately 2- to about 8- fold the MIC values . The test compounds were inoculated with equal volumes of the specified bacteria placed in a 96-well plate. The microtiter plates were incubated at 37 °C and duration (1-120 min) under the fluorescent light (conditions are summarized in the figure legend) . An aliquot of the culture medium was taken from each well and a serial dilution was performed. The diluted culture was incubated at 37 °C and CFU/mL were counted. Bactericidal activity was defined as greater than 3 log-fold decrease in colony forming units [64] .

Anti -biofilm assays.

The biofilms were generated on 12-well plates by incubating each bacterium for five days. The planktonic bacteria in the culture media were gently removed, and fresh media were placed. PV-10 (25 x MIC) or linezolid (200 x MIC) were added into each well and incubated at 37 °C under dark or light for 24h (6.7 KJ/cm ² ) . An aliquot of each culture was diluted (xl0,000 or xl00,000) on agar plates at 37 °C for 24h. CFU/mL was counted [47, 69] .

The bacteria (about 1.01X1 0 ⁴ ) were spread on an agar plate and incubated in an oven at 37 °C for 2 days. The air-exposed biofilms on an agar plate were treated with PV-10. After Ih under fluorescent light (0.57 KJ/cm ² ) , saline (2 mL) was added to the agar surface. The bacterial suspension (100 mL) was taken into a sterile tube, and serial dilutions were performed. The diluted sample (100 mL) was spread on agar plates and incubated at 37 °C for 24h CFU/mL were counted. Mammalian cell lines and culturing

Vero cells (ATCC™ CCL-81) were purchased from the ATCC. HEK cells (SCCE020) were purchased from MilliporeSigma. The cell lines were cultured and maintained in the media as recommended by the suppliers .

Vero cells were cultured in Eagle's Minimum Essential Medium (MEM) (CORNING®, 10-009-CV) supplemented with 10% FBS, 1% Penicillin- streptomycin Solution (cellgro®, 30-002-CI) , 1% HyClone™ MEM Non-Essential Amino Acids Solution (100X) (GE Healthcare Life Sciences, SH30238.01) and 1% HyClone™ Sodium Pyruvate 100 mM Solution, (GE Healthcare Life Sciences, SH30239.01) and incubated in a 37 °C incubator with 100% humidity and 5% CO ₂ . Cultures were refreshed with fresh medium every 2 days until the culture reaches 100% confluence, which takes approximately 5 days depending on the proliferation rate.

Human Epidermal Keratinocytes , Neonatal (MILLIPORE®, Catalog# SCCE020) were cultured in EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (MILLIPORE®, SCMK001) and incubated in a 37 °C incubator with 100% humidity and 5% CO ₂ . Refresh with fresh medium after 2 days and three times weekly until the culture reaches 100% confluence.

Cytotoxicity assay with Vero cells

All testing follows the guidelines set by the Clinical & Laboratory Standards Institute (CLSI) with minor modifications. Cytotoxicity assays for PV-10 were performed in a 24-well plate. Into each well (1 mL medium/well ) , 1 μL of drug concentration was added. After 1, 2, 3 and 4h of incubation under the light at room temperature (r.t.) , the medium was removed, and the cells were washed with PBS (x3) . After adding the medium (1 mL/well) , 10 μL of 3-

(4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added and incubated for another 3h at 37 °C (5% CO ₂ ) . The medium was removed, and DMSO (1 mL/well) was added. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by a BioTek Synergy™ HT Spectrophotometer [43] .

Vero cells [5 x 10 ⁴ cells/well (in 196 μL of the culture medium) ] were plated in a 96-well plate and the cell cultures were incubated for 4 days to form the monolayer (100% confluence) . Into each well RB (0-300 mM) was added.

Images were obtained every hour using an IncuCyte® Live-Cell Imaging System (Essen BioScience, Ann Arbor, MI) . Cell proliferation was quantified using the metric phase object confluence (POC) , a measurement of the area of the field of view that is covered by cells, which is calculated by the integrated software.

Cytotoxicity assay with HEKa cells

We referenced the protocols described in Testing Cell Monolayer Integrity on Transwell Permeable Supports (CLS-AN-047W) . Cytotoxicity of PV-10 against Human Epidermal Keratinocytes (HEKa) were evaluated in a hanging cell culture insert in a 24-well plate. HEKa (4x10 ⁵ cells/mL, 0.5 mL) cell suspension) was placed into the insert where the outside of the inserts was filled with EpiGRO™ Complete Culture Media (1.5 mL) [67] . The cell suspension was incubated at 37 °C overnight (about 18 hours) .

The next day, without removing the original medium, an additional 0.5 mL of the medium was added to the inside of each insert and incubated at 37 °C for 2 days. High quality HEKa approached 100% confluence by the third day of submerged culture. On the 4th day, the media was gently aspirated from each insert. The skin cell cultures were maintained at the air/liquid interface.

The skin culture was incubated at 37 °C for an additional 10 days. During the 10-day incubation, the media was changed every other day and any bubbles were removed from beneath the insert membrane .

After 25 days of 3D skin culture, approximately 6 to 8 layers of live epithelium were produced. HEKa tissues were treated with RB (0-200 mM) for Ih under fluorescent light at r.t., washed with PBS (x3) , and fixed with 4% formalin for Ih at r.t. The skin tissues were released from the insert and embedded into paraffin. Sections of 4 μM were cut, transferred onto slides for hematoxylin and eosin (H&E) staining [71] .

Whole-genome sequencing of M. smegmatis strains

RB-resistant M. smegmatis (ATCC607™) strains were prepared according to the procedures reported previously [72] . To identify single nucleotide polymorphisms (SNPs) that may contribute to the bacterial resistance to RB, the genomic DNAs were purified from the stationary cultures of RB- resistant mutant and its parental control M. smegmatis 607™ according to the procedure reported previously [46] .

The purified genomic DNA was submitted to the University of Minnesota Genomic Center (UMGC) for quality control analysis, the library preparation and DNA sequencing using an advanced Illumina MiSeq™ DNA-seq technology. Sequence reads from the mutant and the control were evaluated for their quality using FastQC. Low quality tails and adapters were removed with Trimmomatic [73] . The whole-genome sequence of M. smegmatis strain FDAARGOS_679 was used as a reference, and SNPs or other variants such as deletion and insertions were called by using a bioinf ormatic tool Snippy [https : //github . com/tseemann/snippy] .

Antibiotics

All antibiotics were purchased from commercial sources (amikacin disulfate salt (Sigma Aldrich, A1774-1G) , capreomycin sulfate (Sigma Aldrich, C4142-1G) , ciprofloxacin hydrochloride monohydrate (TCI, C2227) , ethionamide (TCI, E0695) , isoniazid (Sigma Aldrich, I3377-5G) , linezolid (Chem-Impex, 29723) , meropenem trihydrate (Ark Pharm, AK161987) , rifampicin (Sigma Aldrich, R3501- 1G) ) and used without further purification unless otherwise noted. APPB (aminouridyl phenoxy- piperidinylbenzyl butanamide) was synthesized according to the reported procedure [74] . Cytotoxicity of RB under light

Cytotoxicity assays for RB in Vero cells were performed in a 24-well plate. Into each well (1 mL medium/well ) , 1 μL of drug concentration was added. After 1, 2, 3 and 4 h of incubation with drugs under the fluorescent light at r.t., the medium was removed and the cells were washed with PBS (x3) .

Images were collected by AMG EVOS xl transmitted light imaging microscope. After adding the medium (1 mL/well) , 10 μL of MTT solution (5 mg/mL in PBS) was added and incubated for another 3 h at 37 °C (5% CO ₂ ) . The medium was removed, and DMSO (1 mL/well) was added. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by a BioTek Synergy® HT Spectrophotometer.

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Each of the patents, patent applications and articles cited herein is incorporated by reference. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article .

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting . Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art .