PL243213B1 - Mixture of naphthoquinone and silver and use of the mixture as an antibacterial agent for the control of Pseudomonas aeruginosa - Google Patents

Abstract

The subject of the application is a mixture containing silver and 1,4-naphthoquinone, characterized in that it contains bactericidal doses of silver and 2-hydroxy-1,4-naphthoquinone acting against Pseudomonas aeruginosa. The above mixture can be used as an antibacterial agent against Pseudomonas aeruginosa, preferably as an agent for application to the skin or wounds.

Description

Description of the invention

The invention relates to a method of activating the bactericidal properties of a selected compound from the group of 1,4-naphthoquinones against the naturally resistant Pseudomonas aeruginosa bacillus with silver nanoparticles, and thus with a mixture of these factors. The invention also relates to the medical use of the mixture for combating P. aeruginosa as an antibacterial agent, especially for external use, e.g. on the skin or wounds.

The phenomenon of antibiotic resistance of microorganisms, i.e. the ability to multiply in the presence of an antibiotic, is an increasingly common problem faced by medicine. Along with the growing number of microorganisms showing resistance to a wider range of antibiotics, the pool of possible therapies used to treat infections is decreasing, and thus the threat to human health and life is growing. The World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) indicate in their latest reports that in the post-antibiotic era, sustainable strategies for the prevention and treatment of infectious diseases are essential - CDC, Antibiotic Resistance Threats in the United States, 2019. 2019, U.S. Department of Health and Human Services: Atlanta, GA; WHO, 2019. ANTIBACTERIAL AGENTS IN CLINICAL DEVELOPMENT: an analysis of the antibacterial clinical development pipeline. 2019, WHO: Geneva, Switzerland.

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic pathogen of humans and animals with significant virulence. P. aeruginosa is naturally resistant to many chemical molecules that are active against other pathogens. In the case of burn wound infections, P. aeruginosa is one of the most frequently isolated bacterial species and poses a particular problem in their treatment due to multidrug resistance limiting therapeutic options. In addition to antibiotics, ionic silver, i.e. silver nitrate and silver sulfadiazine, is used with great success in the treatment of burn wounds. Nevertheless, the phenomenon of acquiring resistance to preparations containing silver by pathogens infecting wounds is increasingly observed. It is therefore necessary to develop strategies to delay or abolish the development of bacterial resistance and to expand therapeutic options. Certain 1,4-naphthoquinone chemicals are known to act against certain gram-positive bacteria and fungi. However, gram-negative bacteria show moderate resistance to 1,4-naphthoquinones or remain completely resistant to them as in the case of P. aeruginosa, which has been described only for some compounds of the 1,4-naphthoquinone group. This is described in Kuete, V. et al., Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype. Int J Antimicrob Agents, 2011. 37: pp. 156-61; Church, D. et al. Bum wound infections. Clin Microbiol Rev, 2006. 19(2): pp. 403-34; Atiyeh, B.S. et al., Effect of silver on burn wound infection control and healing: review of the literature. Burns, 2007. 33(2): pp. 139-48; Percival, S.L., Bowler P.G. and Russell D. Bacterial resistance to silver in wound care. J Hosp Infect, 2005. 60(1): pp. 1-7; Widhalm J. R. and Rhodes D. Biosynthesis and molecular actions of specialized 1,4-naphthoquinone natural products produced by horticultural plants. Hortic Res, 2016, 3: 16046.

Silver-only antibacterial mixtures have also been described, such as CN103622995 and R. Salomoni et al. "Antibacterial effect of silver nanoparticles in Pseudomonas aeruginosa". Nanotechnol Sci Appl. 2017; 10:115-121,2017.

In work: Habbal O, Hasson SS, El-Hag AH, Al-Mahrooqi Z, Al-Hashmi N, AlBimani Z, Al-Balushi MS, Al-Jabri AA. Antibacterial activity of Lawsonia inermis Linn (Henna) against Pseudomonas aeruginosa. Asian Pac J Trop Biomed. 2011 Jun;1(3):173-6, the antimicrobial activity of henna extracts that may contain lawson is reported. However, the authors did not present the results of chemical analyzes indicating the presence and dose of lawson in the tested extracts, thus it is impossible to determine whether lawson is responsible for the observed antibacterial activity or whether it is the effect of a mixture of various compounds present in the plant material. In the paper: Amin Pasandi Pour, Hassan Farahbakhsh "Lawsonia inermis L. leaves aqueous extract as a natural antioxidant and antibacterial product, (Formerly Natural Product Letters, Volume 34, 2020 - Issue 23, 05 Feb 2019, Pages 3399-3403), the authors indicate only a list of tested microorganisms, but they do not describe the effective doses of the preparation. The observed antibacterial effect of the extract results from the presence of active ingredients other than the subject invention. In the full version of the scientific article, there is no data on the activity of the extract against P. aeruginosa, and the authors refer to other works indicating the antibacterial effect of henna extracts against P. aeruginosa. The authors of the review paper Maryon Strugstad, Sasko Despotovski "A Summary of Extraction, Synthesis, Properties, and Potential Uses of Juglone: A Literature Review" indicate the activity of juglone against P. aeruginosa, citing the following research papers:

Clark, A.M., T.A. Jurgens, & C.D. Hufford. 1990. Antimicrobial activity of juglone. Phytotherapy Research 4(1): 1-14, where the P. aeruginosa strain is listed as one of the many pathogens tested, however, the authors describe that juglone showed no activity against Gram-negative bacteria, which include P. aeruginosa. For the remaining pathogens, except for P. aeruginosa, the MIC values are shown in Table 5.

Pereira et al. 2007. Walnut (Juglans regia L.) leaves: Phenolic compounds, antibacterial activity and antioxidant potential of different cultivars. Food Chemistry and Toxicology 45:2287-22, where only Juglans nigra tissue extracts, i.e. mixtures of many compounds, including juglone, were tested, showing their activity against selected pathogens, but not against P. aeruginosa, i.e. the tested extracts were inactive against this pathogen (Table 3).

Sharma et al. Microwave-assisted efficient extraction and stability of juglone in different solvents from Juglans regia: Quantification of six phenolic constituents by validated RP-HPLC and evaluation of antimicrobial activity. AnalyticalLetters 42:2592-2609, where the bacterial growth inhibitory concentration, i.e. MIC, was tested, which is in most cases lower than the bactericidal concentration indicated in our application, i.e. MBC, and this is due to the fact that microbial growth is a process that is disturbed in in the first place, and the bactericidal effect requires the activation of different changes in the bacterial cell under the influence of the substance.

The lack of bactericidal activity of 1,4-naphthoquinones against P. aeruginosa is described, for example, in Takis et al. Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials. Antimicrobial Agents And Chemotherapy, Oct. 2002, Vol. 46, No. 10, p.3133-3141. In the same publication, the authors Strugstad and Despotovski cite research results indicating the use of juglone as a carbon source by bacteria of the species Pseudomonas aeruginosa (Schmidt, S.K. 1988. Degradation of juglone by soil bacteria. Journal of Chemical Ecology 14(7): 1561-1571 ).

Krychowiak et al, "Combination of Silver Nanoparticles and Drosera binata Extract as a Possible Alternative for Antibiotic Treatment of Bum Wound Infections Caused by Resistant Staphylococcus aureus", (PloS One 2014 Dec 31;9(12) discloses a mixture of selected naphthoquinones and silver nanoparticles The mixture is for use against P. aeruginosa, which is a naphthoquinone-resistant pathogen unlike the susceptible S. aureus, i.e. it is not affected by the maximum applicable dose of 512 μg/mL.

The phenomenon of synergistic interactions of two antibacterial agents is not common, so it is also not an expected effect, which has been repeatedly described in the literature, for example in: Odds FC (2003). Synergy, antagonism, and what the checkboard puts between them. Journal of Antimicrobial Chemotherapy, 52(1), 1-1. In most cases of combinations of biologically active agents there are no interactions or interactions are insignificant, and therefore the agents used in combination have the same activity as the agents used separately. As indicated, e.g. in: Berenbaum MC (1978). A method for testing for synergy with any number of agents. Journal of Infectious Diseases, 137(2), 122-130, in the case of germicides, this means that a germicidal effect is obtained when the entire germicidal dose of either the first factor or the second factor is used, and when a mixture is used where the first factor is halved We will supplement the bactericidal dose with the second factor also in the bactericidal dose reduced by half, while further lowering the doses of both factors in the mixture will result in the abolition of the bactericidal effect of the mixture. The situation is different in the case of synergistic interactions, i.e. lowering the doses of both factors in the mixture does not result in eliminating the bactericidal effect of the mixture.

The present invention is based on the phenomenon of restoring the sensitivity of resistant microbial cells to molecules of a chemical compound by using a second agent in the mixture that acts as a sensitizing substance.

The mixture developed according to the invention is an example of a two-component system in which the interaction of factors is used to increase their biological potential based on the phenomenon of activation and synergy.

According to the invention, a new mixture of two components was developed in such a way that one naphthoquinone compound belonging to the group of 1,4-naphthoquinones and a second component, i.e. silver, were selected.

The composition thus selected constitutes a mixture having the desired bactericidal effect against P. aeruginosa resistant to this 1,4-naphthoquinone.

The invention therefore relates to a method of activating the bactericidal properties of a selected naphthoquinone belonging to the group of 1,4-naphthoquinones (i.e. 2-hydroxy-1,4-naphthoquinone, called lawson), against the naturally resistant Pseudomonas aeruginosa, by means of silver. According to the invention, the mixture containing silver and 1,4-naphthoquinone is characterized in that it contains bactericidal doses of silver nanoparticles in the mixture in a concentration equal to or greater than 4 μg Ag/mL and 2-hydroxy-1,4-naphthoquinone in dose equal to or greater than 8 μg/mL.

Preferably, it comprises spherical silver nanoparticles.

Preferably, it comprises silver nanoparticles stabilized with 11-mercaptoundecanoic acid.

Preferably, it comprises spherical silver nanoparticles with an average size of 5 nm.

The invention also relates to the medical use of this mixture as an antibacterial agent against Pseudomonas aeruginosa, preferably as an agent for application to the skin or wounds.

In the example of the invention, it is described that the mixture contains spherical silver nanoparticles with an average size of 5 nm stabilized with 11-mercaptoundecanoic acid at a concentration equal to or greater than 2 μg Ag/mL and 2-hydroxy-1,4-naphthoquinone at a concentration equal to or greater than 8 μg /mL. It contains the minimum dose of both ingredients with a bactericidal effect against P. aeruginosa at a concentration of approximately 2.5 χ 105 colony-forming units (JTK)/mL, which means that this dose reduces the number of bacterial cells by 99.9%, i.e. to at least 2.5χ 102 CFU/mL.

The invention is described in more detail in an example confirming the effectiveness of the mixture and its use. The example describes a mixture containing lawson and spherical silver nanoparticles with an average size of 5 nm stabilized with 11-mercaptoundecanoic acid. This naphthoquinone alone has been shown to be ineffective against the reference strain of P. aeruginosa, i.e. its minimum bactericidal concentration is higher than 512 μg/mL.

Fig. 1 shows the chemical structure of lawsone, i.e. 2-hydroxy-1,4-naphthoquinone.

Fig. 2 shows changes in the minimum bactericidal concentrations of lawsone (LAW) and a preparation of silver nanoparticles stabilized with 11-mercaptoundecanoic acid (Ag) simultaneously applied to P. aeruginosa ATCC 27853.

Example: Removal of Pseudomonas aeruginosa resistance to 2-hydroxy-1,4-naphthoquinone with silver nanoparticles.

Ready-made preparations of selected spherical silver nanoparticles (AgNPs) stabilized with 11-mercaptoundecanoic acid (AgCwCOOH) with an average metallic core size of 5 nm (Prochimia Surfaces Sp. z o.o.) were used in the study. The silver concentration in the preparations was determined by elemental analysis using inductively coupled plasma optical emission spectrometry (ICP-OES). The performance of the ICP-OES optical spectrometer (Perkin Elmer ICP-OES Optima 2000 DV) was optimized before each series of measurements. The preparation of silver nanoparticles was initially diluted 10 times with distilled water. Then, 1 mL of pure nitric acid was added to 250 μL of the prepared suspension. (65%), and then supplemented with demineralized water to a volume of 5 mL. The following parameters of the ICP-OES spectrometer were used during the analyses: generator power 1300 W, generator frequency 40 MHz; removable quartz burner; axial view of the plasma; argon gas (Ar): plasma gas flow 15.0 L/min, assist gas flow 0.2 L/min; gas flow in the atomizer 0.8 L/min; glass cyclonic spray chamber; sample flow rate: 1.5 L/min. Measurements of the concentration of silver ions (Ag+) were made at a wavelength of 328.068 nm in 3 repetitions.

The commercially available compound 2-hydroxy-1,4-naphthoquinone, i.e. lawson (Sigma Aldrich), was used in the research. The bactericidal effect of silver nanoparticles AgCwCOOH and lawson (Fig. 1) used separately was tested against the reference strain P. aeruginosa ATCC 27853 using the well-known method of medium microdilution as described in: Krychowiak M, Kawiak A, Narajczyk M, Borowik A, Królicka A: Silver Nanoparticles Combined With Naphthoquinones as an Effective Synergistic Strategy Against Staphylococcus aureus. Front Pharmacol 2018, 9:816. First, solutions of the test factors were prepared in Mueller-Hinton medium supplemented with cations (CA-MHB, Beckton Dickinson) by serial two-fold dilutions, as described below.

Description of the preparation of the mixture.

For the purposes of the experiment, the mixtures were prepared in a microbiological medium, however, the mixture can also be prepared in water or aqueous solutions, e.g. saline. Concentrated aqueous suspensions of silver nanoparticles, after determining the silver (Ag) content, were added directly to the CA-MHB medium to the final concentration of 128 μg Ag/mL, and then serial two-fold dilutions were made in the medium to obtain a mixture with a concentration of 64, 32, 16, 8, 4, 2 or 1 μg Ag/mL.

Before adding lawson to the medium, its concentrated solutions in dimethyl sulfoxide (DMSO) containing 51.2 mg of the compound in 1 mL were prepared. In order to perform the experiment, the solutions prepared in this way were added to the medium to the final concentration of 512 μg/mL, i.e. in a volume of 10 μL to 0.99 mL, and then serial two-fold dilutions were made in the medium to obtain solutions with a concentration of 256, 128, 64, 32 , 16, 8 or 4 μg/mL. From the prepared mixtures of AgCwCOOH and solutions of lawson in the medium, 100 μL were taken and transferred to the wells of a 96-well microtest plate for testing the bactericidal activity of individual factors. In the procedure of testing the interaction of silver nanoparticles and 2-hydroxy-1,4-naphthoquinone, i.e. lawson, the Checkerboard Titration approach was used, which is also described in: Krychowiak M, Kawiak A, Narajczyk M, Borowik A, Królicka A: Silver Nanoparticles Combined With Naphthoquinones as an Effective Synergistic Strategy Against Staphylococcus aureus. Front Pharmacol 2018, 9:816, involving the simultaneous application of two tested factors on a microtest plate, where each factor is used in the following concentration gradient in the medium: 2 χ MBC, 1 χ MBC, 0.5 χ MBC, 0.25 χ MBC , 0.125 χ MBC, 0.06 χ MBC and 0.03 χ MBC, where MBC is the Minimum Bactericidal Concentration (MBC). In this way, each well of the microtest plate contains a unique combination of concentrations of the test factors. In the case of compounds or agents not showing bactericidal activity, as is the case with lawson, a concentration gradient is used starting from the highest possible concentration of the compound, i.e. most often 512, 256, 128, 64, 32, 16 and 8 μg /mL. In the case of silver nanoparticles, the concentration gradient used in the experiment was as follows: 16, 8, 4, 2, 1.0.5 and 0.25 μg Ag/mL.

The mixtures were prepared in such a way that concentrated aqueous suspensions of silver nanoparticles, after determining the silver (Ag) content, were added directly to the CA-MHB medium to a final concentration of 32 μg Ag/mL, and then serial two-fold dilutions were made in the medium to obtain mixtures with a concentration of 16 , 8, 4, 2, 1 and 0.5 μg Ag/mL. Before adding lawson to the medium, its concentrated solutions in dimethyl sulfoxide (DMSO) containing 51.2 mg of the compound in 1 mL were prepared. In order to perform the experiment, the solutions prepared in this way were added to the medium to the final concentration of 1024 μg/mL, i.e. in a volume of 20 μL to 0.98 mL, and then serial two-fold dilutions were made in the medium to obtain solutions with a concentration of 512, 256, 128, 64 , 32 and 16 μg/mL. Then, the mixtures were prepared by combining nanoparticle suspensions and lawsone solutions in a 1:1 volume ratio.

Then, to the wells containing 100 μL of suspensions, solutions or mixtures in the medium, 10 μL of bacterial inoculum containing approximately 2.5 χ 10 ⁵ colony forming units (JTK) in 1 mL was added. The inoculum was prepared by diluting a 6-hour bacterial culture (CA-MHB, 37°C, 150 rpm) in fresh CA-MHB medium to a 0.5 degree McFarland turbidity measured with a densitometer (DensiMeter II, EMO). The microtest plates were incubated for 24 hours at 37°C, and then the contents of the wells in which bacterial growth was inhibited were plated on Tryptic Soy Agar (BTL Polska Sp. z oo). The agar plates prepared in this way were incubated for 24 hours at 37°C in order to count the bacterial cells (JTK) remaining in the wells after treatment with the factor, and thus to determine the minimum bactericidal concentration of the tested factors - MBC. The MBC concentration was defined as the lowest factor concentration reducing the baseline number of CFU in the fovea (approx. 2.5 χ 10 ⁵ CFU/mL) by 99.9%, i.e. 3 logarithms (approx. 2.5 χ * 10) within 24 hours. ² CFU/mL).

As indicated in Table 1, lawson has no bactericidal activity against P. aeruginosa (MBC > 512 μg/mL). Nevertheless, the use of lawson in combination with silver nanoparticles AgCwCOOH (MBC = 8 μg Ag/mL) resulted in a bactericidal effect at its concentration of 8 μg/mL and the concentration of nanoparticles corresponding to 2 μg Ag/mL (Table 1, Fig. 2). The above results indicate that silver effectively interacts with lawson, i.e. 2-hydroxy-1,4-naphthoquinone, eliminating the resistance of P. aeruginosa to this compound. This allows you to achieve a bactericidal effect

PL 243213 Β1 of the mixture with a significantly reduced concentration of the silver preparation (even to 75%) and the concentration of lawson up to 8 pg/mL. This allows for a wide range of possibilities to modulate the biological activity of both factors and to optimize the composition of the mixture.

Table 1

Concentrations of lawsone (2-hydroxy-1,4-naphthoquinone) and silver nanoparticles in mixtures in CA-MHB medium determining the bactericidal effect against the reference strain P. aeruginosa ATCC 27853

Lawson (pg/mL) AgCioCOOH (pg Ag/mL) >512 0 512 2 256 2 128 2 64 2 32 2 16 2 8 2 8 4 0 8

AgCwCOOH - silver nanoparticles stabilized with 11-mercaptoundecanoic acid.

Ag, silver ions.

Claims (5)

1. A mixture containing silver and 1,4-naphthoquinone, characterized in that it contains bactericidal doses of silver nanoparticles in the mixture at a concentration equal to or greater than 4 pg Ag/mL and 2-hydroxy-1,4-naphthoquinone in a dose equal to or greater than 8 pg/mL. 2. A mixture according to claim The device of claim 1, characterized in that it comprises spherical silver nanoparticles. 3. The mixture according to claim 1-2, characterized in that it contains silver nanoparticles stabilized with 11-mercaptoundecanoic acid. 4. The mixture according to claim 1-3, characterized in that it comprises spherical silver nanoparticles with an average size of 5 nm. 5. A mixture as described in any one of claims 1-4 for use as an antibacterial agent against Pseudomonas aeruginosa, preferably as an agent for application to the skin or wounds.