PL243140B1 - Mixture of 3-chloro-5-hydroxy-2-methylo-1,4-naphtoquinone and silver nanoparticles and application of the mixture as antibacterial agent for controlling Pseudomonas aeruginosa - Google Patents

Abstract

The invention relates to a mixture containing a bactericidal dose of silver nanoparticles and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone with a bactericidal effect against P. aeruginosa. The invention relates to the medical use of the mixture as an antibacterial agent against P. aeruginosa, preferably for application to the skin or wounds.

Description

The invention relates to a method of activating the bactericidal properties of 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, called 3-chloroplumbagin, against the naturally resistant Pseudomonas aeruginosa bacillus by means of silver nanoparticles, and thus by means of a mixture of these factors . The invention therefore relates to the medical use of the mixture for combating P. aeruginosa, in particular for external application, e.g. to 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 an increasing range of antibiotics, the pool of possible therapies used to treat infections is decreasing, and thus the threat to human health and life is increasing. The World Health Organization (WHO) and the US Centers for Disease Control and Prevention (CDC) indicate in their latest reports that, due to the advent of the post-antibiotic era, it is necessary to apply sustainable strategies for the prevention and treatment of infectious diseases - 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 commonly isolated bacterial species and poses a particular problem in their treatment due to multidrug resistance limiting therapeutic options - Church, D. et al. Bum wound infections. Clin Microbiol Rev, 2006. 19(2): pp. 403-34. 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 described, among others in: 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.

In the publication of Ogihara et al. (2013) described the activity of extracts from Dionaea muscipula tissues, which are mixtures of various compounds in these tissues synthesized. The authors of the study did not carry out analyzes to determine the activity of the pure compound, but only found its presence in the tested extracts and the activity of these extracts, among others. against P. aeruginosa.

It is therefore necessary to develop strategies to delay or abolish the development of bacterial resistance and to expand therapeutic options.

The action of compounds from the group of 1,4-naphthoquinones alone is known against some gram-positive bacteria and fungi - 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. Nevertheless, gram-negative bacteria show moderate resistance to 1,4-naphthoquinones, including the 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone used, or remain resistant to them completely resistant as P. aeruginosa - Coban, A.Y. et al., Effects of efflux pump inhibitors phenyl-arginine-beta-naphthylamide and 1-(1-naphthylmethyl)-piperazine on the antimicrobial susceptibility of Pseudomonas aeruginosa isolates from cystic fibrosis patients. J Chemother, 2009. 21(5): p. 592-4.].

In the publication of Ravichandiran et al. (2019) describes highly modified derivatives of naphthoquinones, i.e. the structure of naphthoquinone modified in various ways by adding many phenolic rings and other chemical groups to it, which significantly changes the chemical nature and biological properties of the resulting substances in relation to the starting substance, i.e. 1,4-naphthoquinone . Among the results of research on the antibacterial activity of compounds synthesized by Ravichandiran and co-workers, tested by the authors, there is no 1,4-naphthoquinone and there are no simple chemical structures based on two carbon rings and simple substituents specific to 1,4-naphthoquinones, including 3-chloro -5-hydroxy-2-methyl-1,4-naphthoquinone. The lowest inhibitory concentration of 250-500 μg/mL has been described. Concentrations above 250 mg/L are concentrations at the limit of solubility of naphthoquinones in the microbial medium. The indicated publication does not confirm the activity of 3-chloro-2-methyl-1,4 naphthoquinone against P. aeruginosa.

In the publication Krychowiak Maśnicka et. al. (2002), the results of analyzes of the activity of plumbagin, i.e. the pure compound, against two strains of P. aeruginosa were described, which confirmed the lack of such activity. The data contained in the publication indicate that the minimum inhibitory concentration (MIC) of plumbagin is higher than 500 μg/mL for both tested strains, which confirms the resistance of this pathogen to naphthoquinones. The MIC is only lower when plumbagin is used with the efflux inhibitors studied.

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, among others 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 germicidal agents, this means that a germicidal effect is obtained when the entire germicidal dose of the first factor or the entire germicidal dose of the second factor is used, and when a mixture is used where the first factor is reduced by half of the bactericidal dose will be supplemented with the second factor also in the bactericidal dose reduced by half, while further reduction of 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.

The invention concerns a method of activating the bactericidal properties of 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, i.e. 3-chloroplumbagine, against the naturally resistant Pseudomonas aeruginosa bacillus, using silver in a given form - nanoparticles silver. The invention therefore relates to the medical use of the P. aeruginosa control mixture as an antibacterial agent for topical application, i.e. to the skin or wounds. The mixture according to the invention contains a given type of silver nanoparticles and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, called 3-chloroplumbagin, and contains 3-chloro-5-hydroxy-2-methyl-1,4- naphthoquinone at a dose equal to or greater than 16 μg/mL and silver nanoparticles at a concentration equal to or greater than 1 μg Ag/mL. According to the invention, the mixture contains a bactericidal dose of silver nanoparticles (AgCwCOOH) acting bactericidal against P. aeruginosa and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone. The demonstrated mechanism of interaction between silver nanoparticles and 3-chloroplumbagin is a phenomenon specific to compounds from the group of 1,4-naphthoquinones with high potential for combating one of the most dangerous bacterial pathogens of humans - P. aeruginosa.

Preferably, the invention relates to the medical use of a mixture comprising 3-chloroplumbagine (3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone) and spherical silver nanoparticles with an average size of 5 nm stabilized with 11-mercaptoundecanoic acid (AgClwCOOH).

Preferably, it contains silver nanoparticles stabilized with 11-mercaptoundecanoic acid (AgC10COOH).

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

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 1 μg Ag/mL and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone at a concentration equal to or greater than 16 μg/mL. It contains the minimum dose of both ingredients that is bactericidal against P. aeruginosa at a concentration of approximately 2.5 x 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 2.5 x 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 3-chloroplumbagine and spherical silver nanoparticles with an average size of 5 nm stabilized with 11-mercaptoundecanoic acid. Naphthoquinone alone has been shown to be ineffective against this bacterial strain, i.e. MBC is higher than 512 μg/mL.

Fig. 1 shows the scheme of the synthesis of 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone (3-chloroplumbagin). (1) 2,3-epoxy-5-hydroxy-2-methyl-1,4-naphthoquinone. (2) 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone.

Fig. 2 shows the chemical structure of 3-chloroplumbagin, i.e. 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone.

Fig. 3 shows the changes in the minimum bactericidal concentrations of 3-chloroplumbagine and the preparation of silver nanoparticles stabilized with 11-mercaptoundecanoic acid (AgCwCOOH) simultaneously applied to P. aeruginosa ATCC 27853.

Example: Removal of Pseudomonas aeruginosa resistance to 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone with silver nanoparticles.

Ready-made preparations of 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 compound 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone (3-chloroplumbagine) was synthesized according to the method presented below, which is shown in Fig. 1. The chemical structures of the reaction products were confirmed by the spectral method - proton resonance and the purity of the compounds was checked using the TLC method. Chromatography system 10:1 hexane:ethyl acetate. ¹ H NMR spectra were taken on a Varian VXR-S spectrometer operating at 500 MHz. Chemical shifts are given in units of δ in ppm downfield from the inner tetramethylsilane. The abbreviations used in the descriptions of ^1H NMR spectra are as follows: br.s-broad signal, s-singlet, d-doublet, dd-doublet of doublets, t-triplet, k-quartet and m-multiplet.

g 5-hydroxy-2-methyl-1,4-naphthoquinone was dissolved in 250 mL of cold ethanol and added to a solution of sodium perborate (2.5 g) in 270 mL of water. The reaction was carried out for 2 minutes, adjusting the solution to pH 5 with 1M H2SO4. Then it was shaken with saturated NaCl solution and petroleum ether (3 times). The organic layer was dried with Na2SO4 and evaporated under reduced pressure. Reaction products of 2,3-epox-5-hydroxy-2-methyl-1,4-naphthoquinone (1) and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, i.e. 3-chloroplumnagine (2 ) was purified using column chromatography. Starting eluent hexane:ethyl acetate (50:1), then 20:1.

Intermediate: 2,3-epoxy-5-hydroxy-2-methyl-1,4-naphthoquinone (1): 50% yield ¹ H NMR (CDCl 3 , 500 MHz): 1.75 (3H, s); 3.84 (1H, s); 7.28-7.30 (1H, m); 7.60-7.62 (1H, m); 7.66 (1H, t, J=7.8Hz); 11.22 (1H, s).

The 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone was purified again by column chromatography. Eluent hexane : ethyl acetate (20:1).

3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, i.e. 3-chloroplumbagine (2): yield 10% ^1H NMR (CDCl3, 500 MHz): 2.38 (3H, s); 7.30-7.32 (1H, m); 7.65-7.72 (2H, m); 11.80 (1H, s). The bactericidal effect of silver nanoparticles AgCwCOOH and 3-chloroplumbagine (Fig. 2) used separately was tested against the reference strain of 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, in the Mueller-Hinton medium supplemented with cations (CA-MHB, Beckton Dickinson), solutions of the tested factors were prepared by means of serial two-fold dilutions.

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.

In the case of 3-chloroplumbagin (Fig. 2), concentrated solutions in dimethyl sulfoxide (DMSO) containing 25.6 mg of the compound in 1 mL were prepared before adding to the medium. 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 20 μL to 0.98 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 3-chloroplumbagin 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 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, the Checkerboard Titration approach was used, which was 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 x MBC, 1 x MBC, 0.5 x MBC, 0.25 x MBC , 0.125 x MBC, 0.06 x MBC and 0.03 x 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 3-chloroplumbagin, 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 3-chloroplumbagin to the medium, concentrated solutions in dimethyl sulfoxide (DMSO) containing 25.6 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 40 μL to 0.96 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 3-chloroplumbagine solutions in a 1:1 volume ratio.

Then, 10 μL of bacterial inoculum containing approximately 2.5 x 10 ⁵ colony-forming units (JTK) in 1 mL was added to wells containing 100 μL of suspensions, solutions or mixtures in the medium. The inoculum was prepared by diluting a 6-hour bacterial culture (CAMHB, 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 inhibition of bacterial growth was observed were plated on Tryptic Soy Agar (TSA; 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 agent, and thus to determine the Minimum Bactericidal Concentration (MBC) of the tested agents. The MBC concentration was defined as the lowest factor concentration reducing the baseline number of CFU in the well (approx. 2.5 x 10 ⁵ CFU/mL) by 99.9%, i.e. 3 logarithms (approx. 2.5 x 10 ² JTK/mL).

As indicated in Table 1, 3-chloroplumbagine has no bactericidal activity against P. aeruginosa (MBC >512 μg/mL). Nevertheless, the use of plumbagin in combination with silver nanoparticles AgCwCOOH (MBC = 8 μg Ag/mL) resulted in a bactericidal effect at its concentration of 16 μg/mL and the concentration of nanoparticles corresponding to 2 μg Ag/mL (Table 1, Fig. 3). The above results indicate that silver effectively interacts with 3-chloroplumbagin, i.e. 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, eliminating the resistance of P. aeruginosa to this compound. This allows to achieve the bactericidal effect of the mixture with a significantly reduced concentration of the silver preparation (up to 75%) and the concentration of 3-chloroplumbagin to 16 μg/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 3-chloroplumbagin (3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone) and silver nanoparticles in mixtures in CA-MHB medium determining the bactericidal effect against the reference strain P. aeruginosa ATCC 27853.

PL 243140 B1

3-chloroplumbagine (pg/mL) AgC ₁₀ COOH (pg Ag/mL) > 512 0 512 1 256 1 128 1 64 1 32 1 16 2 16 4 0 8

AgCioCOOH - silver nanoparticles stabilized with 11-mercaptoundecanoic acid.

Ag - silver ions.

Claims (4)

1. A mixture containing silver in the form of nanoparticles and 1,4-naphthoquinone, containing a bactericidal dose of silver nanoparticles and 3-chloro-5-hydroxy-2-methyl-1,4-naphthoquinone, with 3-chloro-5- the hydroxy-2-methyl-1,4-naphthoquinone is at or greater than 16 pg/mL and the silver nanoparticles are at or greater than 2 pg Ag/mL for use as an antibacterial agent against P. aeruginosa, preferably for application to the skin or wounds. 2. The mixture for use according to claim The device of claim 1, characterized in that it comprises spherical silver nanoparticles with an average size of 5 nm. 3. The mixture for use according to claim 1-2, characterized in that it contains silver nanoparticles stabilized with 11-mercaptoundecanoic acid. 4. The mixture for use according to claim 1-3, characterized in that it comprises spherical silver nanoparticles with an average size of 5 nm stabilized with 11-mercaptoundecanoic acid.