PL238127B1 - Application of 2'-hydroxychalcone - Google Patents

Abstract

The subject of the application is the use of 2'-hydroxychalcone of formula 1 for the production of medicines and/or as an ingredient of medicinal products for the treatment and prevention of kidney stones, ureteral stones and stones in the lower urinary tract, as well as cystitis and for the production of dietary supplements, nutritional ingredients or as an ingredient of functional food, used to support the treatment and prevention of these diseases.

Description

The subject of the invention is the use of 2'-hydroxychalcone to inhibit the growth and ureolytic activity of urinary tract pathogens for the manufacture of a medicament for the treatment and prevention of urinary tract diseases.

The literature reports to date describe a moderate antibacterial activity of 2'-hydroxychalcone against gram-positive and gram-negative bacteria. (International Letters of Chemistry, Physics and Astronomy 2013; 7: 102-119).

From US patent 3,839,580 the composition of (p-nitrobenzamido) acetohydroxamic acid and its derivatives and ampicillin, sulfamethoxazole and nitrofurantoin for the treatment of urinary tract infections caused by Proteus strains is known.

No. 4,024,256 describes a method of treating infections caused by Proteus strains using a source of hexamethylenetetramine (urotropine) and a source of hydroxamic groups, or only hydroxamic acids. In turn, derivatives of hydroxamic acid have also been characterized as effective urease inhibitors in studies conducted on rats. The results of these studies are disclosed in patents US4,083,996 and US 4,157,396.

U.S. Patent 4,225,526 describes 8 - [(4-aminophenyl) sulfonyl] -amino-2-naphthalenylphosphoric acid amide which inhibits whole cell Proteus mirabilis activity at micromolar concentrations.

In turn, US Patent 4,222948 discloses [(4-aminophenyl) sulfonyl] amino phenyl phosphate amides with antibacterial and anti-ureolytic activity against Proteus mirabilis and Escherichia coli.

The US patent US 8,969,384B2 describes the possibility of using flavonoid derivatives in the prophylaxis and treatment of Helicobacter pylori infections.

Another description of the invention, US 5,192,277 describes the possibility of using flavonoids as absorbent components with bacteriostatic and deodorant properties.

Another description of the invention, US 5,192,277 describes the possibility of using flavonoids as absorbent components with bacteriostatic and deodorant properties.

In the Japanese patent JP4450355 (B2), mixtures of flavones, flavonols (including quercetin), dihydroflavonols and catechins are proposed as an agent preventing nappy rash and ammonia release.

Another Japanese patent application JPS5488289 (A) proposes (-) -? - (chalcone-4'-carbonylamino) -benzylpenicillin as an antibacterial compound effective against Proteus strains.

According to the currently available literature data, it is known that urease is an important factor in the virulence of many bacterial strains. The main examples are Proteus spp. And strains of Providencia stuartii, Morganella morganii (Prokaryotes 2006; 6: 245-269), Pseudomonas spp., Klebsiella spp., Staphylococcus spp., Corynebacterium spp. And Ureaplasma ureolyticum 2000 (Microbesma ureolyticum 2000). ; 2: 533-542). They have the ability to colonize the human urinary system, and their ureolytic activity increases the pH of the urine. The increase in alkalinity is directly related to the precipitation of phosphates, additionally exacerbated by the formation of crystallization centers around bacterial deposits. Infections are a particular problem in long-term catheterization patients, leading in extreme cases to complete loss of catheter patency. Compounds that are active urease inhibitors are proposed as adjunctive therapy, especially due to the increasing resistance of pathogenic bacterial strains to commonly used antibiotics.

Currently, many groups of substances are known to limit the activity of bacterial urease. The best known are phosphoric acid amides, hydroxamic acids, polyphenols, quinones, heavy metal ions and thiols (J Med Chem 2016; 59: 8125). The most potent urease inhibitors are phosphoramidate derivatives with commercially available N- (diaminophosphinyl) -4-fluorobenzamide (Flurofamide). This compound is an analog of the urea hydrolysis transition state with an IC50 at the nanomolar level. Its main disadvantage is low hydrolytic stability, significantly limiting the therapeutic use (J Vet Pharmacol Ther 1986; 9: 280).

Another urease inhibitor used in medicine is acetohydroxamic acid, approved in 1983 by the US Food and Drug Administration for the treatment of urinary tract infections. The inhibitory activity of a compound is based on its ability to complex ions

PL238 127 Β1 nickel, present in the active center of urease and necessary for its enzymatic activity. Acetohydroxamic acid is commercially available under the name Lithostat®, but the possibility of its use is very limited due to a number of side effects, including teratogenic activity.

In addition to synthetic compounds, many naturally derived urease inhibitors are known. In this group of compounds, the most frequently mentioned are terpene derivatives, polyphenols, quinones and sphingolipids (Arch Pharm Chem Life Sci 2016; 349: 519). The best described are flavonoids, with particular emphasis on quercetin, active against Helicobacter pylori urease with IC50 = 11.2 μΜ (J Agric Food Chem 2012; 60: 10575). Flavonoids have been described as non-competitive and reversible inhibitors of bacterial urease. The performed molecular modeling allowed to determine the enzyme inhibition mechanism: hydrogen bonds formed by inhibitors block the position of the loop regulating the access of the substrate to the active site of the enzyme. Many bacterial urease inhibitors have also been identified among chalcones and their derivatives (J Iran Chem, 2014; 11: 1113-1119). 2'-hydroxychalcone has been determined to be inactive with plant urease (Chemistry & Biodiversity, 2005; 2: 487-496). Due to the large variety of naturally occurring flavonoids and the possibility of synthesizing new derivatives, the anti-ureolytic activity of many of them has not yet been known. In recent years, the inhibitory effect of hops extract on Helicobacter pylori has been described, without determining the main active substance (Czech J Food Sci 2015; 33: 302-307). Isoxanthohumol is a known component of such extracts, present in concentrations up to several mg L ^-1 .

The essence of the invention is the 2'-hydroxychalcone of the formula 1 for use in the treatment and prevention of nephrolithiasis, ureterolithiasis and calculus in the lower urinary tract, as well as cystitis.

It is preferred that the action of 2'-hydroxychalcone is that the growth of E. coli strains and / or the growth and ureolytic activity of Proteus spp., P. stuartii, M. morganii, Pseudomonas spp., Klebsiella spp., Staphylococcus spp. , Corynebacterium spp. And Ureaplasma ureolyticum.

Determination of in vitro anti-ureolytic activity of 2'-hydroxychalkon against purified bacterial urease.

Example 1. Ureolytic activity was monitored by measuring the released ammonium ions according to the Watherburn method. The inhibitory activity of the compounds was determined using the indophenol Berthelot colorimetric reaction. All kinetic constants were calculated with the GraphPad Prism 5 computer program using the nonlinear regression method.

Preserved urease activity was determined after 15, 30, 45, 60 and 90 minutes of pre-incubation with 2'-hydroxychalcone of formula 1 at a concentration of 100 μg mL ^-1 . In parallel, a control incubation was carried out without the addition of 2'-hydroxychalkon.

Incubation time [min] —Control —β— 2-hydroxychalcone

Ureaza Proteus mirabilis PCM1360

Ki [pgmL ' ¹ ] 10O 50 [pg mL ^-1 ]

312.1 35.2

204.2 ± 12.8

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Preliminary studies revealed the inhibitory effect of 2'-hydroxychalcone against P. mirabilis PCM1360 urease. Incubation of the purified P. mirabilis PCM1360 urease with 100 μg mL ¹ 2'-hydroxychalkon reduced ureolytic activity by about 25%. The concentration of 2'-hydroxychalkon was determined, allowing for a 50% reduction in urease activity (IC50) and the inhibition constant (Ki), obtaining the values of 204.2 μg mL ¹ and 312.1 μg mL ^{1, respectively} .

Determination of antibacterial properties of 2'-hydroxychalkon.

Example 2. Antibacterial properties of 2'-hydroxychalcone were determined against Proteus mirabilis PCM1360 cells by colorimetric measurement of the change in activity of intracellular bacterial dehydrogenases converting soluble tetrazolium salt (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; MTT) into insoluble crystallizing formazan, the concentration of which was measured spectrophotometrically after dissolving in isopropanol.

The experiment was performed according to the procedure optimized and described for P. mirabilis PCM1360 (AJMB, 2015; 7 (4): 159-167). The bacterial suspension with a density of about 10 ⁸ cfu mL ^{1 was} incubated for one hour with 2'-hydroxychalcone at a concentration of 200 μg mL ¹ , and a control sample without 2'-hydroxychalkon was prepared in parallel. Then both samples were incubated with 0.5 mg mL of ¹ MTT solution for half an hour. Successively, a mixture of isopropanol and 1% hydrochloric acid in the proportion of 1: 1 was added to each sample. After a minimum incubation of 15 minutes, the absorbance was measured at 550 nm. The measurement allowed to indirectly determine the viability of cells in relation to the control sample.

Lifetime [%] (MTT test) min 60 min

69.7 ± 11.8 59.0 ± 2.4

It has been shown that the 15-minute incubation of bacterial cells with 2'-hydroxychalkon at a concentration of 200 μg mL ¹ reduces the metabolic activity of P. mirabilis PCM1360 by approximately 30%. The observed effect intensifies over time - after 60 minutes of incubation, the activity of cells is reduced by nearly 40%.

Example 3. The bactericidal properties of 2'-hydroxychalcone were determined against Escherichia coli PCM2057 cells - a strain intended to determine the antibacterial activity of new chemical compounds. A set of BacLight® fluorescent dyes was used, allowing quantitative and qualitative differentiation of living and dead cells in the mixture. Cells were incubated for 60 minutes with the test compound, then washed and stained according to the manufacturer's instructions. The amount of viable cells in the mixture [%] was determined on the basis of the relative fluorescence of the stained bacterial cells. The tests were performed in the presence of 2'-hydroxychalkon at a concentration of 200 μg mL ¹ .

Viable cells [%] (BacLight®) min

44.9 ± 4.8

According to the method used, a 60-minute incubation of E. coli PCM2057 with 2'-hydroxychalcone at a concentration of 200 μg mL ¹ allows about 50% of the cells present in the mixture to be killed.

Example 4. Bacteriostatic properties against Proteus mirabilis PCM1360 and Escherichia coli PCM2057 cells were tested according to the standard CLSI procedure (CLSI document M07-A9, 2012), allowing the determination of Minimum Inhibitory Concentrations (MIC).

E. coli PCM2057 P. mirabilis PCM1360

MIC50 MICso MIC50 MICgo

[pgmL- ¹ ] [pg mL ^-1 ] [pg mL ' ¹ ] [pg mL' ¹ ]

100> 200> 200> 200

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In the concentration range studied, the values of the Minimum Inhibitory Concentration of 2 '- hydroxychalcone were characterized, which allowed to reduce the growth of E. coli PCM2057 by 50% compared to the control sample (MIC50). 2'-hydroxychalcone showed stronger bacteriostatic activity against E. coli PCM2057 (MIC50 = 50 μg mL ¹ ) than against P. mirabilis PCM1360 (MIC50 => 200 μg mL ¹ ) after 24 h of incubation.

Determination of the inhibitory effect of 2'-hydroxychalcone on the ureolytic activity of whole cells of Proteus mirabilis.

Example 4. The effectiveness of reducing urease activity in whole cells was investigated indirectly by measuring the concentration of ammonium ions generated in the urea hydrolysis reaction (PLoS ONE 2017; 12 (8): e0182437). The incubation samples contained live cells of the Proteus mirabilis PCM1360 strain with a density of 10 ⁷ CFU ml ^and suspended in a phosphate buffer at pH 7.2 ± 0.2 containing 100 mM urea. The retained activity of bacterial cells was determined after 30 minutes of pre-incubation with 2'-hydroxychalkon at the concentration of 100 μg mL ¹ .

Whole cells Proteus mirabilis PCM1360

Preserved activity [%] after 30 min 57.9 ± 6.5

IC50 [pg mL · ¹ ] 190.3 ± 19.8

The concentration allowing the reduction of the ureolytic activity of whole cells of P. mirabilis by 50% compared to the control sample (IC50) was determined. Due to the results obtained on purified urease, the measurement was performed after 30 minutes of incubation with 2'-hydroxychalcone, obtaining an IC50 = 190.3 μϋ mL- ¹ .

The above results present a complete set of in vitro determinations allowing for the precise determination of the antibacterial and antimicrobial properties of 2'-hydroxychalcone of the formula 1 against the most common urinary tract pathogens. Antibacterial characteristics of 2'-hydroxychalcone against E. coli PCM2057 have been demonstrated. The 60-minute incubation of the bacterial suspension with the test compound at a concentration of 200 µg mL ¹ allowed 50% of the cells to be killed. Standard determinations of the Minimum Inhibitory Concentration of 2'-hydroxychalkon allowed to determine the MIC50 value against E. coli PCM2057 at the level of 100 μg mL ¹ . The determined bacteriostatic activity against the reference strain of E. coli PCM2057 is a particularly important characteristic, as it proves the possibility of limiting the activity of this most popular pathogen of the urinary system.

The obtained values are at a level comparable to other known, effective antibacterial compounds of natural origin.

2'-Hydroxychalcone has been identified as a P. mirabilis PCM1360 urease inhibitor in both the purified enzyme and whole bacterial cell system. The IC50 values determined were 204.2 μg mL ¹ for the purified enzyme studies and 190.3 μg mL ¹ for the whole cell studies. The obtained result is comparable with the values obtained for the control substances - quercitin (IC50 = 229.8 ± 18.4 μg mL ¹ ) and naringenin (IC50 = 262.0 ± 35.6 μg mL ¹ ) - flavonoids known as urease inhibitors.

Due to the high conservativeness of the structure of the active site of bacterial ureases and the known mechanism of urease inhibition by flavonoids, 2'-hydroxychalcone can be described as an effective inhibitor of bacterial ureases.

Claims (2)

1. 2'-Hydroxychalcone of the formula 1 for use in the treatment and prevention of nephrolithiasis, ureterolithiasis and calculus in the lower urinary tract, as well as cystitis. 2. 2'-Hydroxychalcone of formula 1 for use according to claim 1 The method of claim 1, wherein the growth of E. coli strains and / or the growth and ureolytic activity of Proteus spp., P. stuartii, M. morganii, Pseudomonas spp., Klebsiella spp., Staphylococcus spp., Corynebacterium spp. And Ureaplasma ureolyticum are restricted. .