KR20160046489A - Pharmaceutical composition for prevention or treatment of infections caused by biofilm, and coating composition for inhibiting formation of biofilm - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating infectious diseases caused by a biofilm and a coating composition for inhibiting the formation of a biofilm, wherein the compositions comprise carvacrol, or carvacrol and eugenol, as active ingredients. The pharmaceutical composition and the coating composition have excellent antimicrobial effects and show excellent effects of inhibiting and removing the formation of a biofilm, thereby making the pharmaceutical composition helpfully used in preventing or treating various infectious diseases caused by the biofilm, and the coating composition for coating helpfully used in inhibiting the biofilm formed on various medical devices, medical materials or medical implants.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating infectious diseases caused by biofilm and a coating composition for inhibiting the formation of biofilm,

The present invention relates to a pharmaceutical composition for preventing and treating infection caused by a biofilm, and a composition for inhibiting biofilm formation.

Streptococcus pneumoniae (Streptococcus pneumoniae , pneumococci) is a major cause of pneumonia, meningitis, bacteremia, and otitis media (OM) in children and the elderly (Ispahani meat al . , 2004; Kadioglu, 2008). Colonization and disease of pneumococci are often associated with the formation of biofilms (Hall-Stoodley L & et al., 2006).

Most bacteria are generally present in planktonic form in the water, but if the survival conditions become poor due to the reduction of nutrients or the attack of antibiotics, the phenotype in the form of a biofilm that forms a community on the surface of the solid Change. Therefore, a biofilm is a collection of microorganisms such as bacteria, yeast, fungi and the like attached to the surface of human body (skin, oral cavity, teeth, etc.) or equipment (piping, storage tank, etc.) , It is also provided as a habitat for microorganisms, which accelerates the formation of bacteria. Such biofilm microorganisms may cause serious problems in the whole industry and the medical field, and therefore research is required.

(Costerton et al . , 1999; Wolcott Rd, 2008). In this study, we investigated the effect of an exopolysaccharide (EPS) on the surface of host cells. That is, the formation of a biofilm is accomplished through a process in which the bacteria form a substrate composed of glycoproteins three-dimensionally around the cells, which attaches to surrounding solids, and the bacteria form a series of quorum sensing (QS) Through the process, insufficient nutrients can be trapped in the matrix and protected from external attack (antibiotics, hypoxia). In addition, the biofilm formation reduces the metabolic rate required for bacterial survival, allowing survival with a small amount of nutrients, thereby increasing the likelihood of survival in harsh environments. Biofilm is a phenomenon that is commonly seen in nature, and bacteria on the surface of rocks are known to live in the form of biofilm even in rivers with a constant flow rate.

Bacteria in biofilms display enhanced resistance to antibiotics and host defense systems, often leading to persistent and difficult-to-treat infections (Kania meat al . , 2008; Vlastarakos meat al . , 2007). A variety of studies have shown that the abiotic bacterial biofilms are significantly more resistant to penicillin, tetracycline, lymphedicine, amocyline, erythromycin, clindamycin and levofloxacin (del Prado meat al . , 2010; Garcia-Castillo meat al . , 2007). Marks et al. (2012) reported that pneumococcal biofilms formed on mouse nasopharyngeal tissue are more resistant to gentamycin and penicillin G than do planktonic biofilms (Marks < RTI ID = 0.0 > meat al . , 2012). Thus, there is an urgent need to search for alternative antimicrobial targets in order to develop antimicrobial compounds against pneumococcal biofilms.

Especially, biofilm in human infectious diseases causes many clinical problems. For example, if bacteria form on the surface of a tooth in the form of biofilm to form tartar, it is known to cause tooth decay and periodontal disease. The most significant factors affecting oral health factors are tooth corrosion or tooth decay, gum disease or periodontal disease, and the occurrence of unpleasant odors, the root cause of which is formed by pathogens living in the mouth Is a biofilm. The pathogens present in the solution state such as saliva in the state where they are not attached to the surface of the oral cavity can be easily removed together with toothpaste and beverage ingestion, and the chance of causing diseases in the oral cavity is very small. However, the pathogenic bacteria are attached to the surface of the oral cavity for effective survival, thereby forming a biofilm. Once the biofilm is formed, it is difficult to remove by conventional physical methods, because the protective ability against external physical and chemical stimuli is remarkably increased. In addition, the use of a highly toxic antimicrobial agent or a large amount of antimicrobial agent . These biofilms, when left untreated, become thicker and stronger to form tartar and become a habitat for many bacteria, resulting in various oral diseases. In addition, when biofilm is present on the surface of the central vein or implant, it is not removed by general antibiotics, and bacterial infection is removed from the biofilm, resulting in periodic fever. In addition, the formation of biofilms on the surfaces of implants and medical materials, such as organ compartments and cochlear implants, has been reported.

To date, treatment of infections by biofilms is known to be the only solution to remove infectious agents or to physically remove the habitat of biofilms.

On the other hand, carbacol is a representative aromatic essential oil extracted from oils such as camphor tree (Cinnamomum camphora Sieb), origanum hirtum, wild bergamot and muskchae (thyme). The IUPAC name of carbacrole is 2-methyl-5-propan-2-yl-phenol or 2-methyl-5- (1-methylethyl) phenol and generally has antimicrobial activity against microorganisms such as fungi and bacteria, It is known to have.

In addition, eugenol is a clear pale yellow essential oil extracted from clove oil, nutmeg, cinnamon, basil and bay leaves, which is a major component accounting for about 72 to 90% of the essential oil extracted from clove. The IUPAC name of eugenol is 4-allyl-2-methoxyphenol, and its activity as an antibacterial and anesthetic agent is known and widely used as a restorant and a prosthetic device in dentistry.

Accordingly, the inventors of the present invention have completed the present invention by confirming that carbacol has an effect of inhibiting the formation of biofilm while studying a substance for treating infectious diseases caused by biofilm, In studying a combination preparation containing eugenol, the present inventors completed the present invention by confirming that the effect of the above-mentioned preparation is inhibited by the synergistic action to inhibit the formation of the biofilm.

It is an object of the present invention to provide a method for preventing or treating infectious diseases caused by a biofilm, which comprises carbacol as an active ingredient, based on the fact that carbacol exhibits an excellent effect on bacterial growth and inhibition of biofilm formation And to provide a coating composition for inhibiting biofilm formation.

Further, based on the fact that the combination of carbacol and eugenol is synergistically effective in enhancing bacterial proliferation and biofilm formation inhibitory effect, it is possible to prevent the infectious diseases caused by biofilms containing carbacol and eugenol as active ingredients A pharmaceutical composition for prevention or treatment, and a composition for coating to inhibit biofilm formation.

In order to solve the above problems, the present invention provides a pharmaceutical composition comprising carbacol or carbacol and eugenol as an active ingredient, for the prophylaxis or treatment of infectious diseases caused by biofilm.

In order to solve the second problem, the present invention provides a coating composition for inhibiting biofilm formation, comprising carbacrol or carbacrol and eugenol as an active ingredient.

The pharmaceutical compositions and coating compositions according to the present invention have excellent antimicrobial effects and show excellent efficacy in suppressing and eliminating biofilm formation. They are useful for preventing or treating various infectious diseases caused by biofilm, And can be effectively used in coating compositions for inhibiting biofilms formed on medical materials, medical implants and the like.

FIG. 1 is a graph showing the degree of growth when S. aureus and pseudomonas were grown with varying concentrations of carbacol. Optical density was measured at 600 nm (OD ₆₀₀ ).
Figure 2 is a graph of the CFU counts of single-species and multi-species biofilms of preformed S. aureus and pseudomonas treated with various concentrations of carbacol.
FIG. 3 is a graph showing the synergistic effect on the biofilm removal that has been performed during the combined treatment of carbacol and eugenol, wherein the pre-established MRSA and MSSA biofilms are treated with various concentrations of carbacol and eugenol, either alone or in combination , Cell viability was determined by CFU count, and the experiment was repeated twice using three samples.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition comprising carbachol as an active ingredient and for preventing or treating infectious diseases caused by biofilm.

The composition may further comprise an eugenol as an active ingredient, wherein the eugenol is preferably contained in a proportion of 1 to 3 parts by weight based on 1 part by weight of carbachol.

As can be seen from the results of the following examples, carvacrol has excellent biofilm formation inhibiting and removing ability, and when the carbacol is complexed with eugenol, synergistic action removes the biofilm And thus can be usefully used for the prevention or treatment of infectious diseases caused by biofilm.

In the present invention, the infectious disease caused by the biofilm may be an infectious disease which can be inhibited by the antimicrobial effect, and includes cases of infectious diseases which are treated with conventional disinfectants, antibiotics and infectious diseases upon diagnosis of diseases. Examples of infectious diseases caused by such biofilm include, but are not limited to, cavities, periodontitis, ear infections, musculoskeletal infections, necrotizing fasciitis, fungal infections, osteomyelitis, bacterial prostatitis, endocarditis of natural valve, cystic fibrosis pneumonia Meloidosis and nosocomial infections such as ICU pneumonia, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis, and biliary stent blockage.

The location at which the biofilm can form includes a suture, an extraction site, an arteriovenous vein, a scleral buckle site, a contact lens, an IUD, an endotracheal tube, a Hickman catheter, a central venous catheter, Other examples are described in a paper by Costerton J et al and Costerton J and Steward (2001 Battling Biofilms, Scientific American pp 75-81) , Which is incorporated herein by reference in its entirety. Other locations where biofilms can form include cavities that can progress to gum disease and cavity, contact lenses that can progress to eye infections, ear infections that can become chronic infections, and pneumonia Includes a lung that can.

In the present invention, the infection may be cystic fibrosis, which may result from skin infections, burns, and / or wound infections. The compositions of the present invention may be particularly suitable for treating infections in immunocompromised individuals.

On the other hand, the biofilm can be formed by pathogenic bacteria. The pathogenic bacteria refers to microorganisms which cause diseases such as bacteria and viruses, though not limited thereto. Examples of the pathogenic bacteria include bacterial infections, respiratory infections, It refers to causative bacteria of infectious diseases. Specific examples of these causative bacteria include Salmonella spp., Shigella spp., Vibrio parahaemolyticus, Vibrio choreae, Escherichia coli O-157, Campylobacter Such as Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica, Helicobacter pylori, Entemoeba histolytica, Bacillus cereus, The present invention relates to a method for the treatment of bacterial infections such as Clostridium botulinum, Haemophilus influenzae, Streptococcus pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae, Branhamella catarrhalis, Mycobacterium tuberculosis, Plasma (Mycoplasma pneumoniae), A-type strain (Storeptococcus pyogenes), Corynebacterium diphtheriae, Bordetella (MSSA), Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Pertussis, Chlamydia psittaci, Pseudomonas aerginosa, methicillin resistant Staphylococcus aureus Enterobacter spp., Proteus spp., Acinetobacter spp., Enterococcus faecalis, Staphylococcus saprophyticus, and B-type microorganisms (for example, Klebsiella pneumoniae, Enterobacter spp., Proteus spp. Storeptococcus agalactiae).

Disinfecting in the present invention means destroying pathogens, and it does not matter whether or not the non-pathogenic microorganisms remain. The disinfecting agent includes, but is not limited to, hydrogen peroxide, chlorine, iodophors, phenol, alcohols, chlorhexidine gluconate, triclosan, and the like.

The diagnosis of the disease includes diagnosis using a medical device or the like, but the medical device whose surface is a polymer material or a metal material is preferable, but is not limited thereto.

In addition, the composition of the present invention may contain at least one known active ingredient that has an effect of preventing or treating infectious diseases caused by biofilm together with carbacol or carbacol and eugenol. Furthermore, the composition of the present invention may contain, in addition to the above-described effective ingredients for administration, one or more further pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be a salt solution, a sterilized water, a Ringer's solution, a buffered saline solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol and a mixture of at least one of these components. If necessary, an antioxidant, a buffer, Other conventional additives may be further included. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it may be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA), in a suitable manner in the art.

The composition of the present invention may be administered orally or parenterally depending on the intended method. The dose may vary according to the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, The range varies. The daily dose of the carbacol is 0.5 to 3.0 mg / kg, preferably about 1.0 to 2.0 mg / kg, and the daily dosage of the combined preparation of carbacol and eugenol is 0.5 to 3.0 mg / kg, About 1.0 to 2.0 mg / kg, but it may be added or subtracted depending on the clinical result, and it is preferable that the divided dose is administered once to several times a day.

The composition of the present invention may be used alone or in combination with conventional disinfectants, antibiotics, hormone therapy, drug therapy, and biological response modifiers for the prevention or treatment of infectious diseases caused by biofilm.

On the other hand, the present invention provides a coating composition containing carbacrol as an active ingredient and inhibiting biofilm formation.

The composition may further comprise an eugenol as an active ingredient, wherein the eugenol is preferably contained in a proportion of 1 to 3 parts by weight based on 1 part by weight of carbachol.

The coating agent is characterized in that it is coated on a medical device, a medical material, or a medical implant.

The medical device may include, but is not limited to, consultation and diagnosis equipment, clinical examination equipment, radiology equipment, surgical apparatus and equipment ), Cure apparatus & equipment, Rehabilitation medicine and physiotherapy appartus, Ophthalmic appartus, Dental appartus, Central supply equipment, Hospital equipment Hospital equipment and emergency equipment, oriental medicine & equipment, pharmaceutical equipment, obesity cure & healthcare equipment, medical supplies, consumables, etc. .

The medical material may be classified into a metal material, an inorganic material, a polymer material, a composite material, and the like depending on the material thereof. The metal material is classified into cobalt, chrome, stainless steel, touch tack, decarburization, etc. Inorganic materials include bioglass and ceramics, and polymeric materials include natural polymer materials, bioplastics, synthetic Polymeric materials, synthetic rubbers, and synthetic fibers. The medical composite materials include, but are not limited to, fiber reinforced plastics, glass fibers, and carbon fibers.

Such medical implants include various implants, from very simple, such as a catheter, to very complex and sophisticated devices such as artificial organs or sensors. However, unlike intrinsic intention, a medical implant implanted in the subcutaneous tissue of a patient to alleviate the inconvenience of treatment or illness of the patient may cause the patient's life to be threatened, This is because serious infections can occur in the process of More than half of hospital acquired infections are associated with implantation of medical devices. Specific examples of medical implants include endovascular prostheses, stents, heart stents, peripheral stents, orthopedic implants, bone or joint implants, Artificial heart valves, subcutaneous and / or intramuscular implants.

The surface of the medical device is preferably a polymer material or a metal material.

The polymer material refers to a polymer used for medical activities, that is, a polymer used for artificial organs in place of a medical device or part of a body used in various medical activities including diagnosis and treatment. Applications include blood transfusion equipment, blood transfusion equipment, syringes, soft contact lenses, artificial kidney filters, urethra, heart valves, artificial blood vessels, bones, skin, tendons, ears, jaws, eyes, . Examples of the material to be used include, but are not limited to, polyvinyl chloride, polyethylene, natural rubber, silicone rubber, polypropylene, and polyurethane.

The metal material may be, but not limited to, stainless steel, cobalt alloy, titanium alloy, metal biomaterial, cobalt chromium alloy, titanium alloy, tantalum, shape memory alloys and the like.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

S. aureus  Wow pseudomonas For Carvacrol  Minimum inhibitory concentration and minimum sterilization concentration

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were calculated from the broth micro-dilution method in TSB medium (tryptic soy broth) supplemented with bovine serum albumin (BSA) -dilution). Stock culture was cultured and 1 × 10 ⁵ CFU of bacteria were added to fresh TSB + 2% BSA medium. Twice-fold diluted serially diluted carbachol (64-512 ug / ml) in TSB + 2% BSA medium was prepared in 96-well plates (200 μl per well). Wells without carbacol were used as positive growth controls. To increase the solubility of carbacol, all wells were treated with dimethyl sulfoxide (DMSO) at a final concentration of 0.1% v / v. Wells without bacteria were used as a negative growth control. Plates were incubated at 37 ° C for 24 hours and bacterial growth was analyzed by measuring the optical density at 600 nm (OD ₆₀₀ ) using a spectrophotometer. 10 [mu] l from all wells showing no visible growth was plated on BHI (brain heart infusion) agar and then cultured overnight at 37 [deg.] C to count the number of colonies. MIC was defined as the lowest concentration of carbacol in the absence of significant growth and MBC was defined as the lowest concentration of carbacol when the 99.9% or more of the initial inoculation was reduced. The experiment was repeated twice using three samples.

For biofilm Carbacol  Effect measurement

To test the effect of carbacol on the pre-established biofilm, the preformed biofilm was washed for 24 hours to remove the planktonic bacteria and incubated at carbacol concentrations of 256, 512, 1024 占 퐂 / 占 퐇 at 37 占 폚 for 6 hours Exposed. The biofilm was then dispersed by sonication. Colony forming units (CFUs) per viable ml were counted from diluted samples plated and cultured using dispersed biofilm cells. To quantify the number of viable cells grown on the biofilm, the bacteria were recovered by sonication at 50 W for 10 seconds and the diluent was successively incubated in selective medium (CN supplemented oxacillin resistance screening agar base (ORSAB) or pseudomonas agar) To count colony forming units (CFU).

About biofilm removal Cava crawl Eugenol  Measure synergy

The synergistic effect of carbacol and eugenol was analyzed by the known Checkerboard test method. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) biofilms constructed over 24 hours were diluted continuously with two-fold dilutions of eugenol, carbacol, The mixture was treated for 6 hours. After incubation at 37 ° C for 6 hours, CFU was counted. The fractional inhibitory concentration index (FICI) was calculated by summing the fractional inhibitory concentrations (FIC) of each component. The FICs were calculated by comparing the MICs when the components were used in combination, MIC of the time. If the FIC index is less than or equal to 0.5, the synergistic effect is defined by the interaction of carbacol and eugenol, and if the FIC index is greater than 0.5 and less than or equal to 1.0, And if the FIC index is greater than 1.0 and less than or equal to 2.0, each component is defined as being independent of each other, and if the FIC index is greater than 2.0, it is defined as having an antagonistic effect. Here, the minimum biofilm eradication concentration (MBEC) is defined as the minimum eugenol concentration that induces a 90% or more reduction in the number of viable cells in a pre-established biofilm. The MIC is converted to MBEC (2 x MIC) to compute FICI and FICs.

result

S. aureus  Wow pseudomonas For Carvacrol  Minimum inhibitory concentration and minimum sterilization concentration

As a result, the MIC of carbacol against S. aureus was measured at 256 / / ㎖, and the MBC was measured at 512 / / ㎖. Similarly, the MIC of carbacol for pseudomonas was measured at 512 [mu] g / ml and the MBC was measured at 1024 [mu] g / ml (Fig. 1).

For biofilm Carbacol  Effect measurement

As a result of the measurement, it was confirmed that carbacrol had an effect of reducing the survival bacteria of pre-constructed biofilm. Carbacol exhibits strong biofilm removal ability against both S. aureus and pseudomonas biofilms. Biomass of biofilm was significantly decreased by carbacol treatment. Similarly, surviving bacteria were also found to be significantly reduced in carbacol-treated biofilms. The minimum biofilm eradication concentration (MBEC) of carbacol to S. aureus was determined to be 512 μg / ml, and the MBEC of carbacol to pseudomonas was determined to be 1024 μg / ml (MBEC is 2 × MIC) (Figure 2).

About biofilm removal Cava crawl Eugenol  Measure synergy

As a result, the combination of eugenol and carbacol showed a synergistic effect on pre - established MRSA and MSSA biofilms. The MBEC of eugenol and carbacol was calculated as 2 × MIC, and the MBEC of eugenol was 0.08% and the MBEC of carbacol was 0.04%. When 0.25 × MBEC (0.02%) eugenol alone was applied to the biofilm, 25% CFU was decreased. Similarly, when the biofilm was treated with 0.25 x MBEC (0.01%) carbacol alone, survival bacteria was reduced by 40%. 99% of the bacteria were significantly reduced (Fig. 3) when the biofilm was combined with the eugenol and carbacol (0.02% eugenol and 0.01% carbacol). When the combination of eugenol and carbacrol is used, it can be confirmed that the MBEC is decreased by 4 times or more and the synergistic effect is exhibited by measuring the FICI of 0.5 or less.

Claims (9)

A pharmaceutical composition comprising carbacol as an active ingredient and for preventing or treating infectious diseases caused by biofilm. The method according to claim 1,
A pharmaceutical composition for preventing or treating infectious diseases caused by biofilm, wherein the composition further comprises an eugenol as an active ingredient. 3. The method of claim 2,
Wherein said composition comprises 1 to 3 parts by weight of eugenol based on 1 part by weight of carbacol. The method according to claim 1,
The infectious diseases include, but are not limited to, cavities, periodontitis, otitis media, musculoskeletal infections, necrotizing fasciitis, fungal infection, osteomyelitis, bacterial prostatitis, endocarditis of natural valve, cystic fibrosis pneumonia, meloidosis, ), ICU pneumonia, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis, and biliary stent blockage (biliary stent blockage). The method according to claim 1,
The biofilm may be selected from the group consisting of Salmonella spp., Shigella spp., Vibrio parahaemolyticus, Vibrio choreae, Escherichia coli O-157, Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica, Helicobacter pylori, Entemoeba histolytica, Bacillus cereus, Botulinum toxin, Clostridium botulinum, Haemophilus influenzae, Streptococcus pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae, Branhamella catarrhalis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, A-type strain (Storeptococcus pyogenes), Corynebacterium diphtheriae, Bordetella pertussis, (Eg, Chlamydia psittaci, Pseudomonas aerginosa, methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), Escherichia coli, Klebsiella pneumoniae ), Enterobacter spp., Proteus spp., Acinetobacter spp., Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus aureus (Storeptococcus agalactiae). Lt; RTI ID = 0.0 > 1, < / RTI > A composition for coating comprising carbacol as an active ingredient and inhibiting biofilm formation. The method according to claim 6,
Wherein the composition further comprises an eugenol as an active ingredient. 8. The method of claim 7,
Wherein said composition comprises 1 to 3 parts by weight of eugenol based on 1 part by weight of carbacol. The method according to claim 6,
Wherein the composition is coated on a medical device, a medical material or a medical implant.

Images