Topical antiseptic formulation containing polyhexamethylene guanidine hydrochloride for oral use in animals

Abstract

The present patent of invention relates to a novel "topical antiseptic formulation containing polyhexamethylene guanidine hydrochloride for oral use in animals" pertaining to the technical field of veterinary medicine. More specifically, it relates to a topical formulation for oral antisepsis aimed at the prevention (prophylaxis) of oral diseases in domestic animals, including caries and periodontitis. It is also intended for antiseptic use prior to and / or during clinical and surgical procedures requiring microbiological control in the oral cavity (such as dental cleaning, dental implants, oral biopsies, tooth extractions, endodontic treatments, and the application of local anaesthetics), based on polyhexamethylene guanidine hydrochloride (PHMGH). The action of this topical formulation is based on the mechanisms of action of the component PHMGH, which exhibits high antifungal and antimicrobial activity even at low concentrations, lacks hepatotoxicity, nephrotoxicity, cytotoxicity, and genotoxicity, and provides rapid and effective action in controlling microorganisms associated with conditions affecting the oral cavity of domestic animals, including dental caries and periodontitis. The proposed formulations therefore address problems arising in the technical field related to oral antisepsis, and serve both as a method for prophylaxis and for treatment or microbiological control in dental procedures, representing an alternative to conventional drugs and / or oral rinses.

Description

DESCRIPTIVE REPORT Topical antiseptic formulation containing polyhexamethylene guanidine hydrochloride for oral use in animals BRIEF DESCRIPTION

[0001] This patent is an unprecedented invention “TOPIC ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS”, belonging to the veterinary sector.In particular, it refers to the formulation for topical application for oral use in animals, aimed at preventing (prophylaxis) oral diseases in domestic animals, including cavities and periodontitis, in addition to use as antisepsis prior to and / or during clinical and surgical procedures that require microbiological control in the oral cavity (dental cleanings, dental implants, oral biopsies, tooth extractions, endodontics, applications of local anesthetics, among others), made from polyhexamethylene guanidine hydrochloride (PHMGH), whose action is based on the mechanisms of action of this synthetic polymer (polyhexamethylene guanidine hydrochloride; PHMGH), compared to commercially established products, such as chlorhexidine digluconate.Additionally, in a pioneering manner, possible hepatotoxic, nephrotoxic, genotoxic and cytotoxic effects of this conservative therapy were verified, associated with the histopathological evaluation necessary for the development of new drugs. FIELD OF APPLICATION

[0002] The present invention belongs to the section of veterinary needs, especially to the field of animal science, as it describes the use of polyhexamethylene guanidine hydrochloride (PHMGH) in the form of oral topical use for asepsis, treatment and prevention of oral conditions, including dental caries and periodontitis, in domestic animals. TECHNIQUE FUNDAMENTALS

[0003] Among oral conditions, periodontitis and dental caries stand out, of a It is multifactorial, progressive, and highly prevalent, and is considered the main cause of tooth extractions (BANIHANI et al., 2018: Outcomes of the conventional and biological treatment approaches for the management of caries in the primary dentition). The oral microorganisms most commonly involved in dental caries are Streptococcus mutans, S. sanguinis, S. salivarius, S. mitis, S. sobrinus, and Lactobacillus spp. (ELAMIN et al., 2018: Dental caries and their association with socioeconomic characteristics, oral hygiene practices, and eating habits among preschool children in Abu Dhabi, United Arab Emirates - the NOPLAS Project). In addition to local changes, due to the rich oral vascularization, microorganisms and their metabolites can enter the lymphatic and blood vessels, leading to bacteremia, and the patient's systemic immune response predisposes to the formation of immune complexes in the bloodstream.Immune complexes can adhere to the walls of the endothelium, causing local inflammation and endothelial lysis, leading to failure of various organs such as the liver, nervous system, kidneys, joints and heart, especially in senile patients (ALMEIDA et al., 2017: Periodontal treatment in patients with chronic kidney disease. A pilot study).

[0004] In this sense, oral hygiene care, including frequent toothbrushing with fluoride toothpastes and the use of antiseptic mouthwashes, is essential for preventing oral diseases. Chlorhexidine digluconate 0.12% is commonly used as a broad-spectrum antiseptic and is effective in the chemical removal of cariogenic biofilms (JEYAKUMAR et al., 2020: Antibiofilm activity of oral health care products containing chlorhexidine diguclonate and citrox). In contrast, although it affects bacterial viability, it does not inhibit biofilm formation and promotes bacterial adhesion at concentrations above its minimum inhibitory concentration (TEIXEIRA, 2010: Physical-chemical processes in dental biofilm related to caries production).Furthermore, there are descriptions of adverse effects when used as a mouthwash, such as changes in tooth color, restorations, prostheses and tongue, formation of supragingival calculus, loss of taste, soft tissue burns, pain and xerostomia, as well as cytotoxicity (GUIMARÃES et al., 2006: Self-perception of side effects by adolescents in a chlochexidine-fluoride-. based preventive oral health program; ZANATTA; ROSING, 2007: Chlorhexidine: mechanisms of action and current evidence of its efficacy in the context of supragingival biofilm; LAWRENCE et al., 2008: Community-Level Assessment of the Effects of the Broad-Spectrum Antimicrobial Chlorhexidine on the Outcome of River Microbial Biofilm Development). Given the common use of chlorhexidine digluconate in the health area, especially as an antiseptic agent, resistance to this product should not be neglected (CRUZ et al., 2012: Evaluation of the cytotoxicity of chlorhexidine solutions at concentrations of 2.5% to 5%).

[0005] Despite advances in the pharmaceutical industry, each drug presents distinct therapeutic responses and side effects, with none being completely effective. Therefore, research into new drugs is justified in dentistry (ARGYRAKI et al., 2018: UV light-assisted antibiotics for eradication of in vitro biofilms; JEYAKUMAR et al., 2020: Anti-biofilm activity of oral health care products containing chlorhexidine diguclonate and citrox), aiming for promising and less costly treatments. In this context, polyhexamethylene guanidine hydrochloride (PHMGH) stands out.

[0006] Thus, considering the high incidence of dental caries and the scarcity of data regarding polyhexamethylene guanidine hydrochloride (PHMGH) in dentistry (human and veterinary), the present study aimed to evaluate the antiseptic effect of topical solutions containing PHMGH, in addition to verifying possible toxic effects of this conservative therapy.

[0007] It is noteworthy that polyhexamethylene guanidine hydrochloride is a cationic synthetic polymer of the guanidine family, highly soluble in water (ZHANG; JIANG; CHEN, 1999: Synthesis and antimicrobial activity of polymeric guanidine and biguanidine salts), with antibacterial (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; OULÉ et al., 2012: Akwaton, polyhexamethyleneguanidine hydrochloride-based sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections), antifungal and antiviral activities, even at low concentrations (OULÉ et al., 2015: Fungicidal activity of AKWATON and in vitro assessment of its toxic effects on animal cells'). Furthermore, polyhexamethylene guanidine hydrochloride is devoid of volatile organic compounds and is odorless (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections), characteristics that may be desirable for the development of topical oral and / or cutaneous formulations.

[0008] The polymer also demonstrates rapid and prolonged performance, low toxicity (CHOI; KUM; LEE, 2016: Antifungal activity of the cationic antimicrobial polymerpolyhexamethylene guanidine hydrochloride and its mode of action; MASH AT, 2016: Polyhexamethylene biguanide hydrochloride: features and applications), and lack of resistance (PAN; XIA; XIAO, 2019: Cationic polymers with tailored structures for rendering polysaccharide-based materials antimicrobial: an overview). However, there are still no scientific descriptions of its in vivo use in oral antisepsis and prevention of oral diseases, including dental caries and periodontitis, nor are there any studies on clinical safety for use in animals.

[0009] The development of this invention was based on the antimicrobial mechanisms of action of polyhexamethylene guanidine hydrochloride (PHMGH), which acts on the cell membrane by inhibiting enzymes essential for germination and growth, in addition to promoting phospholipid degradation, allowing leakage of its contents and coagulation of the cytosol, with consequent cell inactivation. In fungal plasma membranes, it induces the formation of pores, which cause the loss of K ions. +followed by cell contraction and death. There is also evidence that once inside the cell, the polymer binds to DNA and other nucleic acids, damaging or inactivating bacterial DNA (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; ZHOU; ZHENG; ZHONG, 2011: Interactions of biocidal guanidine hydrochloride polymer analogs with model membranes: a comparative biophysical study; VITT et al., 2015: Antimicrobial activity of polyhexamethylene guanidine phosphate in comparison to chlorhexidine using the quantitative suspension method; MASHAT, 2016: Polyhexamethylene biguanide hydrochloride: features and applications).

[0010] In this sense, the present invention falls specifically within the area of topical oral pharmaceutical applications, since it refers to the development of formulations for oral antisepsis and prevention of oral diseases in animals.

[0011] The antiseptic effect of topical oral solutions containing polyhexamethylene guanidine hydrochloride (PHMGH) was evaluated in vitro. Different concentrations of solutions containing PHMGH, alone or in combination with chlorhexidine digluconate (CLX), were evaluated against the main microorganisms responsible for biofilm formation and caries causation, such as Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mitis, Streptococcus sobrinus, Lactobacillus casei, and Enterococcus faecalis. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC75), fractional inhibitory concentration index (FICI), and bactericidal kinetics were determined.In this context, oral solutions containing PHMGH were effective against all microorganisms, with MIC and MBC ranging from 0.31 to 1.24 ppm, and lower values, especially against Streptococcus mitis and Enterococcus faecali, compared to commercial chlorhexidine digluconate (CLX). At concentrations of 9.8 to 2500 ppm, oral solutions containing PHMGH inhibited 75% of biofilms of microorganisms, including Streptococcus mutans; and the MBC75 of PHMGH and CLX were similar for this microorganism. Regarding bactericidal kinetics, solutions containing PHMGH polymer showed higher percentages of reduction of microorganisms when compared to CLX, and at concentrations of 3000 ppm and 1000 ppm and, with 2 and 4 minutes of exposure, respectively, solutions containing PHMGH polymer induced 100% bacterial death.

[0012] The investigation of possible toxic effects was carried out in vivo assays. The study protocol was approved by the Ethics Committee on Animal Use of the University of Franca (Approval No. s 8704160318) and the toxicogenetic evaluation was conducted according to the OECD-474 (2016) recommendations. Topical instillations of solutions containing 0 PHMGH (four drops) at the highest concentration tested (625 ppm) were performed daily for 90 consecutive days, mimicking the use of the product as a mouthwash. Untreated animals (negative controls) were also included in the study. Until day 90, no animals showed local changes in the oral mucosa or tongue. Compared to the control group, analyses of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine showed no hepatotoxic or nephrotoxic effects. Bone marrow samples were evaluated using the micronucleus test, and the absence of genotoxicity and cytotoxicity was reported. Histopathological analysis of fragments of the oral mucosa, tongue, esophagus, stomach, and intestine of the animals was carried out for histopathological analysis (edema, ulceration, necrosis, hyperplasia, hemorrhage, and polynuclear and mononuclear inflammatory infiltrate), suggesting possible ingestion of the oral solutions.In general, no statistical differences were observed between the animals that received the treatment and the negative control group, considering the concentration of the topical oral solutions evaluated.

[0013] The results suggest that oral solutions containing PHMGH demonstrated prominent antimicrobial activity in vitro against oral microorganisms, especially those involved in dental caries, without indicating hepatotoxicity, nephrotoxicity, or genotoxicity in vivo. Furthermore, they did not cause alterations in the oral structures of treated animals. Therefore, the use of PHMGH as an active ingredient in topical oral solutions offers prospects for the development of new oral antiseptic products, representing a promising and less expensive option. BACKGROUND OF THE INVENTION

[0014] In the current state of the art, there are some prior art works that describe in vitro biological activities of polymers of the polyhexamethylene guanidine family, especially polyhexamethylene guanidine phosphate (PHMG-P); however, none of them scientifically described the in vivo use of PHMGH (PHMGH - polyhexamethylene guanidine hydrochloride) for oral antiseptic purposes in animals.

[0015] The activity of polyhexamethylene guanidine phosphate (PHMG-P) against periodontopathogenic and cariogenic microorganisms compared to chlorhexidine was evaluated using the quantitative suspension method (VITT et al., 2015: Antimicrobial activity of polyhexamethylene guanidine phosphate in comparison to chlorhexidine using the quantitative suspension method). Both antiseptics tested at their clinically used concentrations of 0.2% (w / v) and 1% (w / v) provided rapid bactericidal effects against S. aureus, P. aeruginosa, E. coli, C. albicans, A. actinomycetemcomitans, and P. gingivalis with reduction factors greater than 6.0. Diluted PHMG-P solution and 0.05% chlorhexidine continued to exhibit antibacterial activity, but the reduction of peripathogenic microorganisms to amounts below 1.0 x 10 3colony-forming units / ml required prolonged exposure time. To obtain bactericidal effect against S. mutans, both antiseptics at all concentrations required longer exposure times. On the other hand, a 1% PHMG-P solution demonstrated no activity against L. acidophilus. The high reduction factor of polyhexamethylene guanidine phosphate and the retention of bactericidal effects, even at 0.05%, support the use of PHMG-P as a biocide with sufficient antimicrobial activity against peripathogens.

[0016] The in vitro antimicrobial effects of chlorhexidine digluconate (CLX), polyhexamethylene biguanide (PHMB), and octenidine dihydrochloride (OCT) on cariogenic microorganisms were evaluated using the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (CELIK et al., 2016: Antimicrobial activity of different disinfectants Against cariogenic microorganisms). For this, CLX, PHMB, and OCT were diluted in distilled water to the final test concentrations. Different MICs and MBCs were observed for all disinfectants against each microorganism. The lowest MIC and MBC values against S. mutans (60 mg / L) were obtained for PHMB. The lowest values against L. rhamnosus (15 mg / L, 30 mg / L), A. viscosus (30 mg / L), and L. acidophilus (15 mg / L, 30 mg / L) were determined for OCT. In this sense, PHMB and OCT can be considered as alternatives to replace CLX, since they were effective against cariogenic microorganisms.

[0017] Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CLX) to determine the potential cytotoxic and immunomodulatory effects in human gingival fibroblasts (VITT et al., 2017: Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts). At a concentration of 0.00009% PHMG-P, complete loss of fibroblast viability was observed within 24 h, whereas inhibition of fibroblast viability by CLX occurred at significantly higher concentrations than 0.0009%. Short-term exposure to 0.005% PHMG-P led to loss of fibroblast viability after 5 min, whereas cells exposed to 0.005% CLX survived 30 min of treatment. The cytotoxic effects on gingival fibroblasts were triggered by PHMG-P and CLX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1(3)-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, suggesting anti-inflammatory properties.

[0018] Polyhexamethylene biguanide (PHMB) has been used for years as an antiseptic in medicine for various clinical applications (Rosin et al., 2001: Effect of a polyhexamethylene biguanide mouthrinse on bacterial counts and plaque). However, little is known about its antibacterial activity in the oral cavity and its ability to inhibit plaque formation. A mouthrinse containing 0.04% PHMB was compared with a negative control placebo rinse (10% ethanol, flavored) and two rinses containing chlorhexidine as the active ingredient (positive control), one of which was a 0.12% aqueous solution and the other a commercially available mouthrinse (Skinsept mucosa) diluted with 0.12% chlorhexidine. The mouthrinse containing PHMB reduced mucosal bacteria significantly more effectively than the placebo after 4 hours and 5 days.The results indicated that mouthwash with 0.04% PHMB inhibits plaque growth and reduces oral bacterial counts, and can be used in preventive applications in the oral cavity.

[0019] A mouthwash containing 0.12% PHMB (polyhexamethylene biguanide) was also compared with a commercially available product containing essential oils (Listerine®), and no significant difference was found regarding the inhibition of bacterial plaque (ROSIN et al., 2002: The effect of a polyhexamethylene biguanide mouthrinse compared to an essential oil rinse and a chlorhexidine rinse on bacterial counts and 4- day plaque regrowth). On the other hand, reductions in bacterial counts (after 5 days) on the tooth surface were significantly greater with the PHMB-containing mouthwash. Furthermore, the 0.12% PHMB solution was more effective in reducing bacterial counts (after 4 hours) in the mucosa than the commercial product. Thus, the PHMB mouthwash was shown to inhibit plaque recolonization and reduce oral bacterial counts, indicating that PHMB may have applications in the prevention of plaque-associated diseases.

[0020] Mouthrinses containing 0.04% and 0.12% polyhexamethylene biguanide hydrochloride (PHMB) inhibited plaque growth and reduced oral bacterial counts (WELK et al., 2005: The effect of a polyhexamethylene biguanide mouthrinse compared with a triclosan rinse and a chlorhexidine rinse on bacterial counts and 4-day plaque regrowth). Another mouthrinse containing 0.2% PHMB was compared with a positive control (0.12% chlorhexidine aqueous solution), with a commercially available mouthrinse (Colgate Total Plax) whose formulation contains 0.3% triclosan and 2.0% poly(vinylmethyl ether / maleic acid) copolymer, and with a negative placebo control (10% ethanol, flavored). The 0.2% PHMB mouthwash was significantly better at inhibiting plaque than placebo but less effective than 0.12% chlorhexidine aqueous solution.No significant difference was observed between the mouthwash containing PHMB and Colgate Total Plax (commercially available). Reductions in bacterial counts (tooth surface and mucosa) with the 0.2% PHMB solution were significantly greater compared to the placebo and the commercial triclosan-based product (Colgate Total Plax). However, compared to the positive control (0.12% aqueous chlorhexidine solution), the antimicrobial activity of the PHMB-containing rinse was lower on the tooth surface and equally effective on the mucosa.

[0021] Mouthwashes containing PHMB (polyhexamethylene biguanide) and CIO2 (chlorine dioxide) are viable alternatives to CLX (chlorhexidine), as studies have shown that the antimicrobial effects of PHMB are comparable to those of CLX and that the antimicrobial effects of CIO2 are even greater than those of CLX. Additionally, these alternative solutions have few or no reported side effects or side effects. adverse effects (SANTOS et al., 2021: Could polyhexanide and chlorine dioxide be used as an alternative to chlorhexidine? A systematic review).

[0022] The in vitro maximum inhibitory dilution (MID) of two chlorhexidine (CLX)-based mouthwashes: Noplak®, Periogard®, and a polyhexamethylene biguanide (PHMB)-based mouthwash: Sanifill Premium®, was evaluated against 28 strains of Staphylococcus aureus using the agar dilution method (NASCIMENTO et al., 2018: Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary staphylococcus aureus). Sanifill Premium® inhibited the growth of all strains at a 1 / 40 dilution and of 1 strain at a 1 / 80 dilution. Noplak® inhibited the growth of 23 strains at a 1 / 640 dilution and of all 28 strains at a 1 / 320 dilution. Periogard® inhibited growth of 7 strains at a 1 / 640 dilution and of all 28 strains at a 1 / 320 dilution. The data were subjected to the Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated. No significant differences were found between Noplak® and Periogard®.Sanifill Premium® was the least effective. Therefore, it was concluded that CLX-based mouthwashes have better antimicrobial activity against S. aureus than PHMB-based mouthwashes.

[0023] Although the above reports refer to the application of solutions containing polyhexamethylene biguanide (PHMB) as the active ingredient, no studies were found involving the application of topical antiseptic oral solutions aimed at preventing caries with polyhexamethylene guanidine hydrochloride (PHMGH). Antifungal activity against Candida albicans biofilm for denture base acrylic resins (GAMA et al., 2020: Evaluation of guanidine antifungal solutions for denture base resin: an in vitro study; GARCIA et al., 2020: Guanidine derivative inhibits C. albicans biofilm growth on denture liner without promoting loss of materials' resistance). It was concluded that the use of a 0.5% by mass aqueous solution of PHMGH for 5 min was effective as a disinfectant agent against C. albicans biofilm, in addition to maintaining roughness and flexural strength.

[0024] Other documents relate the use of PHMB as antiseptic agents, which will be cited below. However, it should be noted that polyhexamethylene biguanide (PHMB), in the form of its hydrochloride, hydrochloride or other salts, is a synthetic polymer distinct from polyhexamethylene guanidine hydrochloride (PHMGH), the subject of this patent application.

[0025] Document BR102015003322-2, entitled "Pharmaceutical composition containing aminoguanidine derivatives with antiseptic activity", described a drug comprising antiseptic and antimicrobial compounds obtained from aminoguanidine derivatives, for use as a degerming and alcoholic antiseptic for the skin or in aqueous form for the mucosa before carrying out invasive procedures and / or any procedure aiming at antiseptic activity, aiming at another option of antiseptic product and as prevention of healthcare infection, to be used both in human patients and in animals.

[0026] Document PI0305735-6, entitled "Germicidal and antiseptic compositions for multiple applications, and process of disinfection, sanitization and sterilization of objects and surfaces", described new germicidal compositions for use in asepsis and prophylaxis that have as their basic essence the combination of two powerful antimicrobial agents, triclosan and poly(hexamethylene) biguanide or its hydrochloride / hydrochloride, until then, not described in the art of the technique in a combined form, in synergism, with the purpose of acting concomitantly, through different mechanisms of destruction and / or inhibition of pathogenic microorganisms; the prior art also described the process of disinfection, sanitization and sterilization of objects and surfaces.

[0027] Document PI0614927-8, entitled "Antimicrobial Compositions," describes an antimicrobial composition comprising a synergistic mixture of active agents and a primary antimicrobial agent, such as polyhexamethylene biguanide (PHMB), a secondary antimicrobial agent, and optionally an organic acid, against various types of microorganisms. Various additional processing aids, such as alcohols and surfactants, can also be incorporated into the mixture. The described composition allows for the use of a concentration significantly fewer individual constituent antimicrobial agents to obtain the same degree of antimicrobial efficacy, or even greater efficacy.

[0028] Document PI0215517-6, entitled "Antiseptic composition and methods for disinfecting fabric and for producing an antiseptic composition", described a composition containing at least one antimicrobial agent, intended primarily for the antisepsis of fabrics and, more particularly, for the antisepsis of the skin.

[0029] Document BR1020200125400A2, titled "Antiseptic wet wipes with chlorhexidine digluconate and PHMB," addresses the application for patent protection for antiseptic wet wipes with chlorhexidine digluconate and PHMB (polyhexamethylene biguanide). The invention is focused on the cosmetics and hygiene products sector, with application to the skin surface to eliminate, primarily, germs present on the hands. The wet wipes were manufactured with a solution composed of chlorhexidine digluconate and polyhexamethylene biguanide, promoting antiseptic activity, eliminating approximately 99.9% of Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesuis, and Klebsiela pneumoniae.

[0030] Although previous products have been found that contain biguanide in their composition and that these are used for topical asepsis (oral, cutaneous or surfaces), formulations consisting of polyhexamethylene guanidine hydrochloride (PHMGH) in aqueous and / or alcoholic solutions for use in antisepsis and prevention of oral diseases in domestic animals and humans, including tooth decay and periodontitis, are not described. PURPOSE OF THE INVENTION

[0031] The present invention aims to provide a topical oral pharmaceutical composition based on polyhexamethylene guanidine hydrochloride (PHMGH) for oral antisepsis and prevention of oral caries diseases in domestic animals, especially dental caries and periodontitis.

[0032] The choice of the pharmaceutical form was based on the high solubility of the polymer (polyhexamethylene guanidine hydrochloride) in water (ZHANG; JIANG; CHEN, 1999: Synthesis and antimicrobial activity of polymeric guanidine and biguanidine salts; OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections). Furthermore, the topical formulation was prepared quickly and easily, without specialized equipment, making it inexpensive to handle. Furthermore, as it is devoid of volatile organic compounds, the polymer is odorless (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; OULÉ et al., 2012: Akwaton, polyhexamethylene-guanidine hydrochloride-based sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections), representing an advantage for formulations intended for domestic animals. BRIEF DESCRIPTION OF THE INVENTION

[0033] The present invention relates to topical oral formulations based on polyhexamethylene guanidine hydrochloride (PHMGH) for oral antisepsis, aiming at the prevention (prophylaxis) of oral diseases in domestic animals, including caries and periodontitis, in addition to use as antisepsis prior to and / or during clinical and surgical procedures that require microbiological control in the oral cavity (dental cleanings, dental implants, oral biopsies, tooth extractions, endodontics, application of local anesthetics, among others). Furthermore, the formulations demonstrated the absence of morphological alterations in oral structures, as well as the absence of toxic effects (hepatotoxic, nephrotoxic, genotoxic and cytotoxic), characteristics desirable for the development of new drugs. FIGURE DESCRIPTION

[0034] The invention will be described in a preferred embodiment, so for better understanding, reference will be made to the figures: • FIG 1: Minimum Biofilm Inhibitory Concentration (CIMB75) of the polymer polyhexamethylene guanidine hydrochloride (PHMGH) against cariogenic microorganisms. 'Significantly different from the negative control (p<0.05). a Lowest concentration of PHMGH that showed 75% or more inhibition of biofilm formation. • FIG 2: Minimum Biofilm Inhibitory Concentration (MIIC75) of chlorhexidine digluconate (CLX, 0.12 to 59.0 ppm) against cariogenic microorganisms. 'Significantly different from the negative control (p<0.05). a Lowest concentration of CLX that showed 75% or more inhibition of biofilm formation. • FIG 3: Frequencies of micronucleated polychromatic erythrocytes (A) and PCEs / total erythrocytes ratio (B) obtained from the bone marrow of Wistar rats subjected to oral treatment with polyhexamethylene guanidine hydrochloride 625.0 ppm (PHMGH after 90 days of treatment). MNPCEs: micronucleated polychromatic erythrocytes; PCEs: polychromatic erythrocytes; NC: negative control; PC: positive control (methyl methanesulfonate, 40 mg / kg, intraperitoneal, single dose). aSignificantly different from the negative control group (p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0035] The topical oral antiseptic formulation and its use in animals, which is the subject of this patent, has an action based on the mechanisms of action of its component: polyhexamethylene guanidine hydrochloride. Among oral conditions, periodontitis and dental caries stand out, being multifactorial, progressive and highly prevalent, and considered the main cause of tooth extractions (BANIHANI et al., 2018: Outcomes of the conventional and biological treatment approaches for the management of caries in the primary dentition).

[0036] In this sense, oral hygiene care, including the use of antiseptic mouthwashes, is essential for preventing oral diseases. Despite advances in the pharmaceutical industry, the therapeutic response and distinct side effects justify the search for new products and medications, including in dentistry (ARGYRAKI et al., 2018; JEYAKUMAR et al., 2020). In this context, polyhexamethylene guanidine hydrochloride (PHMGH) stands out, a cationic synthetic polymer of the guanidine family with high antibacterial and antiviral activity and low toxicity. The guanidine polymer inhibits bacterial growth by attacking them through the electrostatic attraction between its cationic groups and anionic groups on the bacterial cell surface (QIAN et al., 2011; VITT et al., 2015; BRZEZINSKA et al., 2017).

[0037] PHMGH (Akwaton®) is a cationic synthetic polymer of the guanidine family, synthesized by polycondensation of hexamethylenediamine chloride with dicyandiamide (PAN et al., 2019: Cationic polymers with tailored structures for rendering polysaccharide-based materials antimicrobial: an overview). Cationic polymers such as guanidine-based polymers are of great interest and widely used due to their high antibacterial and antiviral activity and low toxicity, in addition to being odorless (SOWLATI-HASHJIN et al., 2020: Insights into the Polyhexamethylene Biguanide (PHMB) mechanism of action on bacterial membrane and DNA: a molecular dynamics study).

[0038] Considering the high occurrence of dental caries and the scarcity of data regarding polyhexamethylene guanidine hydrochloride in dentistry, the present study aimed to evaluate the antiseptic effect of topical solutions containing PHMGH, in addition to verifying possible toxic effects of this conservative therapy.

[0039] In this context, the following topical oral antiseptic formulation is proposed: 0.01% to 2.0% polyhexamethylene guanidine hydrochloride (PHMGH - polyhexamethylene guanidine hydrochloride); 99.9% to 98% water.

[0040] Optionally the formulation will include in v / v: 0.01% to 2.0% polyhexamethylene guanidine hydrochloride (PHMGH - polyhexamethylene guanidine hydrochloride); 10% to 70% ethanol; and 28.0% to 89.99% water.

[0041] The formulation was prepared by solubilizing, under agitation, the active ingredient polyhexamethylene guanidine hydrochloride (PHMGH - polyhexamethylene guanidine hydrochloride; Akwaton®) using water and / or ethanol as solvent for topical oral use in domestic animals such as dogs, cats, horses, cattle, sheep, goats and pigs in the concentrations of the formulations described above. TESTS PERFORMED.

[0042] The tests performed for the proposed formulation are described below.

[0043] Assessment of antibacterial potential: bacteria used in the tests Microbiological samples were obtained from the American Type Culture Collection (ATCC) and maintained in the culture collection of the University of Franca (Franca, São Paulo, Brazil), under cryopreservation at -80°C. The strains used were: Streptococcus mutans (ATCC 25175), Streptococcus sanguinis (ATCC 10556), Streptococcus salivarius (ATCC 25975), Streptococcus mitis (ATCC 49456), Streptococcus sobrinus (ATCC 33478), Lactobacillus casei (ATCC 11578) and Enterococcus faecalis (ATCC 4082). The strains were resuspended and turbid on a 0.5 McFarland scale, according to the Clinical and Laboratory Standards Institute (CLSI, 2012: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically), resulting in a final concentration of 10 6 CFU / mL / well.

[0044] Minimum Inhibitory Concentration (MIC) Determination: MIC values were determined in triplicate using the broth microdilution method in 96-well microplates, according to CLSI (CLSI, 2012: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically), using resazurin as a bacterial growth indicator. The PHMGH concentrations tested ranged from 0.31 to 625.0 ppm. Sterility controls of the medium, inoculum, and positive (chlorhexidine digluconate, CLX, Sigma-Aldrich, St. Louis, MO, United States; 0.12-59.0 ppm) were also performed. The microplates were incubated in an aerobic incubator at 36°C for 24 hours, and then 30 pL of resazurin (Sigma-Aldrich; 0.02%) was added to each well. Afterwards, the plates were incubated for 30 minutes for macroscopic evaluation of color changes, either blue (absence of microorganism growth) or pink (presence of microorganism growth).

[0045] Determination of Minimum Bactericidal Concentration (MBC): To determine the MBC, a 10 pL aliquot of the inoculum was removed from each well before adding resazurin. The inoculum aliquot was then plated on other plates containing Mueller Hinton agar, which were incubated at 37°C for 24 hours in a microbiological incubator. A substance was considered to have a bacteriostatic effect when its MBC value was greater than its MIC value. However, a substance was considered to have a bactericidal effect when its MBC value was equal to its MIC value. The assays were performed in triplicate.

[0046] Determination of Minimum Biofilm Inhibitory Concentration (MBIC75): to define the lowest concentration of PHMGH that showed 75% or greater inhibition of biofilm formation, the MBIC75 was performed by microplate microdilution (CLSI, 2012: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically), with modifications. For this, several dilutions of the polymer were prepared in a 96-well culture plate containing Brain Heart Infusion broth (BHI, Kasvi®, São José dos Pinhais, Paraná, Brazil). CLX was used as a positive control (0.12–59.0 ppm), and bacterial strains without polymer were used as a negative control. The inocula were adjusted to a concentration of 1 x 10 6CFU / mL. The plates were incubated at 37°C for 24 hours; afterward, the well contents and non-adhered (planktonic) cells were aspirated and removed for washing with 150 pL of Milli Q water (Merck Millipore, Burlington, Massachusetts, USA). The biofilm was fixed with 150 pL of methanol (Synth, Diadema, São Paulo, Brazil) for 20 minutes and dried at room temperature (TRABULSI; ALTERTHUM, 2005: Microbiology; MORAES et al., 2020: Assessment of the antibacterial, antivirulence, and action mechanism of Copaifera pubiflora oleoresin and isolated compounds against oral bacteria). The wells were stained with 150 pL of crystal violet solution (Sigma-Aldrich; 0.2%). Biofilm formation was quantified by adding 150 pL of 33% acetic acid (Merck Millipore) to the wells stained with crystal violet, with readings performed at 570 nm using a microplate reader (ASYS, Eugendorf, Salzvurg, Austria).To determine CIMB75, the inhibition percentage was calculated by the equation: Inhibition (%) = [1 - (A595 of the treated well / A595 of the control well)] x 100. The assays were performed in triplicate.

[0047] Determination of the Fractional Inhibitory Concentration Index (FICI): The FCI was performed to evaluate the synergism of PHMGH with CLX (CLSI, 2012: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically). For this, the cultures were allocated to tubes containing BHI broth and the inocula were standardized in a spectrophotometer. 500 pL were transferred to another tube containing 4.5 mL of broth, then 2 mL of this inoculum was added to a tube containing 10 mL of broth, to provide a concentration of 5x10 5 CFU / mL / well. MIC determination was used by microdilution, similar to a “chessboard”, containing serial dilutions of the CLX, which was tested alone and in combination with PHMGH. The polymer MICs were tested by distributing 100 pL of a 4x concentrated solution into column 1, rows A to G. In row H, from columns 2 to 11, the MIC of CLX alone in a 4x concentrated solution was evaluated. To evaluate the combination of PHMGH with CLX, 50 pL of each substance (8x concentrated solution) were initially distributed into row A of columns 2 to 11 and serially diluted to row G. For CLX, 10 vials with a 4x concentrated solution were initially prepared for each corresponding column in the microplate, with a volume of 50 pL / well being distributed from columns 2 to 11 in rows A to G, totaling a final volume of 100 pL for the combination of PHMGH with CLX. Then, 100 pL of the inoculum was added to all wells, totaling 200 pL of final volume in the microplate wells. The microplates were incubated at 37°C for 24 hours.Subsequently, 30 pL of 0.02% resazurin solution was added to each well and the color change was observed: blue (no bacterial growth) or pink (bacterial growth). The ICIF was obtained by the sum of the fractional inhibitory concentration (CIF) of PHMGH with the CIF of CLX. The procedures were performed in triplicate and the results expressed in ppm, being considered synergistic (ICIF <0.5), additive (ICIF>0.5 and <1), indifferent (ICIF>1 and <4.0) or antagonistic (ICIF>4.0).

[0048] Determination of bactericidal kinetics (“Time Kill Curve”): this experiment analyzes the time required for the death of microorganisms by determining the surviving colonies at time intervals of 1, 2, and 4 minutes, based on the need for the oral hygiene product to be effective in a short exposure time (CAO et al., 2020: Killing Streptococcus mutans in mature biofilm with a combination of antimicrobial and antibiofilm peptides; MORAES et al., 2020: Assessment of the antibacterial, antivirulence, and action mechanism of Copaifera pubiflora oleoresin and isolated compounds against oral bacteria). PHMGH and CLX were prepared in BHI broth at concentrations of 500 ppm, 1000 ppm, and 3000 ppm (JEYAKUMAR et al., 2020: Anti-biofilm Activity of Oral Health Care Products Containing Chlorhexidine Diguclonate and Citrox). Afterwards, 500 pL of these solutions were added to 500 pL of bacterial suspension containing 1x10 6CFU / mL. As a negative control, 500 pL of BHI medium was added to 500 pL of the bacterial suspensions. Each tube was then subjected to a tube shaker. After shaking for periods of 1, 2, and 4 minutes, 50 pL aliquots were transferred to a tube containing 450 pL of broth, and so on until dilution 10' 7 Finally, 50 pL of each dilution was plated on BHI agar plates divided into eight quadrants. The plates were incubated at 37°C for 24 hours, and the number of colonies developed on each plate in CFU / mL was counted, followed by calculation of the percentage reduction of each microorganism at different exposure times and concentrations of PHMGH and CLX.

[0049] In vivo evaluation: Male Wistar rats (Rattus norvegicus), weighing approximately 300g, were used. The animals were provided by the Animal Facility of the University of São Paulo (Ribeirão Preto, Brazil). The rats were kept in plastic boxes in an experimental room under controlled temperature (22±2°C) and humidity (50±10%) conditions under a 12-h light-dark cycle, with ad libitum access to standard chow and water. The study protocol was approved by the Ethics Committee for Animal Use of the University of Franca (Approval no. s 8704160318). Five rats were subjected to topical instillations of PHMGH (four drops) at the highest concentration tested (625 ppm), daily, for 90 consecutive days, mimicking the use of the product as a mouthwash. Untreated animals (negative control, n=5) were included in the study.

[0050] Clinical evaluation: through direct clinical inspection, rats treated with PHMGH were daily evaluated for the presence of changes in the mucosa (gingival, alveolar, jugal and palatine) and tongue, such as sensitivity, edema, ulcer, hemorrhage and necrosis, as well as dysphagia, hyporexia or anorexia resulting from oral sensitivity or loss of taste, followed by weight loss.

[0051] Biochemical analyses of hepatotoxicity and nephrotoxicity: At the end of the experimental period (day 90), the rats were euthanized (Flecknell et al., 2007: Laboratory animals) and blood samples were collected directly from the heart. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed to investigate liver damage, while serum urea nitrogen and creatinine levels were evaluated as indicators of nephrotoxicity.

[0052] Toxicogenetic evaluation: bone marrow samples from animals treated with PHGMH were collected to assess genotoxic potential using the micronucleus test. The experiments were conducted according to the OECD recommendations (OECD-474, 2016: Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4). Negative (untreated) and positive (methyl methanesulfonate, Sigma-Aldrich; 40 mg / kg body weight, a single intraperitoneal dose) control groups were included. The frequency of micronucleated polychromatic erythrocytes (MCPEs) was obtained from the analysis of 4,000 polychromatic erythrocytes (PCEs) per animal, totaling 2,000 per treatment group. The same slides were used to analyze the cytotoxicity of the treatments; for this, 500 erythrocytes per animal were analyzed and the PCEs / total erythrocytes ratio was calculated.

[0053] Histopathological analysis: After macroscopic inspection, fragments of the oral mucosa and tongue were collected for histopathological analysis, as well as samples from the esophagus, stomach, and intestine, assuming possible ingestion of the product. The samples were processed according to classical histological techniques (Carson; Hladik, 2009: Histotechnology - a self-instructional text). Edema, ulceration, necrosis, hyperplasia, hemorrhage, and inflammatory infiltrates were classified using intensity scores: 0 (absent), 1 (mild), 2 (moderate), or 3 (severe).

[0054] Statistical analyses: The microbiological results of CIMB determination and genotoxicity were statistically verified by one-way analysis of variance (ANOVA) for randomized experiments (F statistic and P-value). For p<0.05, the least significant difference was calculated at a = 0.05. The biochemical and histopathological results were analyzed by the nonparametric t-test. EVALUATION OF ANTIBACTERIAL POTENTIAL

[0055] The MIC and MBC values of PHMGH are shown in Table 1.

[0056] PHMGH was effective against all bacteria evaluated, with promising MIC and MBC values ranging from 0.31 to 1.24 ppm. Compared to CLX, the synthetic polymer demonstrated lower values, especially against Streptococcus mitis and Enterococcus faecali (4 to 6 times lower). Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (CBM) of polyhexamethylene guanidine hydrochloride (PHMGH - 625 to 0.31 ppm) and chlorhexidine digluconate (CLX 0.12 to 59.0 ppm), against cariogenic microorganisms (American Type Culture Collection, ATCC). Microorganisms (ATCC) MIC / MBC (ppm) PHMGH CLX Streptococcus mutans (25175) 0.31 / 0.31 0.46 / 0.46 Streptococcus sanguinis (10556) 0.93 / 0.93 0.92 / 0.92 Streptococcus salivarius (25975) 0.62 / 0.62 0.92 / 0.92 Streptococcus mitis (49456) 0.93 / 0.93 3.69 / 3.69 Streptococcus sobrinus (33478) 0.62 / 0.62 0.46 / 0.46 Lactobacillus easel (1 1578) 0.31 / 0.31 0.46 / 0.46 Enterococcus faecalis (4082) 1 .24 / 1 .24 7.37 / 7.37

[0057] The CIMB75 results of PHMGH and CLX for each bacterium are shown in Figures 1 and 2 (FIG. 1 and FIG. 2), respectively. PHMGH inhibited 75% of biofilm formation of the tested microorganisms at concentrations ranging from 9.8 to 2500 ppm, including Streptococcus mutans, a relevant microorganism in oral biofilm formation. The inhibitory activity of CLX was observed at lower concentrations, from 7.37 to 29.5 ppm.

[0058] It is noteworthy that significant inhibition of biofilm formation was observed at the lowest concentration of PHMGH tested (1.22 ppm) for all microorganisms used. The inhibition percentages were: S. mutans - 69.3%, S. sanguinis - 47.6%, S. salivarius - 35.1%, S. mitis - 67.8%, S. sobrinus 62.8%, Lactobacillus casei - 34.4% and Enterococcus facecalis - 36.7%. PHMGH was more effective in inhibiting biofilm for S. mutans and S. mitis. It is interesting to note that the CIMB75 of PHMGH and CLX were similar for S. mutans, being 9.9 and 7.37 ppm, respectively. The ICIF results are shown in Table 2. The combination of PHMGH and CLX against the panel of cariogenic microorganisms tested resulted in indifferent effects. Table 3 presents the bactericidal kinetics results for the cariogenic microorganisms evaluated. Table 2. MIC (isolated and combined, ppm), interaction of methylparaben hydrochloride polyhexamethylene guanidine (PHMGH) with chlorhexidine digluconate (CLX)-Fractional Inhibitory Concentration Index (ICIF, ppm), against cariogenic microorganisms. Microorganisms *Isolated MIC *Combined MIC _ ICIF Result (ATCC) PHMGH CLX PHMGH CLX Streptococcus mutans 0.31 0.46 0.31 0.92 3.0 Indifferent (25175) Streptococcus sanguinis 0.93 0.92 0.93 0.92 2.0 Indifferent (10556) Streptococcus salivarius 0.62 0.92 0.62 0.46 1.5 Indifferent (25975) Streptococcus mitis 0.93 3.69 0.93 7.37 3.0 Indifferent (49456) Streptococcus sobrinus 0.62 0.46 0.31 0.46 1.5 Indifferent (33478) Lactobacillus casei 0.31 0.46 0.31 0.46 2.0 Indifferent (11578) Enterococcus faecalis 1 .24 7.37 0.31 7.37 1 .25 Indifferent (4082) 'MIC: Minimum Inhibitory Concentration

[0059] Considering the concentrations and exposure times employed, PHMGH demonstrated higher percentages of microorganism reduction when compared to CLX, demonstrating antibacterial efficacy. PHMGH, at concentrations of 3000 ppm and 1000 ppm, and with 2 and 4 minutes of exposure, respectively, induced 100% cariogenic bacterial death. Regarding Streptococcus mutans, the results of PHMGH ranged from 67.1 to 100% inhibition; in contrast, those of CLX were less significant (30.8 to 84.7%). Regarding this microorganism, at concentrations of 1000 and 3000 ppm, the percentage of inhibition promoted by PHMGH in the three times evaluated was above 95.5%, thus demonstrating the promising antibacterial activity of this synthetic polymer. Table 3. Percentage reduction of microorganisms after exposure to the polymer polyhexamethylene guanidine hydrochloride (PHMGH) and chlorhexidine digluconate (CLX) for 1, 2 and 4 minutes. Microorganisms (ATCC) Tempo PHMGH 160 (min) (ppm) (ppm) 500 1000 3000 500 1000 3000 Streptococcus mutans 1 67.1 95.5 97.8 30.8 64.4 66.6 (25175) 2 90.8 100.0 100.0 40.5 56.5 78.9 4 98.7 100.0 100.0 56.9 78.2 84.7 Streptococcus sanguinis 1 17.1 74.2 82.3 12.1 41.0 77.1 (10556) 2 29.3 86.1 100.0 26.7 68.5 88.8 4 79.6 100.0 100.0 48.0 93.8 100.0 Streptococcus salivarius 1 33.3 69.5 89.6 30.8 52.4 70.8 (25975) 2 74.0 96.6 100.0 44.4 71.1 88.7 4 86.4 100.0 100.0 77.9 97.9 100.0 Streptococcus mitis 1 41.3 69.2 87.7 17.4 54.5 71.6 (49456) 2 62.8 80.1 94.4 24.9 70.2 94.8 4 74.6 100.0 100.0 54.2 85.0 100.0 Streptococcus sobrinus 1 33.2 64.8 90.1 12.0 42.7 55.7 (33478) 2 89.1 100.0 100.0 14.4 66.8 89.3 4 91 .2 100.0 100.0 39.3 68.8 100.0 Lactobacillus casei 1 9.0 74.2 97.4 20.1 51.9 71.6 (11578) 2 38.3 86.2 100.0 35.6 79.3 89.9 4 66.3 100.0 100.0 56.9 93.7 97.1 Enterococcus faecalis 1 19.7 75.5 94.3 11.0 43.2 69.9 (4082) 2 49.6 86.8 98.6 36.5 68.5 81 ,4 4 59.5 98.2 100.0 48.0 86.2 100.0

[0060] Thus, using the MIC and MBC determination technique, the PHMGH polymer demonstrated effective antimicrobial activity against all cariogenic bacteria tested, particularly against Streptococcus mitis. Furthermore, it also showed promising activity against Enterococcus faecalis, a highly resistant microorganism in the oral cavity. even without the support of another bacterium or substrate (ZHANG et al., 2015: Correlation between Enterococcus faecalis and persistent intraradicular infection compared with primary intraradicular infection: a systematic review). These results corroborate data in the literature, in which PHMGH has a broad in vitro spectrum against Gram-positive bacteria, even at low concentrations, and there are no reports of antimicrobial resistance to PHMGH (Vitt et al. 2015: Antimicrobial activity of polyhexamethylene guanidine phosphate in comparison to chlorhexidine using the quantitative suspension method; Choi et al., 2016: Antifungal activity of the cationic antimicrobial polymerpolyhexamethylene guanidine hydrochloride and its mode of action).Previous studies approve the antimicrobial activity of the polymer against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus bovis and Bacillus subtilis) and negative (Escherichia coli) microorganisms, which favored the healing process of superficial skin wounds in rats (Dias et al., 2021: Evaluation of the antiseptic and wound healing potential of polyhexamethylene guanidine hydrochloride as well as its toxic effects).

[0061] It is noteworthy that PHMGH revealed more evident antibacterial activity against the microorganisms Streptococcus mitis and Enterococcus faecalis when compared to CLX, considered the “gold standard” in the prevention and treatment of oral pathologies (GUIMARÃES et al., 2006: Self-perception of side effects by adolescents in a chlochexidine-fluoride-based preventive oral health program; ZANATTA; ROSING, 2007: Chlorhexidine: mechanisms of action and current evidence of its efficacy in the context of supragingival biofilm; LAWRENCE et al., 2008: Community-Level Assessment of the Effects of the Broad-Spectrum Antimicrobial Chlorhexidine on the Outcome of River Microbial Biofilm Development; CRUZ et al., 2012: Evaluation of the cytotoxicity of chlorhexidine solutions at concentrations of 2.5% to 5%). These results indicate that the synthetic polymer may be a promising adjuvant in the microbial control of oral diseases, especially dental caries.

[0062] The beneficial antimicrobial effects of PHMGH found in the present work by determining MIC and MBC can be attributed to its mechanism of action on the cell membrane in promoting the inhibition of enzymes essential for the growth of microorganisms, in addition to the degradation of phospholipids (MASHAT, 2016: Polyhexamethylene biguanide hydrochloride: features and applications), with coagulation of the cytosol and leakage of its cytoplasmic contents, with consequent cell death (PAN et al., 2019: Cationic polymers with tailored structures for rendering polysaccharide-based materials antimicrobial: an overview; OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; VITT et al., 2015: Antimicrobial activity of polyhexamethylene guanidine phosphate in comparison to chlorhexidine using the quantitative suspension method). Still in relation to the mechanism of action, there is evidence that, once inside the cell, PHMGH binds to DNA, damaging it and leading to bacterial death (ALLEN et al., 2004: Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids).Furthermore, it is possible that PHMGH distinguished itself from CLX in its antimicrobial action due to the presence of the flexible linear alkyl chain in the polymer that improves the partition capacity in the hydrophobic regions of phospholipid membranes, damaging the phospholipid bilayer of microorganisms (LUO et al., 2017: Interactions of biocidal polyhexamethylene guanidine hydrochloride and its analogs with POPC model membranes). PHMG inhibited 75% of biofilm formation of the microorganisms tested, being more effective against Streptococcus mutans, considered the prominent pathogen in the initial process of oral biofilm formation (KRZYSCIAK et al., 2014: The virulence of Streptococcus mutans and the ability to form biofilms; HASIBUL et al., 2018: D-Tagatose inhibits the growth and biofilm formation of Streptococcus mutans).

[0063] At low concentrations, guanidine polymers increase bacterial cell membrane permeability, promoting the loss of potassium ions and, consequently, a slight change in bacterial homeostasis. On the other hand, at higher concentrations, they increase cell wall damage and pore size, generating significant loss of calcium, sodium, lithium, and inorganic phosphate ions, resulting in the complete loss of membrane function, precipitation of intracellular constituents, and bacterial death (ALFEI; SCHITO, 2020: Positively Charged Polymers as Promising Devices against Multidrug-Resistant Gram-Negative Bacteria: A Review). Additionally, polymers of the guanidine family exhibit extremely rapid bactericidal and fungicidal effects when compared with other antiseptic agents (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; OULÉ et al., 2012: Akwaton, polyhexamethylene-guanidine hydrochloride-based sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections; OULÉ et al., 2015: Fungicidal activity of AKWATON and in vitro assessment of its toxic effects on animal cells). It is noteworthy that this patent represents the first study on the kinetics of PHMGH-induced killing of the main bacteria causing dental caries. IN VIVO EVALUATION

[0064] Throughout the experimental period, no rats showed changes in oral mucosa or tongue, such as sensitivity, edema, ulcers, hemorrhage, or necrosis, or in food and water intake. Consequently, this did not cause body weight loss in those treated topically with PHMGH. The means and standard deviations for serum ALT, AST, urea nitrogen, and creatinine levels are shown in Table 4. The solution containing PHMGH showed no evidence of hepatotoxicity or nephrotoxicity, demonstrating that the polymer did not harm the liver and kidney function of topically treated animals, respectively, even considering that the animals may have accidentally ingested the polymer.

[0065] Animals treated topically with PHMGH solution did not show symptoms of clinical toxicity, indicating that the polymer may be harmless to the oral cavity at the highest concentration tested. Furthermore, PHMGH solution is odorless and tasteless (OULÉ et al., 2008: Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight meticillin-resistant Staphylococcus aureus and nosocomial infections; OULÉ et al., 2012: Akwaton, polyhexamethylene-guanidine hydrochloride-based sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections), facilitating its use as a mouthwash. Regarding clinical evaluations, progressive weight gain was observed, indicating that topical treatment did not cause local changes or changes in the animals' taste, as well as constant handling was not a stress factor (Subramanion et al., 2011). Table 4. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine of Wistar rats (n = 5) subjected to oral treatment for 90 days with polyhexamethylene guanidine hydrochloride (PHMGH). Group (n=5) ALT (U / L) AST (U / L) Urea (mg / dL) Creatinine (mg / dL) CN 84.3±21.4 96.3±10.9 59.8±13.1 0.6±0.1 PHMGH 75.0±15.3 105.3±30.8 53.5±2.6 0.5±0.0 NC: negative control. Values are mean ± standard deviation (SD).

[0066] Regarding the toxicogenetic analysis, the animals treated with PHMGH showed no significant difference in the frequency of chromosomal damage when compared to the negative control group (FIG. 3A). No significant differences were observed in the PCEs / total erythrocyte ratios between the groups (FIG. 3B). These data indicate the absence of genotoxicity and cytotoxicity of PHGMH under the experimental conditions used. Macroscopic evaluation of the organs of rodents treated with PHMGH revealed no external or internal signs of edema, ulcerations, necrosis, and hemorrhage after 90 days of topical oral exposure to the tested polymer. Regarding the scores of edema, ulcerations, necrosis, hyperplasia, hemorrhage, and polymorphonuclear inflammatory infiltrate, no statistical difference was observed between the rodents treated with PHMGH compared to the untreated ones (Table 5).

[0067] The absence of hepatotoxicity or nephrotoxicity in topical treatments with PHMGH has already been reported in the scientific literature (ASIEDU-GYEKYE et al., 2014: A preliminary safety evaluation of polyhexamethylene guanidine hydrochloride; DIAS et al., 2021: Evaluation of the antiseptic and wound healing potential of polyhexamethylene guanidine hydrochloride as well as its toxic effects). Biochemical toxicity occurs when values exceed three times the normal range (BERTOLAMI, 2005: Mechanisms of hepatotoxicity; BOTELHO et al., 2010: Subacute effect of high doses of copaiba oil on liver enzyme levels in rat serum). Table 5. Means and standard deviations: histopathological scores (0: absent, 1: mild, 2: moderate or 3: intense) in tissues of Wistar rats subjected to oral treatment for 90 days with polyhexamethylene guanidine hydrochloride (PHMGH). Tissue Group (n=5) Edema Ulcer Necrosis Hyperplasia Oral mucosa NC 0.2±0.4 0±0 0±0 0.2±0.4 PHMGH 0.6±0.5 0±0 0±0 0.2±0.4 Language NC 0±0 0±0 0±0 0±0 PHMGH 0±0 0±0 0±0 0±0 Esophagus NC 0±0 0±0 0±0 0±0 PHMGH 0±0 0±0 0±0 0±0 Stomach NC 0.2±0.4 0±0 0±0 0±0 PHMGH 0±0 0.2±0.4 0±0 0±0 Intestine NC 0.2±0.4 0±0 0±0 0.2±0.4 PHMGH 0±0 0.2±0.4 0±0 0±0 Inflammatory infiltrate Tissue Group (n=5) Hemorrhage - polymorphonuclear mononuclear Oral mucosa NC 0.2±0.4 0.4±0.5 0.2±0.4 PHMGH 0.6±0.9 0.6±0.5 0±0 Language NC 0±0 0±0 0±0 PHMGH 0±0 0±0 0±0 Esophagus NC 0±0 0±0 0±0 PHMGH 0±0 0±0 0±0 Stomach NC 0±0 0.2±0.4 0.2±0.4 PHMGH 0±0 0.4±0.5 0±0 Intestine NC 0±0 0.2±0.4 0.4±0.5 PHMGH 0.4±0.5 1 .0±0.7 1 .8±0.4 a NC: negative control. 3 Significantly different.

[0068] Previous research has demonstrated that 50,050 ppm of PHMGH administered by gavage had an LD50 greater than 200 mg / kg in rats and no genotoxicity or cytotoxicity at doses up to 1500 mg / kg, revealing the absence of significant risk (DIAS et al., 2021: Evaluation of the antiseptic and wound healing potential of polyhexamethylene guanidine hydrochloride as well as its toxic effects). Therefore, accidental ingestion of the polymer (PHMGH), when used as a mouthwash, should not cause toxicogenetic damage, as well as damage to organs of the digestive system. Still on this topic, Flemingson et al. (FLEMINGSON et al., 2008: Effect of three commercial mouth rinses on cultured human gingival fibroblasts: An in vitro study) comparatively evaluated the cytotoxicity of PHMGH with mouth rinses (chlorhexidine, listerine, and povidone-iodine) against human gingival fibroblasts, showing that the three commercial products were more toxic, especially chlorhexidine. Furthermore, PHMGH was characterized by the US Environmental Protection Agency as having a low risk of adverse effects on health and the environment (OULÉ et al., 2012: Akwaton, polyhexamethylene-guanidine hydrochloridebased sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections; LYSYTSYA'S, 2017: Research on the impact of polyhexamethyleneguanidine on the plant component of biocenoses), after analysis by mass spectrometry and photocalorimetry. ADVANTAGES OF THE INVENTION

[0069] The present invention has the following main advantages: • Provide topical formulation for oral antisepsis and prevention / treatment of oral diseases in domestic animals, especially tooth decay and periodontitis; • Provide topical formulation for oral antisepsis, which can be used in situations that require prior microbiological control and / or during clinical and surgical procedures such as dental cleanings, dental implants, oral biopsies, tooth extractions, endodontics, applications of local anesthetics, among others; • Provide effective and low-cost topical formulation for oral antisepsis and prevention / treatment of oral diseases, especially dental caries and periodontitis in domestic animals; • Provide topical formulation for oral antisepsis and prevention / treatment of oral diseases in domestic animals, with absence of hepatotoxicity, nephrotoxicity, cytotoxicity and genotoxicity; • Provide topical formulation for oral antisepsis and prevention / treatment of oral diseases in domestic animals using chloramphenicol hydrochloride. polyhexamethylene guanidine as an active ingredient, which is odorless, making it easier to obtain formulations that are well accepted by domestic animals.

Claims

CLAIMS 1 TOPICAL ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS, characterized by comprising in v / v: 0.01% to 2.0% polyhexamethylene guanidine hydrochloride (PHMGH); 99.9% to 98% water. 2 TOPICAL ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS, characterized by optionally comprising in v / v: 0.01% to 2.0% polyhexamethylene guanidine hydrochloride (PHMGH); 10% to 70% ethanol; and 28.0% to 89.99% water. 3 - USE OF THE TOPICAL ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS, according to claims 01 or 02, characterized by its topical oral use in antisepsis, treatment and prevention of diseases, including caries and periodontitis, caused by microorganisms in the oral cavities of domestic animals. 4 - USE OF THE TOPICAL ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS, according to claims 01 or 02, characterized by its topical oral use for prior antisepsis and / or during clinical and surgical procedures that require microbiological control in the oral cavity (dental cleanings, dental implants, oral biopsies, tooth extractions, endodontics, applications of local anesthetics, among others) of domestic animals. 5 - USE OF THE TOPICAL ANTISEPTIC FORMULATION CONTAINING POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE FOR ORAL USE IN ANIMALS, according to claims 3 and 4, characterized in that the domestic animals are dogs, cats, horses, cattle, sheep, goats and pigs.

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