Compositions for removing necrotic or infected tissues from body surface lesions and from oral cavity

Abstract

A composition, usable for removing a biofilm and necrotic or infected tissues from skin lesions and lesions of the oral cavity, comprises methanesulfonic acid 99.0% and a proton acceptor. The proton acceptor is selected from the group consisting of: anhydrous sodium carbonate, 5 -amino-2-mercaptobenzimidazole, ethylenediaminetetraacetic acid tetrasodium salt, sodium gluconate, sodium tartrate dihydrate, 2-mercapto-5-benzimidazole sodium sulfonate, dimethyl sulfoxide, polyethylene glycol 400, polyethylene glycol 600, silicon dioxide, tetraethoxysilane, and mixtures thereof. The aforesaid composition can be prepared in the form of a solution, gel or cream.

Claims

1. A method of removing biofilm and necrotic or infected tissues from skin lesions and lesions of the oral cavity, the method comprising: applying a composition comprising methanesulfonic acid to a patient in need thereof

2. The method of to claim 1, wherein said methanesulfonic acid is methanesulfonic acid 99.0%.

3. The method of to claim 1, wherein the composition further comprises a proton acceptor.

4. The method of claim 3, wherein said proton acceptor is selected from the group consisting of: anhydrous sodium carbonate, 5-amino-2-mercaptobenzimidazole, ethylenediaminetetraacetic acid tetrasodium salt, sodium gluconate, sodium tartrate dihydrate, 2-mercapto-5-benzimidazole sodium sulfonate, dimethyl sulfoxide, polyethylene glycol 400, polyethylene glycol 600, silicon dioxide, tetraethoxysilane, and mixtures thereof.

5. The method of claim 1, wherein the composition is prepared in a form selected from the group consisting of: solution, gel and cream.

6. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00011 Methanesulfonic acid 70-90% by weight Sodium carbonate 0.5-2% by weight 5-amino-2-mercaptobenzimidazole 1-2% by weight Ethylenediaminetetraacetic acid tetrasodium salt 1-6% by weight Sodium gluconate 1-6% by weight Sodium tartrate dihydrate 2-6% by weight 2-mercapto-5-benzimidazole sodium sulfonate 1-4% by weight

7. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00012 Methanesulfonic acid 70-90% by weight 5-amino-2-mercaptobenzimidazole 1-2% by weight Ethylenediaminetetraacetic acid tetrasodium salt 1-6% by weight Sodium gluconate 1-6% by weight Sodium tartrate dihydrate 2-6% by weight 2-mercapto-5-benzimidazole sodium sulfonate 1-4% by weight Silicon dioxide 0.1-7% by weight

8. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00013 Methanesulfonic acid 70-90% by weight 5-amino-2-mercaptobenzimidazole 1-2% by weight Ethylenediaminetetraacetic acid tetrasodium salt 1-6% by weight Sodium gluconate 1-6% by weight Sodium tartrate dihydrate 2-6% by weight 2-mercapto-5-benzimidazole sodium sulfonate 1-4% by weight Silicon dioxide 0.1-7% by weight Tetraethoxysilane 0.1-2% by weight

9. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00014 Methanesulfonic acid 70-90% by weight Dimethyl sulfoxide 10-30% by weight Silicon dioxide 0.1-7% by weight Tetraethoxysilane 0.1-2% by weight

10. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00015 Methanesulfonic acid 70-90% by weight Sodium gluconate 1-6% by weight Sodium tartrate dihydrate 2-6% by weight Silicon dioxide 0.1-7% by weight Tetraethoxysilane 0.1-2% by weight Polyethylene glycol 400 or polyethylene glycol 600 1-10% by weight

11. The method of claim 4, wherein the composition has the following formulation: TABLE-US-00016 Methanesulfonic acid 70-90% by weight 5-amino-2-mercaptobenzimidazole 1-8% by weight Ethylenediaminetetraacetic acid tetrasodium salt 1-8% by weight 2-mercapto-5-benzimidazole sodium sulfonate 1-8% by weight

12. A method of treatment of a pathology selected from the group consisting of: chronic cutaneous ulcer, periodontal disease, perimplantitis and aphtha, the method comprising applying a composition comprising methanesulfonic acid to a patient in need thereof.

Description

SHORT DESCRIPTION OF THE DRAWINGS

[0032] The invention can be better understood and implemented with reference to the enclosed drawings that illustrate an embodiment thereof by way of non-limiting example in which:

[0033] FIG. 1 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of an embodiment of the composition according to the invention;

[0034] FIG. 2 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of another embodiment of the composition according to the invention;

[0035] FIG. 3 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of a further embodiment of the composition according to the invention;

[0036] FIG. 4 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of another further embodiment of the composition according to the invention;

[0037] FIG. 5 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of a further other embodiment of the composition according to the invention;

[0038] FIG. 6 is a photograph of a Petri dish showing a microbial growth inhibition halo around the depositing zone of a sample of a still further embodiment of the composition according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The composition according to the invention, comprising methanesulfonic acid, can be formulated as a solution, gel or cream and can be easily applied on chronic cutaneous ulcers or on lesions of the mucosa of the oral cavity, such as for example periodontal pockets, perimplantitises or aphthae. A surprisingly unexpected property of the composition according to the invention is that the latter is able to act on the biofilm and on the necrotic or infected tissues, causing a rapid desiccation thereof and enabling the removal thereof (through washing or sterile gauze) only a few seconds after the application and thus avoiding complicated, painful and costly surgical procedures (debridement).

[0040] The action of the composition is due to the release of hydrogen ions (H.sup.+) or protons that, possessing a great hydration enthalpy (−1130 KJ/mole), cause the dehydration of the microbial species that make up the biofilm or proliferate in the infected tissues. This action mechanism is achieved regardless of the microbial species present and makes the composition according to the invention active against any microbial species, whether bacterial, fungal or viral.

[0041] The Inventors have carried out researches to identify the most suitable source of protons, namely a source that is such as to enable an effective release of the protons in contact with the skin or the mucosa of the oral cavity, with resulting denaturation of the microbial proteins present therein, without however causing damages to the healthy tissue surrounding the lesions. This requires the preparation of appropriate formulations where the release of the protons is optimized, for example by varying the degree of viscosity of the formulations on the basis of the type of lesion for which the use of the composition according to the invention is intended.

[0042] The potentially usable sources of protons essentially consist of strong acids (HA), which have to be used in concentrated form, namely in the presence of the least possible amount of water. This is required because the dissociation of the strong acid into H.sup.+ protons and A.sup.− anions has to occur in contact with the microbial species, subtracting molecules of water from the latter, which molecules will form the hydration sphere of the H+ protons with release of thermal energy (−1130 KJ/mole). The dosage of the H+ protons released by the acid species in concentrated form is problematic as it is not possible to dilute with water, which nevertheless represents the best solvent for solubilizing methanesulfonic acid.

[0043] As it is known, the acid dissociation constant (K.sub.a) relating to the reaction HA .fwdarw.H.sup.++A.sup.− is defined by the following relation:

K.sub.a=[H.sup.+][A.sup.−]/[HA]

in which the concentrations in moles/litre of the different species are indicated between square brackets. The negative decimal logarithm of the acid dissociation constant is defined as pK.sub.a(pK.sub.a=−log K.sub.a).

[0044] In the following Table 1, the pK.sub.a values are shown (at a temperature of 25° C.) of the main strong acids, which dissociate completely, or almost completely, in an aqueous environment. From Table 1 it can be inferred that the trifluoromethanesulfonic acid is the strongest acid (namely, the acid having the highest pK.sub.a value), whereas the trifluoroacetic acid is the weakest acid:

TABLE-US-00001 TABLE 1 Acid Chemical formula pK.sub.a Trifluoromethanesulfonic CF.sub.3SO.sub.3H −13 Hydroiodic HI −10 Perchloric HClO4 −10 Hydrobromic HBr −9 Hydrochloric HCl −8 Sulphuric H.sub.2SO.sub.4 −3 Nitric HNO.sub.3 −1.4 Methanesulfonic CH3SO3H −1.2 Trifluoroacetic CF3COOH −0.25

[0045] Considering the volatility of the hydroiodic, hydrobromic, hydrochloric and trifluoroacetic acids, as well as the oxidizing action of the perchloric and nitric acids, which can cause undesired effects on the skin and on the mucosa of the oral cavity, the Inventors focused the attention on the sulphuric and on the methanesulfonic acid and deemed the use of the latter to be preferable. In fact, the methanesulfonic acid (MSA) has a dissociation constant that is about sixty times less than that of the sulphuric acid, which makes the methanesulfonic acid substantially more suitable for use on the skin and in the oral cavity. The methanesulfonic acid is a stable strong acid and a key intermediate in the biogeochemical cycle of the sulphur. The methanesulfonic acid is formed in great quantities in the atmosphere by the chemical oxidation of the atmospheric dimethyl sulphide (DMS) (most of which is of organogenic origin) and is deposited on the earth through rain, snow as well as dry deposition. Therefore, this acid is usually defined as a “green acid” (M.D. Gernon, M. Wu, T. Buszta, P. Janney, Green Chem. 1 (1999) 127-140; S.C. Baker, D.P. Kelly, J.C. Murrell, Nature 350 (1991) 627-628.). The methanesulfonic acid has a low tendency to oxidize organic compounds and a high thermal stability, which makes it usable in liquid form over a wide range of temperatures. The methanesulfonic acid is odourless, due to the very low vapour tension, and does not produce hazardous volatile substances, which enables a safe handling thereof. Furthermore, the methanesulfonic acid is provided with a greater penetration capacity against the biofilm.

[0046] The Inventors further provided for the use of the methanesulfonic acid in combination with small quantities of suitable proton acceptors (or protonic acceptors), which enable the acidity of the composition according to the invention to be adjusted, and more exactly to be reduced. The protonic concentration has to be reduced because the protons, if they are released in an excessive quantity, may cause the dehydration of the epithelial cells with consequent oedema, erythema, desquamation and tissue necrosis with formation of ulcers. By reducing the concentration of the protons released by the methanesulfonic acid, the latter can be effectively used in the treatment of lesions (chronic cutaneous ulcers, periodontal disease, perimplantitises and aphthae) in which the biofilm is present for producing a desired dehydrating effect against the contaminating microbial species without however damaging the surrounding healthy tissues.

[0047] In one embodiment of the composition according to the invention—defined below as “first formulation”—the proton acceptor comprises a weak acid salt that is soluble in the concentrated methanesulfonic acid. The anion of the weak acid, by capturing part of the protons released by the methanesulfonic acid, avoids irritative phenomena, at the same time enabling the dehydration effect to be maintained. This enables any microbial species to be deactivated aspecifically that is responsible for skin, gingival or periodontal infections or aphtha.

[0048] In the following Table 2, the dissociation constants are shown of a plurality of weak acids, the anions of which (contained in the corresponding salts that are soluble in methanesulfonic acid) can be suitably used:

TABLE-US-00002 TABLE 2 Acid Formula K.sub.a1 K.sub.a2 K.sub.a3 Acetic CH.sub.3COOH 1.74 × 10.sup.−5 Ammonium ion NH.sub.4.sup.+ .sup. 5.70 × 10.sup.−10 Anilinium ion C.sub.6H.sub.5NH.sub.3.sup.+ 2.50 × 10.sup.−5 Boric H.sub.3BO.sub.3 .sup. 5.81 × 10.sup.−10 Carbonic H.sub.2CO.sub.3 4.44 × 10.sup.−7 .sup. 4.69 × 10.sup.−11 Citric HOOC(OH)C(CH.sub.2COOH).sub.2 7.45 × 10.sup.−4 1.73 × 10.sup.−5 4.02 × 10.sup.−7 Ethylenediamine- [(HOOC).sub.2CH.sub.2NCH.sub.2].sub.2 10.sup.−2   2 × 10.sup.−3 7 × 10.sup.−7; tetraacetic K.sub.a4, 5 × 10.sup.−11 Phosphoric H.sub.3PO.sub.4 7.11 × 10.sup.−3 6.32 × 10.sup.−8  4.5 × 10.sup.−13 Phosphorous H.sub.3PO.sub.3   3 × 10.sup.−2 1.62 × 10.sup.−7 Formic HCOOH 1.80 × 10.sup.−4 Glycolic HOCH.sub.2COOH 1.47 × 10.sup.−4 Gluconic HOOC(CH.sub.2OH).sub.4CH.sub.2OH  7.4 × 10.sup.−3 Hydrazinium ion H.sub.2NNH.sub.3.sup.+ 1.05 × 10.sup.−8 Hydrazoic HN.sub.3  2.2 × 10.sup.−5 Hypochlorous HClO  3.0 × 10.sup.−8 Lactic CH.sub.3CHOHCOOH 1.39 × 10.sup.−4 Malic HOOCCHOHCH.sub.2COOH 3.48 × 10.sup.−4 8.00 × 10.sup.−6 Mandelic C.sub.6H.sub.5CHOHCOOH  4.0 × 10.sup.−4 Methylammonium ion CH.sub.3NH.sub.3.sup.+  .sup. 2.3 × 10.sup.−11 Succinic HOOCCH.sub.2CH.sub.2COOH 6.21 × 10.sup.−5 2.31 × 10.sup.−6 Tartaric HOOC(CHOH).sub.2COOH 9.20 × 10.sup.−4 4.31 × 10.sup.−5

[0049] Given the low K.sub.a values, the anions of the weak acids listed in Table 1 (appropriately incorporated into suitable salts) can sequester part of the protons of the methanesulfonic acid.

[0050] In another embodiment of the composition according to the invention—defined below as “second formulation”—the proton acceptor further comprises silicon dioxide particles, which can be pretreated with an ammonium hydroxide (NH.sub.4OH) solution and subsequently subjected to anhydrification through heat treatment at 100° C. to increase the neutralizing efficacy thereof. The second formulation can be considered to be substantially complementary to the first formulation.

[0051] In a further embodiment of the composition according to the invention—defined below as “third formulation”—the proton acceptor comprises tetraethoxysilane, which acts as a cross-linking agent as well as a sequestering agent against the protons of the methanesulfonic acid. The third formulation can be substantially complementary to the first and to the second formulation.

[0052] In another further embodiment of the composition according to the invention—defined below as “fourth formulation”—the proton acceptor comprises anhydrous dimethyl sulfoxide (DMSO), which is added to a concentrated solution of methanesulfonic acid. The DMSO enables the acidity to be reduced of the composition according to the invention by protonation of the oxygen atom thereof. The fourth formulation can be substantially complementary to the first, to the second and to the third formulation.

[0053] In a further other embodiment of the composition according to the invention—defined below as “fifth formulation”—the proton acceptor comprises polyethylene glycol. This enables the acidity of the composition according to the invention to be reduced through the protonation of the oxygen present in the chain of the polymers, as well as the viscosity of the composition according to the invention to be increased. The fifth formulation can be substantially complementary to the first, to the second, to the third and to the fourth formulation.

[0054] In a still further embodiment of the composition according to the invention—defined below as “sixth formulation”—the proton acceptor comprises molecular species containing amine groups, or more in general nitrogen atoms, which can bind protons.

[0055] The various embodiments of the composition disclosed above enable an equilibrium to be established between the methanesulfonic acid (indicated by HA) and proton acceptors (indicated generally by B). This equilibrium is disclosed by the reaction HA .sup.+B.Math.A.sup.−+HB and has as a result a reduction of the concentration of methanesulfonic acid without massive introduction of dilution water, maintaining the antimicrobial effect of the composition according to the invention and the efficacy of the latter in removing biofilm and tissues rapidly and painlessly.

[0056] The chemical components that are more suitable for preparing the various embodiments of the composition according to the invention are listed below. Of these, the methanesulfonic acid is the active principle, whilst all the other listed components are suitable proton acceptors:

[0057] Methanesulfonic acid 99.0%; CAS N.75-75-2

##STR00001##

[0058] Anhydrous sodium carbonate or Na.sub.2CO.sub.3; CAS N. 497-19-8

##STR00002##

[0059] 5-amino-2-mercaptobenzimidazole; CAS N. 2818-66-8

##STR00003##

[0060] Ethylenediaminetetraacetic acid tetrasodium salt or EDTANa4; CAS N. 13235-34-4

##STR00004##

[0061] Sodium gluconate; CAS N. 527-07-1

##STR00005##

[0062] Sodium tartrate dihydrate; CAS N. 6106-24-7

##STR00006##

[0063] 2-mercapto-5-benzimidazole sodium sulfonate; CAS N. 207511-11-3

##STR00007##

[0064] Dimethyl sulfoxide or DMSO or (CH.sub.3).sub.2SO, CAS N. 67-68-5

##STR00008##

[0065] Polyethylene glycol 400 or PEG 400 (average molecular weight: 400 Da), CAS. 25322-68-3; polyethylene glycol 600 or PEG 600 (average molecular weight: 600 Da), CAS 9004-74-4

##STR00009##

[0066] Silicon dioxide or SiO.sub.2; CAS 112945-52-5

[0067] Tetraethyl orthosilicate or tetraethoxysilane or TEOS or Si(OC.sub.2H.sub.5).sub.4; CAS N. 78-10-4

##STR00010##

[0068] In the following Tables 3-8, possible qualitative and quantitative compositions of the first, second, third, fourth, fifth and sixth formulation of the composition according to the invention are disclosed in greater detail by way of non-limiting example. The expression “possible qualitative and quantitative compositions” should be understood in the sense that a person skilled in the art can easily and suitably modify each of the formulations of the composition according to the invention (for example, through the addition of pharmacologically acceptable excipients) on the basis of the physical form of administration (solution, cream or gel), provided that the composition always contains methanesulfonic acid as an active principle and at least one suitable proton acceptor.

[0069] Tables 3-8 disclose the qualitative and quantitative composition of six liquid (solution) or gel embodiments of the composition according to the invention:

TABLE-US-00003 TABLE 3 First formulation Component Percentage by weight Methanesulfonic acid 70-90%  Sodium carbonate 0.5-2%.sup.  5-amino-2-mercaptobenzimidazole 1-2% EDTANa4 1-6% Sodium gluconate 1-6% Sodium tartrate dihydrate 2-6% 2-mercapto-5-benzimidazole sodium sulfonate 1-4%

TABLE-US-00004 TABLE 4 Second formulation Component Percentage by weight Methanesulfonic acid 70-90%  5-amino-2-mercaptobenzimidazole 1-2% EDTANa4 1-6% Sodium gluconate 1-6% Sodium tartrate dihydrate 2-6% 2-mercapto-5-benzimidazole sodium sulfonate 1-4% SiO.sub.2 0.1-7%.sup. 

TABLE-US-00005 TABLE 5 Third formulation Component Percentage by weight Methanesulfonic acid 70-90%  5-amino-2-mercaptobenzimidazole 1-2% EDTANa4 1-6% Sodium gluconate 1-6% Sodium tartrate dihydrate 2-6% 2-mercapto-5-benzimidazole sodium sulfonate 1-4% SiO.sub.2 0.1-7%.sup.  TEOS 0.1-2%.sup. 

TABLE-US-00006 TABLE 6 Fourth formulation Component Percentage by weight Methanesulfonic acid 70-90% Dimethyl sulfoxide 10-30% SiO.sub.2  0.1-7% TEOS  0.1-2%

TABLE-US-00007 TABLE 7 Fifth formulation Component Percentage by weight Methanesulfonic acid 70-90%  Sodium gluconate .sup. 1-6% Sodium tartrate dihydrate .sup. 2-6% SiO.sub.2 0.1-7% TEOS 0.1-2% PEG 400 or PEG 600  1-10%

TABLE-US-00008 TABLE 8 Sixth formulation Component Percentage by weight Methanesulfonic acid 70-90%  5-amino-2-mercaptobenzimidazole 1-8% EDTANa4 1-8% 2-mercapto-5-benzimidazole sodium sulfonate 1-8%

[0070] The procedure for preparing the six formulations referred to in the previous Tables 3-8 is not disclosed in detail below, because the chemical components of the various formulations can be added to the methanesulfonic acid according to a variable order and nevertheless without altering the properties of the final solution. In fact, the different chemical components are solubilized without reactions intervening that are able to interfere with the phenomenon of the protonation of the proton acceptors.

[0071] By way of non-limiting example of the invention, two tests of in vitro and in vivo antimicrobial activity of the composition according to the invention (Example 1; Example 2) and two procedures for treating lesions (skin lesions and lesions of the mucosa of the oral cavity) that are based on the use of the composition according to the invention (Example 3; Example 4) are disclosed below.

Example 1—Test of In Vitro Antimicrobial Activity

[0072] The antimicrobial activity of the first, second, third, fourth, fifth and sixth formulation of the composition according to the invention was tested against the following strains of microorganisms (purchased from Diagnostic International Distribution S.p.A.): Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Enterococcus hirae ATCC 10541, Candida albicans ATCC 10231. Mixtures of the different strains of microorganisms were prepared, having concentrations expressed as colony forming units (CFU) comprised between 1.5×10.sup.12−5.5×10.sup.12 for each species. 100 μl samples of the mixture were inoculated on Petri dishes containing TSA (Tryptone Soya Agar) solid culture medium. The inoculation was carried out according to a known and standardized analytical method, namely by depositing the liquid sample on the surface of the agar by a micropipette and distributing the liquid sample on the surface of the agar by using sterile glass beads. Subsequently, 50 μl aliquots of the six formulations of the composition according to the invention were deposited in a central zone of the agar of each Petri dish. The dishes were then incubated at 37° C. for 24 h.

[0073] FIGS. 1 to 6 show six Petri dishes marked 1 to 6, in each of which a 50 μl aliquot was deposited respectively of the first, second, third, fourth, fifth and sixth formulation of the composition according to the invention.

[0074] After the incubation, the dishes were examined in order to evaluate the microbial proliferation (formation of colonies) and the width of the inhibition halo (namely, the width of the portion of medium in which the microbial proliferation had been inhibited) surrounding the zone of agar on which samples of the six formulations had been deposited. As shown in FIGS. 1-6, in all the Petri dishes clear zones of microbial growth inhibition were observed, surrounding the depositing zones of the preparations according to the invention. The analytical result obtained indicates that the six different formulations of the composition according to the invention are able to inhibit the growth of 10.sup.11 CFU of gram-positive bacteria, gram-negative bacteria and of the fungal species Candida albicans.

Example 2—Test of In Vivo Antimicrobial Activity

[0075] The in vivo antimicrobial activity of the composition according to the invention was tested, which composition was prepared in the form of a gel according to the fourth formulation, in which the methanesulfonic acid is supported on silicon. Six volunteers, namely (six) patients diagnosed with chronic periodontal disease (periodontitis) were selected randomly. The selected patients were of an age comprised between 35 and 55 years and had not previously received any surgical or non-surgical periodontal therapy. None of the aforesaid patents was pregnant, had taken antibiotics, had used antibacterial mouthwash in the six previous months, had teeth with furcation involvement or had a story of abuse of alcohol or drug. Before any treatment, at the initial time (day) T1, each of the six patients was subjected to a subgingival microbiological sampling through the use of four sterile paper tips (of the type used to dry the endodontic canals), which were inserted into the periodontal pockets in four quadrants (right maxillary quadrant, left maxillary quadrant, right mandibular quadrant, left mandibular quadrant) and left therein for 20 seconds. The insertion site of each paper tip was isolated by using cylindrical cotton swabs. The gel containing the composition according to the invention was applied with a small brush in each periodontal pocket and left therein for a period of 15 seconds. After this time elapsed, the gel was removed by washing abundantly with physiological solution for 30 seconds and simultaneously sucking the liquid.

[0076] After the aforesaid procedure had been completed and subject to drying with a jet of air, a second subgingival microbiological sampling was carried out (by using sterile paper tips), at the time T2, in the same periodontal pockets examined at the initial time T1. The second subgingival microbiological sampling was carried out through the same procedure used in the initial time T1 . The paper tips used for each patient at the time T1 and at the time T2 were transferred in sterile test tubes and sent to a microbiological laboratory, for subsequent extraction of the DNA and PCR analysis (polymerase chain reaction). The bacterial count through PCR was directed mainly to Tannerella forsythia (TF), Treponema denticola (TD), Fusobacterium nucleatum (FN) and Campylobacter rectus (CR), which are the bacterial species that are more implicated in the periodontitis. The total bacterial load (TBL) was also determined through PCR.

[0077] In the PCR analysis, the primers and the oligonucleotide probes were based on gene sequences of 16S rRNA of the human oral microbiome database (HOMD 16S rRNA RefSeq Version 10.1), which counts 845 items. Absolute quantification assays were carried out through PCR by using the 7500 Sequence Detection System (Applied Biosystems). The amplification profile was started with an incubation period of 10 min at 95° C. to activate polymerase, followed by an amplification in two steps of 15 sec to 95° C. and 60 sec at 57° C. for 40 cycles. All these steps were carried out by including controls without amplification probes to exclude the contamination of the reagents. Plasmides containing specific DNA sequences (purchased from Eurofins MWG Operon, Ebersberg, Germany) were used for the quantitative evaluation. These positive controls were used to plot standard curves (by showing in Cartesian axes the threshold cycle values against the logarithm of the number of copies), which were used to check the amplification efficiency and for the quantification of targets in each sample (J Biol Regul Homeost Agents, 2017, 31(1): 257-262; J Biol. Regul. Homeost. Agents, 2016, 30 (2 Suppl 1): 87-97; J Biol Regul Homeost Agents, 2015, 29(3 Suppl 1): 101-10.).

[0078] The absolute quantities of different bacterial species in periodontal pockets of patients affected by periodontitis, detected through bacterial count at the initial time T1 and at the time T2, are shown in the following Table 9:

TABLE-US-00009 TABLE 9 TF 1 TF 2 TD 1 TD 2 FN 1 FN 2 CR 1 CR 2 TBL 1 TBL 2 357 0 1205 0 126485 1970 1678 0 645280 54501 181 0 327 0 22031 1561 1049 92 162489 63541 9177 0 10059 0 739463 823 51446 0 4430648 52805 400 185 537 202 82289 24180 3211 844 315053 110991 69 0 210 63 137127 28550 11395 3575 979525 302285 0 0 0 0 16985 4887 2104 119 186067 62545 (1: initial sampling; 2: post-treatment sampling; TF: Tannerella forsythia; TD: Treponema denticola; FN: Fusobacterium nucleatum; CR: Campylobacter rectus; TBL: total bacterial load).

[0079] The results of the microbiological analysis were treated statistically, in particular by carrying out a related-samples Wilcoxon non-parametric test by SPSS software. The Wilcoxon test demonstrated a statistically significant reduction (p≤0.05) of the quantity of each bacterial species after the local treatment with the gel containing the composition according to the invention. The results of the Wilcoxon test are shown in the following Table 10:

TABLE-US-00010 TABLE 10 Null hypothesis Sig Decision The median of the differences 0.018 Rejects null hypothesis between TF1 and TF2 is equal to 0 The median of the differences 0.018 Rejects null hypothesis between TD1 and TD2 is equal to 0 The median of the differences 0.012 Rejects null hypothesis between FN1 and FN2 is equal to 0 The median of the differences 0.012 Rejects null hypothesis between CR1 and CR2 is equal to 0 The median of the differences 0.012 Rejects null hypothesis between TBL1 and TBL2 is equal to 0

[0080] It should be further noted that no (immediate or after a lapse of time) side effects or adverse reactions were observed after the local application of the gel containing the composition according to the invention. In fact, the patients reported no sensations of pain, burning, tingling and/or numbness during the entire period of treatment.

Example 3—Treatment of Patients Affected by Chronic Cutaneous Ulcers

[0081] The composition according to the invention was tested on over 20 patients who were volunteers, by applying a treatment protocol comprising the following steps: [0082] Removing with a sterile gauze the easily removable necrotic materials that are present on the bottom of the ulcer; [0083] Drying completely the bottom of the ulcer; [0084] Only in the case of particularly sensitive patients, for whom a painful response is expected, pre-treating the bottom of the ulcer for a time of about 5 minutes with a pack of ointment containing 5% lidocaine; [0085] Removing the pack, washing the ulcer to remove the lidocaine ointment and drying; [0086] Applying the composition according to the invention on the bottom of the ulcer and on the surrounding skin for about 1 cm beyond the edge of the ulcer, by using the finger of a hand covered by sterile single-use glove; [0087] Letting the composition according to the invention to act for about 20-30 seconds; [0088] Washing abundantly with sterile physiological solution; [0089] Drying with sterile gauze; [0090] Rubbing the bottom of the ulcer with sterile gauze to remove the desiccated material; [0091] Leaving on the bottom of the ulcer the possible desiccated material that did not detach itself through the effect of rubbing with the gauze; [0092] Covering the ulcer with a sterile greasy gauze or with another type of suitable medication (according to the choice of the operator); [0093] Bandaging the zone according to known procedure; [0094] Performing subsequent checks at 7-day intervals, or at shorter intervals if deemed necessary.

[0095] During each check, the aforesaid protocol provides for proceeding in the following manner: [0096] Removing bandages and medication; [0097] Removing progressively, by using normal pliers and scissors and starting from the edges of the lesion, the desiccated material left on the bottom of the ulcer; [0098] Covering the ulcer with a sterile greasy gauze or with another type of suitable medication (according to the choice of the operator); [0099] Bandaging the zone according to known procedure.
When the bottom of the ulcer is covered by granulation tissue, upon complete granulation and according to the choice of the operator, it is possible to graft skin and/or skin substitute in the lesion to complete the healing process.

[0100] The treatment protocol disclosed above resulted to be effective in desiccating the bottom of the ulcers in all the treated patients. No complications and/or side effects emerged at the systemic or local level or on the perilesional skin. In all the treated patients the residual desiccated material disappeared progressively at the end of the procedure without the need for an additional intervention and left a granulation tissue on the bottom of the lesion.

[0101] Therefore, the application of the composition according to the invention (containing methanesulfonic acid together with proton acceptors) produced in all the cases a complete restoration of the tissues in the lesion, promoting the healing thereof. The treatment protocol disclosed above can be applied to all the patients, thus avoiding complicated, costly and potentially risky surgical procedures. Furthermore, the treatment with the composition according to the invention can reduce the need to use antibiotic therapies, which are substantially costly and associated with the increasingly rising phenomenon of the antibiotic resistance.

Example 4 —Treatment of Patients Affected by Lesions of the Mucosa of the Oral Cavity

[0102] A non-surgical treatment protocol and a surgical treatment protocol are disclosed below.

[0103] The non-surgical treatment protocol comprises the following steps: [0104] Isolating the affected part with cylindrical cotton swabs; [0105] Drying with a jet of air for a time of 10 seconds; [0106] Through the use of a small brush, applying the composition according to the invention in the periodontal pocket around the tooth (in the case of periodontal disease), around the implant (in the case of perimplantitis) or on the aphtha; [0107] Letting the composition to act for a period of 20 seconds; [0108] Washing the treated zone with a flow of sterile water and simultaneously aspirating the liquid, so as to remove the composition completely;

[0109] The non-surgical treatment protocol comprises the following steps: [0110] Performing local anaesthesia; [0111] Proceeding with periodontal flap, consisting of the lifting of a portion of gingiva at the periodontal pocket; [0112] Performing a surgical periodontal or peri-implant debridement; [0113] Drying with a jet of air for 10 seconds; [0114] Through the use of a small brush, applying the composition according to the invention in around the tooth (in the case of periodontal disease) or around the implant (in the case of perimplantitis); [0115] Letting the composition to act for a period of 20 seconds; [0116] Washing the treated zone with a flow of sterile water and simultaneously aspirating the liquid, so as to remove the composition completely; [0117] Removing possible residues of purulent tissue.

[0118] The result obtained through the treatment protocols disclosed above can be verified both clinically (namely, by measuring the depth of the periodontal pocket in the non-surgically treated patients) and by carrying out microbiological tests before and after the treatment. Surprisingly, in 20 patients non surgically treated an immediate reduction of the periodontal pocket by about 50% was observed after the application of the composition according to the invention.

[0119] As a person skilled in the art will understand, variations on and/or additions to what has been disclosed above are possible. For example, although the previously disclosed compositions were prepared on laboratory scale, the person skilled in the art is able to provide preparation procedures that are suitable for a production on industrial scale.