ITMI20131151A1 - COMPOSITION AND RELATIVE PERIODONTAL, IMPLANTOLOGICAL AND ENDODONTIC TREATMENT KIT WITH OPTIMIZED AND REGENERATIVE ACTION - Google Patents

Description

E S C R I Z I O N E

Attached to the patent application for INDUSTRIAL INVENTION entitled: COMPOSITION AND RELATIVE PERIODONTAL, IMPLANTOLOGICAL AND ENDODONTAL TREATMENT KIT WITH OPTIMIZED ANTISEPTIC AND REGENERATIVE ACTION

FIELD OF THE INVENTION

Hydrogen peroxide solution with limited cytotoxic impact with preservation of the optimal antiseptic action for use associated with laser in dentistry and reactive kit.

FRONT TECHNIQUE

The use of diode lasers in the dental field on soft tissues ensures excellent cutting capacity and haemostasis without the use of sutures, while the associated use of diode lasers associated with dilute solutions of hydrogen peroxide guarantees deep sanitation.

The diode laser, due to its characteristic wavelength (980 nm that used in these methods) is very little absorbed by water and hydroxyapatite, therefore it is able to penetrate into the depth of the irradiated tissues.

The laser at low average energy levels (LLLT) is able to stimulate the activity of cells responsible for the regeneration of tissues lost with bacterial diseases typical of the oral cavity (Leonida, Caccianiga, Laser Med Sci 2012). The diode laser adjustments used in the protocol combine the high peak power necessary for the elimination of pathogenic bacteria of the oral cavity with a low average power typical of LLLT, which allows you to take advantage of all the potential of laser-assisted biostimulation, because the average temperature of the operating field never exceeds 45 ° C, and remains in the bed of tissue vasodilation.

Hydrogen peroxide has its own recognized antibacterial capacity, for which irrigation protocols of the periodontal pockets are in use in order to lower the bacterial load present.

By combining the hydrogen peroxide of a penetrating laser with the correct adjustments, with almost complete suppression of the thermal increase typical of diode lasers, (diode 980 nm), the laser actually increases the effectiveness of hydrogen peroxide for photodynamic action with activation of hydrogen peroxide (Caccianiga G et col. Eur J Inflamm. 2012; 10: 2 (S): 97-100). The energy transfer from the diode laser to the H2O2ne molecule causes homolytic cleavage to OH * (hydroxy radical) or decomposition to H20 and <1> 02 (singlet oxygen).

The usefulness of these radicals with a powerful bactericidal action in odontological applications has been well highlighted by the author (Caccianiga G and coll. Adv Odont. 2007: 23) and by Rey G and coll. (Implantology. 2004: 56-72).

The decontaminating effects of the combined use of hydrogen peroxide and diode laser significantly decrease the possibility of post-operative superinfections, see previous references and also Walsh JL (J Orai Implan. 1992: 38-42) and Missika & Stroumza (J Odontostom . 2003: 215-29).

The peroxide association with diode laser is therefore able to maximize the pre- and post-operative decontamination treatment, where the laser increases the antiseptic action of the indrogen peroxide while, at the same time, it operates the biostimulation of the soft tissues and lasts, promoting regrowth. Such combined capabilities are evident in Rey G's protocol (Impladontie. 2000: 27-34).

Furthermore, hydrogen peroxide is devoid of chromophores, and as such it is completely transparent allowing the complete penetration of the laser beam into the tissues affected by the disease and well oxygenated with hydrogen peroxide irrigation.

However, some limitations have been noted, which can be attributed to the quality of the commonly used hydrogen peroxide. These in fact contain stabilizers as the solution of H2O2 in water requires additives to avoid decomposition.

The most common stabilizers are: coloid tin and sodium pyrophosphate (at 25-250 mg / L); silver nitrate; organophosphates (Dequest ™ line, Monsanto); nitrates; phosphoric acid; colloidal silicates; trisodium phosphate (see patents US2005065052 and US2003151024). The latter suffer from the disadvantage of a high pH, which tends to be aggressive. Recently introduced are the sugar alcohols, eg. mannitol (patent EP 1043273) or sorbitol (patent EP1452484) which are stabilizers believed to have low impact.

However, the contribution of stabilizers on the properties of hydrogen peroxide associated with the diode laser remained to be evaluated. If the literature data show good performance, it seemed appropriate to further increase the balance between antiseptic and regenerating properties.

In this sense, laboratory methods were used to evaluate a hydrogen peroxide composition having the best relationship between stability, antibacterial action and low impact (non-negative contribution) to laser biostimulation.

SUMMARY

It has been discovered that a particular hydrogen peroxide solution in association with the diode laser in dentistry represents an optimal compromise between the bacterial decontamination action and the typical laser biostimulation.

An object of the invention is a solution of hydrogen peroxide stabilized by at least one glycerophosphate, said composition useful in odontological treatments guided by diode lasers.

The inventive composition will preferably contain H202-glycerophosphate in a ratio ranging from 1000: 1 to 10: 1 mol / mol.

A particular inventive object consists of an aqueous solution containing about 3% H2O2 and about 0.5% w / v glycerophosphate.

A further particular inventive object is constituted by an aqueous solution such as the previous one, further containing between 1% and 3% of a phosphate.

Another inventive object consists of a kit comprising:

1) a package with an aqueous solution of hydrogen peroxide as described above in single- or multipurpose packaging;

2) a 980 nm diode laser, preset according to one or more of the modes designated (a) - (f) in the description;

3) a sterile disposable tip with reinforced 400 μ optical fiber and angled tip for maximum irradiation within periodontal and bone defects. A further inventive object consists of a kit as described above where the hydrogen peroxide is stabilized with substances with a low cytotoxic impact. These and other inventive aspects are better illustrated in the following description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 reports the results of the analysis of the expression of keratin-5 and -8 of cultures of keratinocytes, fibroblasts and organotypic co-cultures subjected to contact with the solution according to the invention (A), a commercial hydrogen peroxide solution (B) in addition to H2O2-free control

DETAILED DESCRIPTION OF THE INVENTION

The solution object of the present invention is substantially constituted by a solution of hydrogen peroxide in water in the presence of a glycerophosphate with a stabilizing function with a low cytotoxic impact.

The concentration of hydrogen peroxide in the inventive solution varies from 1% to 10% w / w, preferably 2% -6% w / w, even more preferably around 3% w / w. This concentration is obtained by dilution with demineralized and purified water of the non-stabilized hydrogen peroxide at 30% and more than can be purchased from primary suppliers and / or producers of this substance.

The dilution operation must be carried out in special containers of glass, stainless steel or other inert material that is not a source of activation of the decomposition of the peroxide. All accident prevention regulations must be strictly observed by the operators.

In the inventive solution, the H202-glycerophosphate ratio can be comprised between 1000: 1 and 10: 1 mol / mol, preferably between 100: 1 and 10: 1 mol / mol.

In the present inventive field, the term "glycerophosphate" includes the phosphorylated derivatives of glycerin in position 1 and / or 2. Said glycerophosphates include the following structural categories:

A) products of hydrolysis (lipolysis) of natural phospholipids with preservation of chirality such as: - L-a-glycerylphosphorylcholine (a-GPC)

- L-a-glycerylphosphorylethanolamine (a-GPE)

- L-a-glycerylphosphoryldiemethyletnanolamine (a-GPEMe2)

- L-a-glycerylphosphorylinositol (a-GPI)

- L-a-glycerylphosphorylserine (a-GPS)

B) Racemic 1-glycerophosphates obtained by chemical synthesis such as

- l-rac-glycerylphosphorylcholine

- l-rac-glycerylphosphorylethanolamine

- l-rac-glycerylphosphtidic acid

C) 2-glycerophosphates obtained by condensation such as:

- β-glycerylphosphtidic acid (β-GPA)

Also contemplated are also mixtures such as a- and β-glycerophosphate sodium salt, also referred to as: sodium (2RS) -2,3-dihydroxypropyl phosphate + sodium 2-hydroxy-1- (hydroxymethyl) ethyl phosphate hydrate.

The glycerophosphates can be partially salified with physiologically compatible bases such as soda, potash, magnesium, calcium, ammonia or organic amines such as eg. ethanolamine, diethanolamine, triethanolamine, tripropylamine, deanol, trometamine, choline, betaine, ethylenediamine, piperazine, piperidine, etc. This partial salification operation can be carried out by adding the preformed salt or by neutralization in situ, partial or complete, of the hydrogen peroxide solution with at least one phosphate. The preliminary considered optimal pH is acidic or slightly acidic, for example between 6 and 4.

The association allows the vaporization of the granulation tissue in the periodontal pockets, the sterilization of infected canals in endodontics, the treatment of sensitive stumps and necks as well as minor oral surgery by excision.

Fields of use. The laser-hydrogen peroxide stabilized association according to the invention is particularly useful in the following cases:

a) endodontic treatments (dental devitalizations) where the antiseptic action of the hydrogen peroxide activated by the laser allows an effective and rapid disinfection of the infected canals, after the correct preparation of the dental canals carried out according to the state of the art, favoring the healing of periapical lesions in a short time and reducing relapses and failures, quickly and more effectively;

b) treatment of periodontal disease with the joint effect of biostimulation of the inflamed tissue and elimination of bacteria with arrest of periodontal infection, consequent to the procedures for the elimination of supra- and subgingival tartar deposits according to the state of the art: in addition to the effects mechanical effects (scalers, curettes and ultrasounds) and chemical ones (solutions based on polyvinyl-iodates) are added the physical effects of photodynamic therapy, in which the transparency of the hydrogen peroxide allows a complete penetration of the penetrating beam typical of the diode laser from 980 nm in depth;

c) treatment of dentinal hypersensitivity of vital necks and abutments by crystallization of the dentinal tubules without effects on the dental pulp;

d) minor oral surgery in gingivectomy, gingivoplasty, vestibuloplasty, frenectomy and clinical crown lengthening;

e) treatment of ulcerative canker sores, angular cheilitis and herpes labialis, with immediate relief due to the bactericidal effect;

f) treatment of bacterial peri-implantitis, or in the second surgical phase of uncovering the implant, providing a result without modification of the implant or thermal damage to the surrounding structures (for the ablative action of the diode laser, additional adjustments to those provided for in the machine in question, in order to eliminate any risk of thermal increase beyond the threshold of 45 ° C).

These treatments are optimally performed in the presence of hydrogen peroxide with a low cytotoxic impact.

A further inventive object is constituted by the kit comprising a multi- or single-dose bottle containing hydrogen peroxide as described above in association with a diode laser.

In a particular inventive embodiment said kit will comprise:

Another inventive object consists of a kit comprising:

1) a package containing vials or strips or other small disposable container containing an aqueous solution of hydrogen peroxide as described above;

2) a 980 nm diode laser, preset, as follows:

a) decontamination program: 2.5 watts, t-on 20 μμ, t-off 80 μ, 10,000 Hz b) regeneration program: 2.0 watts, t-on 20 μ, t-off 80 μ, 10,000 Hz c) peri-implantitis program: 2.5 watts, t-on 20 μ, t-off 80 μ, 10,000 Hz d) gentle biostimulation program (curved lens): 4.5 watts, t-on 20 μ, t-off 40 μ , 16700 Hz (postoperative under anesthesia)

e) medium biostimulation program (curved lens): 6 watts, t-on 20 μ, t-off 40 μ, 16700 Hz (periodontal maintenance and implantation)

f) classic biostimulation program (curved lens): 1 watt, continuous (periodontal maintenance and implantation)

3) a sterile disposable tip with reinforced 400 μ optical fiber and angled tip for maximum irradiation within periodontal and bone defects. The hydrogen peroxide package (1) can be in single-dose as the PE strips or from vials-syringes each containing from 1 to 5 mL in 10 or 20 or more units per box. The hydrogen peroxide package (1) may, optionally, consist of a multidose container of 30, 50 or more mL in glass or other inert material.

Hydrogen peroxide can be stabilized with low cytotoxic impact substances. These and other variants of the kit associated with the invention can be easily obtained by those skilled in the art, for example by following the methods described such as to optimize the balance of stability / biocidal potential / cytotoxic effect.

The inventive solution and relative kit can be used in the periodontal field, with different protocols, for example comprising the following steps:

i) supra- and sub-gingival debridement with ultrasound instrument and irrigation with a solution based on polyvinyl jodates and green betadine dilution. 1: 5);

ii) use of the Prophy-jet on the tooth surface with a jet of bicarbonate solution necessary to open the root cement and the dentinal tubules obliterated by ultrasonic activity, and which, once reopened, thus become accessible to the therapy activity laser / hydrogen peroxide photodynamics;

iii) irrigation of the periodontal pockets with the hydrogen peroxide solution according to the invention;

iv) irradiation with fiber optic laser with a diameter of 400 µm by insertion parallel to the surface of the tooth root above the depth of the pocket and directed from top to bottom with repeated and relatively uniform movements, for 30 seconds in each sector of the defect.

The inventive solution and relative kit can be used in the endodontic field with different protocols, for example comprising the following steps:

- after the steps necessary for shaping the root canals, according to the international guidelines of endodontics, irrigation is carried out with the hydrogen peroxide object of the invention and 5 10 "passages are carried out with the regeneration program (the energy is lower due to the reduced size of the channels, and therefore to control the thermal effect which must not exceed 45 ° C).

In a further inventive field, the glycerophosphate of the invention can be replaced in whole or in part by one or more stabilizers with behavior similar to glycerophosphate.

Examples of such stabilizers are inorganic or organic phosphates and polyphosphates such as: mono-di- or tri-sodium or mono-di-potassium phosphate; pyrophosphates such as pyrophosphate (diphosphate), triphosphate, trimetaphosphate and hexametaphosphate, linear and cyclic polyphosphates at n. intermediate condensation; bisphosphonates such as alendronate, risedronate, etidronate, clodronate, pamidronate, tiludronate, ibandonate, zoledronate, incadronate, olpadronate, neridronate, am hydronate, oxidronate and medronate; phytates such as phytic acid and its metabilites with a reduced degree of phosphorylation.

Further examples of such stabilizers include: i) sugar alcohols such as glycerol, erythritol, xylitol, sorbitol, mannitol, lactitol, lactulose; ii) carboxylic alcohols such as lactic, tartaric, etc .; iii) carboxylates such as citric, maleic, formic acid; iv) aminocarboxylates such as EDTA, NTA, DTPA, IDA; v) amino acids such as gliein, beta-alanine, etc .; vi) keto and aldo-oils such as pyruvic acid, fructose, glucose; as well as their salts and solvates and clathrates.

These glycerophosphate substitutes can be used alone or in copies after determining their cytotoxic impact in association with the diode laser.

A wide range of variants of the present invention can be realized without thereby exceeding the inventive scope. Some implementation methods are illustrated by the following examples.

EXAMPLES

Example 1 - Hydrogen peroxide stabilized with glycerophosphate

A solution of H2O2 is prepared by diluting the 30% solution from Sigma-Aldrich, Cat. No. 3% with double distilled water. 95313, with characteristics shown in Table I.

TABLE I - Specifications of the starting hydrogen peroxide

grade ACS reagent

vapor pressure 23.3 mmHg (30 ° C)

grade puriss. p.a.

assay> 30% (RT)

quality not stabilized

impurities <0.002% non-volatile matter

<0.005% free acid (as H2S04)

density 1.11 g / mL at 20 ° C

Then 1 g of Glycerol phosphate disodium salt hydrate from Carbosynth is added

Ltd (Compton, Berkshire, UK).

Stability in 1 L pyrex glass bottle shows a loss of titre in H202

less than -2% of the initial value in 6 months of storage at room T.

Comparative example 1 - Hydrogen peroxide stabilized with acetanilide

The product available from Sigma-Aldrich is considered as the reference solution,

Cat. No. 323381; the characteristics here are presented in Table II.

Table II - Specifications of the comparison hydrogen peroxide solution vapor density 1.1 (vs air)

vapor pressure 23.3 mmHg (30 ° C)

contains ~ 200 ppm acetanilide as stabilizer

concentration 3 wt. % in H20

refractive index n20 / D 1.335

density 1 g / mL at 25 ° C

Example 2 - Comparison test of cytotoxicity

To evaluate the comparative impact on cytotoxicity, an already

tested to measure the biocidal effects of the association with and without

laser irradiation by the team of prof. Caccianiga.

Primary cell lines of fibroblasts and keratinocytes (Matched Set-Cryopreserved Dermal Fibroblasts and Keratinocytes, Tebu-Bio ™) isolated from human skin are placed in culture media, respectively in Euroclone ™ and Tebu-bio ™ (Human Aduli Keratinocyte Growth Medium KM- 2). The lines are cultivated at confluence (70-80% min.) In a 1: 1 mix of the two culture media (+ 5% of final FBS, called “A”). Fibroblasts (0.5x10 <5> cells / well), keratinocytes (1x10 <5> cells / well) or grown together in co-culture, called “organotypic”, are sown in 12-well 1 cm diameter racks in monolayer.

The cultures are then added with 0.3 mL of the H2O2 solution of Example 1 ("A", test solution), of Comparative Example 1 ("B", positive control), or with an equal quantity of distilled water ("C ", Negative control).

The culture media (A) - (C) as described above are replaced at 48 hour intervals in correspondence with the irradiation treatment, carried out with a continuous 980-nm diode laser (Wiser Doctor Smile, Lambda ™) at intervals of 48 hours for 10 days. A device (Onda Piana, Lambda ™) allows to keep the irradiation intensity constant at a distance from 0 to 100 cm, uniforming the amount of energy per cm <2> of area (output power: 1 Watt; t. Exposure: 50 sec .; energy density: 50 J / cm <2>). 2 days after the last irradiation, groups (A) - (C) are analyzed by flow cytometry (FACS) and western blotting to quantify keratins 5 and 8 with monoclonal antibodies reactive to cytokeratins 5 and 8 (KRT 5/8, Antibodies-online ™).

The data obtained from the FACS analysis at the end of the exposure are shown in Fig. 1 to compare the inventive solution (A), the positive (B) and negative (C) control.

As can be seen, solution (A) has a lower impact on the biostimulating capacity than the hydrogen peroxide solution stabilized in a classical way.

Example 3 - Comparison test of bactericidal activity

The assay is carried out with modified methods as per Eur J Inflamm. 2012; 10 (2) S: 97-100 with cultures of typical strains causing infections affecting the oral cavity: - Haemophilus actinomycetemcomitans CIP 52103T ("HA")

- Bacterioides forsytus CIP 105219T ("BF")

- Porphyromonas gingivalis CIP 103683T ("PG")

- Micromonas micros CIP 105294T ("MM")

- Fusobacterium nucleatum CIP 101130T ("FN")

Briefly 30 uL of suspension for each strain is placed in 1.5 ml Eppendorf tubes_ with 5% culture medium and treated with a Wiser 980 mm diode laser (Wiser Doctor Smile, Lambda ™) with irradiation along the test tube. 10 ", of which 5" of vertical movement and 5 "of rotary movement. Washes are carried out with 1 part of hydrogen peroxide solution in 2 parts of the culture solution with a contact time of 3 min. checking for any temperature increase. At the end the population density is measured in CFU (colony forming units)

Table III - Decrease in UFC / 30 uL expressed as -Log _

HA BF PG MM FN

3% H202 of example 1 8.20 3.95 6.60 3.79 3.50

3% H202deN "comparative example 1 8.00 3.50 6.00 4.37 3.45

The above examples have shown how the inventive solution is able to make a significant contribution to the antiseptic action of the diode laser in dentistry without significantly worsening the performance in terms of tissue bio-regeneration, with particular advantage in clinical practice.

The purpose of the invention is however better defined by the claims, rather than by the examples reported above.

Claims (7)

CLAIMS 1. A composition of hydrogen peroxide consisting of an aqueous solution of H2O2 and at least one glycerophosphate for use in dentistry associated with the diode laser. 2. The composition according to claim 1 wherein the H2O2 is present in an amount comprised between 2% and 6% w / v. 3. The composition according to claim 1 wherein said glycerophosphate is selected from the group comprising: α-GPC, a-GPE, a-GPEMe2, a-GPI, a-GPS, 1-racglycerylphosphorylcholine, 1 -rac-glycerylphosphorylethanolamine, 1 -racglycerylphosphtid acid, β-glycerylphosphtidic acid, a- and β-glycerophosphate mixture, and related salts. 4. The composition according to claims 2 and 3 with H202-glycerophosphate in a ratio of between 1000: 1 and 10: 1 mol / mol. 5. The composition according to claim 4 wherein said glycerophosphate is represented by the mixture a- and β-glycerophosphate sodium salt. 6. A kit for dental treatment essentially consisting of: a) a package with an aqueous solution of hydrogen peroxide according to one or more of the preceding claims in a single- or multipurpose configuration; b) a 980 nm diode laser, with presets chosen from those designated as (a) - (f) in the description; c) a sterile disposable tip, with reinforced 400 μ optical fiber. 7. The kit according to claim 6 where the glycerophosphate is replaced in part or in whole by one or more stabilizers with low cytotoxic impact when used in association with the diode laser.