Composition for the treatment of periodontitis and regeneration of interdental papillae

Abstract

Described herein are compositions comprising hyaluronic acid and a mixture of polynucleotides extracted from natural sources for use as an active agent in therapeutic and / or cosmetic treatments for interdental papilla regeneration, or in therapeutic and / or cosmetic treatments for interincisal papilla recession (Black Triangle Syndrome), or in therapeutic and / or cosmetic treatments for periodontitis.

Description

[Technical field]

[0001] The present invention refers to compositions which are active in the therapeutic and / or cosmetic treatment of periodontitis and in the regeneration of interdental papillae. [Background technology]

[0002] Periodontitis is a disease characterized by inflammation of the periodontal tissues. It is one of the most widespread chronic inflammatory diseases in the population of Western countries and in Italy it is estimated that in severe cases it affects about 10-15% of adults, while a proportion of 2-30% is affected by its milder forms. Periodontitis is the main cause of loss of dental elements, which is the cause of serious functional disorders as well as can have a negative impact on interpersonal relationships, having a significant impact on the psychological sphere and impairing the aesthetics of the smile.

[0003] The characteristic sign of periodontitis is the loss of attachment of the teeth to the socket, which leads to the formation of periodontal pockets, loose teeth, bleeding gums, abscesses, suppuration and loss of one or more teeth.

[0004] Traditionally, the treatment of periodontitis involves first suppressing bacteria and inflammation, immediately followed by the essential next step of regenerating lost hard and soft periodontal tissues in order to restore tooth attachment and eliminate gingival and bone pockets.

[0005] The interdental papilla is the part of the free gingiva that occupies the space between two adjacent teeth, causing the free gingival margin to run down the dental arch in a festoon shape. In addition to being important in the biology and coating of the proximal portion of the tooth, the papilla has important aesthetic importance, especially in the anterior region. The explicit focus on preserving all adjacent tissues as an aesthetic objective began with a 1985 article by Evian and coworkers (Evian CI, Com H, Rosenberg ES. Retained interdental papilla procedure for maintaining anterior esthetics. Compend Contin Educ Dent 1985;6(l):58-64), which describes a surgical method with a specific aesthetic objective and defines it as "retained interdental papilla surgery".

[0006] The loss of this part of the gingival tissue is also the subject of research, especially with regard to possible conservation and regeneration techniques. Indeed, dental esthetics are becoming more and more important for both dentists and patients. The presence of interproximal papillae between the maxillary anterior teeth is in fact an important esthetic factor. Recession of the interproximal papillae causes many functional problems as well as esthetic problems such as the formation of the so-called black triangle ("black triangle syndrome" (or "incisal papilla recession").

[0007] In 1998, Nordland and Tarnow classified loss of papillary height using a scale. This scale is still widely used today and identifies four classes: normal, class I, class II, and class III (Nordland WP, ​​Tarnow DP. A classification system for loss of papillary height. J Periodontol 1998;69:1124-6). The classes identified by Nordland and Tarnow are based on the following anatomical references: 1) Tooth contact points, 2) Apical / facial expansion of the CEJ (cemento-enamel junction), 3) using coronal / proximal extension of the CEJ.

[0008] These classes are briefly described below. -Normal: Papillae fill the interdental space up to the point of contact. -Class I: The tip of the papilla is between the contact point and the coronal portion of the CEJ (space is present but the coronal / proximal CEJ is not visible). -Class II: The tip of the papilla is apical to the coronal / proximal CEJ but at the same level as the facial CEJ (visible coronal / proximal CEJ). -Class III: The tip of the papilla is apical to the facial CEJ.

[0009] In cases where interdental tissue loss has already occurred, numerous techniques aimed at reconstruction can be implemented. The methods proposed in the literature that are suitable for solving this tissue defect can be divided overall into surgical and non-surgical techniques, although this distinction is merely descriptive, since surgical and non-surgical techniques can often be combined with each other.

[0010] surgical reconstruction techniques Many surgical strategies have been described in the literature with the aim of correcting interdental tissue defects, however the cited knowledge is often limited to case studies.

[0011] In 1992, Beagle proposed a surgical method, called the pedicle flap technique, to recreate the interdental papilla between the maxillary central incisors (Beagle JR. Surgical reconstruction of the interdental papilla: case report. Int J Periodontics Restorative Dent 1992;12(2):145-51). The applicability of this technique is limited to large interdental areas, where the presence of greater connective tissue may be facilitated by thick biotypes.

[0012] In 1994, Aubert and coworkers proposed the deep rotated connective tissue flap technique, seeking greater preservation of the nutrient supply to the involved tissues (Aubert H, Bertrand G, Orlando S, Benguigui F, Acocella G. Lembo connettivale profondo di rotationazione per ricostruire la papilla interdentale. Minerva Stomatologica 1994;43(7-8):351-357). However, this technique has the disadvantage that it can only be used for one papilla at a time.

[0013] Based on the intuition of the usefulness of connective tissue fillers, but with the aim of simplifying the surgical technique, Han and Takei proposed the semilunar coronary repositioning papilla technique in 1996 (Han TJ, Takei HH. Progress in gingival papilla reconstruction. Periodontol 2000 1996;11:65-8). This method is inspired by the integrated reliability of the bilayer technique, which consists of using a connective tissue graft under a partial thickness flap. One of the advantages of this technique is the dual blood supply from both the connective tissue and the overlying flap, which favors the survival of the graft (Langer Β, Langer L. Subepithelial connective tissue graft technique for root coverage. J Periodontol 1985;56(12):715-20). Despite the intuition that the use of a two-layer technique leads to practical advantages, the coronal shift of the interdental tissue complex introduced by Han and Takei still represents a real technical difficulty compared to the method previously proposed by Aubert et al., 1994.

[0014] In 1998, Azzi et al. proposed the envelope technique, an approach similar to the one described above, but with some modifications that may improve its applicability (Azzi R, Etienne D, Carranza F. Surgical reconstruction of the interdental papilla. Int J Periodontics Restorative Dent. 1998;18(5):466-73).

[0015] Finally, the therapeutic difficulties associated with significant tissue defects and the search for long-term stability led Azzi et al. to propose the use of autogenous bone grafts in 2001 (Azzi R, Takei HH, Etienne D, Carranza FA. Root coverage and papilla reconstruction using autogenous osseous and connective tissue grafts. Int J Periodontics Restorative Dent 2001;21(2):141-7).

[0016] In general, the aforementioned surgical reconstructive therapies create an esthetic-anatomical compromise and alter the physiological relationship between the soft and hard periodontal tissues. The clinical consequence is the formation of periodontal pockets, or at best a long junctional epithelium, between the dental elements and the new interdental tissues. At the same time, the disruption of the normal relationship between the tissues leads in the long term to a high degree of uncertainty of the outcome.

[0017] non-surgical reconstruction techniques Non-surgical reconstructive techniques are essentially aimed at modifying non-gingival anatomical determinants related to the presence of interdental tissues. Their use therefore makes it possible to act on the interdental space and / or the distance between the contact points and the bone crest. Three different types of approaches have been proposed for these techniques: ·Restoration / Prosthetics Orthodontics Involves curettage and injection of fibroblasts

[0018] The possibilities of restorative / prosthetic treatment are the possibility of modifying the coronal morphology of the teeth and thus creating more apical contact points, inducing gap closure or reducing the interdental space (Prato GP, Rotundo R, Cortellini P, Tinti C, Azzi R. Interdental papilla management: a review and classification of the therapeutic approaches. Int J Periodontics Restorative Dent 2004;24(3):246-55). The possibility of reproducing new and more favorable anatomical determinants, with the aim of inducing a proliferative response of the soft tissues, also leads to an esthetic closure of the so-called black triangle. Importantly, the degree of morphological modification of the teeth should be evaluated based on the expected response of the soft tissues. It is therefore not considered good practice to correct interdental areas without papillae with a complete reconstructive closure of the teeth. The resulting overcorrection may in fact cause undesirable tissue reactions.

[0019] Orthodontic treatment also modifies the anatomical determinants, but in this case, this is done with a completely more physiological approach. Periodontal disease, together with problems of bone resorption and soft tissue recession, often causes tooth displacement, especially in the anterior-superior region. When the supporting tissues are reduced and inflamed, the dental elements respond to functional forces with migration, tipping, and flaring movements. This accentuates the loss of adjacent tissues, as the relationships between the anatomical determinants change. Therefore, after adequate periodontal treatment, orthodontic treatment may serve orthodontic purposes (Cardaropoli D, Re S, Corrente G, Abundo R. Reconstruction of the maxillary midline papilla following a combined orthodontic-periodontic treatment in adult periodontal patients. J Clin Periodontol 2004;31(2):79-84; Cardaropoli D, Re S. Interdental papilla augmentation procedure following orthodontic treatment in a periodontal patient. J Periodontol 2005;76(4):655-61; Melsen Β, Agerbaek N, Markenstam G. Intrusion of incisors in adult patients with marginal bone loss. Am J Orthod Dentofacial Orthop 1989;96(3):232-41). However, even orthodontic treatment has application limitations. Regardless of possible fixation difficulties, the obvious limitation actually comes from the inter-arch relationship. For this reason, orthodontic treatment is often supported in its final stages by conservative prosthetic correction.

[0020] In 1985, Shapiro described the procedure of periodic curettage of papilla tissue to stimulate its regeneration (Shapiro A. Regeneration of interdental papillae using periodic curettage. Int J Periodontics Restorative Dent 1985;5(5):26-33). This simple operation, repeated every 15 days for 3 months, has proven to be able to induce soft tissue hyperplasia. However, this technique is a plausible option only in cases of very aggressive periodontal disease and, anyway, positive results were not obtained in all cases treated by the author.

[0021] In 2007, a proposal for the injection of fibroblasts into the interdental tissues was published (McGuire MK, Scheyer ET. A randomized, double-blind, placebo-controlled study to determine the safety and efficacy of cultured and expanded autologous fibroblast injections for the treatment of interdental papillary insufficiency associated with the papilla priming procedure. J Periodontol 2007;78(l):4-17). The authors described a non-surgical method represented by the successive injection of autologous fibroblasts (FBL). The basis of this technique lies in the trophic role attributed to these cells. FBL play an important role in the healing and maintenance of the health of the gums by generating collagen matrix and fibers. It was therefore hypothesized that by using these cells it would be possible to promote the regrowth of the papilla. However, the specificity of the proposed technique limits its applicability on an outpatient basis.

[0022] More recently, in addition to conventional treatments, a less invasive treatment was proposed by infiltrating the gums with hyaluronic acid. The treatment is carried out by filling the lost papilla tissue with a filler. This filling is done by using cross-linked hyaluronic acid, which acts as a filler, creating volume and filling the missing areas of the papilla (Bertl K, Gotfredsen K, Jensen SS, Bruckmann C, Stavropoulos A. Can hyaluronan injections augment deficient papillae at implant-supported crowns in the anterior maxilla? A randomized controlled clinical trial with 6 months follow-up. Clin Oral Implants Res. 2017 Sep; 28(9): 1054-1061 (a)). Cross-linked hyaluronic acid creates a volume effect in the infiltrated tissue, but it can last for many months, since degradation occurs very slowly. In fact, the molecule is protected from hyaluronidase degradation as a result of the chemical modification process carried out by the cross-linking step. However, the results obtained are contradictory and side effects are not absent (Bertl K, Gotfredsen K, Jensen SS, Bruckmann C, Stavropoulos A. Adverse reaction after hyaluronan injection for minimally invasive papilla volume augmentation. A report on two cases. Clin Oral Implants Res. 2017 Jul;28(7):871-876)(b)).

[0023] The present invention is based on the recognition that the simultaneous administration of hyaluronic acid and polynucleotides, preferably via topical and / or systemic routes, exerts a positive effect on both periodontitis and on the regeneration of interdental papillae, exhibiting a synergistic effect between the components and allowing the achievement of better results than the administration of the components used separately.

[0024] The inventors have found that in the context of periodontitis and diseases characterized by recession of the interdental papilla, such as, for example, Black Triangle Syndrome, the combination of polynucleotides and hyaluronic acid has an anti-inflammatory, nutritive and biostimulatory effect in the infiltrated area. It has also been found that the composition for use according to the invention, which comprises a combination of polynucleotides and hyaluronic acid, and in which the hyaluronic acid is preferably not cross-linked, is rapidly resorbed. Thus, in comparison with the cross-linked hyaluronic acid infiltration described in the prior art (see, for example, EP1525011 and the aforementioned publication by Bertl et al., 2017), it is essential to recognize that in this case this is not just a filling treatment.

[0025] Indeed, thanks to the biochemical properties of polynucleotides and hyaluronic acid, the invention makes it possible to achieve an anti-inflammatory effect and a physiological response of the tissue, which can promote a significant and spontaneous regeneration of the interdental papilla and an improvement in tissue nutrition.

[0026] Further advantages are that the administration of the compositions for use according to the invention via topical and systemic routes is well tolerated and not very uncomfortable for the patient, even without the use of local anesthesia, the treatment is fast and non-invasive, the treatment is repeatable and does not cause discomfort to the patient's daily life, instead allowing the patient to immediately resume normal social life and a normal diet, contrary to what happens after surgery, no side effects are reported, the treatment is very effective and therefore the patient is very satisfied. Summary of the Invention [Problem to be solved by the invention]

[0027] The object of the present invention is therefore synergistic compositions comprising a polynucleotide and hyaluronic acid as active ingredients, and their use in the applications defined by the appended claims, which form an integral part of this description. [Means for solving the problem]

[0028] Hyaluronic acid (HA) is a sulfur-free polymer that belongs to the glycosaminoglycan family. It is ubiquitous in mammalian tissues, has an unbranched structure, and consists of regularly repeating disaccharide units composed of glucuronic acid and N-acetylglucosamine.

[0029] The size of HA varies but exceeds 1 million daltons, and in some tissues ranges from 3 to 9 million daltons. Initially, HA was isolated from the vitreous substance of the vitreous body by Mayer in 1934, hence the name, and for many years HA was considered as a packing and structural substance with extraordinary hydrophilicity, without any special biological role. Only in the last decade, especially after the discovery of tissue receptors for HA such as CD44 and RHAMM, has attention been focused on the biological role of this polymer. Thus, knowledge of the role of HA as an active substance in more complex processes of tissue regeneration and repair has been expanded. Thus, HA is no longer considered an inert molecule, but a molecule that actively participates in important biological processes such as cell migration and replication, angiogenesis, and tissue growth and maturation.

[0030] Within the scope of the present invention, hyaluronic acid can be used as such or variously salified, for example with sodium.

[0031] Typically, hyaluronic acid or its sodium salt is used with a molecular weight of 500 to 3000 kilodaltons, preferably 600 to 1600 kilodaltons. Further molecular weights of hyaluronic acid that can be used within the scope of the present invention are 700 kilodaltons, 800 kilodaltons, 900 kilodaltons, 1000 kilodaltons, 1200 kilodaltons, 1400 kilodaltons, 1600 kilodaltons, 1800 kilodaltons, 2000 kilodaltons, 2200 kilodaltons, 2400 kilodaltons, 2800 kilodaltons.

[0032] Non-crosslinked hyaluronic acid is preferably used.

[0033] The term "polynucleotide" is intended to denote a fraction of nucleic acid polymer chains, preferably DNA, having a molecular weight distribution preferably in the range of 0.5 kilodaltons to 3000 kilodaltons, more preferably in the range of 30 kilodaltons to 2000 kilodaltons, for example with a weighted numerical average molecular weight of about 250 kilodaltons. Said fraction of DNA polymer chains is obtained by extraction from natural sources of animals or plants, such as fish sperm or plants, by a process that preferably includes a step of partial fragmentation of the DNA chains to reduce their molecular weight, and a step of partial depurination of the DNA to eliminate its information capacity.

[0034] The preferred percentage of DNA depurination is preferably in the range of 1% to 5% of purine bases removed (i.e., depurination sites) from the total purine bases originally present. The preferred percentage of depurination is 2% to 2.5%. Further percentages of depurination of polynucleotides usable within the scope of the present invention are 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.1%, 2.2%, 2.3%, 2.4%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, and 4.9%.

[0035] From the foregoing, it is apparent that the properties of PNs differ significantly from those of conventional polynucleotides, which are not provided below. Partial depurination, -Virus and prion sterilization techniques, · Purification procedures for products with a purity of more than 98% when dry, less than 0.5% protein, and molecular weight range of 0.2-3000 kilodaltons.

[0036] PNs are known and documented to have the ability to induce mitosis in fibroblasts, endothelial cells, and other cells, they have been used to stimulate tissue repair, and they have shown a remarkable ability to promote exonovo synthesis of extracellular matrix molecules in many experimental models.

[0037] In all wound healing processes, the scientific literature has confirmed that PN has a positive effect on wound repair. PN also has a remarkable ability to modulate the action of many inflammatory factors and to interact with physiological growth factors, which makes these molecules of great importance and biologically very active in the proliferation of various cell lines and their secretory activity.

[0038] The polynucleotides used in the present invention preferably have a molecular weight distribution in the range of 0.2 kilodaltons to 3000 kilodaltons, more preferably in the range of 30 kilodaltons to 2000 kilodaltons, and have a numerical average molecular weight of about 250 kilodaltons. The molecular weight distribution of the polynucleotides used in the present invention may also fall within the following ranges: 5 to 2500 kilodaltons, 50 to 1500 kilodaltons, 100 to 1000 kilodaltons.

[0039] The composition according to the invention comprises -Hyaluronic acid as mentioned above - comprising the polynucleotide (PN) described above.

[0040] In the case of liquid formulations (e.g. formulations suitable for injection or topical application), for example, a polyol is used to obtain an appropriate osmolarity. A preferred polyol is mannitol in a concentration of 0 mg / ml to 50 mg / ml. To ensure an approximately neutral pH (7.0 + / - 1.0), a buffer system is used, preferably a phosphate buffer.

[0041] The mannitol used can be of sterile, pyrogen-free pharmaceutical grade for injection according to the European Pharmacopoeia.

[0042] The phosphate buffer used can be pharmaceutical grade, for injection, sterile, and pyrogen-free.

[0043] In addition to liquid formulations, the PN+HA combination for use according to the invention can also be used in the form of gel formulations which are known per se and are commercially available, for example as Nucliaskin® oral care from Mastelli Srl.

[0044] The compositions used typically include: - 1 mg / ml to 20 mg / ml, preferably 5 mg / ml to 12 mg / ml of polynucleotides; - 1 mg / ml to 20 mg / ml, preferably 5 mg / ml to 12 mg / ml of hyaluronic acid, with a polynucleotide / hyaluronic acid weight ratio in the range of 0.2:1 to 2:1. [Table 1]

[0045] The effectiveness of the above compositions in the treatment of periodontitis has been determined by clinical trials with patients suffering from acute or chronic periodontitis. In these clinical trials, which are described in detail in the experimental section below, a commercially available composition within the scope of the present invention (20 mg / ml state-of-the-art - Class III medical device - CE0373) was administered to patients via local infiltration into the periodontal pocket in the vestibule. The results obtained show that the administered composition was able to reduce the depth of the periodontal pocket in 94% of the cases treated. There was also a significant improvement in subjective parameters such as burning sensation, pain, discomfort, bleeding, swelling and redness.

[0046] The effectiveness of the above composition in the regeneration of the interdental papilla was determined by clinical trials with patients who presented with at least one site with papilla loss in the aesthetic area, a papilla defect commonly called "Black Triangle Syndrome". In these clinical trials, which are described in detail in the experimental section below, a commercially available composition within the scope of the present invention (20 mg / ml Modern - Class III Medical Device - CE0373) was administered to the patients through infiltration into the papilla and its surroundings (vestibule). The results obtained show that the administered composition was able to induce an improvement in the clinical picture of the papilla in 100% of the cases treated. Moreover, as shown by extended clinical studies, administration by osmosis of the aforementioned commercially available compositions, possibly due to their combination with hyaluronic acid polynucleotides, did not cause the side effects or adverse effects described in the state of the art (in particular Bertl K. et al., Clin Oral Implants Res. 2017 Sep;28(9):1054-1061(a) and Bertl K et al., Clin Oral Implants Res. 2017 Jul;28(7):871-876)(b)).

[0047] The synergistic effect of compositions containing PN and HA was determined by in vitro tests, described in detail in the experimental section below, aimed at determining their effect on human gingival fibroblast proliferation and protein matrix deposition. The results obtained show that the effect of HA alone is moderate, but the combination with PN enhances the effect of the latter. It can therefore be concluded that the combination of HA and PN results in a synergistic effect in gingival fibroblasts, both in terms of cell regeneration and protein matrix deposition. This may explain the special effectiveness of the combination of HA and PN, in particular in the treatment of Black Triangle Syndrome and in periodontal therapy.

[0048] Finally, a study was carried out to evaluate local inflammation and the presence of metalloproteinases in the periodontal pocket (assessed through sampling of gingival crevicular fluid and measurement of alpha-2-macroglobulin and MMP-9 markers analyzed by ELISA method) in order to verify the modulatory effect of the product on inflammatory cytokines, as well as the local tolerability of the composition containing the combination of PN and HA.

[0049] In this study, 25 patients with periodontal disease with at least two periodontal pockets were evaluated. All patients were treated with the usual standard of care of hygiene and prevention, and in addition, the test MD was used in one of the two pockets in a randomized manner through a single application in the periodontal pocket.

[0050] The results showed that for alpha-2-macroglobulin, 100% of the periodontal pockets treated with the test MD showed improvement, i.e., an improvement of 68%, whereas in the untreated group, one pocket worsened, with a mean improvement of 63%.

[0051] Concerning the MMP-9 parameters, the results show that the periodontal pockets treated with the test MD showed an improvement of 69%, whereas the six untreated pockets deteriorated (24%), with the mean improvement of the untreated pockets being significantly lower at 49%. These results are also particularly important to exclude possible interference due to individual differences, since each patient acts as an autocontrol, i.e. patients have both treated and untreated pockets.

[0052] Thus, in the laboratory, using objective and blinded parameters, it appears that treatment with polynucleotides + hyaluronic acid brings about an improvement compared to standard hygiene and preventive therapy, especially by regulating and reducing parameters related to inflammation, as well as by reducing MMP9 activity, and also highlights the high tolerability and biocompatibility of the device. [Brief description of the drawings]

[0053] (No corresponding part in the original text) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Experimental design 1. Periodontal Treatment - Clinical Research In this study, 15 patients, aged between 30 and 83 years, including 2 men and 13 women, suffering from acute periodontitis (3 patients) or chronic periodontitis (12 patients) were treated. Periodontitis is defined as an inflammation of the periodontal tissues, leading to the formation of periodontal pockets, loose teeth, bleeding gums, abscesses and suppuration, resulting in the loss of one or more teeth. 12 / 15 patients were familiar with the disease when they participated in the study, and 6 / 15 were smokers. Each patient underwent hygiene and prophylaxis according to the guidelines, then was treated with polynucleotides and hyaluronic acid gel (20mg / ml modern - class III medical device - CE0373). [Table 2]

[0055] material and method Each patient underwent a specific treatment program consisting of three main phases. Three visits were scheduled in total, one visit per phase. Details of the program are described below. 1) Pre-treatment phase: Hygiene and preventive sessions are scheduled and carried out (e.g. manual and ultrasonic therapy aimed at reducing the presence of plaque, tartar and therefore bacteria).

[0056] An average of 2–3 20–30 min hygiene and prevention sessions were conducted for each patient each month. Each patient had their first visit (V1) during this phase.

[0057] In V1, patients were clinically evaluated to record subjective parameters (symptoms reported by the patient) and objective parameters (signs detected by the physician).

[0058] 2) Treatment phase with the test medical device: once cleaned, the patient received polynucleotide and hyaluronic acid gel (20 mg / ml latest) by topical and percutaneous route, this phase was divided into several sessions. -The first session consisted of topical application to the periodontal pocket and topical infiltration into the vestibule. -Subsequent monthly sessions included only localized infiltration of the vestibule.

[0059] Each patient underwent a second visit (V2) at this stage, in which subjective and objective parameters were assessed again.

[0060] 3) Maintenance phase: sessions of hygiene and local infiltration of the vestibule with polynucleotides and hyaluronic acid gel (20 mg / ml latest) are performed every 3-6 months.

[0061] Each patient underwent a final study visit (V3) during this phase, in which subjective and objective parameters were assessed again to evaluate the clinical course of the disease compared with the initial (baseline) situation.

[0062] Application to the periodontal pocket was performed using a blunt tip applicator.

[0063] Local infiltration into the vestibule was performed with a 30G needle at a distance of 0.5–1 cm after appropriate local disinfection.

[0064] result At the end of the study, 27% of treated cases showed a reduction in pocket depth of 2 mm, while 67% of cases showed a reduction of 1 mm. In only 6% of cases was the pocket depth unchanged.

[0065] Burning, pain, discomfort, bleeding, swelling and redness were the subjective parameters evaluated during the study in V1 (pre-treatment phase), V2 (treatment phase) and V3 (maintenance phase). All parameters tested showed improvement over the starting conditions. In detail: Pain reduction, assessed using a VAS scale with scores from 0 to 10, averaged 67.3%. In fact, mean values ​​ranged from 3.7 in V1 to 1.2 in V3, a mean reduction of 2.5 points.

[0066] The graph in Figure 1 shows the decrease in pain sensation felt by each patient from Visit V1 to Visit V3.

[0067] The reduction in burning sensation, assessed using a VAS scale with scores from 0 to 10, averaged 64.4%. In fact, the mean values ​​ranged from 3.9 in V1 to 1.4 in V3, a mean reduction of 2.5 points.

[0068] The graph in FIG. 2 shows the reduction in burning sensation perceived by each patient from Visit V1 to Visit V3.

[0069] The reduction in discomfort, assessed using a VAS scale with scores from 0 to 10, averaged 63.6%. In fact, the mean values ​​ranged from 3.7 at V1 to 1.3 at V3, representing an average reduction of 2.3 points.

[0070] The graph in FIG. 3 shows the decrease in discomfort felt by each patient from Visit V1 to Visit V3.

[0071] Reduction in bleeding was assessed with a score of 0–3, with 0 indicating no bleeding and 3 indicating severe bleeding, averaging 64.7%; in fact, the mean values ​​ranged from 2.3 in V1 to 0.8 in V3, a mean reduction of 1.5 points.

[0072] The graph in Figure 4 shows the reduction in bleeding achieved for each patient from Visit V1 to Visit V3.

[0073] In conclusion, treatment of chronic and total periodontal disease with a gel containing a combination of PN+HA (11 / 15 cases treated) provided excellent results in terms of disappearance of swelling and redness. Signs such as bleeding and symptoms such as burning, pain and discomfort also improved by more than 60%. There was also an objective improvement in terms of reduction of pocket depth.

[0074] Therefore, in the dental field, treatment with gels containing a PN+HA combination presents a valuable and innovative therapeutic option associated with hygiene and preventive sessions to improve and strengthen the response to conventional hygiene and preventive therapies.

[0075] 2. Clinical trials of nipple regeneration (Black Triangle Syndrome) Ten patients, including seven men and three women, with a mean age of 52 years were treated in this study. Two of the patients were smokers at the start of the study. In all cases, patients presented with at least one site of nipple loss in the aesthetic area, a nipple defect commonly referred to as "black triangle syndrome." [Table 3]

[0076] material and method Each patient underwent a specific treatment program consisting of two main phases. One visit was scheduled for each phase during the study, and one follow-up visit was scheduled after the treatment phase, for a total of three visits. The details of the program are described below. 1) Pretreatment phase: the phase during which hygiene and preventive sessions were scheduled and carried out (e.g. manual and ultrasound therapy aimed at reducing the presence of plaque, tartar and therefore bacteria). All patients were instructed to carry out mechanical cleaning at home using an extra-soft toothbrush and a brushing technique with apical-coronal movements.

[0077] An average of 2–3 20–30 min hygiene and prevention sessions were conducted for each patient each month. Each patient had their first visit (V1) during this phase.

[0078] Photographs were taken at V1, and physician clinical evaluation and patient satisfaction were recorded.

[0079] 2) Treatment phase with the test medical device: At least one month after stabilization of the clinical picture after hygiene and prophylactic sessions, patients were administered polynucleotides and hyaluronic acid gel (20 mg / ml latest).

[0080] After disinfection with a mouthwash and a local disinfectant at the infiltration site, the affected area was treated by infiltrating a small amount of the product (generally less than 0.1-0.2 ml) into the nipple and its surrounding area (vestibule). Local infiltration into the vestibule was performed with a 30 G needle at a distance of 0.5-1 cm.

[0081] Treatments were repeated at 3-6 week intervals for a total of 3-10 infiltrations.

[0082] Each patient underwent a second visit (V2) at this stage, during which the effect was documented photographically by comparing it with the one taken before treatment and by completing a data collection form that recorded the physician's clinical evaluation and the patient's satisfaction.

[0083] result The subjective assessment of the patients was very positive, both in terms of pain and compliance with treatment, and satisfaction with the above treatment, with the following results:

[0084] The clinical results were very encouraging. 100% of patients showed improvement as assessed on a quartile scale. Although not all cases showed complete healing, all cases showed clinical improvement of the nipple.

[0085] On average, regrowth began to appear after the second or third session, but in some cases regrowth began after the seventh or eighth session.

[0086] Regarding objective evaluation of the nipple, all patients showed improvement in nipple structure, including a change in classification. - Class III nipples became Class I nipples in 3 cases - Class II nipples became Class I nipples in 6 cases In some cases, Class I papillae became normal (the papillae filled the interdental space again up to the contact point).

[0087] It should be emphasized in particular that the improvement of the nipples occurred very quickly; all patients showed an initial regeneration of the nipples already after one week, and this regeneration process continued over time. In fact, at the follow-up the sites were checked and the improvement that had occurred after application one month after the last treatment was still present. There was no recurrence in any of the cases.

[0088] In conclusion, the trophic / regenerative effect of only locally infiltrated polynucleotides has been shown for the first time in the treatment of Black Triangle Syndrome, a disease that has previously been treated with invasive methods such as orthodontic, restorative and surgical interventions, but with poor clinical outcomes.

[0089] 3. In vitro study of gingival fibroblasts Human gingival fibroblasts were cultured in growth medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (BFS). Cells were cultured at 10,000 / cm 2 After 24 hours from seeding, the cells were incubated under the conditions shown in Table 3 and then resuspended in DMEM + 10% BFS.

[0090] Cells were counted and protein was quantified after 3 days of treatment.

[0091] Cell numbers were measured with a particle counter, and protein was quantified by spectrophotometric reading of stained protein lysates by using a modification of the Lowry method.

[0092] Table 3 presents the concentrations of the compositions tested, as well as the values ​​related to cell proliferation and protein content, expressed as a percentage of the control, which is a fibroblast culture not subjected to any treatment, measured at the same time points. [Table 4]

[0093] Cell proliferation and protein quantification results are also shown in Figures 5 and 6 .

[0094] The results show that hyaluronic acid does not change its action as its concentration increases, but rather constantly maintains a mild effect in terms of both protein expression and cell proliferation. Instead, it is clear that polynucleotides increase their effect as the dose increases. Compared to the individual components, the effect obtained with the mixture (consisting of 50% hyaluronic acid and 50% polynucleotide for each total concentration) was consistently superior to that of PN used alone and of HA used alone.

[0095] Therefore, keeping in mind the low efficacy of hyaluronic acid alone, it is clear that the presence of hyaluronic acid is important to enhance the effect of PN, the most active component in cell proliferation and protein expression. It can be concluded that the combination of HA and PN results in a synergistic effect in gingival fibroblasts, both in terms of cell regeneration and protein matrix deposition. This may explain the special efficacy of the combination of HA and PN, especially in the treatment of Black Triangle Syndrome and in periodontal therapy.

[0096] 4. In vivo studies of gingival crevicular fluid Treatment of periodontitis involves first controlling bacteria and inflammation, followed closely by the next step of regenerating lost hard and soft periodontal tissues in order to restore tooth attachment and eliminate gingival and bone pockets.

[0097] ODONTO-PNHA is a class IH medical device manufactured by Mastelli Srl-Sanremo (IM). It is a proprietary combination of polynucleotides and hyaluronic acid for application in the periodontal pocket.

[0098] The primary objective of the clinical trial with Odonto-PNHA is to evaluate the efficacy and safety of the medical device in non-surgical periodontal treatment. The experimental hypothesis is that the addition of Odonto-PNHA may help to obtain periodontal attachment gain in the periodontal pocket to avoid surgical procedures.

[0099] To evaluate this, a total of 50 patients were recruited, each of whom provided a test site (treated with ODONTO-PNHA) and a control site (untreated). Patients will be followed up for 12 months after treatment. The study is being carried out in a single clinical center at the Department of Periodontology, Universita La Sapienza.

[0100] Primary objectives: To evaluate the performance of ODONTO-PNHA on probing pocket depth (PPD), marginal bleeding index (MB), modified plaque index (mPLI), pain, investigator's global satisfaction with ease of use of the device, and patient's global satisfaction with device performance (5-point Likert scale). Tolerability and safety assessment.

[0101] Aim of biochemical analysis: To evaluate the effect of ODONTO-PNHA on the inflammatory state of the tissues around the teeth in 25 subjects. This was assessed by sampling gingival crevicular fluid and evaluating alpha-2 macroglobulin (a2M) and metalloproteinase-9 (MMP-9) in the fluid at baseline and 6 weeks.

[0102] Gingival crevicular fluid samples adsorbed onto methylcellulose strips were processed and analyzed by ELISA.

[0103] Matrix metalloproteinases are an important group of proteinases involved in collagen degradation during the destructive process of periodontal disease and can be measured in gingival crevicular fluid. Alpha-2 macroglobulin is a plasma protein involved in the regulation of blood clotting and immune response and is increased in the presence of chronic infection. Twenty-five patients with periodontal disease and recruited in the ODONTO-PNHA-01 clinical trial underwent two gingival crevicular fluid samplings, one from periodontal pockets treated with the study MD and one from untreated pockets.

[0104] Collections were performed at baseline (Visit 1) and at week 6 (Visit 2). A total of four samples were assessed for the presence and concentration of inflammatory markers.

[0105] result After 6 weeks, the concentrations of markers in both polynucleotide and hyaluronic acid MD-treated and untreated pockets were significantly lower compared to week 0, indicating a general improvement in periodontal disease thanks to standard hygiene and preventive therapy.

[0106] Analysis of data from periodontal pockets treated with the study MD and untreated periodontal pockets showed further differences to the benefit of patients treated with the study MD, which are explained below.

[0107] In treated pockets, the mean alpha2 macroglobulin value was 1898.71 at time 0 and 605.90 after 6 weeks, with a mean reduction of 1292.81, corresponding to 68.09%. Of the 25 treated pockets, 19 (76%) showed an improvement of greater than 50% (Table 4).

[0108] In untreated pockets, the mean alpha-2 macroglobulin value was 1665 at time 0 and 601 after 6 weeks, a mean reduction of 1064 corresponding to 63.91%. Of the 25 treated pockets, 17 (68%) showed an improvement of greater than 50% (Table 4). [Table 5]

[0109] Even greater differences between treatment and control groups were seen in the MMP-9 parameters. In treated pockets, the mean MMP-9 value was 234.9 at time 0 and 72.9 after 6 weeks, a mean reduction of 162.00 corresponding to 68.96%. Of the 25 treated pockets, 18 (72%) showed an improvement of more than 50% (Table 5).

[0110] In untreated pockets, the mean MMP-9 value was 173.4 at time 0 and 87.4 after 6 weeks, representing a mean reduction of 86 of 49.58%. Of the 25 treated pockets, only 9 (36%) showed an improvement of 50% or more (Table 5). [Table 6]

[0111] The results showed that for alpha-2 macroglobulin, all treated patients showed improvement, whereas in the untreated group, one patient showed deterioration, and for MMP-9 parameters, all but one patient showed improvement, whereas in the untreated group, six patients showed deterioration.

[0112] In general, the improvement was greater in patients treated with test MD, and this improvement was particularly evident in MMP-9 parameters.

[0113] These results are particularly important because each patient served as an autologous control, i.e., patients had both treated and untreated pockets, thus eliminating any possibility of interference due to individual differences.

[0114] conclusion Thus, in the laboratory, using objective and blinded parameters, treatment with polynucleotides + hyaluronic acid also appears to bring about improvements compared to standard hygiene and preventive therapy, in particular by regulating and reducing parameters related to inflammation and reducing MMP9 activity.

[0115] Therefore, the preliminary experimental results presented above support the experimental hypothesis that the combination of PN+HA is useful for obtaining periodontal attachment gain in the periodontal pocket to avoid surgical procedures.

Claims

1. 1. A composition comprising hyaluronic acid and a mixture of polynucleotides extracted from a natural source for use as an active agent in therapeutic and / or cosmetic treatments for the regeneration of interdental papillae, or in therapeutic and / or cosmetic treatments for recession of interdental papillae (Black Triangle Syndrome), or in the therapeutic and / or cosmetic treatment of periodontitis, wherein said mixture of polynucleotides has a molecular weight distribution in the range of 0.5 kilodaltons to 3000 kilodaltons, and said hyaluronic acid has a molecular weight of 500 kilodaltons to 3000 kilodaltons.

2. The composition for use according to claim 1, wherein the periodontitis is acute periodontitis or chronic periodontitis.

3. A composition for use according to claim 1 or 2, wherein the therapeutic and / or cosmetic treatment results in a reduction in periodontal pocket depth and / or reduced pain and / or reduced stinging and / or reduced discomfort and / or reduced bleeding.

4. A composition for use according to any one of claims 1 to 3, wherein said therapeutic and / or cosmetic treatment causes nipple regeneration.

5. A composition for use according to any one of claims 1 to 4, wherein said therapeutic and / or cosmetic treatment results in increased proliferation of gingival fibroblasts and increased deposition of a protein matrix.

6. The composition for use according to any one of claims 1 to 5, wherein the treatment is carried out by topical application and / or topical penetration.

7. A composition for use according to any one of claims 1 to 6, wherein said treatment is effected by local application within said periodontal pocket.

8. A composition for use according to any one of claims 1 to 7, wherein the treatment is carried out by local gingival infiltration in the papilla and / or vestibule.

9. A composition for use according to any one of claims 1 to 8, comprising 1 mg / ml to 20 mg / ml of hyaluronic acid and 1 mg / ml to 20 mg / ml of said mixture of polynucleotides.

10. 10. The composition for use according to claim 9, comprising 5 mg / ml to 12 mg / ml of hyaluronic acid and 5 mg / ml to 12 mg / ml of said mixture of polynucleotides.

11. The composition for use according to claim 9 or 10, comprising said polynucleotide and said hyaluronic acid in a weight ratio of 0.2:1 to 2:

1.

12. The composition for use according to any one of claims 1 to 11, wherein said mixture of polynucleotides is partially depurinated.

13. The composition for use according to any one of claims 1 to 12, wherein the mixture of polynucleotides has a depurination ratio in the range of 1% to 5% of purine bases removed from the total purine bases originally present.

14. The composition according to any one of claims 1 to 13, wherein the mixture of polynucleotides is a fraction of nucleic acid polymer chains having a molecular weight distribution in the range of 30 kilodaltons to 2000 kilodaltons, for example an average molecular weight of about 250 kilodaltons.

15. The composition for use according to any one of claims 1 to 14, wherein said mixture of polynucleotides is obtainable by extraction from natural animal or plant sources, by a process comprising a step of partial fragmentation of said polynucleotide nucleic acid chains and a step of partial depurination of said nucleic acids.

16. The composition for use according to any one of claims 1 to 15, wherein the hyaluronic acid is not cross-linked.

17. A composition for use according to any one of claims 1 to 16 in saline-free liquid form in an aqueous solvent comprising a pharma- ceutically acceptable polyol, preferably mannitol.

18. The composition for use according to claim 17, having an osmolality of from 10 m.o.s. / l to 500 m.o.s. / l, preferably from 250 m.o.s. / l to 350 m.o.s. / l.

19. A composition for use according to any one of claims 1 to 18, which is in a form suitable for topical application or topical penetration.

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