CH710622A2 - topical pharmaceutical formulation containing the butyric ester of hyaluronic acid for the treatment of symptoms associated with primary dentition. - Google Patents

Abstract

The present invention relates to a formulation containing butyrate hyaluronic acid or a pharmaceutically acceptable salt thereof for the use in the topical treatment of the symptoms associated with the deciduous dentition.

Description

[0001] The present invention relates to formulations containing a butyric ester of hyaluronic acid for topical use in the treatment of symptoms associated with deciduous dentition.

State of art

[0002] The term "teething" refers to the process of birth and development of teeth within the mouth. There are 20 milk teeth, or deciduous teeth, they erupt around 6–7 months of life to reach completion around 3 years.

[0003] That of the newborn is also called "first dentition" to distinguish it from the permanent dentition of the adult. Despite being already included and partially calcified in the maxillary bones, originating already from the seventh week of gestation from the ectodermal germinal leaflet, deciduous teeth tend to appear only a few months after birth.

[0004] After birth, the teeth gradually erupt through the gums and tend to start to tend towards the sixth month of life. Normally, the first milk teeth to erupt are the two central incisors due to inflammation and relieves pain.

[0005] Hyaluronic acid butyrate (HABut) in which the hydroxyl groups of hyaluronic acid are esterified with butyric acid residues with different degrees of substitution is known to have anti-proliferative and dermoprotective properties as an elasticising and moisturizing skin.

[0006] EP 0 941 253 and WO 2005/092 929 describe the preparation of the butyric esters of hyaluronic acid with different degrees of substitution. These esters have a high antiproliferative activity, which makes them useful in the treatment of diseases characterized by abnormal cell proliferation.

Description of the invention

[0007] The subject of the present invention is therefore a formulation containing butyrate hyaluronic acid or a pharmaceutically acceptable salt thereof, preferably sodium salt for the topical treatment of the symptoms associated with the deciduous dentition.

[0008] Preferably the hyaluronic acid butyrate salt sodium used in the formulations of the invention has a molecular weight between 10 <3> and 10 <7>, Dalton more preferably between 10 <4> and 10 <6> Dalton, and a degree of substitution (DS) from 0.01 mol / mol to 3, preferably from 0.1 to 2, where the degree of substitution is defined as the ratio between the number of butyric acid residues and the number of disaccharidic units GlcNAc- GlcUA of hyaluronic acid.

[0009] Hyaluronic acid butyrate sodium salt is preferably present in the formulation from 0.01% to 5% by weight with respect to the total weight of the formulation, more preferably from 0.05% to 1%.

[0010] In a preferred embodiment of the invention the formulation contains panthenol, preferably from 0.1% to 5% by weight with respect to the total weight of the formulation, more preferably from 0.5% to 3%.

[0011] The pharmaceutical formulation preferably contains plant extracts with soothing properties such as extract of Mallow sylvestris and Chamomilla recutita or Matricaria chamomilla (Commiphora myrrha, Calendula officinalis, Aloe barbadensis, Glycyrrhiza glabra).

[0012] Other pharmaceutically acceptable agents can be added to the formulation such as carrier agents, emulsifying agents, gelling agents, preservatives, antioxidant agents, buffering agents, sweeteners, flavorings and / or mixtures thereof.

[0013] The formulations of the invention are preferably in the form of a cream, gel or solution for topical use for use in the oral cavity.

[0014] The formulations of the invention can be applied on the gum 1 to 20 times, preferably 1 to 10 times a day depending on the need.

[0015] A preferred embodiment of the invention is the formulation containing: <tb> Purified Water / Demineralised Water <SEP> q.b. 100 g <tb> Panthenol / Panthenol <SEP> from 0.1 to 5 g <tb> Malva sylvestris Extract (Hydroglyceric extract <1%) <SEP> from 0.01 to 5 g <tb> Chamomilla recutita Extract (Hydroglyceric extract <1%) <SEP> from 0.5 to 5 g <tb> Xantana / Xanthan Gum rubber <SEP> from 0.01 to 1 g <tb> Stevia <SEP> from 0 to 0.7 g <tb> Aroma Mela (1: 150) natural <SEP> from 0 to 2 g <tb> <SEP> hydroxyethylcellulose from 0.5 to 5 g <tb> Sodium Hyaluronate Butyrate <SEP> from 0.05 to 1 g <tb> Potassium Sorbate <SEP> 0 to 0.5 g <tb> Sodium Benzoate <SEP> from 0 to 0.5 g <tb> Citric Acid <SEP> from 0 to 5 g <tb> Sodium Citrate <SEP> from 0 to 5 g

Example 1

CHAMODENT BABY GEL

[0016] <tb> Purified Water / Demineralised Water <SEP> q.b. 100 g <tb> Panthenol / Panthenol <SEP> 1.00 g <tb> Malva sylvestris Extract (Hydroglyceric extract <1%) <SEP> 1.00 ́g <tb> Chamomilla recutita Extract (Hydroglyceric extract <1%) <SEP> 2.00 g <tb> Xantana / Xanthan Gum rubber <SEP> 0.20 g <tb> Stevia <SEP> 0.10 g <tb> Aroma Mela (1: 150) natural <SEP> 0.50 g <tb> Hydroxyethylcellulose <SEP> 2.00 g <tb> Sodium Hyaluronate Butyrate (SHB by Sigea S.r.l.) <SEP> 0.10 g <tb> Potassium Sorbate <SEP> 0.10 g <tb> Sodium Benzoate <SEP> 0.10 g <tb> Citric Acid <SEP> 3.60 g <tb> Sodium Citrate <SEP> 1.20 g

Example 2

[0017] <tb> Purified Water / Demineralised Water <SEP> q.b. 100 g <tb> Panthenol / Panthenol <SEP> 1.5 g <tb> Malva sylvestris Extract (Hydroglyceric extract <1%) <SEP> 1.50 g <tb> Chamomilla recutita Extract (Hydroglyceric extract <1%) <SEP> 3.00 g <tb> Xantana / Xanthan Gum rubber <SEP> 0.20 g <tb> Stevia <SEP> 0.30 g <tb> Aroma Mela (1: 150) natural <SEP> 0.40 g <tb> Hydroxyethylcellulose <SEP> 3.00 g <tb> Sodium Hyaluronate Butyrate (SHB by Sigea S.r.l.) <SEP> 0.10 g <tb> Potassium Sorbate <SEP> 0.10 g <tb> Sodium Benzoate <SEP> 0.10 g <tb> Citric Acid <SEP> 3.00 g <tb> Sodium Citrate <SEP> 1.00 g

[0018] The formulation was obtained by the following procedure:

[0019] PHASE 1: In a suitable mixer with blades and turbine the demineralized water was loaded and panthenol, the glycerine extract of mallow, the glyceric extract of chamomile, the potassium sorbate, were added in the order. sodium benzoate, aroma of apple juice, stevia, citric acid and sodium citrate. The mixture was stirred under vacuum.

[0020] PHASE 2: The solution obtained in the previous phase was filtered in series at 0.5 and 0.2 microns.

[0021] PHASE 3: The sodium hyaluronate butyrate, the xanthan gum and the hydroxyethylcellulose were added and the mixture obtained was kept under stirring under vacuum.

[0022] PHASE 4: The product obtained was unloaded into the loading hopper of the tube filling machine, appropriately calibrated for distribution in the primary packaging.

Comparison between Chamodent Baby Gel and commercial products

[0023] To evaluate the influence of the Chamodent Baby Gel formulation on the acute inflammatory process, comparing it with that of commercial products, an in vitro experimental model was used which uses leukocytes that first reach the site of phlogosis: polymorphonuclear neutrophils (PMN). The idea is to simulate an inflammatory environment and evaluate how these cells, stimulated by a pro-inflammatory cytokine (TNF-α, tumor necrosis factor), respond in the presence of the substances under examination.

[0024] The anti-inflammatory effect was analyzed by evaluating two fundamental functional responses of PMNs: the production of reactive oxygen species (ROS) following metabolic activation and the ability to adhere to biological surfaces.

[0025] The acute inflammatory response promoted by treatment with the pro-inflammatory cytokine is considered to be proportional to the number of cells adhered to the quantity of ROS produced.

Evaluation of superoxide anion production

[0026] The production of ROS, index of PMN metabolic activation, was evaluated in terms of superoxide anion (O2 <–>) released in the medium after incubation for 60 minutes of cells with TNF-α in plate wells from microtitulation coated with Hyaluronic Acid (HA), Laluronic Acid Butyrate (HABut), Dentinal, Dentin n. and Alovex. We used a spectrophotometric method that allows us to measure the amount of cytochrome c reduced by the superoxide anion produced by the cells during plate incubation. The wells coated with the various substrates are filled with a solution of cytochrome c 0.18 mM and TNF 0.15 ng / ml in Hepes buffer. The modules thus prepared are heated for 10 to 37 degrees in a humidified incubator; to each well is added a cell suspension of 1.5 x 10 <6> PMN / ml in Hepes buffer. At 15 minute intervals the plate is taken from the incubator and subjected to spectrophotometric analysis in a microplate reader at a wavelength of 550 nm and 540 nm, which correspond to the peak of cytochrome c reduced absorption and the isosbestic point respectively of the reduced and oxidized cytochrome c absorption spectra. The difference between the absorbance values recorded at the two wavelengths is proportional to the quantity of cytochrome c reduced. The quantity of O2 <–> produced by 10 <6> cells is calculated as follows: nmoles O2 <–> / 10 <6> PMN = D.O. x 10 <6> /0.0037 x n where n is the number of cells added to each well.

Evaluation of adhesion to biological surfaces

[0027] The quantity of cells that had adhered to the substrate during the metabolic assay was evaluated by measuring the activity of the enzyme myeloperoxidase, taken as a marker of neutrophils. After performing spectrophotometric readings to measure O2 <–> production, the microplate wells are filled with PBS and centrifuged at 200 g for 5 minutes to remove the unadjusted cells from the surface. The dosage of myeloperoxidase activity is carried out by measuring the oxidation of the substrate 3 ́, 5, 5 ́-tetramethylbenzidine (TMB) by the MPO enzyme in the presence of H2O2. Acetate buffer containing TMB, cetyltrimethylammonium (CTAB) and 3-amino-1,2-4-triazoum (AMT) is added to each well and the plate is stirred for 5 ́ to facilitate cell lysis and promote the release of MPO from the granules . The eosinophiloperoxidase activity deriving from eosinophils that can contaminate PMN preparation is inhibited with ATM. After 2 minutes from the addition of H2O2 the reaction is blocked with H2SO4 and the absorbance of each well is measured at a wavelength of 405 nm. The percentage of adherent cells is calculated by referring to a standard curve constructed, in each experiment, based on the peroxidase activity values calculated for known quantities of cells.

[0028] The results of the experimentation are shown in fig. 1 and fig. 2 .

[0029] Fig. 1: Production of superoxide anion released from neutrophils in contact with surfaces coated with Ha, HABut Chamodent Dentinale, Dentinale Natura and Alovex.

[0030] Resting: PMN not activated with TNF. TNF: PMN activated with TNF-α.

[0031] Fig. 2: Adhesion of PMN to a surface coated with Ha, HABut, Chamodent Dentinale, Dentinale Natura and Alovex.

[0032] Resting: PMNs not activated with TNF. TNF: PMN activated with TNF-α.

Conclusions

[0033] The experiments show a clear anti-inflammatory activity of Chamodent Baby Gel, better also compared to the only sodium hyaluronate butyrate (HABut produced by Sigea), but above all compared to commercial formulated products (Dentinale produced by Montefarmaco, Dentinale Nature produced by Montefarmaco and Alovex produced by Recordati). Compared to the other substances tested, in the presence of Chamodent Baby Gel, the activated neutrophils show a lower release of superoxide and a lower level of adhesion to the surface behavior indicative of the action of a compound with anti-inflammatory activity. As reference we wanted to use sodium hyaluronate (HA) which has the worst performance of Chamodent Baby Gel.

[0034] All the results are consistent with the thesis of a better performance of Chamodent Baby Gel compared to the other substances, pure or formulated, tested.

Claims (13)

1. Formulation containing butyrate hyaluronic acid or a pharmaceutically acceptable salt thereof for use in the topical treatment of symptoms associated with deciduous dentition. 2. Formulation for use according to claim 1 containing hyaluronic acid butyrate sodium salt. 3. Formulation for use according to claim 2 wherein the sodium hyaluronic acid butyrate salt has a molecular weight between 10 <3> and 10 <7> Dalton. 4. Formulation for use according to claim 3 wherein the sodium hyaluronic acid butyrate salt has a molecular weight between 10 <4> and 10 <6> Dalton. 5. Formulation for use according to each of claims 2 to 4 in which the sodium hyaluronic acid butyrate salt has a degree of substitution of 0.1 to 2 mol / mol. 6. Formulation for use according to each of claims 2 to 5 wherein the hyaluronic acid sodium salt is present at a concentration from 0.05% to 1% by weight with respect to the total weight of the formulation. 7. Formulation for use according to each of the preceding claims, further containing panthenol. 8. Formulation for the use according to claim 7 wherein the panthenol is present at a concentration from 0.1% to 5% by weight with respect to the total weight of the formulation. 9. Formulation for use according to each of the preceding claims, furthermore containing one or more plant extracts with soothing and / or anti-inflammatory properties. 10. Formulation for use according to claim 9 wherein the plant extract with soothing properties is selected from Malva sylvestris extract, Chamomilla recutita, Matricaria chamomilla, Commiphora myrrha, Calendula officinalis, Aloe barbadensis, Glycyrrhiza glabra. 11. Formulation for use according to each of the previous claims further containing a carrier, emulsifying, gelling, preserving, antioxidant, buffering, sweetening and / or flavoring agent and / or mixtures thereof. 12. Formulation having the following composition for the use in the topical treatment of the symptoms associated with the deciduous dentition: <tb> Purified Water / Demineralised Water <SEP> q.b. 100 g <tb> Panthenol / Panthenol <SEP> from 0.1 to 5 g <tb> Malva sylvestris Extract (Hydroglyceric extract <1%) <SEP> from 0.01 to 5 g <tb> Chamomilla recutita Extract (Hydroglyceric extract <1%) <SEP> from 0.5 to 5 g <tb> Xantana / Xanthan Gum rubber <SEP> from 0.01 to 1 g <tb> Stevia <SEP> from 0 to 0.7 g <tb> Aroma Mela (1: 150) natural <SEP> from 0 to 2 g <tb> <SEP> hydroxyethylcellulose from 0.5 to 5 g <tb> Sodium Hyaluronate Butyrate <SEP> from 0.05 to 1 g <tb> Potassium Sorbate <SEP> 0 to 0.5 g <tb> Sodium Benzoate <SEP> from 0 to 0.5 g <tb> Citric Acid <SEP> from 0 to 5 g <tb> Sodium Citrate <SEP> from 0 to 5 g 13. Formulation for use according to each of the previous claims in the form of a cream, gel or solution.