ES2671184B2 - FORMULATION FOR MODIFIED RELEASE OF ACTIVE SUBSTANCES OR DRUGS, USE AND PROCEDURE OF OBTAINING - Google Patents

Description

FORMULATION FOR MODIFIED RELEASE OF ACTIVE SUBSTANCES OR

DRUGS, USE AND PROCEDURE FOR OBTAINING

D E S C R I P C I O N

OBJECT OF THE INVENTION

The present invention relates to a pharmaceutical formulation for the modified release of active substances and / or drugs, for the coating of the skin and for the healing of wounds.

BACKGROUND OF THE INVENTION

In the field of biomedicine, hydrogel-type polymer systems have a great potential, having developed systems applicable in drug release, cosmetics, wound treatment, tissue engineering, etc.

Numerous examples of hydrogels with potential applications in different ways are found in the state of the art. For example, WO 2011138484 A1 describes a topical preparation for the treatment of nail disorders based on a thermosensitive hydrogel that is applied as a liquid on the nails and at body temperature gels forming a hydrated polymeric film that releases active principles. WO 2011135150 A1 discloses the composition of ionic hydrogels based on natural anionic polymers and cationic crosslinking molecules for their application as medicaments, medical devices, tissue engineering, cosmetics, regenerative medicine. Patent WO 2010018293 A1 describes the process for obtaining acrylic hydrogels with cyclodextrins for application in contact lenses, such as topical, transdermal or transmucosal delivery systems or in the preparation of cosmetics. WO 2014041231 A1 discloses injectable hydrogels generated in situ by "click chemistry" crosslinking, useful as implants in tissue regeneration and for the controlled release of drugs, WO 2011157880 A1 refers to an antioxidant composition hydrogel type for the treatment of conditions of the skin that occur with the production of reactive oxygen species or as dressings for wounds and administration of drugs to wounds.

A study has also been published on a three-dimensional polysiloxane polymer network cross-linked chemically in situ by a platinum catalyst that is applied to the skin forming an elastic and transparent film with mechanical properties similar to healthy young skin and which is capable of visibly reducing wrinkles and swelling of the bags under the eyes. ( Yu B., Kang SY, et al., An elastic second skin, Nat Mater 2016; 15 ( 8): 911-918.)

As for dressings for wound healing, hydrogels have certain properties that make them suitable for such application. The hydrogels can absorb and retain the contaminated exudate within the gel mass through the expansion of crosslinked polymer chains favoring the isolation of bacteria, detritus and odor molecules in the liquid. Its high water content allows moisture to be maintained in the wound, favoring the transmission of steam and oxygen to wounds and autolytic debridement (which facilitates the elimination of dead tissue).

In this sense, several pharmaceutical companies have developed different formulations based on hydrogels for the treatment of wounds, among which we can mention: Granugel® (ConvaTec) formed by pectin, carboxymethylcellulose and propylene glycol, Intrasite Gel® (Smith & Nephew) composed of carboxymethylcellulose modified and propylene glycol, Purilon Gel® (Coloplast) made with sodium carboxymethylcellulose, etc. ( Blanco MD, Olmo RM, Teijón JM Hydrogels, In: Swarbrick J., editor, Encyclopedia of Pharmaceutical Technology Third Edition: Informa Healthcare, 2006: 2021-2039.)

Given the importance of these polymeric systems, the search for new molecules with crosslinking capacity allows the development of new formulations with different physicochemical characteristics, which may represent an advance in different pharmaceutical applications.

6-phosphogluconic acid (6-PG) is a metabolite of the pentose phosphate pathway in which glucose is degraded to obtain pentose-phosphate for the biosynthesis of nucleic acids. In patent US3639594A, 6-PG and its salts are described as regenerating agents of the liver tissue. However, there is no evidence that 6-PG has been used as a crosslinking agent.

DESCRIPTION OF THE INVENTION

The technical problem that arises is the application of certain substances, either drugs or cosmetic substances, indirectly by a prolonged release of the main substance preferably topically or transdermally. To this end, the formulation that is the object of the present invention is developed, which allows the retention of the drug or substance by entrapment and its release once it is applied to the skin, mucous membranes or cavities, and another of its uses may be protection of the skin against external aggressions or the treatment of wounds.

In the present invention, "hydrogel" is understood to be the three-dimensional polymer network obtained by crosslinking from hydrophilic polymers, natural or synthetic, which can absorb and retain a significant amount of water.

"Crosslinking" or "crosslinking" is understood to be the physical or chemical union between different points of the polymer chains that result in the formation of a hydrogel. The term "crosslinking agent" or "crosslinking agent" refers to that molecule capable of crosslinking between the polymer chains.

The term "drug", "active substance" or "therapeutic agent" refers to any substance that is used in the treatment, cure, prevention or diagnosis of a disease or that is used to improve the physical and mental well-being of human beings and animals. The proportion of this will depend in each case on the drug to be incorporated, the indication for which it is used and the efficiency of administration. When one or more drugs are incorporated into the system of the invention, they can be dispersed at the molecular, particle or systemic level to improve their solubility or control their release. Drugs of interest for the invention, but not limited exclusively to them, are corticosteroids, antifungals, antibacterials, local anesthetics, analgesics, anti-inflammatories, muscle relaxants, etc.

One aspect of the invention relates to the preparation of hydrogels from cationic polymers crosslinked by a crosslinking agent called trisodium salt of 6-phosphogluconic acid (6-PG "Na +.) In particular, the example of hydrogels formed by chitosan is shown. , as cationic polymer, crosslinked with 6-PG "Na +. Another aspect of this invention relates to the obtaining of polymeric networks or matrices hydrogel type, effective for the encapsulation and subsequent controlled administration of therapeutic agents or active ingredients by different routes of administration with local or systemic effect. In particular, the invention relates to obtaining non-toxic and non-irritating hydrogels that can be easily applied and extendable on the skin forming a transparent film with adequate adhesiveness and capable of releasing active substances. In particular, the invention relates to a hydrogel-like formulation that, when applied in the form of a dressing, facilitates the healing of wounds.

This application composition preferably topical or transdermal with possible application by other routes (oral, vaginal, rectal ...) allows the release of active substances in a controlled manner.

According to the invention, a process based on the formation of a hydrogel is detailed, where the polymeric crosslinking of the matrix occurs in situ by the use of 6-PG "Na + as a crosslinking agent in the absence of organic solvents, cosolvents, chemical reagents external or chemical reactions that generate them.

DESCRIPTION OF THE DRAWINGS

To complete the description that is being made and in order to help a better understanding of the characteristics of the invention, a preferred example of the practical realization thereof is included. A series of figures is included as an integral part of said description.

Figure 1. We can observe the 31P-NMR spectrum in solution for 6-PG "pure Na +.

Figure 2. We can observe the 31P-NMR spectrum in the solid state for sample M60-275.

Figure 3. In the graph we can see the results of TGA of chitosan and hydrogels without drug with different percentages of crosslinking with 6-PG-Na +.

Figure 4. In this graph we can see the results of DTG of chitosan and hydrogels without drug with different percentages of cross-linking with 6-PG "Na +.

Figure 5. The graph shows the TGA results of the hydrogels with Piroxicam with different percentages of crosslinking with 6-PG "Na +.

Figure 6. The graph shows the DTG results of the hydrogels with Piroxicam with different percentages of crosslinking with 6-PG-Na +.

Figure 7. X-ray diffractogram of 6-PG-Na +, chitosan and cross-linked hydrogels from 50 to 275% without drug and hydrogels cross-linked to 50 and 275% with Piroxicam.

Figure 8. We observed the swelling profiles of samples M60-100 and M60-275 in aqueous medium buffered at pH 1.2.

Figure 9. We observed the swelling profiles of samples M60-100 and M60-275 in aqueous medium buffered at pH 4.5.

Figure 10. We observed the swelling profiles of samples M60-100 and M60-275 in aqueous medium buffered to pH 6.8.

Figure 11. We observed the swelling profiles of samples M60-100 and M60-275 in aqueous medium buffered to pH 7.5.

Figure 12. Piroxicam release profiles according to the pH of the medium (1.2, 4.5, 6.8 and 7.5) for the M6P-100 hydrogel.

Figure 13. Release profiles of Piroxicam as a function of the pH of the medium (1.2, 4.5, 6.8 and 7.5) for the M6P-275 hydrogel.

Figure 14. We observed the cellular survival percentages of Caco-2 cultures at 24, 48 and 72 h in the absence (control) and presence of: 6-PG-Na +, chitosan, M60-100 and M60-275.

Figure 15. Extensibility values of the commercial gel Salvacam® and the hydrogels M60-100 and M60-275 depending on the weight applied.

Figure 16. Percentage of drug permeated in skin and receptor for M6P-275.

Figure 17. Permeated drug per cm2 as a function of time for the M6P-275

PREFERRED EMBODIMENT OF THE INVENTION

The present invention consists of a hydrogel composed of a cationic polymer crosslinked with 6-PG-Na + and the process for its preparation.

In a particular embodiment, the polymer is chitosan; preferably in concentrations of 1 to 3% w / v. Chitosan can be used in a wide range of molecular weights, preferably 50 to 375 kDa, and with a different degree of deacetylation. The reaction between 6-PG "Na + and chitosan gives rise to a hydrogel-type formulation that is able to house active substances inside and release them after application.The structure of these compounds is as follows:

Trisodium salt of 6-phosphogluconic acid (6-PG-Na +) Chitosan

The preparation of the hydrogel is carried out with a solution of the polymer in an acidic medium to which a solution of 6-PG "Na + in water is added." This mixture is stirred at the vortex for 1 to 3 minutes, forming the hydrogel and lyophilizing The molar ratio of this formulation is 1: X moles of repetitive units of chitosan in relation to the moles of 6-PG-Na +, where X = 0.5-5.

After obtaining the lyophilized product it can be rehydrated with water. The result is a viscous formulation that can be spread on the skin. After a few minutes the formulation dries sticking to the skin, mucous membranes or cavities and begins to release the compound trapped inside.

Next, for a better understanding of the invention, the following example is provided, without this implying a limitation to the invention.

In a particular embodiment, the active substance is Piroxicam between 0.1% and 10% by weight of the formulation. This drug is useful in the anti-inflammatory treatment. In a standard preparation, a certain amount of a stock solution of Piroxicam dissolved in acetone which was allowed to evaporate (0.5% by weight of the formulation) was added to a glass vial. Then, chitosan (MW = 190-310 kDa) dissolved at 2% (15 mg dry weight) was incorporated on the Piroxicam, and the sample was homogenized by vortexing and ultrasound sonication. Finally, different volumes of a stock solution of 6-PG-Na + in water (100 mg / ml) were added, whose molar amounts were 0.02, 0.05, 0.10, 0.13 mmoles, to obtain a percentage of crosslinking (moles of 6-PG'Na + / moles of chitosan repeat units) of 50%, 100%, 200%, 275%, respectively. This mixture was vortexed to favor the reaction and finally lyophilized. The nomenclature used for these formulations is shown in the following table:

Crucifixion percentage

Hydrogels based Hydrogels with piroxicam (6-PG "Na + / chitosan)

50% M 60-50 M 6P-50

100% M 60-100 M 6P-100

200% M 60-200 M 6P-200

275% M 60-275 M 6P-275

The lyophilized sample can be ground and rehydrated to obtain an extensible formulation on the skin that forms a polymeric film or can be applied without rehydrating as a dressing in the treatment of wounds.

According to the first way of use we extend the formulation on the skin in such a way that it remains stuck to the skin (like a patch). One of the advantages is that you can apply the desired amount on the area to be treated to a greater or lesser extent. The adhesion of the hydrogel formulation to the skin increases the residence time of the drug in contact with it, allowing to reduce the number of applications.

As preferred embodiments, this formulation can also be used as a cosmetic for the release of active ingredients, or without containing drugs, for application on wounds or skin surfaces acting as a protective film.

The formulations were characterized by elemental analysis, mass spectrometry with inductively coupled plasma (ICP-MS), thermogravimetric analysis, X-ray diffraction, nuclear magnetic resonance (NMR); Likewise, its swelling, release, extensibility, toxicity properties in Caco-2 cells, adhesion strength to the skin, skin irritation, skin permeability and wound healing were determined.

Elementary analysis The determination of the content of carbon, hydrogen and Nitrogen from the solid state samples was carried out in a LECO CHNS-932 kit, using sulfanamide as a reference. The results are shown in the following table:

N% C% H% P% Chitosan 7.23 40.42 6.94 ^- M60-50 5.92 36.74 6.25 1.3

M 60-100 6.23 38.65 6.79 2.4

M 60-200 5.14 37.35 6.13 5.2

M60-275 4.97 36.47 6.83 5.7

ICP-MS. The phosphorus content was determined by dissolving the compound in 3-10 ml of aqua regia, diluting with bidistilled water to 30-50 ml and measuring the solution in a mass spectrometer with inductively coupled plasma Varian 715-ES. The results are shown in the table mentioned in the previous paragraph.

NMR The 31P-NMR spectrum in solution for the 6-PG "Na + molecule was recorded at 25oC in a Bruker Advance 300 MHz instrument using MeOD as solvent, Figure 1. The 31P-NMR spectrum in solid for sample M60-275 was obtained using a Bruker AV-400-WB spectrometer at 79.5 MHz, figure 2.

Thermogravimetric analysis. It was performed on a Perkin-Elmer Diamond electrobalance, with platinum as a reference material, and consisting of a Pt / Pt-Rh thermocouple (10%). The powder samples, with weights between 15 and 30 mg, introduced in platinum sample holders were heated in air or argon flow (100 ml / min) from room temperature to 900 ° C at a heating rate of 10 ° C / min. , figures 3, 4, 5 and 6.

X-ray diffraction. The sprayed samples were placed and pressed into a sample holder to obtain a flat surface. A Philips X'Pert Plus diffractometer with CuKa radiation (A = 1.5406 Á, 40 kV), scanning speed in the range of 0.6 ° to 10.0 ° (20) with a step of 0.02 ° (20) and a time of 5 second analysis, figure 7.

Swelling It was determined in different media at pH 1.2, 4.5, 6.8 and 7.5 for 48 hours at 370C. The normalized swelling value for each time is calculated according to the equation:

Where mo is the initial weight of the dry hydrogel, that is, the weight at time t = 0 and mt is the weight of the hydrogel at time t, figure 8, 9, 10 and 11.

Release of the drug. It was determined in different media at pH 1.2, 4.5, 6.8 and 7.5 in a thermoregulated orbital shaker (Stuart orbital incubator SI50) at 370C and 100 oscillating movements per minute for 48 hours, figures 12 and 13.

Toxicity. The MTT assay was performed on Caco-2 cells, the hydrogels, chitosan and 6-PG "Na + in PBS, 5 mg / ml, figure 14.

Extensibility. It was carried out according to the standardized procedure of work PN / L / CP / 003/00 for determination of extensibility collected in the second edition of the National Form (Order SSI / 23/2015). The hydrogels were rehydrated in water (120 mg dry hydrogel / ml water) and 0.5 g of sample was used for the assay. The commercial topical formulation Salvacam® was included as reference, figure 15.

Permeability in skin. It was carried out in Franz cells using pig's ear skin as a membrane and phosphate buffer at pH 7.4. The formulation M60-275 rehydrated in water, figure 16 and 10% propylene glycol was tested, figure 17.

Adhesion to the skin. It was measured in terms of the force required to detach the polymer film from the skin, using a modification of the Loop Tack test, with a separation speed of 500 mm / s. The hydrogels were rehydrated in water (120 mg dry hydrogel / ml), spread 0.4 g on the skin with a thickness of 1 mm and an area of 1.9 x 4.5 cm, allowed to dry for 30 minutes and finally measurements were made. Pig ear skin was used.

Skin irritation Acute dermal irritation in rabbits was evaluated according to the protocol described in the OECD 404 guide. Hydrogel M60-275 rehydrated (120 mg hydrogel / ml water) was applied on different areas of the shaved skin of the back (area of 6 cm2) and retired at 15 minutes, 1 hour and 4 hours. The skin was observed during 14 days to evaluate the existence of signs of irritation.

Cicatrization. Surgical wounds with a total thickness of 3 cm in diameter were made in Wistar rat females of 12 weeks of age. The animals were divided into two groups, the first one was not treated and the second was treated with M60-275 without rehydration, which was reapplied every 7 days. The areas of the wounds were measured at 7 and 21 days after the excision. Twenty-three days after the surgical excision the animals were sacrificed and the skin samples from the wound were treated and stained with hematoxylin-eosin. These histological samples were observed through a vertical optical microscope (Leica brand DMR model) recording the digital images with a camera (Leica model DFC450C).

Results:

The formation of the hydrogel is produced by ionic interaction between the positive charges of the amino groups of the polymer and the anions of the phospho-acid.

Characterizations: The ICP of the hydrogels shows that the percentage of phosphorus incorporated into the organic network increases as the proportion of added crosslinking agent increases. The 31P-NMR spectra of the crosslinker and the hydrogel, figure 1 and 2, show a slight shift from 4.8 to 2.3 ppm, respectively. This displacement is associated with the non-covalent interaction of the hydroxyl groups of the phosphoric group with the amines of the chitosan. Regarding the thermogravimetric study, in the TGA graphs, figure 3 and 5, it is observed that the loss of mass in the hydrogels is more gradual and incomplete than in the untreated polymer and, the higher the percentage of crosslinking, the greater the final waste. In the graphs of DTG, Figure 4 and 6, it is observed that the maximum decomposition temperature is higher the lower the percentage of crosslinking, both in the presence and absence of drug, concluding that the higher the percentage of crosslinking, the lower the stability Thermal Regarding the X-ray diffractograms, figure 7, it is shown that 6-PG "Na + is an organic compound with a crystalline structure, however, chitosan has two characteristic wide peaks due to the absence of order of the polymer. addition of 6-PG "Na + produces a loss of generalized crystallinity in the structure, reflected in the flattening of both peaks at 100 and 200; nevertheless, clearly defined peaks are observed that could be interpreted as specific zones of the network with greater ordering against chitosan in its original state.

Comparing the hydrogels with different percentages of crosslinking it is observed that the increase in the proportion of 6-PG "Na +" supposes a slight increase in the crystallinity in these specific zones of the system, although in general a depreciation of the structure occurs. drug in the network does not influence the crystallinity of the structure.

The swelling study shows the ionic character of the crosslinking of the polymer network, since at pH <4.5 the hydrogel loses its integrity in a few hours (from 2 to 4 hours), figure 8 and 9. On the other hand, at values of pH close to neutrality, that is, pH 6.8 and 7.5, an over-swelling effect is observed, since initially it retains a larger amount of water and from 5 hours the swelling is reached in equilibrium, figure 10 and 11. As for the cross-linking, it is observed that the higher this percentage, the smaller the swelling, because the links between chains limit the relaxation and expansion of the polymer network.

Regarding the release results, figure 12 and 13, we compared the profiles obtained by calculating the similarity factor, f2, concluding that there are statistically significant differences between the release of hydrogels with different degree of crosslinking at any pH and , in general, between the release profiles of the same hydrogel at different pH values. The higher the percentage of crosslinking and the higher the pH of the longer medium is the release. For M6P-275 90% of the dose is released at 7 hours. The release profiles, studied by the Korsmeyer-Peppas equation, agree with a release influenced both by the diffusion and by the relaxation of the polymer chains, called "anomalous transport" and they adjust to a kinetic of order one.

The toxicity results in Caco-2, figure 14, show that both the starting components (polymer and cross-linking agent) and the hydrogels obtained lack toxicity, since the percentage of cell survival in all cases is approximately 100%.

Regarding the extensibility, figure 15, the evaluated hydrogels have a similar extensibility to that of the commercial gel, which is why they are considered suitable for topical application.

The adhesion force of the polymeric film to the skin was 3.17 ± 0.57 Newton, a value similar to those obtained in other types of skin patches. The rehydrated hydrogel applied to the skin forms a consistent and transparent thin film. Regarding the study of irritation in rabbits, it was shown that the formulation does not cause skin irritation.

In skin permeability studies, figure 16 and 17, it is concluded that the formulation is capable of releasing the drug and being retained mainly in the skin, so it is useful as a topical release system, reducing the systemic effects derived from the transdermal absorption.

Regarding its application as wound dressings, at 7 days the area of the wound is lower for the group treated with the hydrogel than for the group without treatment, with a percentage of wound closure of 40% compared to 26% of the group without treatment. At 21 days after the surgical excision of the skin, both wounds present a similar closing percentage, approximately 95%. However, microscopy images reveal that healing is more effective in the group treated with the hydrogel than in the non-treated group. The hydrogel exerts several actions that help the regeneration of the wound, since it creates a permanent moist environment that stimulates cellular activity at all stages of the healing process, also absorbs exudate and secretions. The moist medium that creates the hydrogel allows the dehybridization of necrotic tissue, helps tissue regeneration in the granulation stage and favors cell division and activity. Also, the polymeric network serves as an extracellular matrix that facilitates reepithelialization. In conclusion, the freeze-dried M60-275 hydrogel can be used as a dressing for wound healing, either alone or as a system for the application of therapeutic substances that promote healing.

Claims (8)

1a.- Formulation for modified release of active substances or hydrogel-type drugs characterized in that it comprises at least one cationic polymer crosslinked with the trisodium salt of 6-phosphogluconic acid. 2 .- Formulation according to claim 1 characterized in that the cationic polymer is chitosan. 3a.- Formulation according to claim 2, characterized in that the molar ratio is 1: X moles of repetitive units of chitosan in relation to the moles of trisodium salt of 6-phosphogluconic acid, where X has a value between 0.5 and 5. 4a.- Process for obtaining the formulation described in claims 1 to 3, characterized in that it comprises, at least, the following steps: - A first stage of preparation of the compound with a solution of chitosan with a range of molecular weights of 50 to 375 kDa dissolved in acid medium of 1 to 3% to which is added a solution of trisodium salt of 6-phosphogluconic acid in water , in a molar ratio 1: X moles of repetitive units of chitosan in relation to the moles of trisodium salt of 6-phosphogluconic acid, where x = 0.5-5. - A second stage in which the obtained mixture is stirred for 1 to 3 minutes. 5a.- Method for obtaining the formulation according to claim 4, characterized in that it also comprises a lyophilization step. 6a.- Use of the formulation described in any of claims 1 to 3 and prepared according to the method described in claims 4 and 5 as hydrogel of topical or transdermal application. 7a.- Use of the formulation according to claim 6 as a film to cover wounds, skin damages and combinations thereof. 8a.- Use of the formulation according to claim 6 as a composition for the Ċ release of drugs or active compounds in a controlled manner.