Method for producing a mucoadhesive galenic preparation

Abstract

The invention relates to a method for producing a mucoadhesive galenic preparation, wherein the galenic preparation comprises a hydrogel comprising modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification. The invention also relates to a method for producing a galenic preparation, preferably a mucoadhesive galenic preparation, a galenic preparation, a kit for providing a galenic preparation, and a use of the galenic preparation.

Description

[0001] 17 . 03 . 2026 / TH / NL

[0002] Method for producing a mucoadhesive galenic preparation

[0003] The invention relates to a method for producing a mucoadhesive galenic preparation, a galenic preparation, a kit for providing a galenic preparation, and a use of the galenic preparation.

[0004] General mucoadhesive drug delivery systems for oral applications are described in Recent Advances in biopolymer-based mucoadhesive drug delivery systems for oral application, J. Drug. Deliv. Sci . Technol . 91 (2024 ) 105227. Therein characteristics and challenges of ideal mucoadhesive drug delivery systems comprising a polymer are described. Specific challenges of oral topical application of known systems are physicochemical stability, mucoadhesive properties, biodegradability, toxicity, safety and biocompatibility, patient compliance and comfort, drug stability, drug release, and especially for drug release over several days : salivary flow, drinking / eating and dental hygiene .

[0005] Natural Agarose is a natural high-molecular polymer consisting of repeating units of a disaccharide of B-D-galactopyranose and 3, 6-anhydro-L-galactose, which form a hydrogel via hydrogen bonds at concentrations of approx . 0.2 to approx . 4 wt . % . Natural agarose is non-toxic, safe and highly biocompatible . Natural agarose dissolves only in hot water at temperatures above 90°C, it is usually heated to 95°C and forms a hydrogel on cooling. An agarose hydrogel has a high physicochemical stability over a broad temperature and pH range . It has no anionic or cationic groups, even in a wide pH range and is particularly chemically inert . An agarose hydrogel therefore is particularly well suited as drug delivery device .

[0006] However, main drawbacks of a natural agarose hydrogel specifically with regard to oral topical application over several days are the lack of mucoadhesive properties and specifically with regard totopical application in the sulcus gingivalis a balanced behavior in terms of strength and elasticity or rheological behavior .

[0007] The present invention is therefore based on the task of providing a mucoadhesive galenic preparation comprising a hydrogel and a method for their production, which overcomes the disadvantages of hydrogels of natural agarose without losing its advantages .

[0008] The invention solves this task by independent claim 1. The invention solves this task in particular by providing a method for producing a mucoadhesive galenic preparation, wherein the mucoadhesive galenic preparation comprises a hydrogel comprising modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification, wherein the method comprises the following steps :

[0009] A) providing a modified agarose, preferably in the form of a powder, wherein the modified agarose comprises at least vinyl sulfone groups as modification,

[0010] B) mixing the modified agarose of step A) with an aqueous solution to form a hydrogel, preferably at a temperature below 50°C, more preferably at a temperature below 40°C,

[0011] C) mixing at least one active ingredient or a combination of active ingredients with the provided modified agarose of step A) , and / or the aqueous solution or the hydrogel of step B) , preferably at a temperature below 90°C .

[0012] Preferred embodiments can be found in the dependent claims .

[0013] Firstly, some terms used in the context of the invention are explained .In the present case, natural agarose is understood to mean an unmodified agarose which is consisting of repeating units of a disaccharide of B-D-galactopyranose and 3 , 6-anhydro-L-galactos . The term modified agarose refers to, in contrast to natural agarose, to agarose, which comprises at least vinyl sulfone groups as modification. In a particular preferred embodiment, the vinyl sulfone groups are ethyl vinyl sulfone groups, which can be obtained by the following chemical reaction:

[0014]

[0015] A microparticle is understood to be a particle, which has a largest diameter of between 1 and 1000 micrometers . Approximately spherical and spherical particles are preferred.

[0016] A loaded particle is understood to be a particle comprising an active ingredient . Loaded particles comprising a polymeric or inorganic matrix in which the active ingredient is distributed. A polymeric matrix is particularly preferred, more preferably a polymeric matrix wherein an active ingredient or a combination of active ingredients is / are preferably uniformly distributed and the polymeric matrix at least partially surrounds the active ingredient or a combination of active ingredients and thus at least partially envelops it . In addition to particles which may completely envelop the active ingredient or a combination of active ingredients, particles are therefore also understood to be particles which only partially envelop the active ingredient or a combination of active ingredients and / or optionally have pores or holes in the polymer matrix . In addition, the active ingredient or a combination of active ingredients may be contained in pores of the particles . Loaded microparticles are particularly preferred.Suitable polymers may react with divinyl sulfone to produce a cross-linked polymer, for example in form of a hydrogel . Divinyl sulfone is well-known for its reactivity, but also for its toxicity. For a biocompatible and non-toxic galenic composition, it is therefore highly desirable that the galenic composition does not contain any reactive divinyl sulfone .

[0017] For example, J. Andrade del Olmo et al . (Biocompatible hyaluronic acid-divinyl sulfone inj ectable hydrogels for sustained drug release with enhanced antibacterial properties against Staphylococcus aureus, Mater . Sci . Eng. C 125 (2021 ) 112102 ) describes the modification of hyaluronic acid with divinyl sulfone to produce a crosslinked hyaluronic acid. But although the toxic divinyl sulfone was completely removed and no more vinyl groups at all were detectable, the hydrogels showed cytotoxicity at high degree of modification .

[0018] The galenic preparation according to the invention comprises a hydrogel, wherein the hydrogel comprises modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification. However, test results unexpectedly showed non cytotoxicity, even at high degree of modification of the natural agarose with vinyl sulfone groups .

[0019] Furthermore, the modified agarose shows adjustable behavior in terms of strength and elasticity or rheological behavior, biocompatibility, drug stability, drug re-lease, improved gelation temperature, improved flow properties for application, and especially for drug release over several days : physicochemical stability and excellent mucoadhesive properties . In addition, the hydrogel comprising modified agarose shows improved solubility in contrast to a natural agarose hydrogel .

[0020] The gelation curves reveal that the storage modulus is decreasing with increasing degree of modification of the agarose, that is, the hydrogels become softer . The modified agarose hydrogel istherefore softer than natural agarose hydrogel, with a comparable maximum breaking strength of both. For example both may break at a strain of 30% .

[0021] The method according to the invention makes it possible to provide a mucoadhesive galenic preparation, wherein the mucoadhesive galenic preparation comprises a hydrogel comprising modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification, which is particulate and has mucoadhesive properties and is particularly suitable for use for example in the oral cavity of a patient .

[0022] Modified agarose according to the invention having a degree of modification with sulfone vinyl groups is limiting the ability of the polymer chains to self-reorganize in a secondary structure like helices, double helical structures and / or bundles of helices, and at least therefore has improved properties according to the invention, e . g. improved solubility, which allows higher concentrated aqueous solutions or gels and / or lower temperatures of the aqueous solutions, improved mucoadhesion and improved adaptability of the rheological properties of a desired hydrogel .

[0023] In summary, the galenic preparation according to the invention shows improved properties, which overcomes the disadvantages of natural agarose without losing its advantages .

[0024] Preferably step B) is performed at a temperature between below 50°C and the gelation point of the hydrogel, more preferably below 40°C and the gelation point of the hydrogel .

[0025] Preferably step C) is performed at a temperature between below 90°C and the gelation point of the hydrogel .

[0026] Preferably the modified agarose according to the invention is produced comprising the following steps :Al ) dissolving natural agarose in an aqueous solution at a temperature above 90°C, more preferably at a temperature of about 95°C or higher, to produce an aqueous natural agarose solution, such that the polymer chains have essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel,

[0027] A2 ) cooling the aqueous natural agarose solution from step Al ) under stirring to a temperature below 90°C and above the gelation temperature of the dissolved natural agarose, preferably to a temperature below 50°C, such that the polymer chains forming essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel,

[0028] A3) adjusting the pH value of the agarose solution from step A2 ) to 10-14, preferably 11-13, more preferably to about 12,

[0029] A4 ) mixing divinyl sulfone to the agarose solution from step A3) to form modified agarose, such that the polymer chains forming essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel ,

[0030] A5) separating the modified agarose of step A4 ) , comprising at least vinyl sulfone groups as modification by precipitation, preferably by mixing with at least one precipitating agent and / or cooling, and preferably removing the precipitated modified agarose from the aqueous solution, e . g. by means of centrifugation and / or filtration,

[0031] A6) preferably cleaning of the removed modified agarose with a suitable cleaning agent,A7 ) preferably drying of the removed modified agarose, preferably by means of freeze-drying.

[0032] Preferably the method according to the invention may comprise one or more of the following:

[0033] - the aqueous natural agarose solution, resulting at least from step Al ) to step A3) comprises between 0.01-0, 5 wt . % natural agarose, preferably between 0.1-0, 5 wt . %, more preferably between 0.2-0. 4 wt . % natural agarose, in relation to the whole aqueous natural agarose solution,

[0034] - the precipitated modified agarose of step A5) forms liquid droplets and / or solid particles, preferably by stirring the mixture,

[0035] - the mixture of step B) comprises at least 0.5 wt . % modified agarose, preferably between 0.5-20 wt . %, more preferably from 1 to 5 wt . % modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification, in relation to the whole composition of the hydrogel and / or the galenic composition,

[0036] - the modified agarose of step A) is provided in the form of solid particles, preferably in the form of microparticles, more preferable spherical microparticles,

[0037] the aqueous solution of step B) comprises mixing of a cross- linking agent to the modified agarose .

[0038] The cross-linking agent used may be preferably selected from any suitable cross-linking agent having groups that are reactive with a vinyl group under physiological conditions to form a covalent bond, preferably by Michael addition reaction, such as an amine group, a hydroxyl group or a thiol group, e . g. lysine . In addition, suitable cross-linking agents are agents that are able todeprotonate hydroxyl groups of agaroses, such as metal hydroxides like sodium hydroxide in aqueous solution under physiological conditions, e . g. at a temperature at about 37 °C without needing or producing cytotoxic products .

[0039] The cross-linking preferably comprises a step of at least partially crosslinking the modified agarose . Particularly preferred are those cross-linking agents having a Michael-addition type reactivity and have appropriate biocompatibility. This can for example be a preferred embodiment if a greater stiffness is desired. The cross-linking preferably comprises the following steps :

[0040] i) providing a powder or an aqueous suspension of solid modified agarose particles, preferably microparticles,

[0041] ii) providing an aqueous solution of a cross-linking agent that preferably has a Michael-addition type reactivity or is able to deprotonate hydroxyl groups of the modified agarose,

[0042] iii) mixing of the powder or the aqueous suspension of solid modified agarose particles of step i) and the aqueous solution of the cross-linking agent of step ii) .

[0043] In a preferred embodiment of the invention the method contains a step of mixing particles, preferably microparticles loaded with an active ingredient or a combination of active ingredients with the modified agarose .

[0044] The modified agarose according to the invention is suitable for the dispersion of various active ingredients . Preferably, the active ingredients are loaded into a particle and can be released into the surrounding hydrogel comprising the modified agarose . Depending on the particle itself, the release rate of an active ingredient or a combination of active ingredients can be influenced.Preferably, the active ingredients are loaded into a microparticle .

[0045] In a preferred embodiment, the active ingredient is or the combination of active ingredients are not contained in a particle but in the hydrogel itself . Depending on the desired release rate and stability of the active ingredient or a combination of active ingredients, the active ingredient or a combination of active ingredients can be loaded into a particle or directly in the modified agarose . It is also conceivable to arrange the active ingredient or a combination of active ingredients in the modified agarose in addition to an active ingredient which is loaded into a microparticle . Advantageously, other active ingredients are dissolved or dispersed directly in the modified agarose as are loaded in the microparticles . An arrangement of the active ingredient in the modified agarose can provide the advantage of a faster release or faster biological availability of the active ingredient or a combination of active ingredients .

[0046] In principle, the same active ingredients that can also be arranged in microparticles are suitable for arrangement in the modified agarose . Advantageously, active ingredients introduced directly into the modified agarose are water-soluble . The water solubility may be greater than 0.01 percent in aqueous solution, preferably greater than 1 percent or 10 percent in aqueous solution, especially in extracellular body fluids such as saliva .

[0047] The loaded particles can be dispersed in the modified agarose hydrogel, in order to obtain a dispersion of the particles in the flowable hydrogel . By dispersing the particles, the particles are distributed or dispersed in the modified agarose and agglomeration of the individual particles with one another can thus be prevented or at least significantly reduced. By suppressing agglomeration, a more constant and thus more predictable release of the active ingredient or a combination of ingredients can be achieved. The useof the modified agarose further prevents or reduces the swelling capacity of the particles, which results in a lower decomposition of the particles and thus a lower released amount of the active ingredient or a combination of ingredients . The release can therefore be better controlled via the size of the individual particles, microparticles are particularly preferred here, especially over nanoparticles .

[0048] Preferably the microparticles can be Polylactid-co-Glycolid (PLGA) microparticles, more preferably the PLGA microparticles are loaded with at least a Flavonoid and or a flavonoid derivate as an active ingredient . Other active ingredients may be selected from natural or synthetic active ingredients, naturally occurring active ingredients are preferred. Preferred other active ingredients or combinations of other active ingredients are those that synergistically enhance the intended effect, e . g. without damaging the mucous membrane, especially the oral microbiology. One specific example here is lactof errine .

[0049] By adding the microparticles to the modified agarose, which acts as a matrix, the microparticles are well distributed or dispersed in the modified agarose and agglomeration can be prevented. This results in a more constant release of the at least one flavonoid and / or flavonoid derivative .

[0050] In a preferred embodiment, the dispersion contains microparticles loaded with an active ingredient as described in DE 10 2024 134 253. 6.

[0051] In addition, the method according to the invention can be further developed with further features that are described in connection with the galenic preparation, in connection with its further and additional components and properties . Likewise, the galenic preparation according to the invention can be further developed with further features which are described in connection with the methodaccording to the invention, in particular in connection with its further and additional components and properties .

[0052] Another embodiment of the invention is a galenic preparation comprising a hydrogel, wherein the hydrogel comprises modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification.

[0053] Preferably, the galenic preparation is a mucoadhesive galenic preparation and has improved mucoadhesive properties compared to those based on natural agarose gels or essentially cross-linked natural agarose particles . In this way, an improved treatment of a patient, for example in the oral cavity, can be achieved through improved adhesion of the galenic preparation, in which the galenic preparation adheres longer to the treatment site and thus remains there longer .

[0054] Preferably the galenic preparation is in the form of a dispersion, preferably a dispersion of at least partly soluble solid particles of the modified agarose . Preferably, the dispersed modified agarose is forming a hydrogel, which further improves the mucoadhesive properties .

[0055] Caries and gingivitis are regularly caused by a persistent plaque adhering to the tooth, containing, among others, the pathogenic bacterial species Streptococcus mutans and Porphyromonas gingi-valis, the increased occurrence of which is primarily associated with caries and gingivitis . Streptococcus mutans in particular produces glucosyltransferases (GTFs) that catalyze the synthesis of water-insoluble polysaccharides (EPS) , which contribute significantly to the stability of the biofilm on the tooth in which the bacterial species can continue to multiply.

[0056] Preferably the dispersion comprises particles loaded with an active ingredient or a combination of active ingredients . Preferably the particles are microparticles . Preferably the active ingredientis at least selected from the groups comprising flavonoids or flavonoid derivates . It is known that flavonoids or flavonoid derivatives can inhibit the glucosyltransferase activity of Streptococcus mutans and thus counteract a biofilm that favors caries and inflammation. This applies in particular if dental hygiene is restricted due to other (dental) medical treatment . One example of this is the treatment of marginal periodontitis . The treatment of marginal periodontitis can include the application of a gel to promote wound healing (at least closure) , for example after dental subgingival curettage .

[0057] However, the clinical utility of flavonoids or flavonoid derivatives is compromised by their rapid metabolism and limited bioavailability. Their solubility and / or their structural instability due to autoxidation may limit their applicability if they strongly reduce bioavailability at the site of interest over a prolonged treatment period.

[0058] Preferably, the loaded particles are dispersed in a flowable hydrogel, preferably in modified agarose, to obtain a dispersion of the particles in the flowable hydrogel . Preferably the loaded particles are loaded microparticles . By dispersing the particles, the particles are distributed or dispersed in the modified agarose and agglomeration of the individual particles with one another can thus be prevented or significantly reduced. By suppressing agglomeration, a more constant and thus more predictable release of the at least one flavonoid and / or flavonoid derivative can be achieved. The use of, in particular, a modified agarose hydrogel further prevents or reduces the swelling capacity of the particles, which results in a lower decomposition of the particles and thus a lower released amount of the at least one flavonoid and / or flavonoid derivative .

[0059] Preferably, the modified agarose is flowable at body temperature and / or room temperature, preferably in a range from about 20°C toabout 40°C, in such a way that it can be dispensed from known can-nulae used in dentistry. The flowability of the composition according to the invention is important for the safe and easy application of the composition in the oral cavity, in particular for inj ecting the composition between the neck of the tooth and the gum using a thin cannula and a syringe, i . e . for filling the gingival sulcus directly in depth with the composition. It is particularly advantageous if the user has to apply little force to extrude the composition from the cannula . This extrusion force, for example to actuate the plunger of a syringe, is preferably less than 200 Newtons, more preferably less than 150 Newtons, more preferably less than 100 Newtons, more preferably less than 50 Newtons, more preferably less than 30 Newtons, more preferably less than 15 Newtons . This extrusion force, for example to actuate the plunger of a syringe, is preferably greater than 0.01 Newton, more preferably greater than 0.1 Newton. This extrusion force, for example to actuate the plunger of a syringe, is most preferably greater than 0.1 Newton and less than 10 Newtons .

[0060] The modified agarose preferably has a degree of modification of the vinyl sulfone groups between 10-50%, preferably between 10-45%, and more preferably between 15-40%, of the repeating units of a natural agarose . The degree of modification may be determined by any suitable method known to the skilled person, e . g. by determining the unreacted divinyl sulfone remaining in the separated reaction solution and / or the quantitative determination of the total sulfur content of the separated polymer and / or infrared spectroscopy. Preferred is the method of proton nuclear magnetic resonance spectroscopy ( 1H-NMR) as described by J. Andrade del Olmo et al . (Biocompatible hyaluronic acid-divinyl sulfone inj ectable hydrogels for sustained drug release with enhanced antibacterial properties against Staphylococcus aureus, Mater . Sci . Eng. C 125 (2021 ) 112102 ) and as described further below. The galenic preparation according to the invention is nevertheless non-toxic evenat high degrees of modification, as is regularly observed, for example for other polymers comprising vinyl sulphone groups .

[0061] The galenic preparation has preferably a pH value greater than 4, more preferably between 5 and 11, more preferably between 6 and 11, more preferably between 7 and 11, more preferably of 7.5 to 10.5.

[0062] Preferably the galenic preparation has a total work of adhesion of greater than about 40 mN mm, preferably greater than 50, more preferable greater than 100, and more preferable greater than 200 or 300 mN mm. The total work of adhesion or mucoadhesion of the galenic preparation can advantageously be determined by the method described further below. There is no specific upper limit to the total work of adhesion, as the hydrogel and / or the galenic preparation is supposed to degrade within a predetermined period of time under human body conditions, especially under oral conditions .

[0063] Preferably the galenic preparation is stable in storage in the ready-for-use condition in a primary packaging, preferably a prefilled syringe up to 6 months, preferably up to 12 or 24 months .

[0064] Preferably the galenic preparation according to the invention is produced by the method according to the invention.

[0065] In addition, the method sand / or galenic preparations according to the invention can be further developed with further features that are described, in connection with its further and additional components and properties . Likewise, the galenic preparation and its method for production according to the invention can be further developed with further features which are described in connection with a kit according to the invention, in particular in connection with its further and additional components and properties .It is also an obj ect of the present to provide a kit for providing a galenic preparation comprising of a first component and a second component, wherein the first component comprises modified agarose, preferably as solid particles, wherein the modified agarose comprises at least vinyl sulfone groups as modification, and wherein the second component comprises an aqueous solution or dispersant, preferably comprising a cross-linking agent for the formation of a hydrogel, preferably for the additional formation of covalent bonds with some of the vinyl groups of the modified agarose .

[0066] The first component and the second component can be applied in sequence, preferably with first component being applied first, or the first component and the second component being mixed immediately before application, preferably applied to a mucous membrane, preferably comprising natural or artificial mucus, preferably to the gingival tissue that contacts the tooth.

[0067] It is also an obj ect of the present invention to provide a use of the galenic preparation in treatment and / or prophylaxis of inflammation of the periodontium and / or caries, preferably including a subgingival scaling or gingival curettage .

[0068] The galenic preparation according to the invention make it possible, for example, to reduce a bacteria-specif ic biofilm when used in the oral cavity of a patient . Preferably, by additionally releasing an active ingredient or a combination of active ingredients, a biofilm favoring caries and inflammation can be counteracted. This applies in particular if dental hygiene is restricted (due to other (dental) medical treatment) . An example of this is the treatment of marginal periodontitis . The treatment of marginal periodontitis can include the use of the product to promote wound healing (at least closure) , for example after dental subgingival curettage . Intensive cleaning of the teeth, for example with den-tai floss and similarly effective cleaning agents, is then temporarily restricted, which can lead to the formation of initial caries or worsening of existing caries .

[0069] I . Measured values and methods

[0070] 1. Chemicals used

[0071] The following chemicals were used in the presented examples :

[0072] Agarose was bought from NEEO ultra-quality, Carl Roth (Germany) , Span®80 for synthesis from Carl Roth (Germany) ; mucin type II from porcine stomach, sodium chloride, phosphate buffered saline (PBS) tablets, and Polylactid-co-Glycolid (Resomer® RG 503 H, L : G 50 : 50, 24-38 kDa (PLGA) from Sigma-Aldrich (Germany) ) ; divinyl sulfone (DVS) , sodium hydroxide (NaOH) , cysteine hydrochloride dihydrate, dichloromethane, and hydrochloric acid (HC1) from Fisher-Scientific (USA) ; cyclohexane from VWR Chemicals (USA) ; methanol (MeOH) and acetone, technical grade, from Fisher-Scientific (USA) ; deuterated dimethyl sulfoxide (DMSO-d6) from Deutero GmbH (Germany) ; L-Lysine hydrochloride monohydrate > 98%, from Sigma-Aldrich (USA) ; dithiol-PEG from Tokyo Chemical Industry (Japan) ; ; 2R, 3R) 2 (3, 4, 5-Trihydroxyphenyl ) -3, 4-dihydro-l (2H) benzopyran-3 , 5, 7-triol-3- ( 3 , 4 , 5trihydroxybenzoat ) (EGCG) from BLD Pharmatech Ltd.

[0073] (China) ; gelatine Type A, pork skin, Bloom: 238-282 from VWR Chemicals (USA) ; Poly ( vinylalkohol ) (PVA) from VWR International (Germany) .

[0074] All chemicals were used as purchased without further cleaning.

[0075] 2. Degree of modification, DM

[0076] Nuclear magnetic resonance spectroscopyNuclear magnetic resonance (NMR) spectra were recorded on an Avance 300 MHz NMR spectrometer (Bruker, USA) at 25°C . The polymers were dissolved in deuterated dimethyl sulfoxide (DMSO) at 16 mg / mL and transferred into an NMR tube . Analysis of obtained spectra was done using Topspin software version 4.2.0 (Bruker, USA) . All spectra were calibrated to residual DMSO peak at 2.5 ppm.

[0077] The degree of modification (DM) per repeating unit was evaluated by integration from the respective 1H-NMR spectra . The signals from 5 to 5.3 ppm are hereby referring to the anomeric protons of agarose (2 protons per repeating unit) and the signals around 6.2 and 6.9 ppm are attributed to the double bond of the vinyl sulfone group (3 protons) . Another peak at 4.1 ppm can be observed with increasing DM that corresponds to the methylene group neighboring the sulfone functionality proving successful coupling of DVS to the NA polymer chains / backbones .

[0078] The degree of modification of vinyl sulfone-modif led agarose samples was done by integration and comparison of anomeric protons of agarose ( Ic, 5.0 - 5.3 ppm) and double bond signals ( la, 7.0 ppm; lb, 6.2 ppm) of vinyl sulfone . The following equation was then used for calculation:

[0079] DM_NMR= (2 x ( la + Ib) ) / (3 x ic ) ( 1 )

[0080] Fourier-transform infrared spectroscopy

[0081] Spectra of Fourier-transform infrared spectroscopy (FTIR) were recorded with a Cary 630 FTIR spectrometer (Agilent Technologies, USA) in transmission mode equipped with an attenuated total reflection (ATR) unit . Spectra were obtained with a resolution of 4 cm-1with 8 background scans and 8 scans for the sample in a range from 4000 cm-1to 650 cm-1.

[0082] Elemental analysisElemental composition analysis was used to calculate the degree of modification of the different polymers . Elemental analysis (EA) was performed using a vario MICRO cube (Elementar Analysensysteme GmbH, Germany) equipped with a thermal conductivity detector . Combustion temperature was set to 1150°C and reduction tube was set to 850°C . Results were obtained using vario MICRO software version 4.02 (Elementar Analysensysteme GmbH, Germany) .

[0083] 3. Particle size distribution

[0084] The mean diameter and particle size distribution were determined using an LA-900 laser scattering particle size analyser (Horiba, Japan) . EGCG-loaded PLGA microparticles (5 mg) were weighed into a 2 mL reaction vessel and dispersed in 1 mL of deionised water . Approximately 400 pL of the PLGA particle dispersion was then added to the instrument ' s quartz cuvette and measured at a refractive index of 1.46. The mean diameter of the particles was determined from three individual batches with the same manufacturing conditions .

[0085] 4 . Active ingredient loading, DL

[0086] The actual drug loading DL of the microparticles were evaluated by UV-Vis measurements using a Nano-Drop 2000c spectrophotometer (Thermo Fisher Scientific, USA) . The PLGA microparticles loaded with EGCG were accurately weighed into a 2 mL reaction vessel and dissolved in DMSO ( 0.8 mL) . The amount of EGCG was then determined by comparing the absorbance at 280 nm with a previously established calibration curve and used to calculate the experimental drug loading.

[0087]

[0088] II . Examples and experiments

[0089] 1. Preparation of vinyl sulfone-modif ied agarose

[0090] Examples 1 to 5

[0091] Agarose ( 960 mg) was weighed into a round bottom flask and dissolved in 400 mL of deionized water under heating in the microwave ( 1000 W, 4 minutes) . The agarose solution was subsequently cooled down to 40°C under stirring in an ice bath. The solution was then further stirred at 40°C and the pH of the solution was adjusted to 12 by addition of NaOH ( 1 M, 6 mL) . 1800 pL divinyl sulfone (DVS) were added to the agarose solution. The mixture was let react for 5 minutes before the reaction was quenched by adding HC1 (5 M, 1.2 mL) to adjust the pH to 5. After the reaction, the solution was cooled in an ice batch for 15 minutes, before the product was precipitated in 500 mL of cold MeOH stirring at 1000 rpm. The precipitate was given 15 minutes to settle, before it was collected by vacuum filtration (cellulose filter MN 615) . The crude product was then washed with 200 mL of MeOH and 150 mL of acetone . Finally, the product was transferred into a glass vial and dried at 50°C in a hot air oven for 4 days . The product was obtained as a withe powder (Yield: 80%, degree of modification: 35%) .

[0092] To obtain different degrees of modification, respective amounts of DVS (252 pL, 590 pL, 1000 pL, 1400 pL, and 1800 pL) were added to the agarose solution and mixture was let react for 5 min before the reaction was quenched.

[0093] Tabel 1 : Different degrees of modification auf the modified agarose

[0094]

[0095] 1H-NMR results showed that higher agarose concentrations are unfavorable . At 0.5% (w / v) ) and higher concentration of the natural agarose the degree of modification decreased. Working at dilute concentrations is therefore essential for the synthesis of the vinyl sulfone-modif ied agarose .

[0096] 2. Preparation of vinyl sulfone-modif ied agarose solid particles with a water-in-oil emulsion method

[0097] Example 6

[0098] The modified agarose from example 5 (300 mg) was dissolved in deionized water ( 18 mL) in a water bath at 95°C . After complete dissolution the samples are incubated at 50°C in the oven. Cyclohexane ( 60 mL) and Span®80 (4 mL) were given into a 250 mL glass bottle and stirred with 400 rpm at 50°C . The modified agarose solu-tion was then added to the Cyclohexane / Span8 0 . The mixture was stirred with 400 rpm at 50°C for 15 minutes . The resulting emulsion then was cooled for 15 minutes in an ice bath while stirring at 400 rpm to trigger the thermal gelation of the polymers . Aftergelation, acetone ( 150 mL) was added to dehydrate the particles . The particles were then recovered by vacuum filtration and additionally washed with acetone ( 100 mL) . After drying at room temperature, the particles were stored under inert gas atmosphere until further use .

[0099] 3. Preparation of PLGA microparticles (2.98% DL)

[0100] Example 7

[0101] PLGA microparticles loaded with EGCG were prepared by a water-in-oil-in-water emulsion solvent evaporation / extraction technique . PLGA ( 170 mg) was dissolved in DCM ( 1 mL) and EGCG ( 19 mg) was dissolved in deionized water ( 0. 6 mL) . The EGCG solution was added to the organic phase and emulsified using a VCX130 ultrasound probe (Sonics & Materials Inc . , USA) for 30 sec . at 30% amplitude . This primary emulsion was then dropwise added to a PVA solution (4% (w / v) , 6 mL, 0.9% (w / v) NaCl) that was mixed on a uniTEXERl vortex shaker (Lab Logistics Group, Germany) . After 1 minute of mixing on the vortex shaker, the emulsion was diluted by adding more PVA solution (4% (w / v) , 10 mL, 0.9% (w / v) NaCl) and further vortexed for 2 minutes . The emulsion was then transferred into a NaCl hardening solution ( 0.9% (w / v) , 40 mL) and stirred for 3 hours at 400 rpm to evaporate the organic solvent . The samples were protected from light by wrapping in aluminum foil . After the particles have been solidified, the dispersion was transferred into 50 mL falcon tubes and collected by centrifugation ( 1900 rpm, 5 min. ) . The particle pellet was re-suspended in 50 mL of ice-cold deionized water and again collected by centrifugation ( 1900 rpm, 5 min. ) . Active ingredient loaded PLGA microparticles were washed a total of three times, before they were dispersed in a small amount of deionized water and snap-frozen in liquid nitrogen. Samples were freeze-dried with a Beta 2-8 LD plus freeze-drier (Christ, Germany) by snap freezing in liquid nitrogen and the water was sublimed for 24 hours at room temperature under reduced pressure(< 2 mbar) . After freeze-drying, the EGCG-loaded PLGA microparticles were obtained with a yield of 65% . PLGA microparticles had 2.98 ± 0.14% (w / w) and a unimodal size distribution with a mean diameter (D50) of 50.73 ± 2.91 pm.

[0102] 4. Preparation of an inj ectable periodontal material from modified agarose

[0103] Example 8

[0104] Solid particles of modified agarose from example 6 (40 mg) were weighed into a weighing bowl . The microparticles were then covered with 128 pL of lysine-crosslinking solution (5 mg / mL of lysine in 0.1 M carbonate buffer with pH 10) . Solid particles of modified agarose and the crosslinking solution were then thoroughly mixed with a spatula and loaded into a compule . The formulation was then extruded into a mold (5 mm diameter, 6 mm depth) using a compule dispensing gun. Within 5 minutes the extruded solid particles of modified agarose established sufficient inter-particle crosslinking points to yield one stable device . Stability of the extruded material was shown by immersing and stirring in water .

[0105] 5. Gelation behavior of bulk hydrogels

[0106] The sol-gel transition

[0107] Polymer solutions (2% and 3% (w / v) were prepared by dissolving the respective polymer in deionized water at 95°C . After complete dissolution the samples were equilibrated to 50°C and then transferred into the sample holders of ElastoSens-Bio2 non-invasive rheometer (Rheolution, Canada) . Measurements were taken every degree with a rate of 2 °C / min.

[0108] To compare the stiffness of the modified agarose the storage modulus G' was recorded. The gelation curves revealed that the storage modulus is decreasing with increasing degree of modification, thatis, gels become softer . Natural agarose hydrogel showed a G' of over 100 kPa at 3% (w / v) , modification of 35% reduced the storage modulus to about 3 kPa at the same concentration. For 2% (w / v) hydrogels the storage modulus decreased from around 55 kPa for NA to about 1 kPa for a vinyl sulfone-modif led agarose with a DM of 35% .

[0109] The decrease in G' was accompanied with reduced sol-to-gel transition temperature (when the solution cools down) . We therefore performed temperature sweep analysis for the gelation of modified agarose samples with different degrees of modification at 2% (w / v) . The gelation temperature was then calculated from the temperature where tan (5) showed a maximum. The gelation temperature for natural agarose hydrogel was 38.7 ± 0.3°C, for a degree of modification of 35% it was 25.8 ± 1.2 °C (n = 3) .

[0110] The gel-sol transition

[0111] Hydrogels were prepared by dissolving the respective polymer at 2% (w / v) in deionized water at 95°C . The polymer solutions were transferred into vials and incubated at 4 °C in the fridge for 30 minutes . Melting temperatures ( TM) of the hydrogels were evaluated by a tilted vial method, by incubating the vials in a hot water bath with the temperature increasing in 5°C steps . After 15 minutes of incubation at every temperature the hydrogel samples were taken out of the water bath, tilted and a photo was taken to validate the flow of the samples . The results are summarized in Table 2 .

[0112] In addition, Gel-to-sol transition was analyzed by measuring change of shear storage modulus with increasing temperature on a ElastoSens-Bio2 non-invasive rheometer (Rheolution, Canada) . The polymer solutions (2 mL) were transferred into the sample holders and incubated at 4 °C in the fridge for gelation for 30 minutes and then shear storage modulus was measured while heating at l °C / min with a temporal step of 45 sec . TM was obtained from shear storage modulus (G' ) curves where dG' / dt becomes zero .The results of the methods were in good agreement . Natural agarose hydrogels are melting only at temperatures > 90°C . The melting temperature ( TM) of modified agaroses decrease with increasing degree of modification.

[0113] Table 2 : Gel-to-sol transition of hydrogels by tilted vial method, TM was in the range between TGel and TSol .

[0114]

[0115] 6. Mechanical behavior of bulk hydrogels

[0116] Compression testing

[0117] Hydrogels were prepared by loading the respective polymer solution (2% (w / v) ) into a cylindric mold with a height of 9 mm and a diameter of 10 mm and incubating at 4 °C to solidify.

[0118] Unconfined compression tests were performed in air using an EZ-SX texture analyzer (Shimadzu, Japan) equipped with a 50 N load cellat a constant rate of 20%*min ~1with respect to the original height of the hydrogel cylinder . The stress was calculated dividing the compressive force by the cross-section of the cylinder . Youngs' s modulus was calculated by linear fitting to the stressstrain curve in a range from 0 - 5% strain.

[0119] Stress strain curves showed a similar trend as it was seen for the gelation curves in rheological experiments . Mechanical stability is decreasing with increasing degree of modification. Maximum stress until breakage is exponentially decreasing with an increasing degree of modification. Natural Agarose hydrogel cylinders withstand a maximum stress of 138.5 ± 10.2 kPa (n=3) . Hydrogel cylinders from agarose with a degree of modification of 35% withstand a maximum stress of 6.8 ± 0.1 kPa (n=3) .

[0120] As a measure for the stiffness of the hydrogels, Young' s modulus was calculated from the linear elastic region in the range from 0 to 5% strain. Similar to the maximum stress, also the Young' s modulus is exponentially decreasing with increasing modification. Cylinders from Agarose with a degree of modification of 35% had a Young' s modulus of 9.2 ± 1.3 kPa while natural agarose hydrogel cylinders had a modulus of 320.4 ± 29.0 kPa (n=3) .

[0121] While the maximum stress and Young' s modulus varied with the degree of modification, the maximum strain until break did not show significant differences . All hydrogel samples broke at a strain of around 30% . With increasing degree of modification hydrogels are getting softer without getting more elastically deformable before break .

[0122] 7. Modulation of mechanical properties by crosslinking modified agarose hydrogels

[0123] Stress strain curves of covalently self-crosslinked modified agarose hydrogels exhibited higher maximum stress compared to solelyphysical crosslinked samples . Although a strong increase in maximum stress was observed, the self-crosslinked hydrogels (pH 12, 2% (w / v) were very brittle and broke around 25% of strain.

[0124] Crosslinked hydrogels were prepared by incorporating gelatin ( 0.5% (w / v) ) into agarose solutions (2% (w / v) at pH 9. The hydrogels showed increased maximum stress and strain. The maximum stress was 36. 6 ± 4.0 kPa and the maximum strain was 48.2 ± 1.1% (n=3) . Comparable results were obtained when hydrogels were chemically crosslinked with dithiol-PEG (MW: 300 g / mol) in PBS (pH 7 ) . Molar ration between vinyl sulfone-functionality and thiol was kept equimolar . The maximum stress was 41.2 ± 4.8 kPa and maximum strain was 49.1 ± 1.5% (n=3) .

[0125] 8. Mucoadhesive properties of the (periodontal) materials

[0126] To access mucoadhesive properties an EZ-SX texture analyzer (Shi-madzu, Japan) was used, equipped with a 50 N load cell with a stationary and a movable platform in tensile mode .

[0127] Hydrogels were prepared by dissolving the respective polymer in deionized water (2 % (w / v) ) and subsequent equilibration at 50 °C before measurement, and a mucin dispersion was prepared by dispersing mucin in PBS ( 10% (w / v) ; Dissolving 1 PBS tablet in 1 L deionized water (gives 140 mM NaCl, 10 mM phosphate buffer, and 3 mM KC1) ) to simulate mucus (pH ca . 6, 5-7, 5. A 2% (w / v) .

[0128] The Hydrogel ( 150 pL) was evenly deposited on a filter paper circle (20 mm diameter, Merck WHA23016160) with inert backing which was attached to the stationary platform with double-sided tape . The movable platform was equipped with the same filter paper, which was soaked with either 150 pL mucus (pH 4 ) , 150 pL mucus plus 30 pL NaOH (pH 10) , or 150 pL PBS and equilibrated for 3 minutes . The movable platform was lowered onto the surface of the hydrogel at 1 mm / s until a force of 0.2 N and further at 0.05mm / sec until a force of 2 N is reached which is kept constant for a total of 3 minutes .

[0129] For hydrogel particles the stationary platform 20 mm diameter filter paper was also equilibrated with mucin dispersion ( 150 pL, 10% (w / v) ) or PBS ( 150 pL) and was then covered with 30 mg of hydrogel microparticle preparation. NaOH (30 pL, 1 - 0.25 M) was added onto the mucin-soaked filter paper to validate the mucoadhesive properties at different pH (5.5, 7.5 and 10) . For analysis on how interparticle crosslinking is influencing the mucoadhesive properties, the hydrogel particles were covered with crosslinking solution ( 96 pL, 0.5% (w / v) gelatin, pH 9) or PBS ( 96 pL, pH 7.4 ) .

[0130] Detachment force was recorded at 0.05 mm / sec, until the filter papers were completely detached from each other, at room temperature (22 °C) . From the resulting curves the maximum pull off force, Fmax [mN] and total work of adhesion, Wt [mN x mm] trough integration were obtained. Each hydrogel was tested in triplicate and the average values were calculated. To subtract the influence of unspecific interactions between samples and the filter paper a negative control was measured by substituting the mucin dispersion with PBS . The results are shown in tables 3 to 5 .

[0131] Table 3 : Maximum pull off force, Fmax [mN] and total work of adhe-sion, Wt [mN x mm] for samples tested on filter papers with PBS . VS -AG is vinyl sulfone-modif led agarose, while NA is natural aga-rose .

[0132]

[0133]

[0134] A hydrogel of natural agarose exhibits very low, practically no adhesion to the filter paper, as does the hydrogel with a modification level of 20% . The hydrogel with a modification level of 35% shows improved adhesion, although its shear storage modulus G' is significantly lower than that of natural agarose . The microparticles show practically no adhesion to the filter paper .

[0135] Table 4 : Maximum pull off force, Fmax [mN] and total work of adhesion, Wt [mN x mm] for samples tested on filter papers with mucin at pH 10. VS-AG is vinyl sulfone-modif led agarose, while NA is natural agarose .

[0136]

[0137]

[0138] Table 5 : Maximum pull off force, Fmax [mN] and total work of adhesion, Wt [mN x mm] for samples tested on filter papers with mucin at pH 4. VS-AG is vinyl sulfone-modif led agarose, while NA is nat-ural agarose .

[0139]

[0140] Table 6 : Maximum pull off force, Fmax [mN] and total work of adhesion, Wt [mN x mm] for samples tested on filter papers with mucin at different pH values . VS-AG is vinyl sulfone-modif led agarose, while NA is natural agarose .

[0141]

[0142] The mucoadhesion increases with the pH value . Hydrogels / prepara-tions with a pH value greater than 4 are at least therefore preferred, further preferred are hydrogels with a pH value between 5and 11, further preferred between 6 and 11, further preferred between 7 and 11, particularly preferred are pH values of 7.5 to 10.5.

[0143] 8. Cell toxicity determination of a hydrogel with a 35% MoD agarose

[0144] The agarose polymers were extracted according to ISO-10993-12 in extraction medium (Dulbecco ' s Modified Eagle Medium F-12

[0145] (DMEM) from Thermo Fisher Scientific Inc . , USA) with 5% (v / v) fetal calf serum (FCS) from Sigma Aldrich, Germany, and with 100 U / mL Penicillin and 0.1 mg / mL Streptomycin) . The cell toxicity test was carried out using the human Gingival Fibroblast (hGF) (product 300703 from CLS Cell Lines Service GmbH, Germany) . Cells were cultured in DMEM with 5% (v / v) FCS . Cells were exposed to extracts, divinyl sulfone (DVS) 100 pM, 200 pM or respective controls and incubated for 24±1 h at 37±1 °C and with 5% carbondioxide content in a humidified incubator atmosphere . After exposure, the cell viability was determined using WST-8 reagent from TCI Deutschland GmbH, Germany, which was incubated for 1 . 5± 0.5 h at 37 °C and 5% CO2 in a humidified incubator until color development . Absorbance was measured with dual-beam photometer at A = 450 nm and normalized to vehicle or solvent control . According to the ISO 10993-5, extracts / chemicals reducing cell viability to <70% were classified as cytotoxic . None of the modified agarose samples showed cytotoxicity. The cell viability was as high as the control group ( 100%) or even higher ( 110%) . The cell viability of a 0, 2 mM DVS solution was zero and of a 0, 1 mM was 10% .

Claims

Patent claims1. Method for producing a mucoadhesive galenic preparation, wherein the mucoadhesive galenic preparation comprises a hydrogel comprising modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification, wherein the method comprises the following steps :A) providing a modified agarose, preferably in the form of a powder, wherein the modified agarose comprises at least vinyl sulfone groups as modification,B) mixing the modified agarose of step A) with an aqueous solution to form a hydrogel, preferably at a temperature below 50°C, more preferably at a temperature below 40°C,C) mixing at least one active ingredient or a combination of active ingredients with the provided modified agarose of step A) , and / or the aqueous solution or the hydrogel of step B) , preferably at a temperature below 90°C .

2. Method according to claim 1, wherein the modified agarose, comprising at least vinyl sulfone groups as modification, provided in step A) is produced comprising the following steps :Al ) dissolving natural agarose in an aqueous solution at a temperature above 90°C, preferably at a temperature of about 95°C or higher, to produce an aqueous natural agarose solution, such that the polymer chains have essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel,A2 ) cooling the aqueous natural agarose solution from step Al ) under stirring to a temperature below 90°C and above the gelation temperature of the dissolved natural agarose, preferably to a temperature below 50°C, suchthat the polymer chains forming essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel,A3) adjusting the pH value of the agarose solution from step A2 ) to 10-14, preferably 11-13, more preferably to about 12,A4 ) mixing divinyl sulfone to the agarose solution from step A3) to form modified agarose, such that the polymer chains forming essentially no secondary and / or tertiary structure, in particular that they do not form a hydrogel ,A5) separating the modified agarose of step A4 ) , comprising at least vinyl sulfone groups as modification, by precipitation, preferably by mixing with at least one precipitating agent and / or cooling, and preferably removing the precipitated modified agarose from the aqueous solution, preferably by means of centrifugation and / or filtration,A6) preferably cleaning of the removed modified agarose with a suitable cleaning agent,A7 ) preferably drying of the removed modified agarose, preferably by means of freeze-drying.

3. Method according to claim 1 or 2, wherein the method may comprise one or more of the following:- the aqueous natural agarose solution, resulting at least from step Al ) to step A3) comprises between 0.01-0, 5 wt . % natural agarose, preferably between 0.1-0, 5 wt . %, more preferably between 0.2-0. 4 wt . % natural agarose, in relation to the whole aqueous natural agarose solution,the precipitated modified agarose of step A5) forms liquid droplets and / or solid particles, preferably by stirring the mixture,- the mixture of step B) comprises at least 0.5 wt . % modified agarose, preferably between 0.5-20 wt . %, more preferably from 1 to 5 wt . % modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification, in relation to the whole composition of the hydrogel and / or the galenic composition,- the modified agarose of step A) is provided in the form of solid particles, preferably in the form of microparticles, more preferable spherical microparticles,- the aqueous solution of step B) comprises mixing of a crosslinking agent to the modified agarose .

4. Method according to claims 1 to 3, characterized in that the at least on active ingredient or a combination of active ingredients is in the form of particles, more preferable is a component of loaded particles, more preferable is a component of microparticles .

5. Galenic preparation comprising a hydrogel, wherein the hydrogel comprises modified agarose, wherein the modified agarose comprises at least vinyl sulfone groups as modification.

6. Galenic preparation according to claim 5, characterized in that the galenic preparation is a mucoadhesive galenic preparation .

7. Galenic preparation according to claims 5 or 6, characterized in that the galenic preparation is in the form of a dispersion, preferably a dispersion of solid particles .

8. Galenic preparation according to claim 7, characterized in that the dispersion comprises particles loaded with an active ingredient or a combination of active ingredients .

9. Galenic preparation according to claim 8, wherein the active ingredient is at least selected from the groups comprising flavonoids or flavonoid derivates .

10. Galenic preparation according to any previous claims, characterized in hat the modified agarose has a degree of modification of the vinyl sulfone groups between 10-50%, preferably between 10-45%, and more preferably between 15-40%, of the repeating units of a natural agarose .

11. Galenic preparation according to any previous claims, characterized in that the galenic preparation has a total work of adhesion of greater than about 40 mN mm, preferably greater than 50, more preferably greater than 100, and more preferable greater than 200 or 300 mN mm.

12. Galenic preparation according to any previous claims, characterized in that the galenic preparation is stable in storage in the ready-for-use condition in a primary packaging, preferably a pre-filled syringe, up to 6 months, preferably up to 12 or 24 months .

13. Galenic preparation according to any previous claim, characterized in that the galenic preparation is produced by the method according to claims 1 to 4 .

14. Kit for providing a galenic preparation according to claims 5 to 12 and / or produced by the method according to claims 1 to 4, comprising of a first component and a second component, wherein the first component comprises modified agarose, preferably as solid particles, wherein the modified agarose comprises at least vinyl sulfone groups as modification, andwherein the second component comprises an aqueous dispersant, preferably comprising a cross-linking agent for the formation of covalent bonds with the vinyl groups of the modified agarose .

15. The use of the galenic preparation according to claims 5 to 12 in treatment and / or prophylaxis of inflammation of the periodontium and / or caries, preferably including a subgingival scaling or gingival curettage .

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