Ocular insert for use in the treatment of dry eye

Abstract

A tear-fluid insoluble, ocular insert adapted for insertion into the eye of a subject, which is a mammal, for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture thereof, wherein the insert comprises a bacterial cellulose hydrogel and an aqueous solution comprising the therapeutic agent and / or balanced saline solution.

Description

[0001] Ocular insert for use in the treatment of dry eye

[0002] This application claims the benefit of European Patent Application EP24383158 filed October 21st, 2024.

[0003] Technical field

[0004] The present invention relates to an ocular insert useful in the treatment of ocular conditions, in particular a bacterial cellulose ocular insert for the treatment of dry eye. Background Art

[0005] In ophthalmology, the conventional method of treating eye conditions or diseases involves the repeated administration of solutions, suspensions or emulsions containing drugs or therapeutic agents in the form of eye drops or ophthalmic ointments. In some cases, administration must be carried out repeatedly throughout the same day, and even, in severe cases, every hour, which may result in a lack of patient adherence to treatments. In addition, efficiency may decrease due to blinking, dilution with tear fluid and drainage of tear fluid, thus decreasing the therapeutic effect of the drug.

[0006] For this reason, ocular inserts can be developed for the prolonged administration of a drug or therapeutic agent on the surface of the eye. Ocular inserts are devices, both soluble and insoluble, that contain therapeutic agents or drugs which are released into the ocular surface of the eye. They may be placed on the conjunctival space or on the cornea in the case of therapeutic contact lenses.

[0007] Insoluble ocular inserts typically consist of a body comprising a central reservoir containing the drug to be dispensed, in which the reservoir is surrounded by one or more surface layers typically consisting of an insoluble polymer permeable to the passage of the drug. Once placed in the eye, the insoluble inserts release the drug through mechanisms such as diffusion or osmosis, and once the treatment is completed, they are removed. An example of an insoluble ocular insert is Ocusert® (Alza Corporation, United States), a pilocarpine delivery system designed for the chronic treatment of glaucoma. It consists of three layers, two outer layers composed of ethylene vinyl acetate (EVA) and an inner layer with pilocarpine immobilized in alginate. Around the inner layer there is a ring with ethyl acetate impregnated with titanium dioxide to allow visualization of the systems during placement.

[0008] On the other hand, soluble systems are usually monolithic constructs with the drug dispersed or dissolved throughout the matrix that makes up the body of the insert. The matrix is typically made up of a soluble polymer or polymer blend. These inserts release the drug as they dissolve in the tear fluid, so unlike insoluble inserts, soluble inserts do not need to be removed from the eye after completion of treatment. Lacrisert® (Bausch & Lomb Inc., USA) is the best known soluble ocular insert, which consists of hydroxypropyl methylcellulose and is used in the treatment of dry eye syndrome.

[0009] However, the ocular inserts developed so far have certain disadvantages, resulting in low patient acceptance. Both the body of Ocusert® and Lacrisert® are rigid or semi-rigid, which can cause discomfort when placed in the eye or when they do not conform to the anatomy of the eye. For instance, Ocusert®, which is made of ethylene vinyl acetate presents expulsion and discomfort as the two greatest problems associated with their use (see L. Land et al.; Sizes and shapes of conjunctival inserts. International Contact Lens Clinic, .1994). vol. 21(11-12), pp. 212-217). In addition, in the case of Lacrisert®, adverse reactions such as blurred vision, ocular discomfort, foreign body sensation, ocular stinging, ocular irritation, swollen lids, excessive tearing, eyelash crusting may occur due to the resulting debris during the dissolution / degradation process.

[0010] US3828777A describes an ocular device for the controlled and continuous administration of a drug to the eye, comprising a microporous material which is insoluble in tear fluid, suggesting the use of a cellulose derivative. Such ocular devices are inserted in the portion of the eye bounded by the surface of the bulbar conjunctiva of the lid. It has been reported that such placement of the device would, however, be subject to eye movement and would not provide an anchored position, and also that this movement of the device causes pain, irritation, foreign body sensation and watering (see for instance US5147647, column 1, lines 45-55).

[0011] Anton-Sales, I., et al., in “Opportunities of Bacterial Cellulose to Treat Epithelial Tissues”, 2019, Current Drug Targets, 20, pp. 808-822, describes the potential use of bacterial cellulose (BC) to treat damaged epithelial tissues, such as wounds in the cornea and conjunctiva of the eye. Although the cellulose may be loaded with active ingredients and / or its natural water content may be modified, the authors allege that BC-based drug delivery systems have been mainly reported for dermal applications.

[0012] US2012231038A1 describes wound healing compositions comprising biocompatible cellulose hydrogel membranes to cover the wound, which are insoluble in water, and methods of use thereof. Ocular wounds are cited, and, similarly to US3828777A, both the healing and pain-alleviating effects are associated with the thickness of the hydrogel membrane, thus depending on the type of wound. Two types of membranes are described in this document, the “wet cellulose hydrogel membrane” containing bacterial cellulose in a 2 to 9% weight of the total hydrogel and having a Young’s modulus from about 100 kPa to about 700 kPa, and a “re-wet” membrane, which is preferred, containing bacterial cellulose in a 40 to 65% weight of the total hydrogel and having a Young’s modulus from about 4000 kPa to about 15000 kPa (15 MPa).

[0013] Thus, there is a need to provide new ocular inserts with sustained release of therapeutic agents that adapt to the anatomy of the eye, offering comfort for the patient and increasing treatment efficacy.

[0014] Summary of Invention

[0015] The inventors have surprisingly found that a hydrogel composed of a small amount of bacterial cellulose affords a soft, insoluble ocular insert with the appropriate characteristics for sustained drug release, as well as offering comfortability to the patient. Advantageously, the bacterial cellulose is biocompatible and free of endotoxins.

[0016] Furthermore, as a result of the nanometric size and hydrophilicity of its fibres, the inventors have found that bacterial cellulose forms a hydrogel in the form of a continuous open matrix that can be loaded with therapeutic agents and / or a balanced salt solution that are released in a prolonged manner. Thus, the ocular insert of the present invention avoids the problems associated with the periodic administration of drugs in the form of eye drops.

[0017] As such, a first aspect of the present invention relates to a tear-fluid insoluble, ocular insert adapted for insertion into the lower or upper fornix of the eye of a subject, which is a mammal, including a human, for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture of them, wherein the insert comprises: a) a bacterial cellulose hydrogel which is a matrix of cellulose nanofibers hydrated with water, comprising a percentage of dry fraction (i.e., solid fraction) of bacterial cellulose equal to or less than 1.8% by weight with respect to the total hydrogel, and b) an aqueous solution comprising the therapeutic agent and / or the balanced salt solution, wherein the solution is dispersed within the matrix for its sustained release. Advantageously, given the low weight percentage of the bacterial cellulose with respect to the total weight of the hydrogel, the inventors have surprisingly been able to fill the insert with a greater amount of treating solution. That is, the bacterial cellulose membrane acts merely as a delivery system, wherein the treatment solution is dispersed. As mentioned, the composition and nanofibrillated structure of the insert advantageously enables for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture of them for at least 8 hours, hence increasing treatment adherence of patients and with it, treatment efficacy.

[0018] Thus, in a second aspect, the present invention refers to an insert as defined above for use in the treatment and / or prevention of an ocular disease in a subject.

[0019] Ocular devices, such as inserts, are particularly prone to get contaminated or infected by diverse agents and, as such, must be produced, packed and preserved under aseptic conditions. Thus, in a third aspect, the present invention refers to a kit comprising: a) a sealed, sterile first container containing an insert as defined above, and b) optionally, means for inserting and / or removing the insert from the lower or upper fornix of a subject’s eye.

[0020] Brief Description of Drawings

[0021] FIG. 1a shows an image (above) of the bacterial cellulose hydrogel, a scanning electron microscopy image (below) showing the spatial arrangement of the cellulose nanofibers in the hydrogel.

[0022] FIG. 1b shows a scanning electron microscopy image of bacterial cellulose nanofibers, where "D" refers to the mean thickness or diameter of the cellulose nanofibers.

[0023] FIG. 2A and FIG. 2B show a representation of the rectangular prism-shaped insert of the invention.

[0024] FIG. 3 shows a representation of the insert of the invention in the shape of an ovoid prism. FIG. 4 shows a representation of the spheroid-shaped insert of the invention, with a diameter ranging from diameter 2-4 mm, and a length 4-16 mm.

[0025] FIG. 5 shows osmolarity variation of bacterial cellulose hydrogel containing balanced salt solution (BSS).

[0026] FIG. 6 shows instructions for inserting the ocular insert of the present invention.

[0027] FIG. 7 shows device insertion in the inferior conjunctival fornix of a subject.

[0028] FIG. 8 shows the ophthalmologist’s hyperemia assessment in the volunteers using the VBR scale, ranging from 0 to 100.

[0029] FIG. 9 shows Oxford grading system, ranging from 0 to V, to assess both corneal and conjunctival abnormalities after device removal, by staining the ocular surface with fluorescein and Lissamine green respectively.

[0030] FIG. 10 shows noninvasive tear break-up time test using Sirius +, CSO, Italy.

[0031] FIG. 11 shows NIBIIT results (where each dot is the mean of 3 values) for the study eye and control eye using Sirius +, OftalTech Solutions and IDRA® Ocular surface analyzer SBM SISTEMI. FIG. 12 shows different weights of the ocular insert after different extraction times (15 min (N=20), 1 h (N=18), 3 h (N=15), 6 h (N=15)), as well as the mean weight for each time. FIG. 13 A-E includes the responses from the comfort questionnaires of the clinical study. FIG. 14. Scatter plot showing the cumulative release of hyaluronic acid (pg / g of bacterial cellulose) from Hylo Comod® and Hylo Gel® impregnated into bacterial cellulose hydrogels. The data represent three independent experiments for each formulation, with three replicates taken per experiment. Error bars indicate the standard deviation. The release was monitored over 24 hours: for the first 8 hours, the system remained static (no stirring), followed by vigorous stirring from 8 to 24 hours. Complete release was assumed at 24 hours based on the application of vigorous stirring.

[0032] FIG. 15. Scatter plot showing the cumulative release of hyaluronic acid (%) from Hylo Comod® and Hylo Gel® impregnated into BC hydrogels. Data represent three independent experiments, with three measurements taken per experiment. Error bars indicate the standard deviation. The release was monitored over 24 hours: for the first 8 hours, the system remained static (no stirring), followed by vigorous stirring from 8 to 24 hours. Complete release was assumed at 24 hours based on the application of vigorous stirring.

[0033] FIG. 16. Scatter plot showing the cumulative release of hyaluronic acid (pg / g of BC) from Hylo Comod® and Hylo Gel® impregnated into bacterial cellulose hydrogels. Data represent the average of three independent experiments, with three measurements taken per experiment. Error bars indicate the pooled standard deviation across the experiments. The release was monitored over 24 hours: for the first 8 hours, the system remained static (no stirring), followed by vigorous stirring from 8 to 24 hours. Complete release was assumed at 24 hours based on vigorous stirring and long immersion time. Mean values of the three replicates are those described in FIG. 14 and FIG. 15 are plotted below, where the error bars are the pooled standard deviations.

[0034] FIG.17. Scatter plot showing the cumulative release of hyaluronic acid (%) from Hylo Comod® and Hylo Gel® impregnated into bacterial cellulose hydrogels. Data represent the average of three independent experiments, with three measurements taken per experiment. Error bars indicate the pooled standard deviation across the experiments. The release was monitored over 24 hours: for the first 8 hours, the system remained static (no stirring), followed by vigorous stirring from 8 to 24 hours. Complete release was assumed at 24 hours based on vigorous stirring and long immersion time.

[0035] Detailed description of the invention

[0036] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply throughout the description and claims.

[0037] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the,” also include the plural of the noun.

[0038] The word “comprise” for the purposes of the present invention encompasses the case of “consisting essentially of” and “consisting of”.

[0039] The term "eye insert" as used herein refers to an object or device adapted for placement or insertion into the lower or upper fornix of the eye of a subject, wherein said eye insert further comprises a therapeutic agent and / or a balanced salt solution that is released by the eye insert into the anterior segment of the eye. The insert of the invention is insoluble in aqueous solutions, and more particularly in tear fluid, because the insert consists of a bacterial cellulose hydrogel.

[0040] The term "soft eye insert" refers to the fact that the insert of the invention is applied in a wet state and has low resistance to deformation or compression. Thus, the ocular insert of the invention adapts to the anatomy of the eye when inserted into the upper or lower fornix of a subject's eye. Thus, unlike rigid eye inserts, the insert of the invention is soft and causes very minimal discomfort when worn by the subject in the fornix and does not cause irritation.

[0041] The terms "insert", "insertion" or "inserted" refer to the process by which the insert of the invention is placed or deposited in the lower or upper fornix of a subject's eye.

[0042] The terms "tear fluid", "tears" or "tear film", used interchangeably in the invention, refer to the fluid secreted by the lacrimal glands, and which lubricates the eyes of a subject. Tears are composed mainly of water comprising electrolytes, proteins, lipids and mucins. The osmolarity of tear fluid is between 290 mOsmol / L and 360 mOsmol / L, approximately 310 mOsmol / L.

[0043] The term "osmolarity" refers to the concentration of total solute(s) in a volume of solution, where the volume is expressed in liters.

[0044] In the present invention, the expression "adapted for insertion into the lower or upper fornix of a subject's eye" refers to the fact that the insert of the invention has dimensions which allow it to be inserted and adapted to the anatomical space of the lower or upper fornix of a subject's eye. Thus, the insert of the invention can be placed and retained in the lower or upper, preferably lower, fornix of the eye of a subject for a prolonged period of time, causing very minimal discomfort to the wearer and being retained despite the movement of the eye or eyelids. The wet insert can be easily removed by the subject. As known to the person skilled in the art, the expression "lower or upper fornix of a subject's eye" refers to the lower or upper space, respectively, of a subject's eye between the bulbar and palpebral conjunctivae.

[0045] The term "subject" refers to a human or animal, of any sex and age, the animal being a non-human mammal having an anatomy of the eye where the insert of the invention can be placed. Examples of animals with optimal anatomy for insertion of the insert of the invention include, without limitation, non-human primates, dogs, horses, cows, pigs, rabbits, goats or sheep. Thus, the insert of the invention is optimal for both human and veterinary use.

[0046] The terms “treat” or “treatment” are meant to include alleviating, inhibiting, reducing or eradicating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease or condition, or alleviating, reducing or eradicating the cause(s) of the disorder, disease, or condition itself.

[0047] The terms "prevent" or "prevention" are meant to include preventing further symptoms, preventing the underlying causes of symptoms, inhibiting the disease or condition, for example halting the development of the disease or condition, and are intended to include prophylaxis. The terms also include achieving prophylactic benefit. For prophylactic benefit, the compositions are optionally administered to an individual at risk of developing a particular disease, to an individual reporting one or more of the physiological symptoms of a disease, or to an individual at risk of disease recurrence.

[0048] In the context of the present invention, the term "therapeutic agent" refers to a substance or mixture of two or more substances which produces a physiological effect or modulates a biological process, producing a beneficial or therapeutic effect on the subject, for example, a human, to whom it is administered. As known to the person skilled in the art, examples of therapeutic agents in the context of the present invention include, without limitation, hyaluronic acid or its sodium salt; plasma rich in growth factors, autologous serum, a mydriatic drug, an antibacterial, an antiviral, antifungal, antiparasitic a antiinflammatory, hypotensive , miotic; peptide; protein, carbomer, carboxymethyl cellulose, hydroxypropyl guar, and combinations thereof. Other examples of proteins are growth factors or insulin.

[0049] When referring to hyaluronic acid herein it may be understood as covering hyaluronic acid or its sodium salt form, both within the scope of the present invention. The term “mydriatic drug” refers to a type of medicine that make the pupil of the eye dilate (open up). Examples of mydriatics are cyclopentolate, tropicamide, phenylephrine, atropine, homatropine, cyclopentolate, or atropine.

[0050] The term "bacterial cellulose hydrogel", in the context of the present invention, refers to a three-dimensional network or matrix of cellulose nanofibers produced by bacteria, hydrated with a liquid which is typically water or an aqueous solution such as a physiological solution. It may also be named as biosynthetic cellulose, microbial cellulose, bacterial nanocellulose, biocellulose, microfibrillar cellulose, microbial nanocellulose, biosynthesized cellulose, or biosynthesized nanocellulose.

[0051] The term "pure bacterial cellulose", in the context of the present invention, refers to cellulose free of other substances such as, but not limited to, lignin, pectin, carboxymethylcellulose and cross-linking agents.

[0052] According to the IIIPAC definitions, the term "mesopore" refers to pores with a diameter between 2 and 50 nm, and the term "macropore" refers to a pore with a diameter greater than 50 nm. The same definition applies in the present invention for the pores formed by the bacterial cellulose nanofibers.

[0053] In the present invention the term "aqueous solution", refers to a solution comprising at least one dissolved substance, and the solvent is water or mainly water. The aqueous solution may be a therapeutic agent or an aqueous solution comprising a therapeutic agent.

[0054] In the present invention, the term "saline solution" refers to an aqueous solution comprising salts present in physiological fluids such as sodium chloride. In particular, the term ■ “balanced salt solution (BSS)”- refers to a solution made to a physiological pH and isotonic salt concentration. Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. In medicine, balanced salt solutions can be used as an irrigation solution during intraocular and extraocular surgeries.

[0055] The term "autologous serum" refers to a fraction of the blood of the same subject to which it is to be administered, being the upper fraction obtained after coagulation and centrifugation of the blood.

[0056] The term “plasma rich in growth factors” refers to a fraction of blood, usually from the same subject to which it is to be administered, this fraction being the top fraction obtained after centrifugation of unclotted blood.

[0057] The term "antibacterial" refers to a substance that inhibits the growth of bacteria or kills them. Antibacterials are used for the treatment and / or prevention of infections caused by bacteria. Examples of antibacterials known to the person skilled in the art include, without limitation, Amikacin, Azithromycin, Cefazolin, Ceftazidime, Cefuroxime, Ciprofloxacin, Clindamycin, Chloramphenicol, Gentamicin, Imipenem, Moxifloxacin, Netilmicin, Ofloxacin Tobramycin, and Vancomycin.

[0058] The term "antifungal" refers to a substance that inhibits the growth of fungi or kills them. Antifungals are used for the treatment and / or prevention of infections caused by fungi. Examples of antifungals include, but are not limited to, Amphotericin, Fluconazole, Natamycin or Voriconazole.

[0059] The term "antiparasitic" refers to a substance that inhibits the growth of parasites or kills them. Dewormers are used for the treatment and / or prevention of infections caused by parasites. Examples of dewormers include, but are not limited to, propamidine or hexamidine.

[0060] The term "anti-inflammatory" refers to a substance that prevents or decreases inflammatory processes. Examples of anti-inflammatory drugs include, but are not limited to, Bromfenac, Dexamethasone, Diclofenac, FluoromethoIone, Ketorolac, Medroxyprogesterone or Prednisolone.

[0061] The term "hypotensive" refers to a substance with the ability to reduce tension in blood vessels. Since the placement of the insert of the present invention is in the eye, the hypotensive is an ocular hypotensive that has the ability to reduce intraocular pressure. Examples of ocular hypotensives include without limitation Apraclonidine, Bimatoprost, Brimonidine, Brinzolamide, Carteolol, Dorzolamide, Latanoprost or Timolol.

[0062] In the present invention, the term "eye disease" refers to a disease or condition in which the eye, or a part of the eye, is affected.

[0063] As used herein, the term "ocular surface" comprises the conjunctiva and cornea, together with elements such as the lacrimal apparatus, including the lacrimal puncta, as well as the lacrimal canaliculus and associated palpebral structures. In the context of the invention, the ocular surface also encompasses the aqueous humour. As understood by a person skilled in the art, diseases affecting the surface of the eye include, without limitation, inflammatory conditions of the cornea (keratitis) and conjunctiva (conjunctivitis), deformations of the corneal structure, ocular erosion, ocular abrasion, corneal ulcers or alterations of the tear film such as dry eye.

[0064] The term "dry eye" refers to a disease or condition of the surface of the eye, caused by a failure or deficiency in tear film production leading to a lack of lubrication of the eye. As used herein, the term "bilaterally" or "bilateral" refers to an insertion of at least one insert of the invention into the lower or upper fornix of each of the subject's eyes.

[0065] "Unilaterally" or "unilaterally" thus refers to an insertion of at least one insert of the invention into the lower or upper fornix in one of the eyes of the subject.

[0066] The term "eye drops" refers to a solution suitable for ophthalmic use.

[0067] For the purposes of the invention, the expression “room temperature” means a temperature from 20 to 25 °C.

[0068] As mentioned above, a tear-fluid insoluble, ocular insert adapted for insertion into the lower or upper fornix of the eye of a subject, which is a mammal, including a human, for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture thereof, wherein the insert comprises: a) a bacterial cellulose hydrogel which is a matrix of cellulose nanofibers hydrated with water, comprising a percentage of dry fraction of bacterial cellulose equal to or less than 1.8% by weight with respect to the total hydrogel, and b) an aqueous solution comprising the therapeutic agent and / or the balanced salt solution, wherein the solution is dispersed within the matrix for its sustained release. In a particular embodiment, the percentage of dry fraction of bacterial cellulose is from 0.1 to 1.8% by weight with respect to the total hydrogel. In another particular embodiment, the percentage of dry fraction of bacterial cellulose is from 0.4 to 1.5% by weight with respect to the total hydrogel.

[0069] Unlike rigid eye inserts, the wet insert of the invention is soft and causes minimal discomfort when worn by the subject in the fornix. Accordingly, in a particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the insert adapts to the anatomy of the eye when inserted into the upper or lower fornix of a subject's eye.

[0070] In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the insert has a deformation value of between 20 % and 30 %. In a more particular embodiment, the insert has a deformation value of between 22 % and 26 %. In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the insert has a tear strength value (Fmax) of between 4.8 N / cm and 5.4 N / cm. In a more particular embodiment, the insert has a tear strength value of between 5.0 N / cm and 5.2 N / cm. The values of deformation and tear strength are determined by axial tension test on a hydrated hydrogel. The determination of strain and tear strength values is conducted as indicated in Example 8. The amount of bacterial cellulose in the hydrogel insert of the invention can be determined by techniques known to a person skilled in the art, such as gravimetry, namely by weighing the dehydrated material, or by thermogravimetry.

[0071] Bacterial cellulose has a large surface area that allows for high retention of water or aqueous solutions due to its hydrophilic character, porosity and thickness in the nanometer range of cellulose fibers. The bacterial cellulose of the insert of the invention is a pure polymer that does not comprise other compounds such as lignin, hemicellulose or pectins, unlike other cellulose fibres such as those produced by plants, from 10 pm to 50 pm thick measured by digital micrometer. It is a hydrophilic and insoluble polymer of 1,4-P-glucan, synthesized and excreted by bacteria in the form of cellulose nanofibers.

[0072] Accordingly, in a particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose has a high water or aqueous solution retention capacity. In another particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the bacterial cellulose consists of is free from lignin, hemicellulose, or pectins.

[0073] The bacterial cellulose hydrogel of the insert of the invention is formed by randomly intertwined cellulose nanofibers in the three spatial directions (Fig. 1A), forming a porous structure with high water holding capacity, thus forming a hydrogel, where the amount of water retained can be up to 200 times the weight of the polymer. Thus, the bacterial cellulose of the insert of the present invention advantageously forms hydrogels without the need for crosslinking or gelling agents.

[0074] As such, in a particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose does not need further cross-linking agents. In a more particular embodiment of the insert of the present invention, no purification steps of the bacterial cellulose nanofibers are necessary.

[0075] In a particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose can be produced by different bacteria including, but not limited to Komagataeibacter, in particular, Komagataeibacter xylinus (synonyms: Gluconacetobacter xylinus and Acetobacter xylinus) and Komagataeibacter hansenii. Methods for producing bacterial cellulose are known (see for instance US10829567B2)

[0076] In a more particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose is produced by a coculture of the Komagataeibacter xylinus and Komagataeibacter hansenii.

[0077] In an even more particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterium of the genus of Komagataeibacter used is Komagataeibacter xylinus. In an even more particular embodiment, the bacteria strain is Komagataeibacter xylinus CECT 473.

[0078] In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose nanofibers have a thickness of between 5 nm and 100 nm. In a particular embodiment, the nanofibers have a thickness of between 30 nm and 70 nm. In a more particular embodiment, the nanofibers have a thickness of between 40 nm and 60 nm.

[0079] In another particular embodiment, the nanofibers have a thickness of between 5 nm and 60 nm. In another particular embodiment, the nanofibers have a thickness of between 5 nm and 50 nm. In a more particular embodiment, the nanofibers have a thickness of between 5 nm and 30 nm. In a more particular embodiment, the nanofibers have a thickness of between 5 nm and 20 nm. In a more particular embodiment, the nanofibers have a thickness of between 12 nm and 18 nm.

[0080] In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose nanofibers are formed under static or stirred conditions. In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the bacterial cellulose nanofibers form mesopores of size between 10 nm and 50 nm and macropores of size greater than 50 nm. In a more particular embodiment, the macropores are of size between 50 nm and 2000 nm.

[0081] In another particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the insert is composed of either one or multiple layers.

[0082] The bacterial cellulose of the insert of the present invention can be produced by static or stirred culture methods known to a person skilled in the art. Suitable conditions for the growth are an appropriate medium, temperatures around 20 to 40 °C, and a relative humidity around 40-70%. In a particular embodiment, the temperature is around 25-35°C, preferably, 30 °C. In another particular embodiment, the humidity is around 40-60%. Once produced, the hydrogel of the invention can be molded during BC biosynthesis, in particular, using custom made molds and / or cut according to the dimensions of the ocular insert of the invention, in any shape suitable for its retention in the lower or upper fornix of a subject's eye.

[0083] The hydrogel of the invention may also be incorporated into a sealed container and be sterilized by autoclave or gamma ray. Preferably autoclave. The bacterial cellulose hydrogel used in the ocular insert of the present invention is obtainable from a bacteria strain Komagataeibacter xylinus (synonyms:

[0084] Gluconacetobacter xylinus and Acetobacter xylinus) or Komagataeibacter hansenir, and a co-culture of both by a process which comprises: a) Growing the corresponding bacteria on a medium such as Hestrin-Schramm medium and at conditions such as at 30 °C, 60 % relative humidity; to form either films or spheroids; b) collecting the bacterial cellulose hydrogel formed at the interface, washing them with appropriate solutions; and optionally, sterilize them in an autoclave; c) impregnating the bacterial cellulose films or the bacterial cellulose spheroids with the desired therapeutic agent with at least twice their own volume for an appropriate period of time (for instance: 24 h); and d) cutting the bacterial cellulose hydrogel in a laminar flow hood under sterile conditions using a scalpel or laser to give the desired shape. An example of the detailed procedure is set forth in the Examples. In a particular embodiment, the Hestrin-Schramm medium comprises D-glucose, peptone, yeast extract, citric acid, Na2HPC>4-12 H2O, and agar. In another particular embodiment, the bacteria is Komagataeibacter xylinus species. In another particular embodiment, the bacteria is Komagataeibacter xylinus strain CECT 473.

[0085] In a particular embodiment of the insert of the present invention, in combination with any of the embodiments above or below, the insert may be molded during BC biosynthesis and / or cut into a rectangular or ovoid prism. In a more particular embodiment, the insert is molded during BC biosynthesis and / or cut into a rectangular parallelepiped or ovoid prism with dimensions between 4 mm and 16 mm in length, 2 mm to 4 mm in width and 0.5 mm to 2.5 mm in thickness, as shown in FIG.2 and FIG.3.

[0086] In another particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the insert is in the form of a spheroid. In a more particular embodiment, in combination with any of the embodiments above or below, the insert in the form of a spheroid is one where both the width and the thickness have the same dimensions, in that they both refer indistinctly to the diameter of the spheroid at its minor axis. In an even more particular embodiment, the insert may be cut and / or molded into a spheroid with dimensions between 4 mm and 16 mm in length, 2 mm to 4 mm in width and 2 mm to 4 mm in thickness, as shown in FIG.4.

[0087] In a particular embodiment, the ocular insert may be rectangular prism-shaped insert or a ovoid prism. In another particular embodiment, the prism-shaped insert has the following sizes: 12 mm x 3 mm x 1 ,5 ± 1 mm.

[0088] As mentioned above, the hydrogel of the insert of the present invention can be loaded with different substances, such as therapeutic agents, which are released from the hydrogel when they are placed, for example, in contact with a tissue or organ of a subject, in particular the eye, and the effect achieved may be local and / or systemic.

[0089] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the bacterial cellulose hydrogel comprises a therapeutic agent. In a more particular embodiment, in combination with any of the embodiments above or below, the therapeutic agent is comprised in or evenly distributed throughout the bacterial cellulose hydrogel. In an even more particular embodiment, the therapeutic agent is distributed through the hydrogel after exchange with the liquid in hydrogel. In another more particular embodiment, the therapeutic agent is absorbed into the bacterial cellulose hydrogel when the two are in contact.

[0090] In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the therapeutic agent comprised within the aqueous solution is selected from the list consisting of hyaluronic acid, plasma rich in growth factors, autologous serum, an antibacterial, an antifungal, an antiparasitic, an antiinflammatory, a hypotensive, a peptide, a protein, and any combinations thereof.

[0091] In a particular embodiment, the insert of the invention is for the sustained release of an aqueous saline solution allowing tissue hydration, i.e. a balanced salt solution. As known to a person skilled in the art, saline solutions may comprise salts in a concentration similar to that of biological fluids and may be isotonic (salt concentration equal to that of biological fluids) or hypotonic (salt concentration lower than that of biological fluids).

[0092] In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the saline solution is a hypotonic saline solution comprising sodium chloride in a concentration lower than that of biological fluids or tissues, being 0.9 % w / v sodium chloride concentration in biological fluids. Preferably the concentration of sodium chloride in the hypotonic saline solution is between 0.32 % w / v and 0.64 % w / v. In a more particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises potassium chloride in a concentration lower than that found in biological fluids or tissues, preferably the concentration of potassium chloride in the hypotonic saline solution is between 0.0375 % w / v and 0.075 % w / v.

[0093] In a more particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises at least one of the salts selected from the list consisting of calcium chloride dihydrate (between 0.024 % w / v and 0.048 % w / v in the hypotonic saline solution), magnesium chloride hexahydrate (between 0,015 % w / v to 0,03 % w / v in the hypotonic saline solution), sodium acetate trihydrate (between 0,015 % w / v and 0,03 % w / v in the hypotonic saline solution), sodium acetate dihydrate (between 0,085 % w / v and 0,17 % w / v in the hypotonic saline solution), and any combination thereof.

[0094] In another particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hyaluronic acid solution is an aqueous solution comprising hyaluronic acid in an amount of between 0.1 % w / v and 0.4 % w / v, wherein said hyaluronic acid may be cross-linked or not.

[0095] In a more particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises hyaluronic acid in an amount of between 0.1 % w / v and 0.4 % w / v.

[0096] In another particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises a compound selected from a carbomer, carboxymethyl cellulose, hydroxypropyl guar, dextran, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

[0097] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the antibacterial is selected from the list consisting of Amikacin, Azithromycin, Cefazolin, Ceftazidime, Cefuroxime, Ciprofloxacin, Clindamycin, Chloramphenicol, Gentamicin, Imipenem, Moxifloxacin, Netilmicin, Ofloxacin, Tobramycin and Vancomycin.

[0098] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the antifungal is selected from a list consisting of Amphotericin, Fluconazole, Natamycin and Voriconazole.

[0099] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the antiparasitic is selected from the list consisting of propamidine and hexamidine.

[0100] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the anti-inflammatory agent is selected from the list consisting of Bromfenac, Dexamethasone, Diclofenac, FluoromethoIone, Ketorolac, Medroxyprogesterone, Prednisolone, Loteprednol, Cyclosporin A, Lifitegrast, and tacrolimus.

[0101] In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotensive agent is selected from the list comprising Apraclonidine, Bimatoprost, Brimonidine, Brinzolamide, Carteolol, Dorzolamide, Latanoprost and Timolol. In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the antibacterial, antifungal, antiparasitic, antiinflammatory, hypotensive, peptide and protein are formulated in solution form, preferably aqueous solution.

[0102] In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the protein is insulin.

[0103] The therapeutic agent comprised within the insert of the invention must have an osmolarity lower than the osmolarity of the tear fluid of the subject in order for the therapeutic agent to be released from the bacterial cellulose hydrogel of the insert of the invention, producing its effect on the surface of the eye of the subject to whom it is administered.

[0104] Thus, in a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the therapeutic agent has an osmolarity of between 150 mOsmol / L and 300 mOsmol / L measured with an automatic cryoscopic osmometer (Gonotec® OSMOMAT™ 030). In a more particular embodiment, the therapeutic agent has an osmolarity of between 150 mOsmol / L and 280 mOsmol / L. The difference in osmolarity between the therapeutic agent and the tear fluid allows the release of the therapeutic agent once the insert of the invention is in contact with the eye of the subject. The hydrogel of the invention loaded with the different substances mentioned above, may also be incorporated into a sealed container, and be sterilized by autoclave or gamma ray. Preferably, by autoclave.

[0105] In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the ocular insert is maintained in the lower or upper fornix of the eye of the subject for at least 2 hours. In a more particular embodiment, the ocular insert is maintained in the lower or upper fornix of the eye of the subject for a period of time between 2 hours and 24 hours. In another more particular embodiment, the ocular insert is maintained for a period of time of between 2 hours and 12 hours. In an even more particular embodiment, the ocular insert is maintained for a period of time of between 2 hours and 9 hours. After the period of time in which the insert is in the subject's fornix has elapsed, the insert is removed, and a new insert can be inserted. The insertion of new inserts is performed periodically, depending on the needs of each subject.

[0106] Once the ocular insert of the invention is inserted or placed in the fornix of the subject's eye, said therapeutic agent is released in a prolonged manner thereby producing a beneficial or therapeutic effect. In a particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the insert releases the therapeutic agent for at least 8 hours. In a more particular embodiment, the ocular insert releases the therapeutic agent for at least 7 hours. In an even more particular embodiment, the ocular insert releases the therapeutic agent for at least 6 hours. In an even more particular embodiment, the ocular insert releases the therapeutic agent for at least 5 hours.

[0107] As the therapeutic agent is released, the amount thereof in the insert of the invention decreases. However, due to the high capacity of the bacterial cellulose hydrogel of the present invention for the retention of water or aqueous medium, the insert of the invention can be recharged by application of the therapeutic agent in the form of eye drops, when the insert is in the fornix of the subject’s eye.

[0108] Advantageously, as shown in Example 9, the insert of the invention has proven to be safe, even when worn for more than 12 hours, in that it does not affect tear film.

[0109] In a second aspect, the invention relates to an insert as defined above, for use in the treatment and / or prevention of an ocular disease in a subject.

[0110] This aspect can also be formulated as the use of a bacterial cellulose hydrogel matrix containing less than 1.8% weight of bacterial cellulose with respect to the total hydrogel as defined above for the preparation of an insoluble, soft ocular insert adapted for insertion into the lower or upper fornix of the eye of a subject for the prevention and / or treatment of an ocular disease.

[0111] The invention also relates to a method of treatment of a subject suffering from or being susceptible of suffering from an ocular disease, said method comprising the administration to said patient of a therapeutically effective amount of an agent comprised within the bacterial cellulose hydrogel as defined above.

[0112] All the particular embodiments of the use of the invention are also embodiments of the method to prevent and / or treat an ocular disease.

[0113] In a particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the ocular disease is a disease of the surface of the eye. That is, a disease where the affected part of the eye is its surface.

[0114] In a more particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the disease affecting the surface of the eye is selected from the list comprising dry eye, ocular perforation, ocular chemical burn, ocular thermal burn, limbal stem cell deficiency, corneal infection, neurotrophic keratopathy, ocular erosion, ocular abrasion, keratitis, conjunctivitis, keratoconus, bacterial ulcer, fungal ulcer, uveitis, glaucoma and post keratoprosthesis transplantation.

[0115] In another more particular embodiment, in combination with any of the embodiments above or below, the disease affecting the surface of the eye is dry eye. In an even more particular embodiment, the disease is dry eye, and the therapeutic agent comprised within the aqueous solution is selected from the list consisting of hyaluronic acid, plasma rich in growth factors and autologous serum.

[0116] In a particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the subject is a human or an animal. In a more particular embodiment, the subject is a human.

[0117] In a particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the saline solution is a hypotonic saline solution. In a more particular embodiment, the hypotonic saline solution comprises sodium chloride in a concentration lower than that of biological fluids or tissues. In an even more particular embodiment, the concentration of sodium chloride in the hypotonic saline solution is between 0.32 % w / v and 0.64 % w / v. In a more particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises potassium chloride in a concentration lower than that found in biological fluids or tissues. In an even more particular embodiment, the concentration of potassium chloride in the hypotonic saline solution is between 0.0375 % w / v and 0.075 % w / v.

[0118] In a more particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises at least one of the salts selected from the list consisting of calcium chloride dihydrate (between 0.024 % w / v and 0.048 % w / v in the hypotonic saline solution), magnesium chloride hexahydrate (between 0.015 % w / v and 0.03 % w / v in the hypotonic saline solution), sodium acetate trihydrate (between 0.015 % w / v and 0.03 % w / v in the hypotonic saline solution), sodium acetate dihydrate (between 0.085 % w / v and 0.17 % w / v in the hypotonic saline solution), and any combination thereof.

[0119] In a particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the hyaluronic acid solution is an aqueous solution comprising hyaluronic acid in an amount of between 0.1 % w / v and 0.4 % w / v.

[0120] In a more particular embodiment of the insert of the invention, in combination with any of the embodiments above or below, the hypotonic saline solution further comprises hyaluronic acid in an amount of between 0.1 % w / v and 0.4 % w / v.

[0121] In a particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the treatment and / or prevention of an ocular disease comprises: a) introducing the insert into the fornix of the eye that contains the therapeutic agent

[0122] In a more particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the ocular insert is inserted into the lower or upper fornix. In a more particular embodiment, the insert is inserted into the lower fornix of the subject's eye. In another particular embodiment of the use of the insert of the invention for the treatment and / or prevention of an ocular disease, in combination with any of the embodiments above or below, the insertion may be bilateral or unilateral.

[0123] It is part of the invention a sealed, sterile first container containing an insert as defined above. In a particular embodiment, the sealed, sterile first container containing an insert as defined above may be sterilized by autoclave or gamma ray. Preferably, by autoclave. In a third aspect, the invention relates to a kit comprising: a) a sealed, sterile first container containing an insert as defined above, and b) optionally, means for inserting and / or removing the insert from the lower or upper fornix of a subject’s eye. The kit may also comprise instructions on how to use the elements of the kit.

[0124] In a particular embodiment of the kit of the invention, in combination with any of the embodiments above or below, the kit comprises a plurality of packaging units, where each packaging unit comprises an eye insert. In an even more particular embodiment, the packaging units are in the form of a blister.

[0125] The insert of the invention can be easily inserted and removed with the fingers, and furthermore, the insert of the invention can be inserted and removed by using means such as an applicator. As shown in Example 9, volunteers found the insertion process and wearing of the insert to be comfortable.

[0126] In a more particular embodiment of the kit of the invention, in combination with any of the embodiments above or below, the means for inserting the insert is in the form of an applicator and / or a system for removing the insert from the subject's fornix without direct contact with the hands.

[0127] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

[0128] Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0129] Clauses:

[0130] Clause 1. A tear-fluid insoluble, ocular insert adapted for insertion into the lower or upper fornix of the eye of a subject, which is a mammal, for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture thereof, wherein the insert comprises:

[0131] a) a bacterial cellulose hydrogel which is a matrix of cellulose nanofibers hydrated with water, comprising a percentage of dry fraction of bacterial cellulose equal to or less than 1.8% by weight with respect to the total hydrogel, and

[0132] b) an aqueous solution comprising the therapeutic agent and / or the balanced salt solution, wherein the solution is dispersed within the matrix for its sustained release.

[0133] Clause 2. The insert according to clause 1, wherein the bacterial cellulose is obtainable from a Komagataeibacter bacteria.

[0134] Clause 3. The insert according to clause 2, wherein the bacteria is selected from Komagataeibacter xylinus and Komagataeibacter hansenii, and a co-culture of them.

[0135] Clause 4. The insert according to clause 3, wherein the hydrogel is composed of nanofibers having a thickness of between 5 nm and 100 nm.

[0136] Clause 5. The insert according to any of the clauses 1-4, wherein the therapeutic agent comprised within the aqueous solution is selected from the group consisting of hyaluronic acid, plasma rich in growth factors, autologous serum, a mydriatic, an antibacterial, antiviral, antifungal, antiparasitic, anti-inflammatory, hypotensive, peptide, protein, carbomer, carboxymethyl cellulose, hydroxypropyl guar, and combinations thereof.

[0137] Clause 6. The insert according to clause 5, which is for the sustained release of the balanced salt solution.

[0138] Clause 7. The insert according to any of the clauses 1-6, wherein the aqueous solution has an osmolarity between 150 mOsmol / L and 300 mOsmol / L measured with an automatic cryoscopic osmometer (Gonotec® OSMOMAT™ 030).

[0139] Clause 8. The insert according to any of the clauses 1-7, wherein the sustained release of either the therapeutic agent and / or the balanced salt Solution, lasts for at least 8 hours.

[0140] Clause 9. An insert as defined in any of the clauses1-8, for use in the treatment and / or prevention of an ocular disease in a subject.

[0141] Clause 10. The insert for use according to clause 9, wherein the ocular disease is a disease of the surface of the eye.

[0142] Clause 11. The insert for use according to clause 10, wherein the ocular disease of the surface of the eye is selected from the list comprising dry eye, ocular perforation, ocular chemical burn, ocular thermal burn, limbal stem cell deficiency, corneal infection, neurotrophic keratopathy, ocular erosion, ocular abrasion, keratitis, conjunctivitis, keratoconus, bacterial ulcer, fungal ulcer, uveitis, glaucoma, and post keratoprosthesis transplantation.

[0143] Clause 12. The insert for use according to clause 11 , wherein the ocular disease is dry eye.

[0144] Clause 13. The insert for use according to any of the clauses 9-12, wherein the subject is a human.

[0145] Clause 14. The insert for use according to any of the clauses 9-13, wherein the treatment and / or prevention of an ocular disease comprises:

[0146] a) introducing the insert into the fornix of the eye.

[0147] Clause 15. A kit comprising:

[0148] a) a sealed, sterile first container containing an insert as defined in any of the clauses 1-8, and b) means for inserting and / or removing the insert from the lower or upper fornix of a subject’s eye.

[0149] Examples

[0150] Abbreviations:

[0151] BSS (balanced salt solution), RH (relative humidity), MQ (Milli-Q), NaOH (sodium hydroxide), CECT (Spanish Culture Collection), HS (Hestrin-Schramm), m (mass), BET (Brunauer-Emmet-Teller),7SD (standard deviation), OD (oculus dexter), OS (oculus sinister), VBR (validated bulbar redness), NIBIIT (noninvasive tear break-up time), secs (seconds),

[0152] Example 1: Production of bacterial cellulose hydrogel under static conditions Komagataeibacter xylinus strain CECT 473, is commercially available and is deposited in the Spanish type Culture Collection) is grown on solid Hestrin-Schramm medium (HS, see composition Table 1) at 30 °C, 60 % RH

[0153] Table 1: Composition of the Hestrin-Schramm culture medium

[0154] Reagent Mass (g) for 1 L of MQ-water

[0155] D-glucose 20

[0156] Peptone 5

[0157] Yeast extract 5

[0158] Citric acid 1.15

[0159] Na2HPO4-12H2O 6.8

[0160]

[0161] Agar 15

[0162] Colonies are then expanded in liquid HS medium (i.e. without agar) using a sterile loop and incubated at 30 °C, 60 % RH. The bacteria are then diluted in fresh HS medium and grown on sterile plates at 30 °C, 60 % rh.

[0163] Bacterial cellulose films (bacterial cellulose hydrogel) formed at the interface (HS mediumair) are collected and cleaned: in a 50 % (v / v) ethanol-water solution, with boiled water, and with 0.1 M NaOH. Finally, the films are washed with MQ water (Milli-Q) until the pH is neutral and autoclaved.

[0164] The bacterial cellulose films are impregnated with the desired therapeutic agent (at least twice their own volume) for 24 h and cut in a clean room in a laminar flow hood under sterile conditions using a scalpel to give the desired shape. The bacterial cellulose hydrogel can be cut to the dimensions and shape of the insert of the invention.

[0165] Example 2: Production of bacterial cellulose hydrogel under stirred conditions Komagataeibacter xylinus strain CECT 473 (deposited in the Spanish Culture Collection) is grown on solid Hestrin-Schramm medium (HS, see composition Table 1) at 30 °C, 60 % RH. Colonies were then expanded in HS liquid medium (i.e. without agar) using a sterile loop and incubated at 30 °C, 60 % RH. Bacterial cellulose spheroids were prepared by mixing the bacteria in fresh HS medium in an Erlenmeyer flask and the system is placed on an orbital shaker inside the incubator (30 °C, 60 % RH). Finally, the bacterial cellulose spheroids (bacterial cellulose hydrogel) were collected and cleaned: in a 50 % (v / v) water-ethanol solution, with boiled water, and with 0.1 M NaOH. Finally, the spheres were washed with MQ water (Milli-Q) until the pH was neutral and autoclaved.

[0166] The bacterial cellulose spheres were impregnated with the desired therapeutic agent (at least twice their own volume) for 24 hours.

[0167] Example 3: Determination of the dry weight of the hydrogel

[0168] The wet hydrogels obtained in example 1 were weighed after the cleaning step. They were then dried between two Teflon plates in a drying oven at 70 °C for 24 h and weighed once dry. The dry fraction, corresponding to the bacterial cellulose, was calculated from the wet and dry weight.

[0169] mCdry)

[0170] dry fraction (%) = — - — —

[0171]

[0172] Where "m (dry)" refers to the mass of the dry hydrogel, whereas the mass (wet) refers to the mass of the wet hydrogel. The amount of dry residue obtained in three measurements (Table 2) was found to be between 0.1 % and 10% by weight of the total weight of the hydrogel before drying, where the dry weight corresponds to the bacterial cellulose.

[0173] Table 2: Determination of dry fraction of bacterial cellulose hydrogel

[0174] Wet mass (mg) Dry mass (mg) Dry fraction (%)

[0175] 1426 7 0.5

[0176] 1142 7 0.6

[0177]

[0178] 1104 7 0.6

[0179] Example 4: Determination of microporosity, by means of microscopy

[0180] Supercritically dried samples of bacterial cellulose (as explained in the section "Determination of mesoporosity and macroporosity"), which have the same microstructure and porosity as the hydrogel, were placed on an aluminum SEM substrate on a carbon adhesive tape. Scanning electron microscopy images (FEI Magellan 400LXHR SEM) were taken under high vacuum conditions, an accelerating voltage of 2 kV, current of 0.10 nA and at a distance of 5 mm.

[0181] The interwoven structure of cellulose nanofibers was determined (FIG.1A), and the cellulose nanofibers were found to have a diameter (or thickness) in the range between 5 nm and 50 nm. Specifically, the diameter of the cellulose nanofibers was 15±3 nm (Fig.1 B), average of 100 measurements.

[0182] Example 5: Determination of porosity

[0183] The wet hydrogels obtained in Example 1 underwent a solvent exchange phase with ethanol solutions with increasing concentrations from 50 % to absolute ethanol at 4 °C and were loaded into a high-pressure autoclave vessel (300 mL) filled with pure ethanol for supercritical drying. First, the autoclave was pressurized to 100 bars at room temperature. Secondly, the ethanol was exchanged for liquid CO2 at a flow rate of 1.5 kg / h for 2 h. While maintaining the liquid CO2 at a flow rate of 1.5 kg / h, the reactor was heated at 45 °C for 1 h to transform the liquid CO2 to the supercritical state and remove unreacted species. This supercritical state was maintained for 3 h maintaining the same parameters and stopping the CO2 flow. Finally, the autoclave was slowly depressurized, and the dried hydrogels were removed.

[0184] The thickness of the cellulose nanofibers is determined by scanning electron microscopy (SEM).

[0185] The mesoporosity is determined from nitrogen adsorption / desorption measurements using the B.E.T. method. Nitrogen adsorption / desorption measurements were performed using a particle size analyzer (ASAP 2000 V2.04; Micromeritics) after a degassing phase of 20 h at 60 °C.

[0186] The mesoporous structure of the bacterial cellulose was determined to have a pore size between 10 nm and 50 nm.

[0187] The proportion of macroporous was calculated from the total porosities and mesopore volume (adsorption):

[0188] Porosity = (1 -W*V*p) x 100

[0189] where W is the weight of the dry hydrogel (g), V is the volume of hydrogel taken for measurement (cm3), and p is the density of the cellulose (1.4 g / cm3).

[0190] The macroporous structure of the bacterial cellulose was found to have a pore size between 50 nm and 5000 nm. Example 6: Determination of endotoxin concentration

[0191] In accordance with FDA recommendations, endotoxin extraction was carried out by placing autoclaved bacterial cellulose samples in depyrogenated Falcon tubes containing 40 mL of endotoxin-free water for 72 h at 30 °C. To assess the endotoxin content of the bacterial cellulose eluates, a Thermo Fisher Pierce LAL endotoxin quantification chromogenic kit was purchased, and the assay was performed according to the kit instructions. The absorbance of the reaction products was measured at 405 nm in a microplate reader (Infinite 200 PRO, TECAN) at 37 °C.

[0192] After extraction, the amount of exotoxins was less than 5 endotoxin units per sample. Example 7: Determination of osmolarity variation

[0193] Bacterial cellulose hydrogel patches (0 = 16 mm) obtained according to Example 1 were embedded in a 1:1 (v / v) solution of Balanced Salt Solution BSS® (composition in Table 3) and Milli-Q water, making the content embedded in the hydrogel more hypotonic (150 mOsmol / L) than human tears (-310 mOsmol / L).

[0194] Table 3: Composition of salts in BSS 1:1 (v / v)

[0195] Component Concentration range (m / v) Sodium chloride (NaCI) 0.32 % - 0.64 % Potassium chloride (KCI) 0.0375 % - 0.075 % Calcium chloride dihydrate (CaCh^FLO) 0.024% - 0.048 % Magnesium chloride hexahydrate (MgCh^JFLO) 0.015 % - 0.03 % Sodium acetate trihydrate (C2H3NaO2*3H2O) 0.195 % - 0.39 %

[0196]

[0197] Sodium citrate dihydrate (CeHsNasOy^FLO) 0.085 % - 0.17 %

[0198] Each patch was then inserted for 1 min in a well under agitation with 100 pL of undiluted BSS (320 mOsmol / L), after which the insert was transferred to a new well with 100 pL of undiluted BSS for another minute under agitation and so on up to 6 wells for a total of 600 pL. Afterwards, 50 pL from each well were analyzed using an automatic cryoscopic osmometer (Gonotec® OSMOMAT™ 030), in order to evaluate the change in osmolarity upon release of the patch contents.

[0199] Based on the results, it was observed that despite diluting the insert in 600 pL of BSS, it still slightly changed the osmolarity of the last well, indicating that there is still release of its contents. Considering that the tear film turnover (7 pL) is approximately 16% every minute or -1.12 pL / min, it would take approximately 9h for the human eye to renew the 600 pL needed for the insert to release most of its contents (FIG. 5). Example 8: Determination of strain and tear strength values

[0200] A 20 x 50 x 0.5 mm3bacterial cellulose hydrogel with a surface weight of 650 ± 150 g / m2was measured on a Zwick Z2.5 dynamometer with a 2.5 kN load cell for tensile experiments. The test speed was 100 mm. min-1, the gripper pressure was 6 bar and the distance between the grippers was 20 mm. The applied preload was 0.05 N. The strain and tear strength (Fmax) values were 24 ± 2% and 5 ± 1 N / cm, respectively.

[0201] Example 9: Clinical investigation to evaluate feasibility, safety and comfort of the ocular insert of the invention comprising hypotonic saline solution as therapeutic agent

[0202] The study was a first-in-human clinical investigation, designed as a single-center intervention study. The study protocol was approved by the local ethics committee and the Spanish Agency for Medicines and Medical Devices (number: 1106 / 23 / EC-R). The study procedures were performed in accordance with the tenets of the Declaration of Helsinki. Healthy volunteers were included at the Barraguer Ophthalmology Center, Barcelona, Spain. From October 2023 until July 2024, twenty participants were enrolled in the study. The mean age ± SD (standard deviation) of the participants was 42.0 ± 13 years (median 41.5 years, ranging from 26 to 63 years). 50% were men and 50% were women. The right eye was studied in 70% of the subjects (14 out of 20) and the left eye was studied in 30% of the subjects (6 out of 20). All subjects gave written informed consent before inclusion. One eye per subject was included for the insertion of the device.

[0203] Exclusion criteria were:

[0204] • pregnant or breastfeeding women

[0205] • allergies to the cellulose

[0206] • use of systemic medications that affect the stability of the tear film

[0207] • clinically significant blepharitis, Meibomian gland dysfunction, eyelid margin inflammation with or without treatment, punctal occlusion, or a history of severe ocular trauma

[0208] • ocular surgery < 6 months before recruitment

[0209] • concurrent use of topical ocular medications

[0210] • ocular or systemic infections with possible ocular involvement

[0211] The screening of participants included extensive ophthalmologic examination for dry eye condition: osmolarity measurements (TearLab Osmolarity System, TearLab Corporation, USA), noninvasive tear film break-up time (Sirius +, CSO, Italy; IDRA® Ocular surface analyzer, SBM Sistemi, Italy), slit lamp evaluation to score hyperemia, lissamine green (5 mg / ml, Xalabarder Farma, Spain) and fluorescein staining (BioGio™ Fluorescein Strips, Madhu Instruments, India) to score epithelial cells damage, Schirmer’s tear production test I (TearFlo™ Sterile Test Strips, Madhu Instruments, India).

[0212] At all visits, slit lamp evaluation to score hyperemia according to the validated bulbar redness scale (VBR, ranging from 0 to 100) was performed before insertion and after insert extraction. Furthermore, after device extraction, noninvasive tear film break-up time was performed, corneal and conjunctival punctate staining were scored using lissamine green and fluorescein staining using the Oxford scale (ranging from 0 to 5). Additionally, volunteers were asked to complete a customized questionnaire.

[0213] A trained ophthalmologist inserted the ocular insert in the inferior conjunctival fornix using forceps (Vitlab™ PMP Forceps, Vitlab, Germany). The lower eyelid was pulled down by pinching it between the thumb and index finger, and the ocular insert was gently placed in the lower conjunctival sac (FIG. 6 and 7) for four intended durations: 15 minutes, 1 hour, 3 hours, and 6 hours, each on a different day.

[0214] This study was conducted over a period of 6 days, which were not necessarily consecutive.

[0215] • Visit 1: Screening and eligibility (0.75 hours)

[0216] • Visit 2: Device insertion for 0.25 hours

[0217] • Visit 3: Device insertion for 1 hour

[0218] • Visit 4: Device insertion for 3 hours

[0219] • Visit 5: Device insertion for 6 hours

[0220] • Visit 6: Final follow-up visit (0.75 hours)

[0221] Table 4: Study visits and procedures.

[0222] Measurements & Screening Before After device Final processes visit device removal follow-up insertion visit Informed consent y /

[0223] Demographics y /

[0224] Medical History y /

[0225] Osmolarity y

[0226] measurements

[0227] Noninvasive tear film y y y

[0228] break-up time

[0229] Slit lamp evaluation to y y y y

[0230] score hyperemia

[0231]

[0232] Lissamine green y

[0233] staining to score

[0234] epithelial cells damage

[0235] Fluorescein staining to y

[0236] score epithelial cells

[0237] damage

[0238] Subjective ratings y

[0239] (questionnaires)

[0240] Device weighting y

[0241]

[0242] The study materials were three insoluble soft inserts are listed in Table 5. During the clinical investigation, only large inserts and medium inserts were used, as none of the volunteers required the smaller insert.

[0243] Table 5: Devices used during the clinical study.

[0244] Large insert Medium insert Small insert Composition Bacterial cellulose impregnated with a 1:1 (v:v) solution of balanced salt solution (BSS®, Alcon) and ultrapure water Osmolarity 150 mOsmol / L

[0245] Sterilization Autoclaved 121 °C for 40 min

[0246] Weight 105 mg 39 mg 13 mg Dimensions 16x4 mm 8x 3 mm 4x2 mm Thickness 1.6 mm

[0247]

[0248] The following aspects were evaluated:

[0249] • Feasibility: Application and retention of the insert in the lower fornix without being lost up to a maximum of 6h.

[0250] • Safety: Variation in the state of the corneal epithelium pre- and post-insertion, and variation of pre- and post-insertion tear quality and quantity.

[0251] • Comfort: Level of discomfort, blurred vision, dryness, irritation, foreign body sensation based on questionnaire after each test.

[0252] • Insert weight after each test.

[0253] 1. Feasibility: Volunteers were instructed to continue their regular daily activities while wearing the ocular insert. In the event of insert loss, the test was repeated. Table 6 provides details on the time retention, while Table 7 summarizes the causes of device dislodgement.

[0254] Table 6: Retention times of the insert, where time of loss is indicated in brackets.

[0255] volunteer 0,25 h 1 h 3 h 6h

[0256] 1 a (2h30)

[0257] 2 yf b (1h15) /

[0258] 3 yf g (unknown)

[0259] 4 yf a (1h)

[0260] 5 c (4h30)

[0261] 6 d (50 min) / b (2h30) / (medium) 7

[0262] 8 yf yf yf

[0263] 9 yf yf yf

[0264] 10 yf yf yf

[0265] 11 yf yf yf

[0266] 12 yf

[0267] 13 yf V (medium) V (medium, 12h)* 14 yf

[0268] 15 yf yf yf

[0269] 16 yf yf

[0270] 17 e (45 min) yf

[0271] 18 a (2h15) yf

[0272] 19 V (medium) / (medium) V (medium)

[0273]

[0274] 20 f (2h30)

[0275] Table 7: Reasons for loss of the ocular insert.

[0276] Reasons for loss Frequency Letter Eye rubbing 3 a Insert not optimally placed according to volunteer 2 b Use of saline solution 1 c Laughing 1 d Volunteer closed the study eye tightly

[0277]

[0278] e Volunteer washed his face 1 f Unknown 1 g

[0279] During 84 tests, the insert fell out 10 times. Tests were repeated 4 times for volunteers 1, 2, 4, and 5. 11 completed the study without device loss during the intended time (2 used the medium device). * Volunteer 13 wore the insert for 12 hours, exceeding the intended 6 hours. No symptoms or ocular surface irritation were reported.

[0280] Ophthalmologists reported that the device was easy to manipulate, and volunteers found the insertion process and wearing it to be comfortable. It is important to note that the number of dislodgments decreased substantially towards the end of the study, probably due to both the ophthalmologist’s increased experience and to better guidance and management of the volunteers.

[0281] 2. Safety:

[0282] 2.1. Variation in the state of the corneal epithelium pre- and post-insertion.

[0283] Before device insertion and after its removal, the ophthalmologist assessed hyperemia in the volunteers using the VBR (validated bulbar redness) scale, ranging from 0 to 100 (FIG. 8). If hyperemia in the study eye exceeded a score of 20, the insertion of the ocular insert was postponed to another day.

[0284] Both before device insertion and after device removal, the ophthalmologist evaluated ocular surface condition. Both corneal and conjunctival abnormalities were evaluated by staining the ocular surface with fluorescein and Lissamine green, respectively, and results are summarized in Table 8. Fluorescein staining is the standard method used for the diagnosis of dry eye disease. Severity of staining was quantified using Oxford scale (FIG.

[0285] 9) which comprises a series of panels, labeled A-E, of increasing severity of dry eye: mild (grade 0 or I), moderate (grade II or III), and severe (grade IV or V). In each panel, fluorescein staining is represented by punctate dots.

[0286] Table 8: State of the corneal epithelium pre- and post- insertion. Hyperemia and ocular surface abnormalities were evaluated using a slit lamp and VBR (0 to 100) and Oxford scale (0 to V), respectively.

[0287] BDI ADR H H F LG V T OD OS OD OS OD OS OD OS T O N T O N

[0288]

[0289] 1 1 0 - 0 0 0 0 0 BDI ADR

[0290] H H F LG V T OD OS OD OS OD OS OD OS T O N T C N

[0291] 2 0 0 0 0 0 I _ _ _ 3 0 0 0 0 0 0

[0292] 4 0 0 0 0 0 0

[0293] 5 0 0 0 0 0 I

[0294] 1 0 0 0 0 0 0

[0295] 2 10 - 10 0 0 0 0

[0296] 2 3 10 20 0 0 0 0

[0297] 4 10 10 I 0 I 0

[0298] 5 10 10 I 0 I 0

[0299] 1 0 0 0 0 0 0 _ _ 2 0 0 0 0 0 I

[0300] 3

[0301] 3 0 0 0 0 0 0

[0302] 4 0 0 I 0 0 0

[0303] 1 0 0 0 _ _ 0 0 I 2 0 0 0 0 0 0 4

[0304] 3 0 0 0 0 0 0 4 0 10 0 0 0 0 1 0 0 0 I 0 0 _ _ 2 0 0 0 0 0 0

[0305] 5

[0306] 3 10 10 0 0 0 0

[0307] 4 0 0 0 0 0 0

[0308] 1 10 10 0 0 0 0

[0309] 2 10 - 10 0 0 0 0

[0310] 6 3 0 0 0 0 0 0

[0311] 4 0 0 0 0 0 0

[0312] 5 0 0 0 0 0 0

[0313] 1 0 - 0 0 0 0 0 _ _ 2 10 10 0 0 0 0

[0314] 7

[0315] 3 0 0 0 0 0 0

[0316] 4 10 10 0 0 0 0

[0317] 1 10 10 0 0 0 0 _ _ 2 0 10 0 0 0 0

[0318] 8

[0319] 3 10 10 0 0 0 0

[0320]

[0321] 4 10 10 0 0 0 0 BDI ADR

[0322] H H F LG V T OD OS OD OS OD OS OD OS T O N T C N

[0323] 1 0 0 0 I 0 0 _ _ 2 0 0 0 0 0 0

[0324] 9

[0325] 3 0 0 0 0 0 0

[0326] 4 0 0 0 0 0 0

[0327] 1 0 0 0 0 0 0 _ _ 2 0 0 0 0 0 0

[0328] 0

[0329] 3 0 0 0 o o o o 0 0 0

[0330] 4 0 0 0 0 0 0

[0331] 1 0 0 0 0 0 0 _ _ 2 10 10 0 0 0 0

[0332] 1

[0333] 3 0 0 0 0 0 I

[0334] 4 10 0 0 0 0 0

[0335] 1 0 0 0 I 0 I

[0336] 2 0 0 0 0 0 I - 2

[0337] 3 0 0 I I 0 I

[0338] 4 0 10 I 0 0 II

[0339] 1 10 10 0 0 0 0 2 0 0 0 0 0 0 3

[0340] 3 10 10 0 - 0 0 0 4 0 0 0 0 0 0 1 0 0 _ _ 0 0 0 3 10 0 0 0 I 4

[0341] 4 10 10 0 0 0 5 10 10 0 0 I 1 0 0 0 0 0 0 _ _ 2 0 0 0 0 0 0

[0342] 5

[0343] 3 0 0 0 0 0 0

[0344] 4 0 0 0 0 0 0

[0345] 1 10 10 0 _ _ 0 0 0 2 10 10 0 0 0 0 6

[0346] 3 10 10 0 0 0 0 4 10 0 0 0 0 0 1 - 10 10 0 - 0 0 0 7

[0347]

[0348] 2 0 10 0 0 0 0 BDI ADR

[0349] H H F LG V T OD OS OD OS OD OS OD OS T O N T O N

[0350] 3 - 10 0 0 - 0 0 0 4 10 10 0 0 0 0 1 0 0 0 0 0 0

[0351] 2 10 10 0 0 0 0

[0352] 18

[0353] 3 0 0 0 0 0 0 - 4 0 0 0 0 0 0

[0354] 1 10 10 0 0 0 I

[0355] 2 0 0 0 0 0 I

[0356] 19

[0357] 3 0 0 0 0 0 I - 4 0 0 0 0 0 I

[0358] 1 10 0 0 _ _ 0 0 0 2 0 0 0 0 0 0 20

[0359] 3 0 0 0 0 0 0

[0360]

[0361] 4 0 0 0 0 0 0

[0362] Where:

[0363] “V” is volunteer, “T” is test, “OD” is oculus dexter, “OS” is oculus sinister, “BDI” is before device insertion, “ADR” is after device removal, “H” is hyperemia, “F” is fluorescein, “LG” is lissamine green, “T” is temporal, “C” is cornea, and “N” is nasal.

[0364]

[0365] Out of 84 tests, an increase in hyperemia was observed in one case (20 / 100, VBR scale), while in another case a slight staining with Lissamine green was reported in the nasal area (ll / V, Oxford scale), indicating minor irritation possibly due to device insertion. Thus, the results confirm that the device is safe.

[0366] 2.2. Variation of pre- and post-insertion tear quality and quantity.

[0367] After device removal, tear quality and quantity were assessed using the noninvasive tear break-up time test ((Sirius +, OSO, Italy, FIG. 10). This device measures the time it takes for the tear film to break by projecting a pattern of circles onto the ocular surface. FIG. 11 shows a schematic summary of the NIBUT (noninvasive tear break-up time) results, using Sirius +, OftalTech Solutions and IDRA® Ocular surface analyzer SBM SISTEMI, which includes the mean value for both study and control eye.

[0368] As shown in FIG. 11, the mean NIBUT values of the results align with those reported fora healthy population in the TFOS DEWS II report (Ocular Surface, 2017, 15(3), 276-283), which cites means of 11.2 ± 6.82 and 10.4 ± 4.23.

[0369] The difference between the study eye and control eye was analyzed using the Shapiro-Wilk test, which yielded a p-value of 0.61. It is suggested by this p-value that the differences are normally distributed, indicating that the paired t-test is appropriate to use. The p-value from the paired t-test was found to be 0.498. Since this p-value exceeds the common significance level of 0.05, it is indicated by this result that no statistically significant difference is present between the means of the study eye and the control eye. Thus, in view of these results, which show that the tear film of the volunteers was not affected by the insert of the invention, the latter is also considered safe in this respect.

[0370] 3: Comfort:

[0371] A questionnaire, based on the Contact Lens Dry Eye Questionnaire developed by Begley et al., 2012, and adapted to the ocular insert of the present invention, was distributed to the volunteers. Scales ranging from 0 to 5 were used to measure the presence, frequency, and severity of common ocular symptoms, among which were included blurry vision, dryness, irritation, discomfort and foreign body sensation. The questionnaire was answered after extraction of the insert, at different times (i.e., after the insert was worn for 15 minutes, 1 hour, 3 hours, and 6 hours) and results are shown in Table 9, 10, and 11.

[0372] Table 9: Questionnaire results for large insert once the expected duration (T) was reached and the device was extracted.

[0373] I (% of V) F (% of V)

[0374] N S T(h)

[0375] 0 1 2 3 4 5 0 1 2 3 4 5 20 0.25 95 5 95 5

[0376] 18 1

[0377] B / H V

[0378] 3 100 100

[0379] 15

[0380] 6

[0381] 20 0.25 100 100

[0382] 18 1 83 11 6 83 11 6

[0383] DR

[0384] 3 100 100

[0385] 15

[0386] 6 93 7 93 7

[0387] 20 0.25 90 5 5 90 5 5

[0388] 18 1 89 6 6 89 6 6

[0389] IR

[0390] 3 93 7 93 7

[0391] 15

[0392]

[0393] 6 87 7 7 87 7 7 20 0.25 65 20 10 5 65 25 5 5

[0394] 18 1 72 17 6 6 72 17 6 6

[0395] DI

[0396] 3 87 7 7 87 13

[0397] 15

[0398] 6 67 20 13 73 13 13

[0399] 20 0.25 30 50 15 5 30 40 5 10 5 10 18 1 44 44 6 6 39 39 11 11 FBS

[0400] 3 53 47 53 40 7

[0401] 15

[0402] 6 67 27 7 67 27 7

[0403] Where:

[0404] “N” is the total number of volunteers, “S” is the symptom, “B / H V” is blurred or hazy vision at any time, “DR” is dryness of the eye, “IR” is sensation of irritation, “DI” is sensation of discomfort, “FBS” is foreign body sensation, “T” is the time in hours (h) during which the insert was worn before extraction, “V” is the volunteers, “I” is the intensity and “F” is the frequency felt by the volunteers of the given symptom in a range of 0 to 5, from nothing to very intense.

[0405] Note that both the intensity and frequency results are given in percentage (%) of volunteers that reported feeling the given symptom in a scale of 0-5.

[0406]

[0407] Table 10: Questionnaire results for the medium insert (worn / tested by 3 volunteers: 6, 13, and 19) once the expected duration (T) was reached and the device was extracted.

[0408] T DI B / H V DR IR FBS V

[0409] (h) I F I F I F I F I F 6 6 1 1 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 1 1 13

[0410] 6 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 19 3 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0

[0411] Where:

[0412] “V” is the volunteer (6, 13 or 19), “T” is the time in hours (h) during which the insert was worn before extraction, “DI” is sensation of discomfort, “B / H V” is blurred or hazy vision at any time, “DR” is dryness of the eye, “IR” is sensation of irritation, “FBS” is foreign body sensation, “I” is the intensity and “F” is the frequency felt by the volunteers of the given symptom in a range of 0 to 5, from nothing to very intense.

[0413]

[0414] Table 11 : Results following insert dislodqement, that is, for those volunteers who lost the insert before completing the indicated duration, as well as volunteer 13 who wore the insert longer than indicated (12 hours).

[0415] DI B / H V DR IR FBS V L T (h)

[0416] I F I F I F I F I F

[0417] 1 a 2h30 0 0 0 0 0 0 0 0 1 1 2 b 1h15 0 0 0 0 0 0 0 0 2 5 3 c N.A. 2 1 0 0 0 0 1 1 1 1 4 d 1h 0 0 0 0 0 0 0 0 0 0 5 e 4h30 0 0 0 0 0 0 0 0 1 1

[0418] 50

[0419] 6 f 2 4 0 0 2 2 0 0 0 0 min

[0420] 6 g 2h30 2 3 0 0 2 3 0 0 0 0 13 h 12h 0 0 0 0 0 0 0 0 1 1

[0421] 45

[0422] 17 i 0 0 0 0 0 0 0 0 0 0 min

[0423] 18 j 2h15 0 0 0 0 0 0 0 0 0 5 20 k 2h30 0 0 0 0 0 0 0 0 1 1

[0424] Where:

[0425] “V” is the volunteer who wore the insert for a different period of time than instructed, “L” is the letter assigned to this event (loss or overworn) in Table 6, “T” is the retention time in hours (h), “N.A.” is not available, “DI” is sensation of discomfort, “B / H V” is blurred or hazy vision at any time, “DR” is dryness of the eye, “IR” is sensation of irritation, “FBS” is foreign body sensation, “I” is the intensity and “F” is the frequency felt by the volunteers of the given symptom in a range of 0 to 5, from nothing to very intense.

[0426]

[0427] Results of the questionnaire are included in FIG.13.

[0428] The data retrieved from the questionnaires indicates that the insert does not significantly cause blurred vision or dry eye sensation, as these symptoms were reported with very low intensity. Additionally, feelings of irritation and discomfort were also minimal. The primary complaint was a sensation of a foreign body, but as shown in Table 9 for FBS results, this sensation diminishes with increased user experience. It is thus concluded that this study of comfort demonstrates the high comfort profile of the insert of the present invention which moreover improves with user experience. Example 10: In vitro hyaluronic acid release

[0429] The objective of the study was to quantitatively analyze the release profile of hyaluronic acid (using commercial formulations) from bacterial cellulose hydrogels and to characterize the molecular weight of the hyaluronic acid. Sustained release was intended to provide continuous daily lubrication of the ocular surface at least for 8 h.

[0430] Artificial tears quantified: Hylo Comod® and Hylo Gel® were selected due to their high molecular weight hyaluronic acid, simple formulations, and widespread use among patients with dry eye as advised by an ophthalmologist from the Barraquer Ophthalmology Center.

[0431] Table 12. Summary of the main characteristics of the hyaluronic acid-based commercial formulations evaluated in this study. The information provided includes the manufacturer, hyaluronic acid concentration (mg / mL), molecular weight (kDa), pH, osmolarity (mOsm / L), and additional components for each formulation. As manufacturers generally do not share this information, molecular weight values were obtained from HPLC / SEC-MALS measurements. pH and osmolarity were determined experimentally.

[0432] HA

[0433] Cone. HA Mw Osmolarity

[0434] Product Manufacturer (mg / mL) (kDa) pH (mOsmol / L) Other Components Hylo Ursapharm, 1 1948 7,4 278 anhydrous citric Comod® DE acid

[0435] sodium citrate sorbitol

[0436] Hylo Ursapharm, 2 2032 7,2 277 anhydrous citric Gel® DE acid

[0437] sodium citrate sorbitol

[0438] Release solution: Artificial lacrimal system (6.78 g / L NaCI, 2.18 g / L NaHCO3, 1.38 g / L KCI, 0.084 g / L CaCh.2H2O) was used as a media for the release studies.

[0439] Method used: Release studies were conducted using the carbazole assay approved by the European Pharmacopoeia, and molecular weight was determined by high-pressure liquid chromatography coupled with a size exclusion column and multi-angle light scattering (HPLC / SEC-MALS). Procedure: Loading: 50 % of the water was removed from square BC hydrogels (16 x 16 mm, -410 mg) by blotting. Six hydrogels were used per experiment. Each hydrogel was placed in a well of a 12-well plate with 2 mL of the respective eye drops on top and left at RT overnight to allow for impregnation.

[0440] Release’. Each triplet of impregnated BC hydrogels was transferred to 10 mL of artificial lacrimal system. The release was monitored over 24 hours: for the first 8 hours, the system remained static (no stirring), followed by vigorous stirring from 8 to 24 hours. Complete release was assumed at 24 hours based on the extended immersion period up to 24 h before quantifying the HA amount.

[0441] Each experiment consisted of three replicates, and the experiment was repeated three times on different days.

[0442] The release was determined after 30 min, 1 h, 2 h, 3 h, 4 h, 5h, and 8 h of both samples from bacterial cellulose without stirring. After 8 h, vigorous stirring was applied to ensure complete release.

[0443] Results:

[0444] Weight recovery of 72 % was reached after loading. See also FIG. 1 -FIG. 4.

[0445] Sustained release from both HA eye drop formulations is experimentally validated. After 8 h, more than 80 % of the total hyaluronic acid amount is released in the artificial lacrimal system.

[0446] Hyaluronic acid molecular weight determination

[0447] The objective was to determine the molecular weight of hyaluronic acid in Hylo Gel® eye drops.

[0448] Procedure: samples of Hylo Comod® and Hylo Gel® were diluted in distilled water to a final concentration of 0.5 mg / mL and submitted to the Automated Crystallographic Platform at the Institute of Molecular Biology of Barcelona (IBMB-CSIC) for HPLC / SEC-MALS analysis.

[0449] Results: The mean molecular weight of hyaluronic acid was estimated at 1948 kDa and 2032 kDa, which aligns with values reported in the scientific literature. Sample Mean Mw Polydispersity Concentration (mg / mL) (kDa)

[0450] '1HPLC Expected Hylo Comod® 1948 974 (26 %) 0.978 1

[0451] 1952 (55 %)

[0452] 3242 (19 %)

[0453] Hylo Gel® 2032 1215 (31 %) 1.898 2

[0454] 1693 (13 %)

[0455] 2403 (34 %)

[0456] 3363 (18 %)

[0457] The results show that HPLC coupled with SEC-MALS is a reliable and effective technique for determining the molecular weight distribution and quantifying the concentration of hyaluronic acid in ophthalmic formulations.

[0458] Conclusions

[0459] The bacterial cellulose hydrogels absorbed 767 pg / g of hyaluronic acid from Hylo Comod® and 1366 pg / g from Hylo Gel®. In an 8-hour in vitro test, 86% and 80% of the hyaluronic acid were released from Hylo Comod® and Hylo Gel®, respectively. Both products contain high molecular weight hyaluronic acid (1948 kDa for Hylo Comod® and 2032 kDa for Hylo Gel®), and HPLC / SEC-MALS analysis showed they are polydisperse. Bacterial cellulose loaded with high molecular weight hyaluronic acid demonstrated sustained release in an artificial lacrimal system. These results support the viability of this system as a medical device for the treatment of dry eye.

[0460] Citation List

[0461] Patent Literature

[0462] - US2012231038A1

[0463] - US3828777A

[0464] - US5147647A

[0465] Non-Patent Literature

[0466] - Anton-Sales, I., et al., in “Opportunities of Bacterial Cellulose to Treat Epithelial Tissues”, 2019, Current Drug Targets, 20, pp. 808-822.

[0467] Craig, J. P., et al., “TFOS DEWS II Definition and Classification Report”, Ocular Surface, 2017, 15(3), pp. 276-283. Begley, C. G., et al., “Contact Lens Dry Eye Questionnaire-8 (CLDEQ-8) and opinion of contact lens performance”, Optom Vis Sci., 2012, 89(10), pp. 1435-42. L. Land et al.; Sizes and shapes of conjunctival inserts. International Contact Lens Clinic, .1994). vol. 21(11-12), pp. 212-217

Claims

1. Claims1. A tear-fluid insoluble, ocular insert adapted for insertion into the lower or upper fornix of the eye of a subject, which is a mammal, for the sustained release of an agent selected from a therapeutic agent, a balanced salt solution, and a mixture thereof, wherein the insert comprises:3.a) a bacterial cellulose hydrogel which is a matrix of cellulose nanofibers hydrated with water, comprising a percentage of dry fraction of bacterial cellulose equal to or less than 1.8% by weight with respect to the total hydrogel, and4.b) an aqueous solution comprising the therapeutic agent and / or the balanced salt solution, wherein the solution is dispersed within the matrix for its sustained release;5.wherein:6.the hydrogel is composed of nanofibers having a thickness of between 5 nm and 100 nm measured by scanning electron microscopy (SEM);7.the aqueous solution has an osmolarity between 150 mOsmol / L and 280 mOsmol / L measured with an automatic cryoscopic osmometer; and8.the therapeutic agent comprised within the aqueous solution is selected from the group consisting of hyaluronic acid, plasma rich in growth factors, autologous serum, a mydriatic, an antibacterial, antiviral, antifungal, antiparasitic, anti-inflammatory, hypotensive, peptide, protein, carbomer, carboxymethyl cellulose, hydroxypropyl guar, and combinations thereof.

2. The insert according to claim 1, wherein the bacterial cellulose is obtainable from a Komagataeibacter bacteria.

3. The insert according to claim 2, wherein the bacteria is selected from Komagataeibacter xylinus and Komagataeibacter hansenii, and a co-culture of them.

4. The insert according to any of the claims 1-3, bacterial cellulose is from 0.4 to 1.5% by weight with respect to the total hydrogel.

5. The insert according to any of the claims 1-4, wherein the nanofibers have a thickness of between 5 nm and 50 nm.

6. The insert according to claim 1 , which is for the sustained release of the balanced salt solution.

7. The insert according to claim 1, wherein the hyaluronic acid solution is an aqueous solution comprising hyaluronic acid in an amount of between 0.1 % w / v and 0.4 % w / v,8. The insert according to any of the claims 1-7, wherein the aqueous solution has an osmolarity between 150 mOsmol / L and 280 mOsmol / L measured with an automatic cryoscopic osmometer (Gonotec® OSMOMAT™ 030).

9. An insert as defined in any of the claims 1-8, for use in the treatment and / or prevention of an ocular disease in a subject.

10. The insert for use according to claim 9, wherein the ocular disease is a disease of the surface of the eye.

11. The insert for use according to claim 10, wherein the ocular disease of the surface of the eye is selected from the list comprising dry eye, ocular perforation, ocular chemical burn, ocular thermal burn, limbal stem cell deficiency, corneal infection, neurotrophic keratopathy, ocular erosion, ocular abrasion, keratitis, conjunctivitis, keratoconus, bacterial ulcer, fungal ulcer, uveitis, glaucoma, and post keratoprosthesis transplantation.

12. The insert for use according to claim 11 , wherein the ocular disease is dry eye.

13. The insert for use according to any of the claims 9-12, wherein the subject is a human.

14. The insert for use according to any of the claims 9-13, wherein the treatment and / or prevention of an ocular disease comprises:21.a) introducing the insert into the fornix of the eye.

15. A kit comprising:23.a) a sealed, sterile first container containing an insert as defined in any of the claims 1-8, and24.b) optionally, means for inserting and / or removing the insert from the lower or upper fornix of a subject’s eye.

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