WO2014174544A1

WO2014174544A1 - Riboflavin formulations for trans-epithelial cross-linking

Info

Publication number: WO2014174544A1
Application number: PCT/IT2014/000114
Authority: WO
Inventors: Sergio Mangiafico; Danilo Aleo; Maria Grazia Antonietta Saita; Barbara MELILLI; Melina Cro
Original assignee: Medivis S.R.L.
Priority date: 2013-04-24
Filing date: 2014-04-24
Publication date: 2014-10-30
Also published as: ITRM20130248A1

Abstract

The invention concerns preparations of riboflavin (vitamin B2) for use in the treatment of keratoconus with a variant of the non-invasive technique of corneal cross-linking, called trans-epithelial cross-linking, wherein the corneal epithelium is not removed and the preparation of riboflavin to be instilled on the cornea during the treatment is able to pass through the corneal epithelium. The preparations comprise, in aqueous solution, non-phosphorylated riboflavin and a boron compound selected from tetraborates, orthoborates and metaborates of alkali metals and alkaline earth metals, wherein the concentra¬ tion of riboflavin is between 0.01 and 2.0 % by weight and the weight ratio of the boron compound to riboflavin is from 1 :2.5 to 1:25.

Description

RIBOFLAVIN FORMULATIONS FOR TRANS-EPITHELIAL

CROSS-LINKING

DESCRIPTION

Field of the invention

The present invention relates to riboflavin formulations for trans- epithelial cross-linking. More specifically, the invention relates to preparations based on riboflavin (vitamin B₂) for use in the treatment of keratoconus with a variant of the non-invasive technique already known as corneal cross-linking, which consists of inducing a cross-linking in the collagen of the central area of the cornea by irradiation with UV-A in the presence of riboflavin. In the variant of this technique called trans-epithelial cross-linking, in which the corneal epithelium is not removed, the preparation of riboflavin to be instilled on the cor- nea during the treatment is able to pass through the corneal epithelium.

Background of the invention

Keratoconus is a non-inflammatory ocular disease of uncertain etiology, characterized by a bulging (ectasia) of the central part of the cornea following a progressive thinning of said central part and its consequent weakening. With the progression of the disease, the central part of the cornea is curved outwards, progressively losing its spherical shape and becoming conical while projecting outwards. Due to the irregular curvature of the cornea a significant visual distortion occurs, as well as a progressive loss of vision due to the loss of corneal refractive power, and cornea may ultimately get to be punctured with a possible risk of losing not only vision, but also the integrity of the eyeball.

The treatment of keratoconus involves, in the initial phase and in most moderate cases, the use of soft contact lenses to correct a moderate astigma- tism and protect the corneal surface. With the progress of the disease, which is irreversible, it becomes necessary to use rigid contact lenses. In more severe cases, where the vision is seriously jeopardized, the only option provided until a few years ago was to have recourse to surgery, namely corneal transplantation or other techniques of ophthalmic surgery, such as lamellar keratoplasty or the insertion of intracorneal rings.

Starting from 1997, a new technique for the treatment of keratoconus has been developed, which has in recent years become the treatment of choice because of its being less traumatic than surgical treatments: cross- linking of the cornea. Based on the observation that the reduction of biome- chanical strength of the affected corneal tissue is caused by a reduction in intra- and interfibrillar bonds in collagen fibers of the corneal stroma, the new technique is intended to strengthen and increase those bonds, so as to render the cornea more rigid, thus counteracting the bulging and averting the danger of perforation. The cross-linking of corneal collagen with the above technique, at present indicated by the acronym CXL (corneal cross-linking, or also, according to some authors, C3-R - corneal collagen cross-linking with riboflavin), is obtained by irradiating the corneal tissues concerned with UV-A rays of 370 nm of wavelength in the presence of riboflavin-5'-phosphate (flavin mononucleotide, also referred to hereinafter with the acronym FMN) working as a photosensitizing agent. Riboflavin-5'-phosphate is the phosphorylated form of riboflavin or vitamin B₂, whose role in corneal cross-linking is, more precisely, to protect the internal structures of the eye from UVAs and, in addition, to promote the formation of bonds and thus the cross-linking of collagen within corneal stroma (Spoerl, E., Huhle, M., Seiler T. Induction of cross-links in corneal tissue . Exp Eye Res 66, 1998, 97-103; Wollensak G., Spoerl, E., F. Reber et al. Corneal endothelial cytotoxicity of ribofavin/UVA treatment in vitro. Oph- thalmic Res 35, 2003, 324-328; Wollensak G., Spoerl, E., Seiler T. Ribofla- vin/ultraviolet-A-induced collagen cross-linking for the treatment of keratoconus. Am. J. Ophthalmol. 135, 2003, 620-627 ) .

The application of a photosensitizing agent such as riboflavin-5'- phosphate to a tissue, such as cornea, skin, tendons, cartilage or bone tissue, followed by photoactivation by means of irradiation from an electromagnetic energy source with wavelengths of from 350 and 800 nm (i.e. the range which includes the entire visible spectrum, as well as ultraviolet and near infrared) is also described in U.S. patent 7,331 ,350 (assigned to The General Hospital Corporation of Boston, USA). The document concerns a technique of tissue repair which also includes various ophthalmic applications, including corneal transplants, keratoplasty, refractive surgery, and the treatment of corneal lac- erations. Also in the case of ophthalmic applications the object of this treatment is to accelerate the repair of lesions and to weld tissues while avoiding the use of sutures.

When the corneal cross-linking procedure is applied to the treatment of keratoconus, the operating parameters necessary to achieve the desired re- suit without damaging the underlying eye tissues are precisely calibrated, so that only one front layer of the corneal stroma of about 300 μηι is reached by the treatment (Seiler, T., Hafezi, F., Corneal cross-linking-induced stromal demarcation line. Cornea 25, 2006, 1057-1059). This technique of irradiation in the presence of riboflavin increases the corneal biomechanical strength by about 300%. However, owing to the damage made by UVA treatment in the inner ocular structures, this technique is not recommended for patients with a stroma thickness of less than 400 μηι.

As it is known, riboflavin or vitamin B₂ (7,8-dimethyl-10-ribityl- isoalloxazine, molecular weight 376 Da) is a heterocyclic compound obtained by one molecule of flavin which is linked to a chain formed by a sugar, i.e. ribi- tol.

Riboflavin (RF)

It is a yellow substance practically insoluble in water (solubility at 20° C 0.08-0.12 mg/ml, that is 0.008 to 0.012%, depending on the crystal struc- ture). If hit by the light, riboflavin undergoes photolysis, resulting in the cleavage of a ribitol radical. This photoreaction is the basis of link formation between collagen fibrils, thus it is the basis of the of corneal cross-linking technique.

Riboflavin is found in foods mainly in its soluble phosphorylated form. It is only thanks to the soluble phosphorylated form of riboflavin that it is currently possible to formulate this compound in water for carrying out the corneal cross-linking technique.

Riboflavin-5'-phosphate (FMN)

As shown by the scientific and patent literature in the field, riboflavin, or rather its phosphorylated sodium salt form (molecular weight 456 Da, negatively charged), is the hydrophilic photosensitizing molecule currently used in the implementation of the corneal cross-linking technique. For instance, an ophthalmic solution containing approximately 0.1 % of riboflavin as riboflavin phosphate (0.125 g of FMN per 100 ml of solution) and about 20% by weight of dextran is marketed under the brand name Ricrolin^® for the treatment of keratoconus with the CXL procedure. In the cited formulation dextran performs the function of ensuring the mucoadhesivity of the product, so that the product can adhere to the ocular surface and ensure good impregnation of the corneal stroma with the photosensitizing agent.

As noted before, riboflavin-5'-phosphate (or FMN) is a water-soluble molecule which has a poor ability to penetrate through the epithelium so as to reach the corneal stroma in a sufficient amount, thereby allowing an efficient imbibition of the stroma in the exact site where riboflavin phosphate acts to form new bonds. Thus, the treatment is to be preceded by the removal of the epithelium, an operation that must be carried out under local anesthesia.

The corneal epithelium is a lipophilic layer of a thickness that can vary from 50 to 100 μΜ, which contributes by 90% to the resistance towards hydrophilic drugs and only by the 10% to the resistance to lipophilic drugs (Washington, N., Washington, C, Wilson, CG, Ocular Drug Delivery. Physiological Pharmaceutics: Barriers to Drug Absorption, 2nd ed., CRC Press: Boca Raton, FL, 2001 , 249-269; Wilson, CG, Zhu, YP; Kumala, P.; Rao, LS; Dhillon, B., Ophthalmic Drug Delivery. In Drug Delivery and Targeting for Pharmacists and Pharmaceutical Scientists, Hillery, AM, Lloyd AW, Swarbrick, J., Eds., CRS Press: Boca Raton, FL, 2001 , 329-353). Therefore, the hydrophobic molecules, due to their lipophilicity, can permeate the corneal epithelium through the trans-cellular route, in a much more efficient way than hydrophilic molecules. For their feature of lipophilicity such molecules cannot be formulated in the aqueous environment of an ophthalmic solution.

The de-epithelialization step, which is necessary to allow FMN penetration, exposes the underlying stroma to the harmful action of pathogens, as well as to pain symptoms, which are always present in the first 24-48 hours after surgery. Several studies have shown that after de-epithelialization visual acuity, the aberrometric and the topographic index begin to increase until after the sixth month.

Due to the problems related to epithelium removal it would be desira- ble, therefore, to perform the corneal cross-linking using compositions in which riboflavin is capable of crossing the corneal epithelium without the need to have it removed. This technique of cross-linking carried out without de- epithelialization of the cornea, also called trans-epithelial cross-linking (also referred to hereinafter by the acronym TE-CXL, trans-epithelial corneal cross- linking), would also allow access to the treatment for those patients whose corneal stroma is reduced to less than 400 μητι.

In order to obtain ophthalmic compositions based on riboflavin phos- phate (FMN) useful for the technique of trans-epithelial cross-linking, penetration promoting agents (i.e., corneal permeation enhancers) are currently added in the photosensitizing formulations. Said substances, such as, e.g., eth- ylenediaminetetraacetic acid (EDTA), benzalkonium chloride, trometamol (tris(hydroxymethyl)aminomethane) and vitamin E TPGS, are capable of favoring the trans-corneal passage of FMN. For example, in an attempt to provide substances useful in the treatment of keratoconus with the CXL technique without having to remove the corneal epithelium, the use of benzalkonium chloride has been suggested as a surfactant to increase the permeability of the corneal epithelium (Italian patent application MI2007/ A002162 in the name of R. Pinelli ). Subsequently, a hypo-osmolar eye drop preparation containing benzalkonium chloride and FMN has been proposed for the treatment of keratoconus through the technique of trans-epithelial cross-linking (international patent application publ.. No. WO2012/004726, in the name of R. Pinelli ).

In the same trend, the international patent application publ. No. WO2011/012557 (Salvatore Troisi et al.), as well as the publication of Carmine Ostacolo and coll. (Ostacolo, C. et al., Enhancement of corneal permeation of riboflavin-5'-phosphate through vitamin E TPGS: A promising approach in trans-epithelial corneal cross-linking treatment. Int J Pharm. 440, 2013 148 - 153) propose an ophthalmic solution containing a derivative of vitamin E, i.e. d-alpha-tocopheryl polyethylene glycol 1000 succinate, also known as vitamin E TPGS or TPGS, as promoter of the permeation of riboflavin-5'-phosphate through the corneal epithelium, in procedures of trans-epithelial cross-linking without removal of the corneal epithelium.

The use of similar agents as promoters of the permeation of FMN through the corneal epithelium involves a cytotoxic action on the epithelium and on intercellular junctions, which results in a temporary and reversible weakening of the barrier function of the epithelium, thus allowing the passage of FMN through the cornea. It should be noted, however, that the effect of "corneal permeation enhancers" is always proportional to their toxicity (Robert Gurny et al. Ocular Tolerance of absorption Enhancers in Ophthalmic Prepa- rations, AAPS PharmSciTech 4 (1), 2002, 1-5).

In the light of the foregoing, it would be highly advantageous to be able to perform a treatment of trans-epithelial cross-linking wherein riboflavin can penetrate through the cornea, overcoming the corneal epithelium barrier with- out having to remove it, and without the use of permeation enhancers. To this regard, Morrison et al. have studied the possibility of using the technique of solubilization with cyclodextrins to vehiculate through the corneal epithelium non-phosphorylated riboflavin (RF, vitamin B₂) (Peter WJ Morrison, et al. Cy- clodextrin-Mediated Enhancement of Riboflavin Corneal Solubility and Per- meability. Mol. Pharm. 10(2), 2013, 756-62). As it is well known, cyclodextrins are oligosaccharides of overall toroidal shape, with hydrophilic outer surfaces and a lipophilic inner cavity, capable of forming inclusion complexes of the "guest -host type " with hydrophobic compounds, thus making them more water-soluble. According to the results of this work, in the presence of cyclodex- trins RF passes from a solubility of 0.008 % to a solubility of 0.012 to 0.019 %: although this is still a remarkable achievement, similar concentrations are unsuitable for trans-epithelial cross-linking .

Therefore, the studies connected with the present invention have focused on the realization of systems to formulate riboflavin (RF), i.e. a lipophilic molecule practically insoluble in water, in a non-phosphorylated form that is water-soluble and bioabsorbable, so that it can pass the corneal epithelium barrier of and can be used directly in the treatment of keratoconus by trans- epithelial cross-linking. Summary of the invention

In the frame of the studies related to the present invention, a work of Douglas V. Frost of 1942 has been considered (Douglas V. Frost. The water- soluble riboflavin-boron complex. J. Biol . Chem. 145, 1942 693-700), which reports that the solubility of RF in water can be increased by heating the same RF in the presence of boric acid (H₃B0₃ or B(OH)₃), metaboric acid (HB0₂) or borax (Na₂B₄0₇ · 10 H₂O, sodium tetraborate decahydrate). Such solutions of riboflavin are only obtained through the continuation of a lengthy heating at 95° C, temperature at which a significant amount of RF decomposes. Also, when the solutions thus obtained are stored at room temperature they are physically unstable, due to the formation of a crystalline precipitate which may occur even after several weeks or months. In the same paper it is shown that these solutions obtained at alkaline pH are more stable, However, when they are added with acid to provide solutions with physiological pH, that is neutral or slightly acid, they give rise to precipitation of RF as well.

Of the previous year is the British patent GB 560431 in the name of Winthrop Chemical Co., which describes water-soluble compositions of ribo- flavin with an alkali metal borate salt, wherein the concentration of riboflavin exceeds 0.02%, the compounds obtained being double salts of riboflavin and an alkali metal tetraborate. In that case, however, the preparations concerned are obtained in solid form, and the dissolution in water is used only in a temporary manner to homogenize the components, and is followed by drying. The products are stored in solid form, for administration by injection after reconsti- tution with water.

Exploiting the hint given by Frost to the possible formation of unstable soluble complexes between RF and the various borates under the effect of prolonged heating, the possible experimental conditions for the preparation at room temperature of a formulation for ophthalmic use containing RF have been studied, which formulation is stable from the physico-chemical standpoint and may be used in the treatment of keratoconus using the technique of the trans-epithelial cross-linking.

According to the present invention, it has now been found that by using sodium tetraborate in weight concentrations at least 2.5 times higher, and preferably at least 5 times higher than the concentration of RF the solubilization of RF as boron-complex, already at room temperature and without any heating can be achieved. Moreover, the further addition of acid to the solution containing the RF-boron-complex in order to bring this solution to pH neutral or acidic, contrary to expectations, does not give rise to a precipitate of riboflavin even when the solution is placed at a temperature of 4° C for a period of time of 18 months. Using the above solution as a base numerous variants of ophthalmic formulations have been made, in the presence of various excipients normally used in similar formulations, such as viscosifying and/or mucoadhesive polymers, osmotizing agents, buffers and substances able to increase the trans- corneal passage, and all formulations were stable from the physico-chemical point of view. In addition to ensuring the storage stability, the preparations according to the invention have been shown to be remarkably effective in terms of capacity of penetrating through the corneal epithelium, which makes them particularly suitable as photosensitizing preparations for the treatment of kera- toconus with the technique of cross trans-epithelial-linking.

Detailed description of the invention

Thus, the present invention specifically provides an ophthalmic preparation for use in the treatment of keratoconus by the trans-epithelial cross- linking technique (TE-CXL), comprising, in aqueous solution, non-phosphor- ylated riboflavin (7,8-dimethyl-10-ribityl-isoalloxazin or vitamin B₂ - referred to herein as RF) and a boron compound selected from the group consisting of: tetraborates, orthoborates and metaborates of alkali and alkali-earth metals, wherein the concentration of riboflavin is comprised between 0.01 and 2.0% by weight, and the weight ratio of boron compound to riboflavin is comprised between 1 :2.5 and 1 :25.

As it will become more evident with reference to the experimental details reported further on, the results have shown that the RF contained in preparations formulated according to the present invention penetrates through the intact cornea in an amount which is significantly higher than the amount by which the corresponding phosphorylated derivative, FMN, penetrates.

According to some preferred embodiments of the invention, the boron compound used is the tetraborate salt of an alkaline or alkaline-earth metal, preferably tetraborate of alkali metal decahydrate, and even more preferably such compound is sodium tetraborate decahydrate, of the formula Na₂B₄07 · According to some specific embodiments ot the invention, the riboflavin concentration is comprised between 0.01 and 0.1 % by weight, and the weight ratio of the boron compound to riboflavin is comprised between 1:2.5 and 1 :5. According to some further embodiments of the invention the concentration of riboflavin is comprised between 0.1 and 1.5% in peso, e detto rapporto ponde- rale tra il composto del boro e la riboflavina e compreso tra 1 :5 e 1 :15, il rapporto preferito essendo pari a 1 :10.

Besides the non-phosphorylated riboflavin complexed with the boron salt, the preparation according to the invention also contains, firstly, one or more ionic salts selected from potassium, sodium, magnesium or calcium salts, such as chlorides, borates, citrates or sulfates, such as sodium chloride or sodium borate, through which the osmolarity of the preparation may be controlled. The preparation may also contain the corresponding acids, which are necessary to adjust the pH of the final product to a value in the physiologic range, comprised between 6.7 and 7.4.

The riboflavin preparation for TE-CXL according to the invention can also contain, in the aqueous solution, further excipients suitable for its application, in particular one or more viscosifying (rheoloy modificators) and/or muco- adhesive polymers. These are preferably selected from

carboxymethylcellulose, hydroxypropylcellulose, hyaluronic acid, chondroitin sulfate, alginic acid, natural polysaccharides, dextran, carbomers, carbopol, polyvinyl alcohol, polyethylene glycols (preferably PEG400), xanthan gum.

According to some embodiments of the invention, in addition, the prep- aration may further contain a trans-corneal penetration promoting compound which is non toxic for the corneal tissues, e.g. a compound selected from the group consisting of dimethylsulfoxide, ethanol and mixtures thereof.

Further possible excipients include simple sugars such as glycerol, trehalose, mannitol or sorbitol, and anti-oxidants, such as ascorbic acid, sodium metabisulfite, tocopheryl acetate. The composition may include, in addition, antimicrobials and preservatives such as lactoferrin, sodium edetate (sodium salt of ethylene diamine tetraacetic acid, EDTA), benzalkonium chloride, poly- hexanide, TPGS (d-a-tocoheryl polyethylene glycol 1000 succinate, or vitamin E TPGS),

The preparation of the riboflavin-based product according to the invention can be produced as follows.

The appropriate desired amount of riboflavin is suspended in distilled water, i.e. between 0.01 and 2 g per 100 g of water, adding a quantity of sodium tetraborate decahydrate equal to n times the weight of riboflavin (where, according to the invention, n is a value between 2.5 and 25), and the mixture is left under stirring at room temperature until dissolution of the riboflavin sus- pension (approximately 30 minutes).

At this point the other ingredients provided in the formulation can be added, with the exception of the viscosifying and/or mucoadhesive polymer, and then the pH is adjusted to between 6.7 and 7.4 with hydrochloric acid or with boric acid. Then, after a few minutes, the viscosifying and/or muco- adhesive polymer is added until complete dissolution.

By applying the above procedure, it is possible to obtain the solubilization of RF without any heating. Furthermore, the addition of acids to the boron- complex, which is made to bring the solution to neutral or acidic pH, in the range required for a good ocular tolerability, does not give rise to a precipitate of riboflavin even when the solution is placed at a temperature of 4 ° C for storage.

According to the invention it is possible to thereby obtain stable solutions of RF up to the surprising concentrations of 1% by weight and more. These concentrations, in addition to being far superior to the solubility of ribo- flavin in distilled water, are also quite higher than the best result obtained so far to increase the solubility of riboflavin in water, which is represented by the previously cited work of Peter W.J. Morrison, et al., where in the presence of cyclodextrins the solubility of RF increases from 0.008% to from 0.012 to 0.019% by weight.

As it will be apparent from the following experimental data, the formulations according to the present invention have also shown a surprising advantage in corneal permeability compared to similar formulations containing riboflavin-5'-phosphate (FMN). In fact, the experimental permeability obtained for RF was 10-15 times higher than that obtained for FMN, even when the latter is formulated with dextran as in the commercial formulations currently used for corneal cross-linking.

The specific features of the invention, as well as the advantages of the same compared to the solutions of the known art, will become more apparent with reference to the detailed description of some embodiments thereof, shown in the following for illustrative purposes only, and with reference to the results of the relevant experimentation.

EXAMPLE 1

Standard ophthalmic formulation

A base formulation, containing the essential ingredients for the production of the preparation according to the invention is disclosed in the following table.

The preparation is obtained by suspending 0.1 g of riboflavin in distilled water and adding 1 g of sodium tetraborate decahyrate to the suspension, and then leaving under stirring at room temperature until complete dissolution of the riboflavin suspension (approximately 30 minutes). At this point, 0.05 g of EDTA and 0.14 g of sodium chloride are added, and then the pH of the solution is adjusted to a value between 6.0 and 7.8 with hydrochloric acid, to ob- tain the standard preparation.

EXAMPLE 2 Ophthalmic formulation added with polymers

A series of preparations similar to those of Example 1 but added with a polymer compound with function of viscosifying and/or mucoadhesive agent have been obtained according to the formulation shown below.

^* carbopol 0.2%. sodium hyaluronate 0.18%. dextran 15%

The preparation procedure is similar to that of Example 1. Once the standard preparation is obtained carboxymethylcellulose is added, and the mixture is kept under stirring until complete dissolution.

As shown in the preceding table, similar compositions can contain in place of carboxymethylcellulose, for instance, 0.2% by weight of carbopol, or 0.15% of sodium hyaluronate, or also 15% of dextran. EXAMPLE 3

Ophthalmic formulation added with ethanol

A preparation similar to that of Example 2 containing, in addition, ethanol with function of penetration enhancer has been obtained according to the formulation shown below.

Ingredients % w/w

RF 0.1

sodium tetraborate x 10H₂O 1.0

carboxymethylcellulose 0.3 ethanol 12

HCI 2N q.s. to pH 6.0-7.8

distilled water q.s. to 100

The preparation procedure is similar to that of Example 2.

EXAMPLE 4

Ophthalmic formulation added with DMSO

A preparation similar to that of Example 2 containing, in addition, dime- thtlsulfoxide (DMSO) with function of penetration enhancer has been obtained according to the formulation shown below.

The preparation procedure is similar to that of Example 2.

EXAMPLE 5

Ophthalmic formulation added with DMSO and ethanol A preparation similar to those of Exampls 3 and 4 containing, in addi- tion, both DMSO and ethanol has been obtained according to the formulation shown below

Ingredients % w/w

RF 0.1 sodium tetraborate x 10H₂O 1.0

disodium EDTA x 2H₂0 0.05

carboxymethylcellulose 0.3

ethanol 6.0

dimethtlsulfoxide 1.0

HCI 2N q.s. to pH 6.0-7.8

distilled water q.s. to 100

The preparation procedure is similar to that of Example 2.

EXAMPLE 6

Standard ophthalmic formulation with high RF/tetraborate ratio

A preparation similar to that of Example 1 with a lower concentration of sodium tetraborate has been obtained according to the formulation shown below.

The preparation procedure is similar to that of Example 1.

EXAMPLE 7

Standard ophthalmic formulation with high RF/tetraborate ratio added with polymers

A series of preparations similar to those of Example 6 but added with a polymer compound with function of viscosifying and/or mucoadhesive agent have been obtained according to the formulation shown below. Ingredients % w/w

RF 0.1

sodium tetraborate x IOH₂O 0.5

disodium EDTA x 2H₂0 0.05

carboxymethylcellulose * 0.15

NaCI 0.2

HCI 2N q.s. to pH 6.0-7.8

distilled water q.s. to 100

* carbopol 0.2%. sodium hyaluronate 0.18%. dextran 15%

The preparation procedure is similar to that of Example 2. EXAMPLE 8

Ophthalmic formulation with high concentration of RF A preparation similar to that of Example 2 with a higher concentration of riboflavin has been obtained according to the formulation shown below.

* carbopol 0.2%. sodium hyaluronate 0.18%. dextran 15%

The preparation procedure is similar to that of Example 2.

EXAMPLE 9

Ophthalmic formulation with low RF/tetraborate ratio A preparation similar to that of Example 2 with a lower concentration of sodium tetraborate has been obtained according to the formulation shown below.

* carbopol 0.2%. sodium hyaluronate 0.18%. dextran 15% The preparation procedure is similar to that of Example 2.

EXAMPLE 10

Ophthalmic formulation with low RF/tetraborate ratio without EDTA

A preparation similar to that of Example 2 with a lower concentration of sodium tetraborate has been obtained according to the formulation shown below.

EXAMPLE 11 Standard ophthalmic formulation without EDTA

Another standard preparation, similar to that of Example 1 but without sodium edetate has been obtained according to the formulation shown below.

The preparation procedure is similar to that of Example 2. To this standard formulation polymer compounds with functions of viscosifying and/or mucoadhesive agents may be added, with the same procedure shown in Example 2.

Trans-corneal permeability tests

The proposed formulations according to the invention for the treatment of keratoconus by cross-linking the trans-epithelial reported in the examples above were subjected to testing to verify its ability to permeate through the in- tact cornea.

The corneal permeability was evaluated using Franz cells according Reichl and coll. (Reichl, S. et. Al., Human corneal equivalent as cell culture model for in vitro drug permeation studies, Br J Ophthalmol. 88, 2004, 560- 565), on which were placed porcine corneas. In the donor chamber was placed the test formulation and in the receiving room was instead placed an isotonic saline solution containing a phosphate buffer at pH 7.2 (PBS, pho- psphate-buffered saline).

For each series of tests, the samples taken every 30 minutes from the receiving room were analyzed by HPLC. Quantification by HPLC has allowed to establish the amount of riboflavin that has permeated the cornea over time.

In the following tables, as well as in Figures 1 and 2 of the accompany- ing drawings, are reported the results of trans-corneal permeability tests for the solutions according to the invention referred to in Examples 1-4, respectively, in comparison with similar solutions but containing riboflavin-5 '- phosphate (FMN) in place of the non-phosphorylated riboflavin (RF).

The four solutions of comparison defined above are hereinafter referred to, respectively, Ex 1 FMN, FMN Example 2, Example 3 and Example 4 FMN FMN. As a further comparison, was subjected to the same tests the commercial solution cited above, containing 0.1 % of riboflavin as riboflavin phosphate (0.125 g of FMN per 100 ml of solution) and 20% by weight of de-odd, named here FMnd (FMN-dextran).

Table 1 below shows the permeation data of various formulations in comparison, obtained by plotting the amount of drug permeated ( g/cm2) in function of time (min).

TABLE 1

Trans-corneal permeation data - RF boron-complex vs. FMN in formulations corresponding to Examples 1-4 FMND

The same data of Table 1 are shown in Figure 1 attached to the group corresponding to the formulations of Examples 1 and 2, and for the prepara- tion FMnd, and in Figure 2 for the group corresponding to the formulations of Examples 3 and 4.

With reference to the diagrams shown, from the linear part of each curve were calculated permeability coefficient K_p, dividing the flow J (amount permeated per unit area) the concentration of the agent (RF or FMN). The related results are shown in Table 2 below.

TABLE 2

Vauest of flux J and permeability coefficient K_p from transcorneal permeability data of Table 1

As is apparent from an analysis of the above data and from an examination of Figures 1 and 2 attached, with the same excipients with the ophthalmic formulations containing RF-boron complex according to the invention showed a permeability through the cornea that is not denuded approximately 10-15 times higher than that shown by formulations containing FMN, obtained in the same experimental conditions.

The corneal permeability of the products according to the invention was also considerably higher than the corneal permeability of the commercial for- mulation of reference FMnd.

The products of the invention, containing riboflavin not phosphorylated conveyed through the corneal epithelium by means of the formation of a boron-complex, thus represent an effective and real advantage for the treatment of keratoconus with the technique of trans-epithelial cross-linking.

The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.

Claims

1. An ophthalmic preparation for use in the treatment of keratoconus with the trans-epithelial cross-linking technique, which preparation comprises, in aqueous solution, non-phosphorylated riboflavin and a boron compound selected from the group consisting of: tetraborates, orthoborates and meta- borates of alkali and alkali-earth metals, wherein the concentration of riboflavin is comprised between 0.01 and 2.0% by weight, and the weight ratio of boron compound to riboflavin is comprised between 1 :2.5 and 1 :25.

2. An ophthalmic preparation according to claim 1 , wherein said boron compound is the tetraborate salt of an alkali or alkali-earth metal.

3. An ophthalmic preparation according to claim 1 , wherein said tetraborate salt is sodium tetraborate decahydrate.

4. An ophthalmic preparation according to any one of claims 1-3, wherein the concentration of riboflavin is comprised between 0.01 and 0.1 % by weight, and said weight ratio of boron compound to riboflavin is comprised between 1 :2.5 and 1 :5.

5. An ophthalmic preparation according to any one of claims 1-3, wherein the concentration of riboflavin is comprised between 0.1 and 1.5% by weight, and said weight ratio of boron compound to riboflavin is comprised between 1 :5 and 1 : 5.

6. An ophthalmic preparation according to claim 5, wherein said weight ratio of boron compound to riboflavin is 1 :10.

7. An ophthalmic preparation according to claim 1 comprising, in addi- tion, a viscosifying and/or mucoadhesive compound selected from the group consisting of: carboxymethylcellulose, hydroxypropylcellulose, hyaluronic acid, chondroitin sulfate, alginic acid, natural polysaccharides, dextran, carbomers, carbopol, polyvinyl alcohol, polyethylene glycols, xanthan gum.

8. An ophthalmic preparation according to claim 7 comprising, in addi- tion, a compound enhancing trans-corneal permeation selected from the group consisting of: dimethyl sulfoxide, ethanol and mixtures thereof.

9. An ophthalmic preparation according to claims 7 or 8 comprising, in addition, one or more anti-oxidants, antimicrobials and or preservatives selected from the group consisting of: ascorbic acid, sodium metabisulfite, to- copheryl acetate, lactoferrin, sodium edetate, benzalkonium chloride, poly- hexanide, TPGS.