WO2025181000A1

WO2025181000A1 - Composition for the treatment of keratoconus

Info

Publication number: WO2025181000A1
Application number: PCT/EP2025/054840
Authority: WO
Inventors: Farhad HAFEZI
Original assignee: Emagine Sa
Current assignee: Emagine Sa
Priority date: 2024-02-26
Filing date: 2025-02-24
Publication date: 2025-09-04
Anticipated expiration: 2026-08-26

Abstract

The present invention refers to medical treatment and in particular to a novel epi-on CXL protocol and the treatment of keratoconus. In particular the present invention discloses a composition comprising benzalkonium chloride, ethylenediaminetetraacetic acid, and an emulsifier for use in therapy of keratoconus and other ectatic diseases.

Description

COMPOSITION FOR THE TREATMENT OF KERATOCONUS

FIELD OF THE INVENTION

The present invention refers to a composition for a medical treatment and in particular to a novel epi-on CXL protocol and the treatment of keratoconus,

BACKGROUND OF THE INVENTION

Keratoconus is a common corneal disorder, affecting around 1 in 1,500 of the general population, Keratoconus is a degenerative disease, characterized by progressive thinning and steepening of the cornea with corneal astigmatism. Without treatment, the disease progresses, resulting in further vision impairment and eventually in corneal blindness. The disease typically has a young age of onset, and this has two main implications: significantly impaired vision-related quality of life, and the lifetime economic burden of its treatment. If the cornea's cohesion is disrupted, it becomes cone-shaped and progressively thinner. This progressive deformation of the cornea mainly affects young people and is one of the most common reasons for legal blindness in the young. Once far progressed, the cornea becomes so thin that only a corneal transplant (keratoplasty) can save the patient from blindness. However, often, the transplant must be repeated after 15 to 20 years and a third keratoplasty usually is not possible. Along with a global shortage of donor corneas, m patients have until now faced the certain fate of partial or even total disability. At least 5,000 people are affected by keratoconus in Switzerland, around 50,000 in Germany and more than 5 million worldwide. Keratoconus belongs to the family of comeal ectatic diseases, comprising also Keratoglobus, pellucid marginal degeneration and ectasia after refractive laser surgery. These diseases all share very similar characteristics and mainly differ in the age of onset and local distribution of comeal thinning within the cornea. Of all ectatic diseases, keratoconus is the most frequent one.

Comeal cross-linking (CXL), the application of ultraviolet A (UV-A) light and riboflavin to create covalent bonds between collagen and the proteoglycans of the extracellular matrix to increase comeal biomechanical strength, is currently the only technique to halt the progression. Since the introduction of CXL, the number of keratoplasties has decreased by 50%, because the disease can be stabilized before vision deteriorates to an extent making a transplantation necessary. To guarantee effective cross-linking, CXL treatment usually involves removal of the comeal epithelium prior to riboflavin application and ultraviolet light illumination - “epi-off ’ CXL.

The first established CXL protocol was the Dresden protocol, which applies UV-A light after the comeal epithelium has been removed to expose the stroma, which almost entirely consists of collagen. Although its long-term success rate is high, CXL is timeconsuming and was initially limited to the application of corneas with a stromal thickness of more than 400 pm. Therefore, other accelerated epithelium-off CXL protocols were proposed. However, the epithelium-off CXL may cause potential complications, such as serious postoperative pain, delayed epithelium recovery, longterm haze, and infection. Therefore, patients receive antibiotics, steroid drops and lubricants postoperatively until re-epithelialization is complete and for up to 3 months after the procedure. Keeping the corneal epithelium intact should be less painful and help avoid other CXL-associated adverse events, Several methods of “epi-on” (transepithelial) CXL have been proposed. The evidence so far is that epi-off CXL remains the most effective method of strengthening the cornea and slowing keratoconus progression - but transepithelial methods are gaining ground. There are a number of treatment options for keratoconus and other corneal ectatic diseases, but only corneal cross-linking (CXL) is able to halt the progression of the disease.

The reason the Dresden protocol involved removal of the epithelial cells was the fact that riboflavin is a large hydrophilic molecule that cannot penetrate an intact epithelium being composed of 5 to 6 layers of epithelial cells. The intact epithelium reduces UV transmission, consumes oxygen, and acts as a barrier to stromal saturation with riboflavin and oxygen, two essential elements for successful cross-linking.

To address the riboflavin limitations, several approaches have been taken to try and get the riboflavin to the stroma. One approach in 2012 to 2016 were penetration enhancers to increase the penetration rate of the riboflavin. Described compositions were too aggressive to the epithelium. Following such an “early” epi-on approach, the corneas often displayed a destroyed epithelium on day 1 after the procedure, making this a de facto epi-off procedure. Gatzioufas et al. showed that transepithelial CXL method administering a modified riboflavin solution (0.25% riboflavin and 0.01% benzalkonium chloride) has a high incidence of epithelial defects (46%) and punctate corneal epitheliopathy/loose corneal epithelium (23%), which was observed in the immediate postoperative period. (J Refract Suig. 2016;32(6):372-377).

Further approaches include pharmacological cleavage of epithelial tight junctions, intrastromal application of riboflavin through injections or femtosecond laser-created pockets, and iontophoresis, whereby molecules are transported by electrophoresis and electroosmosis due to an applied electric current. However, the requirements for additional devices and the preparation of various forms of riboflavin for different stages and status of KC increase the complexity of CXL operations and the cost of CXL treatment. Leaving the corneal epithelium intact should eliminate wound-related complications and pain associated with epi-off CXL,

To address the element of insufficient oxygen saturation, CXL protocols utilizing pulsed light and an increase in the partial oxygen pressure via oxygen goggles have been introduced.

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a novel, epithelium-on CXL protocol which can achieve a similar corneal biomechanical stiffing effect as an accelerated epithelium-off CXL protocol which being widely applied now. Said objective is solved by the technical teachings of the independent claims. Further advantageous embodiments, aspects and details of the invention are evident from the dependent claims, the description, and the examples.

Surprisingly, it has been found that a composition comprising benzalkonium chloride, ethylenediaminetetraacetic acid, and an emulsifier as disclosed herein accelerates effectively the transepithelial administration of an active agent and especially the transepithelial administration of riboflavin within the eye while keeping the integrity of the comeal epithelial cells intact.

Thus, the present invention relates to a composition comprising benzalkonium chloride, ethylenediaminetetraacetic acid, and a emulsifier for use in therapy and in particular for use in a method for the treatment of keratoconus and other corneal ectatic diseases.

One embodiment of the present invention is directed to the composition for use as a penetration enhancer for transepithelial administration of an active agent and in particular of riboflavin. Riboflavin, also known as vitamin B2, is a water-soluble vitamin. Unlike folate and vitamin B6 riboflavin is only one chemical compound having the CAS Registry Number® 83-88-5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention relates to the use of benzalkonium chloride, ethylenediaminetetraacetic acid to improve the efficiency of topical drug delivery to the eye and in particular as penetration enhancer for riboflavin within a method for the treatment of keratoconus and other corneal ectatic diseases. The riboflavin or other active agents may be administered themselves or in form of a pharmacologically acceptable salt. In addition, the riboflavin or other active agents may be administered in a separate composition following the administration of the penetration enhancer or in one composition together with the penetration enhancer. Penetration enhancers (also called absorption enhancers or sorption promotors) are chemical compounds that can facilitate the penetration of active pharmaceutical ingredients into or through poorly permeable biological membranes. They typically penetrate biological membranes and reversibly decrease their barrier properties. Topical administration to the eye is usually characterized by very poor drug bioavailability due to several natural defense mechanisms, including nasolacrimal drainage, blinking, and poor permeability of the cornea. Enhancement of the corneal permeability to drug molecules is one of the strategies to improve the efficiency of topical drug delivery to the eye. In the example described herein, it is proven that the composition as described herein effectively enhances epithelium-on CXL protocols. The study shown herein proves that, without the application of iontophoresis, by using the composition described herein only, the epithelium-on CXL protocol could achieve a similar biomechanical effect as currently wide applied accelerated epithelium-off CXL protocols. These results hold the promise of unifying and simplifying the current complex epithelium- on CXL protocols.

Benzalkonium chloride (BAC), also known as alkyldimethylbenzylammonium chloride (ADBAC) and by the trade name Zephiran is a type of cationic surfactant having the CAS Registry Numbei® 8001-54-5. BAC is a nitrogenous cationic surfaceacting agent belonging to the quaternary ammonium group that has an extremely wide range of applications. BAC is an active substance from the group of antiseptics with antibacterial and antiviral properties. Benzalkonium chloride is mainly used in the pharmaceutical industry as a preservative for multi-dose containers, especially for eye drops, nasal sprays and inhalation solutions. Herein, BAC is used as penetration enhancer. The inventors assume that the effects are based on BACas a surfactant solubilizing the intercellular junctions within the corneal epithelium to enhance drug delivery. Surprisingly, the inventors have now succeeded in finding a concentration and composition of BAC with other components that achieves optimum absorption of the active ingredient. At the same time, the cornea is not damaged or at least not damaged to the extent that it is destroyed. The composition of the invention makes it possible to administer riboflavin in sufficient quantities while leaving the cornea intact. Consequently, the composition as described herein can be an ophthalmic formulation. The ophthalmic formulation may be administered in any form suitable for ocular drug administration, e.g., as a solution, suspension, ointment, gel, liposomal dispersion, colloidal microparticle suspension, or the like. A suitable ophthalmic formulation is an eye drop formulation preferably a sterile eye drop formulation. In one embodiment, a composition is provided that comprises benzalkonium chloride, ethylenediaminetetraacetic acid, and an emulsifier. The benzalkonium chloride may represent 0.005 wt.% to 0.020 wt.%, preferably 0.0075 wt.% to 0.015 wt.%. The ethylenediaminetetraacetic acid can represent 0.05 wt.% to 0.20 wt.%, preferably 0.075 wt.% to 0.15 wt.%. The emulsifier and in particular the cellulose derivative can represent 0.2 wt.% to 1.5 wt.%, preferably 0.25 wt.% to 1 wt.% and even more preferred 0.5 to 0.7 wt.%. Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid and is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes even at neutral pH. It is thus used to bind metal ions in the practice of chelation therapy, such as for treating mercury and lead poisoning. It is used in a similar manner to remove excess iron from the body. This therapy is used to treat the complication of repeated blood transfusions, as would be applied to treat thalassaemia. EDTA and ophthalmologic ally acceptable EDTA salts are both suitable components. In case EDTA salts are used for mixing the composition as described herein, the concentrations as mentioned refer to the EDTA ion only.

The composition as described herein can be a colloidal suspension or an emulsion. Therefore, it can comprise an emulsifier. An emulsifier is a substance that stabilizes an emulsion by reducing the oil-water interface tension. The emulsifier can be selected from the group comprising or consisting of ascorbyl palmitate, lecithin, trisodium phosphate, tri potassium phosphate, alginates, mono- and diglycerides of fatty acids, and cellulose derivatives, wherein cellulose derivatives are preferred and hydroxypropyl methylcellulose is particularly preferred. Other possible cellulose derivatives are hydroxy ethyl methyl cellulose, carboxy-methyl-cellulose.

The composition as described herein can be slightly hypo-osmolaric. This means the osmolarity of the composition is reduced compared to the stroma osmolarity, below the physiological normal value. In particular it can have a osmolarity of 350 ± 10 mosmol/kg H2O. Without wishing to be bound by theory, it appears that the combination of the benzalkonium chloride, ethylenediaminetetraacetic acid, and an emulsifier, in particular the hydroxypropyl methylcellulose play a significant role. Comparison to similar compositions showed that the advantageously effect (enhancing the penetration of riboflavin, stiffening effect similar to the most commonly used epi-off CXL protocol, no corneal defects, arrest keratoconus progression) is only given for the combination of these three components. Furthermore, the administration regime may be optimized to further increase these effects. Therefore, one aspect of the present invention refers to the therapeutical use of a composition comprising:

0.005 - 0,02% per weight benzalkonium chloride

0.05 - 0,3% per weight Ethylene diaminetetraacetic acid, and 0.2 to 1.5% per weight hydroxypropyl methylcellulose.

Another aspect of the present invention refers to the therapeutical use of a composition comprising:

0.005 - 0,02% per weight benzalkonium chloride

0.05 - 0,3% per weight Ethylenediaminetetraacetic acid, and

0.5-1.0% per weight hydroxypropyl methylcellulose.

The emulsifier and in particular the hydroxypropyl methylcellulose has influence on the viscosity of the composition. The viscosity using between 0.5 and 1.2 % per weight hydroxypropyl methylcellulose is advantageous because it supports the application of the composition, However, for sterilization by filtration, viscosity using less than 0,7% per weight hydroxypropyl methylcellulose is beneficial. Therefore, the range between 0.5 and 0.7 % per weight hydroxypropyl methylcellulose is most suitable. Of course, the viscosity is also influenced by the amount of further ingredients. And there are further technologies for sterilization. Therefore, a broader range 0.2 to 1.5% per weight of hydroxypropyl methylcellulose is feasible.

The composition can be filled to 100 % per weight with a suitable solvent such as water or a buffered solution. Therefore, the composition of the present application can be an aqueous solution. The buffered solution may be selected from the group comprising or consisting of (tris(hydroxymethyl)aminomethane, (4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid), (2-[[ 1 ,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid), (3-(N-morpholino)propanesulfonic acid), (piperazine-

N,N'-bis(2-ethanesulfonic acid)) or a balanced salt solution enriched with bicarbonate, dextrose, and glutathione. It is preferred that the solvent comprises 6.4 mg sodium chloride (NaCl), 0.75 mg potassium chloride (KC1), 0.48 mg calcium chloride dihydrate (CaCl₂-2H₂O), 0,3 mg magnesium chloride hexahydrate (MgCl₂*6H₂O), 3.9 mg sodium acetate trihydrate (C₂H3NaO2’3H₂O), 1,7 mg sodium citrate dihydrate (C6HsNa3O7’2H₂O), sodium hydroxide and/or hydrochloric acid (to adjust pH), per 1 mL water for injection.

Therefore, one embodiment of the present invention refers to a composition, wherein the composition contains

O.008 - 0,012% per weight benzalkonium chloride

0.05 - 0,15% per weight ethylenediaminetetraacetic acid,

0.4 - 1 % per weight hydroxypropyl methylcellulose,

0.6 - 1 ,1 % per weight sodium chloride,

0.6 - 0.85 % per weight potassium chloride,

0.06 - 0,85 % per weight calcium chloride dihydrate,

0.3 mg % per weight magnesium chloride hexahydrate,

3,9 mg sodium acetate trihydrate,

1.7 mg sodium citrate dihydrate and

0.06 - 0.85 % per weight tris(hydroxymethyl)aminomethan.

The pH of the composition described herein may be about 7.5, in particular between 7.4 and 7.6. The pH may be calibrated using (Tris(hydroxymethyl)aminomethan (TRIS). The composition as described herein may contain further the active agent such as riboflavin. The riboflavin can have a concentration of 1 to 2 % per weight. Apart from the components disclosed above and possibly the active ingredient, the solution can be composed to contain no other ingredients.

However, it is preferred that the active agent is administered in a separate composition subsequent to the penetration enhancer. The use of the composition as described herein without riboflavin (subsequently administered) allows for use of one solution in different corneal cross-linking protocols (epi-off and epi-on CXL). Thus, it is very easy for practitioners as they need only one solution which is suitable for all protocols. The concentration of riboflavin within the solution administered subsequently can be between 0.05 and 2.5 %, and is preferably between 0.1 and 0.5 % per weight.

In addition, the composition as described herein allows to use an epi-on protocol which does not need additional instrumentation beside the UV-irradiation device. Therefore, the protocol can be used in any medical facility. Other epi-on protocols require instruments for oxygenation or electrophoresis respectively electroosmosis.

Another aspect refers to the use of the composition as described herein in a therapeutic method comprising repeated application of the composition. The composition may be administered at regular intervals of 10 sec to 2 minutes for total 5 to 30 minutes. Here it is preferred that the composition can be administered at regular intervals of 20 sec to 1 minute for total 8 to 15 minutes. The application of the composition as described herein may be administered directly to the epithelium, such as the cornea, and may be followed by rinsing of the epithelium after waiting for 5 minutes. Thus, the composition may be administered at regular intervals of 10 sec to 2 minutes for total 5 to 30 minutes, followed by an incubation step of 3 to 10 min. Within this incubation step, the last drop of the composition to be administered stays on the cornea. At the end of the incubation step, the comeal surface can be rinsed off. It is preferred to use balanced salt solution (BSS) to rinse off the composition of the present invention.

Another aspect of the invention is the use within an epithelium-on protocol to treat keratoconus and other corneal ectatic diseases. Thus, the application of the active agent, such as riboflavin, can be subsequent to the administration of the penetration enhancer. It can be administered at regular intervals of 10 sec to 1 minute for total of 10 to 30 minutes, and preferably at regular intervals of 15 sec to 30 sec for total of 15 to 25 minutes. The concentration of riboflavin in the composition of the invention or the solution administered subsequently can be between 0.05 to 1 % per weight, in particular 0.075 to 0.5 %, 0.1 to 0.2 %, or 0.1 to 0,175 % or 0,12 to 0.16 % per weight, in a buffer solution, such as sodium phosphate buffer. It is preferred that no carrier is used in the subsequently used riboflavin solution. Higher concentration of riboflavin lead to a quicker saturation but they also limit the treatment depth to a shallower treatment, because more riboflavin molecules per volume of tissue are present and can be bleached by the riboflavin. Less UV light will reach the deeper layers. Therefore, it seems to be an advantageous of the present composition that it allows to use lower riboflavin concentrations in the treatment method.

Thereafter irradiation with UV-A follows. That good results are achieved using of rather low-powered UVA devices is an advantage of the present composition, In one embodiment the UV-A radiation can be applied with 9 to 30 mW/cm² UV-A light for 5 to 25 minutes, resulting in a total irradiation energy of 4 to 20 J/cm². The UV-A light may be applied as pulsed irradiation, such as 0.5 to 2 seconds on and 0.5 to 2 seconds off. In one embodiment the UV-A radiation can be applied with 18 to 30 mW/cm² pulsed UV-A light for 10 to 20 minutes, resulting in a total irradiation energy of 5.4 to 18 J/cm². It is preferred that the radiation step is performed using 18 mW/cm² pulsed UV-A light (1 second on/ 1 second off) for 15 to 20 minutes, resulting in a total irradiation energy of about 10.0 J/cm² (8 to 12 J/cm2). Further preferred is that the radiation step is performed as using 18 mW/cm² pulsed UV-A light (1 second on/ 1 second off) for 18,5 minutes, resulting in a total irradiation energy of 10,0 J/cm

In a further aspect the present invention refers to the use of the composition as described herein as a penetration enhancer for an epithelium. The present invention refers thus to a method of treating a kerato conus patient with the composition,

The method of treating keratoconus may comprise the following steps: a) Applying a composition containing

0,005 - 0,02% per weight benzalkonium chloride

0.05 - 0,3% per weight ethylenediaminetetraacetic acid, and 0.2 % - 1.5 % per weight and preferably, 0.4 - 1 % per weight hydroxypropyl methylcellulose to the comeal surface b) Applying riboflavin, preferably without drug carrier, to the comeal surface; and c) Irradiating the corneal surface with UV-A.A11 aspects and features of the composition and the use of the composition described herein refers also to the method described here. The method may further comprise the following step between step a) and step b): aa) rinsing off the corneal surface with a solution made to a physiological pH and isotonic salt concentration (balanced salt solution). In addition or alternatively, the method may comprise the application of a local anesthetic, such as oxybuprocaine, to the eye, before step a) is conducted. The local anesthetic can be applied every 60 seconds 3 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the distributions and comparisons of porcine corneas’ mean elastic modulus among three study groups. CXL = corneal cross-linking.

Figure 2 shows comparison of mean riboflavin fluorescence intensity grades in the cornea (at 300 pm depth) using different riboflavin formulation which are currently available for purchase. I : epi-off protocol; 2: Paracel™ by Avedro; 3: composition according to the invention; 4: Ricrolin®; 5: Ribo-Cross TE SERVImed EXAMPLES

Example 1

An eye drop formulation of the invention, was prepared as follows:

EDTA-containing benzalkonium chloride 0.1 % stock solution NRF, HPMC, and NaCl, were added to sterile irrigating solution (balanced salt solution, BSS Alcon), and mixed until visual transparency was achieved, indicating dissolution. The pH value of the mixture was set at 7.5 using Trometamol. The formulation had the following composition:

EDTA-containing benzalkonium chloride 0.1 % stock solution NRF: 0.3000g, HPMC: 0.0210 g,

NaCl: 0.0084 g,

Trometamol (TRIS): 0.0021 g balanced salt solution (BSS Alcon) ad 3 g, Example 2

Freshly enucleated porcine eyes with an intact epithelium and no scars were obtained from a local slaughterhouse (Zurich, Switzerland) and used within 8 hours. Eyes were randomly assorted into different study groups.

Group 1 epi-off protocol; Group 2 Paracel™ by Avedro;

Group 3 composition according to the invention;

Group 4 Ricrolin®;

Group 5 Ribo-Cross TE by SERVImed

Control Groups as described in the following For all eyes in epithelium-off group, a hockey knife was used to remove the epithelium. After the preparation of the porcine corneas, 0.4% Oxybuprocaine (Thea Pharma, Schaffhausen, Switzerland) once a minute for 3 minutes was applied.

For group 3 and the respective control group, the formulation of example 1 was applied to the corneal surface every 30 seconds for total 10 minutes. Then, after waiting for 5 minutes, the corneal surface was rinsed off with balanced salt solution (BSS), and 0.15% hypo-osmolaric riboflavin solution without carrier (Ribo-Ker, EMAGine, Zug, Switzerland) was applied to the corneal surface every 20 seconds for 20 minutes for group 3. While for the group 1, without the application of the composition according to example 1, the same riboflavin-dropping protocol was directly applied.

ParaCelTM by Avedro (now Glaukos) refers to a 2-step trans-epithelial riboflavin protocol using two different solutions specifically formulated for direct application on the intact epithelium which was used according to manufacturer’s protocol. For group

1 solution 1 (0.25% riboflavin) was applied to the corneal surface every 60 seconds for total 4 minutes. Thereafter the treating was immediately continued using solution

2 (0.22% riboflavin) which was applied to the corneal surface every 20 seconds for 6 minutes.

Group 4 treatment involves application of Ricrolin+ using iontophoresis. Therefore, a iontophoresis suction ring was placed and centred on the corneal surface. Riboflavin was administered and transferred by the I-ON CXL electric delivering system: 1mA.

After 5 or 10 minutes of imbibition, the exceeding solution was aspired by a syringe and the ocular surface was rinsed abundantly with sterile saline Na-Cl solution, eliminating all the residual riboflavin on the corneal surface. The corneas of Group 5 were treated with RiboCrossTE for 15 min, every 5 to 10 seconds, Subsequently, excess of the solution was removed with a dry triangular sponge.

A negative control group (cornea, no riboflavin solution) and a positive control group (20 minutes administration of riboflavin in epi-off protocol) was used for each group and measured at the same time.

The amount of riboflavin that can be detected at a depth of 310 pm of the cornea immediately after the end of the treatment is used as primary outcome to be measured, Therefore, the treated corneas were rinsed off with PBS, the epithelium was removed and a biopsy punch was placed into 5 mm chamber with the anterior side up. Detection window used was 500-550 nm. The data obtained when using the composition of the invention are good compared to other established compositions. The results are shown in Fig. 2. For Group 3 different parameters were changed: Using 0.12 % riboflavin does not change the outcome significantly. Without incubation after the pretreatment with the composition of the present invention, the amount of riboflavin detected was less, however the difference was not significant. In addition, incubation of 10 minutes instead of 5 minutes shows only a minor increase of detected riboflavin, which was not a significant change. 20 minutes of incubation did not further influence the result.

Example 3

The inventors could prove that, without the application of iontophoresis, by using the epithelium penetration enhancer only, the epithelium-on CXL protocol could achieve a similar biomechanical effect as the currently wide applied accelerated epithelium- off CXL protocol. One hundred fifty freshly enucleated porcine eyes with an intact epithelium and no scars were obtained from a local slaughterhouse (Zurich, Switzerland) and used within 8 hours. Eyes were randomly assorted into three study groups (n = 50 for each group). One group was used for the epithelium-on protocol, another the epithelium-off protocol, and third was the control group. For all the eyes, anterior segment optical coherence tomography (AS-OCT) combined with corneal Placido-based topography was performed (MS-39, CSO Italia, Firenze, Italy), the corneal epithelial and total thickness, as well as the corneal curvature information, were recorded.

The composition according to the present invention was prepared by mixing 0.01 % per weight BAC, 0.1 % per weight EDTA and 0.7 % per weight hydroxypropyl methylcellulose (HPMC). The solution was filled to 100 % per weight with BSS having 350 mosmol and the pH was set at 7.5 using TRIS buffer.

For porcine eyes in the epithelium-on and control groups, the excimer laser phototherapeutic keratectomy (PTK) was performed using the Amaris 750S excimer laser (Schwind eye-tech solutions GmbH, Kleinostheim, Germany). Based on the measured epithelial thickness, the epithelium ablation depth of PTK was calculated, resulting in the remaining central epithelial thickness reaching at 55 pm. The PTK ablation scope was set at a maximum of 10 mm for all ablated eyes. For all eyes in epithelium-off group, the hocky knife was used to remove the epithelium.

After the preparation of all the porcine corneas, 0.4% Oxybuprocaine (Thea Pharma, Schaffhausen, Switzerland) once a minute for 3 minutes was applied. For the epithelium-on and control groups, the composition according to the present invention was applied to the corneal surface every 45 seconds for total 10 minutes. Then, after waiting for 5 minutes, the corneal surface was rinsed off with balanced salt solution (BSS), and 0.1% hypo-osmolar riboflavin solution without further carrier or drug vehicle (Ribo-Ker, EMAGine, Zug, Switzerland) was applied to the corneal surface every 20 seconds for 20 minutes. While for the epithelium-off group, without the application of the composition according to the present invention, the same riboflavin- dropping protocol was directly applied.

All corneas (except for the corneas in the control group) were then irradiated with 365 nm UV-A light using a same cross-linking device (C-eye; EMAGine AG, Zug, Switzerland). For the epithelium-on group, 18 mW/cm² pulsed UV-A light (1 second on/ 1 second off) for 15 minutes was applied, resulting in a total irradiation energy of 8.1 J/cm²; While for the epithelium-off group, 9 mW/cm² continuous UV-A light for 10 minutes was applied, resulting in a total irradiation energy of 5.4 J/cm².

Before the biomechanical measurements, the corneoscleral button was taken in all the corneas. In each cornea, two corneoscleral strips of 5 mm width were prepared centrally in the horizontal axis. To standardize the hydration of all corneas, all the corneoscleral strips were put in 400 mOsmol/L phosphate buffered saline (PBS) solution for 15 minutes.

Stress-strain extensiometry was performed. In brief, 4 mm of the ends of each corneoscleral strip were dedicated to fixation, leaving approximately 11 mm of central corneal strip length to undergo extensiometry, A stress-strain extensometer (Z0.5; Zwick GmbH & Co., Ulm, Germany) was used to perform tensile strength measurements, calibrated with a distance accuracy of 2 mm and a tensile sensor with ≤ 0.21% of measurement uncertainty between 0.25 Newton (N) and 50 N. The extensometer has a linear holder extension ann that moved with a controllable speed, and the instrument was able to measure the real-time force in N exerted by the arm on the held specimen. The force to stress conversion was calculated from the width and thickness of the specimen. In the conditioning cycles, the arm speed was 2 mm/ minute; during the test phase, the position was controlled at the point where load was applied. The biomechanical characterization included elastic testing up to 4 N standard force. For the analysis, the stress-strain curve was considered, as its slope corresponds to the tangent elastic modulus and was determined between 5% to 10% of strain. Data analysis was performed using the Xpert Il-Testing Software (Zwick GmbH & Co., Ulm, Germany).

Statistical analysis was conducted using R software (version 4.2.0, R Foundation for Statistical Computing, Vienna, Austria). A Shapiro-Wilks test was applied to verify the normality of data distribution. Descriptive statistics were described as mean ± standard deviation. Either a one-way analysis of variance (ANOVA) or a Kruskal- Wallis H test was conducted for continuous variables to analyze the equivalence among all groups, A value of P < 0,05 was considered statistically significant.

The mean elastic modulus as a function between 5% and 10% of strain was 5,21 ± 1.58 N/mm, 4.95 ± 1.50 N/mm, and 4.01 ± 1.41 N/mm in the epithelium-on, epithelium- off, and control groups, respectively. The distributions and comparisons between groups are shown in Figure 1. There were no significant differences in the elastic modulus between the epithelium-on and epithelium-off groups (P = 0.45), but significant differences were found between the two cross-linked groups with the control group (P < 0.001 and = 0.001, respectively). Thus, one can conclude that the composition of the present invention allows an epi-on CXL protocol that provides a stiffening effect similar to the most commonly used epi-off CXL protocol and has the potential to clinically replace the latter.

The inventors could further show that the clinical outcome was very good, The epithelia of the treated patients stay intact directly and 1 day after administration. The secondary outcome shows improvements in visual acuity after patients received a treatment comprising the composition of the present invention (as described above).

Claims

WHAT IS CLAIMED IS:

1. A composition comprising

0.005 - 0,02% per weight benzalkonium chloride 0.05 - 0,3% per weight Ethylenediaminetetraacetic acid, and

0.2 - 1.5 % per weight hydroxypropyl methyl cellulose for use in therapy,

2. The composition according to claim 1 for use in a method for the treatment of keratoconus and other corneal ectatic diseases.

3. The composition according to claim 1 or 2, for use as a penetration enhancer for transepithelial administration of riboflavin.

4. The composition according to anyone of claims 1 to 3, wherein the composition is hypo-osmolar.

5. The composition according to claim 4, wherein the composition contains a balanced salt solution as solvent,

6. The composition according to anyone of claims 1 to 5, wherein the composition contains 0,008 - 0,012% per weight benzalkonium chloride

0.05 - 0, 15% per weight ethylenediaminetetraacetic acid,

0.4 - 1 % per weight hydroxypropyl methyl cellulose,

0.6 - 1.1 % per weight sodium chloride,

0.5 - 0.75 % per weight potassium chloride, 0.06 - 0.85 % per weight calcium chloride dihydrate and

0.06 - 0.85 % per weight tris(hydroxymethyl)aminomethan.

7. The composition according to anyone of claims 1 to 6, wherein the composition does not contain riboflavin.

8. The composition according to anyone of claims 1 to 7 for use in a therapeutic method comprising repeated application of the composition,

9, The composition according to anyone of claims 1 to 8, wherein the composition is administered at regular intervals of 10 sec to 2 minutes.

10. The composition according to claim 9, wherein the composition is administered for total 5 to 30 minutes.

11. The composition according to claim 10, wherein the composition is administered at regular intervals of 10 sec to 2 minutes for total 5 to 30 minutes followed by an incubation interval of 5 minutes before the corneal surface is rinsed off

12. The composition according to anyone of claims 1 to 11, wherein riboflavin is administered subsequently.

13. The composition according to claim 12, wherein the riboflavin is administered as 0.1 to 0.5 % per weight in a buffer solution at regular intervals of 10 sec to 1 minute for total of 5 to 30 minutes. 14, The composition according to claim 12 or 13, wherein the riboflavin is administered without carrier.