RU2841327C1 - Hypo-osmotic ophthalmic agent for ultraviolet cross-linking of thin corneas - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to ophthalmology, namely to a hypoosmotic ophthalmic agent for ultraviolet cross-linking. Hypo-osmotic ophthalmic agent for ultraviolet cross-linking in keratectasias with a corneal thickness of 350-400 mcm, containing riboflavin mononucleotide, hydroxypropyl methyl cellulose as a base and purified distilled water 100 ml, wherein riboflavin mononucleotide in amount of 0.075 g and hydroxypropyl methyl cellulose in amount of 0.025 g.

EFFECT: use of said invention provides a stable artificial intraoperative stroma oedema and an increase in the corneal thickness to 400 mcm or more when performing standard UV cross-linking in patients with thin cornea 350-400 mcm, maintaining the required concentration of the active substance due to the optimum composition of the components.

1 cl, 1 ex

Description

The invention relates to the field of medicine, in particular to ophthalmology, namely to medical products used as a photosensitizing riboflavin-containing agent for performing ultraviolet (UV) crosslinking in progressive keratectasia with a corneal thickness of 350-400 μm.

It should be noted that the standard UV crosslinking procedure involves the removal of the corneal epithelium to ensure the diffusion of riboflavin into the stroma, respectively, this reduces the corneal thickness by an average of 40-50 μm from the initial state. One of the main safety requirements for standard (with de-epithelialization) UV crosslinking in patients with keratectasia is the permissible corneal thickness of at least 400 μm. While in some patients this figure may be significantly less and average 350-400 μm. Performing UV crosslinking in such patients requires the introduction of new approaches with the development of tools and methods for performing the procedure.

A product for standard UV crosslinking of a cornea with a thickness of 450 µm is known, consisting of 0.1% riboflavin mononucleotide, dextran with a molecular weight of 450-550 kDa, sodium chloride, tris-(hydroxymethyl)-methylamine, nipagin, trilon B and purified water [Bikbov M.M., Khalimov A.R., Bikbova G.M. // Patent for invention RU No. 2412707 dated 02/27/2011].

An ophthalmological agent for standard UV crosslinking of the cornea in keratectasia is known, which contains 0.1% riboflavin mononucleotide, hydroxypropyl methylcellulose (HPMC), sodium chloride, tris-(hydroxymethyl)-methylamine, nipagin, trilon B, purified water. This agent is recommended for UV crosslinking of the cornea with a thickness of 400 µm or more [Bikbov M.M. et al. // Patent for invention RU No. 2560669 dated 20.08.2015].

The use of riboflavin mononucleotide solutions with dextran or HPMC for standard UV crosslinking in patients with thin corneas (less than 400 µm) is not possible.

The prototype is an ophthalmic solution intended for UV crosslinking in keratectasia with a corneal thickness of less than 400 μm, containing 0.1% riboflavin mononucleotide in an amount of 0.09-0.11 wt. %, and a hypo-osmotic base: HPMC in an amount of 0.04-0.06 wt. %, tris-(hydroxymethyl)-methylamine, nipagin and purified water [Bikbov M.M. et al. // Patent for invention RU No. 2631604 dated September 25, 2017]. This hypo-osmotic ophthalmic agent, in addition to the photosensitizing effect, is intended to form artificial intraoperative edema of the cornea, contributing to an increase in the thickness of the cornea to the required 400 μm or more. However, our studies show that during the installation of this hypo-osmotic agent, the intraoperative dynamics of changes in corneal thickness are variable, and its hypo-osmotic effect does not always induce acceptable stromal edema with the required increase in the size of the cornea, apparently due to the insufficiently low osmotic pressure of the solution.

The objective of the invention is to develop an ophthalmological hypoosmotic solution for standard (with de-epithelialization) ultraviolet corneal crosslinking, to expand the arsenal of means for UV crosslinking and indications for the use of the procedure.

The technical result of using the invention is the creation of a stable artificial intraoperative stromal edema and an increase in corneal thickness to 400 μm or more when performing standard UV crosslinking in patients with a thin cornea of 350-400 μm, maintaining the required concentration of the active substance due to the optimal composition of the components.

The proposed hypoosmotic ophthalmological agent for UV crosslinking in keratectasia with a corneal thickness of 350-400 μm contains riboflavin mononucleotide in an amount of 0.075 wt. %, hydroxypropyl methylcellulose in an amount of 0.025 wt. % as a hypoosmotic base, and water purified to 100.

Characteristics of components.

Riboflavin mononucleotide (sodium riboflavin-5'-monophosphate) is a yellow-orange crystalline powder. The aqueous solution fluoresces in ultraviolet light. It is introduced into the composition of the proposed agent in a concentration of 0.075 wt. % as a photosensitizer and corneal protector.

Hydroxypropyl methylcellulose (with a viscosity of 2% solution of 4000 Pa⋅sec at 20°C) is a natural polymer that dissolves in water to form a transparent viscous or gel-like solution. It is used as a hydrophilic base and has no toxic effect. It is introduced into the composition of the claimed product at a concentration of 0.025 wt.%.

The proposed ophthalmological agent is obtained as follows. 0.075 g of riboflavin mononucleotide is dissolved in 100 ml of purified freshly prepared water. 0.025 g of hydroxypropyl methylcellulose is added in portions to the surface of the resulting solution, heated to 40-50°C, with constant stirring until it is completely dissolved. The resulting solution is filtered through a membrane filter and packaged in vials, then sealed with rubber stoppers, which are rolled with aluminum caps. Vials with the finished agent are sterilized by autoclaving at 110°C and 0.5 atm. for 30 minutes. The solution is stored in a place protected from light.

The claimed hypo-osmotic ophthalmological agent was studied in experiments on rabbits, which were carried out under general anesthesia (solutions of "Xylazine" 20 mg/kg and "Zoletil" 15 mg/kg, intramuscularly) and local anesthesia (installation of eye drops "Inokain") in accordance with the standards and rules of good laboratory practice.

The first series of studies included two groups of animals (8 rabbits). In the 1st group (4 rabbits, 4 eyes), a hypo-osmotic solution of the following composition was used for instillations on the cornea: 0.1% riboflavin mononucleotide, HPMC, tris-(hydroxymethyl)-methylamine, nipagin and purified water [Bikbov M.M. et al. // Patent for invention RU No. 2631604 dated September 25, 2017]; in the 2nd (4 rabbits, 4 eyes) - the proposed hypo-osmotic solution. The frequency of instillation in both groups was 1 drop per 30 seconds.

The dynamics of corneal thickness changes in rabbits were studied during in vivo installations of the studied solutions for 60 minutes; measurements were taken at discrete time intervals every 5 minutes. Epithelium was removed with a microsurgical spatula on a 9 mm diameter corneal area marked with a marker using a Carl Zeiss surgical microscope (Germany). Optical coherence tomography (Vizante OST, Carl Zeiss, Germany) was used to determine the intravital corneal thickness in real time. Corneal thickness values in rabbits were recorded at 3 points: in the center of the cornea and 3 mm from the center.

In the second series of experiments, the quantitative content of riboflavin in the anterior chamber fluid (ACF) samples of rabbits was determined. The studies were conducted in 2 groups of animals: in the 1st (6 rabbits, 6 eyes), ACF was collected after instillation of a hypo-osmotic solution: 0.1% riboflavin mononucleotide, HPMC, tris-(hydroxymethyl)-methylamine, nipagin and purified water [Bikbov M.M. et al. // Patent for invention RU No. 2631604 dated 09.25.2017]; in the 2nd (6 rabbits, 6 eyes) - the claimed hypo-osmotic solution. The frequency of instillation in both groups was 1 drop per 30 seconds.

The VPK was collected from all rabbits using a 30 G needle in a volume of about 0.2 ml after 30 and 60 minutes of instillation. The concentration of riboflavin was determined using an automatic enzyme immunoassay analyzer "Multiscan" (Finland) at a wavelength of 610 nm using the ID-Vit test system (Immundiagnostik, Germany).

Statistical processing of the results was carried out using Microsoft Excel 2010 and Statistika 6.0 programs.

A comparative analysis of the results of the first stage of the study showed the following. The corneal thickness of intact rabbits averaged 370.25±24.1 μm. During de-epithelialization and instillations of the ophthalmic solution in group 1, the dynamics of corneal thickness changes during 60 minutes of observation had some fluctuations from 352.3±9.1 μm to 408.66±12.1 μm and averaged 386.5±10.2, which generally exceeded the size of the cornea even taking into account the removal of the epithelium. In group 2, the trend of corneal thickness changes after instillations of the proposed solution was similar to the first. In this case, the hypoosmotic effect with the appearance of stromal edema was more pronounced. The dynamics (60 minutes) of growth of the corneal thickness of rabbits was higher than in group 1 and varied in the range from 361.3±12.3 µm to 422.3±9.7 µm, averaging 392.3±10.8 µm.

Thus, changing the composition of the solution and decreasing the concentration of riboflavin and hydroxypropyl metal cellulose made it possible to reduce the osmotic pressure of the resulting solution, which, accordingly, led to more pronounced and stable stromal edema with a more significant increase in corneal thickness.

At the second stage of the experiments, the efficiency of saturation of the stroma with hypoosmotic solutions was studied based on the level of riboflavin in the anterior chamber fluid. The 30th and 60th minutes from the start of the installations were chosen as control points for determining the concentration of riboflavin, which corresponds to the stage of ultraviolet irradiation (370 nm) of the cornea during the standard UV crosslinking procedure.

In order to impart greater hypoosmoticity to the claimed ophthalmic solution, we, in comparison with the prototype, reduced the concentration of riboflavin to 0.075% (versus 0.1%) and HPMC to 0.025%) (versus 0.05%).

The concentration of riboflavin in the VPC of rabbits in group 1 was: after 30 minutes of instillation - 604±22.3 μg/l, after 60 minutes - 620±18.2 μg/l; in group 2: after 30 minutes - 588±16.2 μg/l, after 60 minutes - 601±19.5 μg/l, which generally corresponded to stably high values, which are comparable with the results when using other solutions for UV corneal crosslinking, in particular with riboflavin/dextran [Bikbov M.M. et al. Dynamics of the riboflavin level in the aqueous humor of the anterior chamber of the eye of experimental animals with standard saturation of the stroma with solutions for UV corneal crosslinking // Bulletin of Ophthalmology. - 2016. - V. 132, No. 6. - P. 29-35]. At the same time, there were no statistically significant differences between the groups of animals and the observation periods.

Thus, the reduction of the content of riboflavin and hydroxypropyl metal cellulose in the claimed solution made it possible to impart additional hypoosmoticity to the solution, which caused the required diffuse edema of the stroma and an increase in the thickness of the cornea.

The concentration of riboflavin in the anterior chamber fluid was within the range of values observed when performing standard UV crosslinking.

Conclusion. The proposed composition of the base and active components of the claimed hypoosmotic ophthalmological agent during eye instillations with a frequency of 1 drop per 30 seconds caused induced stromal edema with an increase in the size of the cornea by more than 400 μm, with its initial thickness of 350-400 μm, maintained an optimal concentration of riboflavin in the stroma, and ensured safe ultraviolet crosslinking in a standard way (with de-epithelialization).

Claims (1)

Hypo-osmotic ophthalmological agent for ultraviolet crosslinking in keratectasia with a corneal thickness of 350-400 μm, containing riboflavin mononucleotide, hydroxypropyl methylcellulose as a base and 100 ml of purified distilled water, characterized in that it contains riboflavin mononucleotide in an amount of 0.075 g, and hydroxypropyl methylcellulose in an amount of 0.025 g.