WO2018007457A1

WO2018007457A1 - Ophthalmic dye

Info

Publication number: WO2018007457A1
Application number: PCT/EP2017/066807
Authority: WO
Inventors: Detlef Gabel; Andreas Matthias MOHR; Frank RUSELER
Original assignee: Vitreq B.V.
Priority date: 2016-07-08
Filing date: 2017-07-05
Publication date: 2018-01-11

Abstract

The present invention relates to the field of medicine, specifically to ophthalmic surgery, more specifically to an novel ophthalmic dye for surgery with excellent staining properties combined with minimal toxicity in the dark and minimal toxicity during illumination.

Description

OPHTHALMIC DYE

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Dyes are used frequently in ophthalomoligical procedures to stain tissues of interest. In ocular surgery of the anterior chamber and the posterior chamber, dyes are being used for enhancing the contrast and to make the structures visible which are to be removed during the surgery. The conditions to be treated in the anterior chamber are, without being exhaustive: deep lamellar keratoplasty dissection, keratoplasty, Descemet's membranes, cataract surgery, conjunctival surgery, and anterior vitreous surgery. In the posterior chamber, conditions are, again without being exhaustive: macular hole, epiretinal membrane, retinal detachment, vitreomacular traction, macular hole development, unroofed cysts, residual membranes, pit-associated maculopathy, retinopathy of prematurity.

In vitreo -retinal surgery, two different structures need to be removed in certain pathological situations in order to restore vision: the epiretinal membrane (ERM) (an abnormal structure generated e.g. when the patient suffers from chronic diabetes) and the internal limiting membrane (ILM), which separates the vitreous from the retina, and is firmly attached to the latter.

For surgery, the vitreous is removed, and the eye is filled either with gas or with a salt solution in preparation for removal of the ERM and the ILM from the retina. The state of the art is to visualize the structures by staining them with dyes and then to remove them.

The first dye used in ILM staining was indocyanine green (ICG) (CAS number 3599-32- 4). Its spectral properties with an absorption maximum outside the range visible to the eye has limited its usefulness [2]. Staining of the ERM is often done with TB. TB does, however, not stain the ILM strongly [2, 3]. Therefore Brilliant Blue G (BBG) (CAS number 6104-58-1) has been added to TB, in order to visualize also the ILM [4]; it is also used alone. The staining which can be achieved with TB and with BBG, when compared, have been reported as equal, and inferior to that of ICG [5]. Recently, Brilliant Blue G (BBG) (CAS number 6104-58-1) has been combined with Bromophenyl Blue (BP) (CAS number 115-39-9). Acid Violet 17 (CAS number 4129-84-4), which was also recently introduced, has been reported as toxic [8]. All of these dyes and dye combinations thus appear to have drawbacks and risks involved, and there is a continued search for better dyes [9 - 12]. Concentrations of the dyes must be high enough to allow appropriate staining; a reduction in the concentration will usually lead to staining reduced to such low levels that they are of little value for the surgeon.

As an alternative to a dye, or in combination with a dye, the highly insoluble Triamcinolone Acetonide (TA) has been used for aiding the surgeon in locating the ILM [3]. The opaque crystalline flakes of this colorless substance sediment to the membrane, and areas which have been removed can be recognized by the absence of the white flakes. When the eye is filled with salt solution, the dye solution is injected into the salt solution and should then sink, by gravity, onto the retina. Different additives are used to increase density and viscosity. One additive is a polymer, such as polyethylene glycol (PEG). PEG has the disadvantage that it reduces the staining and thereby the visibility of the ILM. Other additives are sugars or sugar alcohols. These have the disadvantage that they contribute to the osmotic pressure and thus to cell death if not balanced by a reduction in other osmotically active substances. Therefore, their maximum concentration, and as a consequence the density of the solution which can be applied safely, is limited. More importantly, the staining effect of the dye can be reduced severely by some of these additives, especially polymeric additives.

A good additive should therefore not reduce the staining. Ideally, an additive for increasing the density should rather enhance the effect of the staining. Finally, any solution used to stain the ILM and the ERM should exhibit low toxicity, in order to not to endanger the results of the surgery.

Accordingly, at present, there is only a limited number of dyes, mostly with blue color, used for staining. These dyes include, but are not limited to, trypan blue (TB) (CAS number 72-57-1), brilliant blue G (CAS number 6104-58-1), bromophenol blue (CAS number 115-39-9), Chicago skye blue (CAS number 2610-05-1), either alone or in combination with another dye. These dyes are not optimal for the purpose, as they show weak staining or are toxic. One dye, indocyanine green (ICG) (CAS number 3599-32-4), has its maximum light absorbance in a wavelength range not visible to the eye, and in a wavelength range where illumination sources such as xenon or LED lights emit no light. Thus, its green color is very faint when compared to other green dyes, despite the fact that it has a very high molar extinction coefficient. Altogether, there is a strong need to develop further green dyes. A green dye would be ideal, as its color cannot be confounded with the reddish fundus of the eye and offers maximal contrast, and therefore would allow optimal identification of the structures to be removed.

The ideal dye would have a good staining ability, a minimal cellular toxicity in the dark, and a minimal cellular toxicity in the strong illumination necessary for surgery.

It is the aim of this invention to provide such dyes.

SUMMARY OF THE INVENTION

In an aspect, the present invention provides for a composition suitable for staining an ophthalmic structure comprising a compound according to Formula I:

, or a pharmaceutically acceptable salt or hydrate thereof, wherein:

a. R is selected from the group consisting of: H; CH₃; (CH₂)_nH, wherein n is an integer from 1 - 6; (CH₂)_nA, wherein n is an integer from 1 - 6 and A is selected from C0₂H, S0₃H, OH, NH₂, and a functional derivative of C0₂H, such as an ester, an amide or a hydrazide;

b R' is selected from the group consisting of: H; CH₃; (CH₂)_nH, wherein n is an integer from 1 - 6; (CH₂)_nA, wherein n is an integer from 1 - 6 and A is selected from C0₂H, S0₃H, OH, NH₂, and a functional derivative of C0₂H, such as an ester, an amide or a hydrazide;

c selected from the group consisting of: H and S0₃H;

d, selected from the group consisting of: H and S0₃H;

e. R"" is selected from the group consisting of: H; (CH₂)_nH, wherein n is an integer from 1 - 6; Br; CI; I; (CH₂)„CHO and (CH₂)„C0₂H, wherein n is an integer from 0 - 6; and a functional derivative of C0₂H, such as an ester or an amide or a hydrazide; f. R^v is selected from a group consisting of H; (CH₂)_nH, wherein n is an integer from 1 - 6; and

g. R^VI is selected from a group consisting of H; 0-(CH₂)_nH, wherein n is an integer from 1 - 6; C6¾; and

h. wherein each of the double bonds can be a Z or and E stereoisomer.

In said composition according to the invention, preferably,

a. R = CH₃ and R' = (CH₂)₅C0₂H and R" = R' " = R" " = R^v = R^VI = H; b. R = (CH₂)₄C0₂H and R' = (CH₂)₅C0₂H and R" = R'" = R"" = R^v = R^VI = H; or,

c. R = R' = (CH₂)₄S0₃H and R" = R" ' = R" " = R^V=R^VI= H.

In said composition according to the invention, preferably, the concentration of the compound according to Formula I, or a pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.005% (w/v) to about 1% (w/v).

In said composition according to the invention, preferably, the concentration of the compound according to Formula I, or a pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.025%) (w/v) to about 0.1 % (w/v), more preferably within the range of about 0.05% (w/v) to about 0.1% (w/v).

Preferably, said composition according to the invention preferably further comprises a dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue and their respective pharmaceutically acceptable salts and hydrates thereof.

In said composition according to the invention, preferably, the concentration of the dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue or a respective pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.005% to about 0.5% (w/v).

Preferably, a composition according to the invention further comprises a perfluorocarbon liquid, preferably a perfluorocarbon liquid selected from the group consisting of perfluorooctane, perfluoroperhydrophenanthrene, perfluorodecalin, perfluorotributylamide and perfluorooctylbromide, more preferably perfluorooctane or perfluorodecalin.

Preferably, a composition according to the invention further comprises an agent that increases the density and/or viscosity of the composition, such that the density and/or viscosity of the composition is increased compared to the density and/or viscosity of H₂0.

Preferably, said agent is selected from the group consisting of: heavy water, diglycerol, triglycerol, a Gadolinium complex, a sugar, a sugar alcohol, a polymer and a polysaccharide. More preferably, said agent is selected from the group consisting of: diglycerol, triglycerol and a Gadolinium complex.

Preferably, in said composition according to the invention the total concentration of the diglycerol and/or triglycerol, if present, is within the range of about 0.1% to about 25% (v/v), preferably within the range of about 2% to about 20% (v/v), more preferably within the range of about 3% to about 6% (v/v), or wherein the concentration of a Gadolinium complex is within the range of about 0.01 M to about 0.1 M, preferably within the range of about 0.01 M to about 0.05 M, more preferably in the range of about 0.02 M to about 0.04 M.

The invention further provides for a kit of parts comprising a composition according to any of claims 1 - 11, wherein the kit comprises the compounds within a single composition or comprises the compounds in multiple compositions.

The invention further provides for a composition according to the invention, or a kit of parts according to the invention for use as a medicament, preferably for use in a method of ophthalmic surgery. Preferably, the ophthalmic surgery comprises staining of an ophthalmic structure.

Preferably, the ophthalmic structure is the internal limiting membrane (ILM) and/or the epiretinal membrane (ERM) and/or the anterior cavity.

The invention further provides for the use of a composition according to the invention or of a kit of parts according to the invention as a surgical adjuvant, preferably in ophthalmic surgery.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been demonstrated that dyes of the general structure of Formula I show excellent staining properties, minimal toxicity in the dark, and minimal toxicity during illumination compared to prior art dyes.

Accordingly, in a first aspect the present invention provides for a composition suitable for staining an ophthalmic structure comprising a compound according to Formula I:

, or a pharmaceutically acceptable salt or hydrate thereof, wherein:

b. R' is selected from the group consisting of: H; CH₃; (CH₂)_nH, wherein n is an integer from 1 - 6; (CH₂)_nA, wherein n is an integer from 1 - 6 and A is selected from C0₂H, S0₃H, OH, NH₂, and a functional derivative of C0₂H, such as an ester, an amide or a hydrazide;

c. R" is selected from the group consisting of: H and S0₃H;

d. R" ' is selected from the group consisting of: H and S0₃H;

e. R"" is selected from the group consisting of: H; (CH₂)_nH, wherein n is an integer from 1 - 6; Br; CI; I; (CH₂)„CHO and (CH₂)„C0₂H, wherein n is an integer from 0 - 6; and a functional derivative of C0₂H, such as an ester or an amide or a hydrazide;

f. R^v is selected from a group consisting of H; (CH₂)_nH, wherein n is an integer from 1 - 6; and

h. wherein each of the double bonds can be a Z or and E stereoisomer. Said compound and a pharmaceutically acceptable salt and hydrate thereof are herein referred to as a compound according to the invention. Said composition is herein referred to as a composition according to the invention.

An exemplary compound of the invention (depicted as "Dye 1 ") is a compound according to Formula 1, wherein: R = R' = (CH₂)₄S0₃H, R" = R' " = R"" = H (CAS number 120768-44-7) has an extinction in a solution of 0.0001% (w/v) in water of 2.8 at 639 nm, while that of TB (CAS number 72-57-1) of the highest commercially available quality at 0.0001% is only 0.7 at 582 nm. Thus, for the same intensity of staining, four times more trypan blue is necessary. ICG at a concentration of 0.0001% in water has an absorbance of 0.1 at 650 nm and is thus much less visible to the eye; its maximum absorbance is beyond 850 nm, a wavelength invisible to the eye.

Preferably, in a composition according to the invention, the functional derivative of

C0₂H is an ester, an amide or a hydrazide.

Preferably, in a composition according to the invention,

a. R = CH₃ and R' = (CH₂)₅C0₂H and R" = R'" = R" " = R^v = R^VI = H; b. R = (CH₂)₄C0₂H and R' = (CH₂)₅C0₂H and R" = R'" = R"" = R^v = R^VI = H; or,

c. R = R' = (CH₂)₄S0₃H and R" = R" ' = R" " = R^v = R^VI = H. Preferably, in a composition according to the invention, the concentration of the compound according to the invention, is within the range of about 0.005% (w/v) to about 1%) (w/v), more preferably within the range of 0.005%) (w/v) to 1% (w/v), more preferably within the range of about 0.01% (w/v) to about 1% (w/v), more preferably within the range of 0.01% to 1%. More preferably, the concentration of the compound according to the invention, is within the range of about 0.025%) (w/v) to about 0.1% (w/v), even more preferably within the range of 0.025%) (w/v) to 0.1% (w/v). Even more preferably, the concentration of the compound according to the invention, is within the range of about 0.05% (w/v) to about 0.1% (w/v), even more preferably within the range of 0.05% (w/v) to 0.1% (w/v).

The compound according to the invention in combination with further dyes was surprisingly found to be even better in staining properties of the ILM than alone. Especially the combination with Trypan Blue results in exceptional staining.

Accordingly, preferably, a composition according to the invention further comprises a dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue and their respective pharmaceutically acceptable salts and hydrates thereof. Preferably, a composition according to the invention further comprises Trypan Blue or a pharmaceutically acceptable salt or hydrate thereof.

Preferably, the concentration of such further dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue or a respective pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.001%) to about 1% (w/v), more preferably within the range of 0.001% (w/v) to 1% (w/v), more preferably within the range of about 0.005%) (w/v) to about 0.5%> (w/v), more preferably within the range of 0.005%) (w/v) to 0.5%) (w/v), more preferably within the range of about 0.01% (w/v) to about 0.5%) (w/v), more preferably within the range of 0.01% (w/v) to 0.5%> (w/v), even more preferably within the range of about 0.05%> (w/v) to about 0.15% (w/v), even more preferably within the range of 0.05%> (w/v) to 0.15% (w/v).

Preferably, in a composition according to the invention:

- Janus Green, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1%) (w/v), preferably within the range of about 0.005%) (w/v) to about 0.01% (w/v),

- Methyl Green, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1 % (w/v), preferably within the range of about 0.01% (w/v) to about 0.05%>

(w/v),

- Methylene Blue, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1%) (w/v), preferably within the range of about 0.01% (w/v) to about 0.1 %

(w/v),

- Crystal Violet, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1%) (w/v), preferably within the range of about 0.01% (w/v) to about 0.05%>

(w/v),

- Methyl Violet, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1%) (w/v), preferably within the range of about 0.01% (w/v) to about 0.05%>

(w/v),

- Ethyl Violet, if present, has a concentration within the range of about 0.001% (w/v) to about 0.1%) (w/v), preferably within the range of about 0.01% (w/v) to about 0.05%> (w/v),

- Evans Blue, if present, has a concentration within the range of about 0.001% (w/v) to about 0.4%) (w/v), preferably within the range of about 0.0025%) (w/v) to about 0.1 %

(w/v),

- Indocyanine Green, if present, has a concentration within the range of about 0.001% (w/v) to about 0.4%> (w/v), preferably within the range of about 0.0025%) (w/v) to about

0.1% (w/v), - Methyl Blue, if present, has a concentration within the range of about 0.001% (w/v) to about 0.4%) (w/v), preferably within the range of about 0.0025%) (w/v) to about 0.1 % (w/v), and,

- Trypan Blue, if present, has a concentration within the range of about 0.001% (w/v) to about 0.25%) (w/v), preferably within the range of about 0.01% (w/v) to about 0.15%

(w/v).

More preferably, in a composition according to the invention:

- Janus Green, if present, has a concentration within the range of 0.001% (w/v) to 0.1% (w/v), preferably within the range of 0.005% (w/v) to 0.01 % (w/v),

- Methyl Green, if present, has a concentration within the range of 0.001% (w/v) to 0.1 % (w/v), preferably within the range of 0.01% (w/v) to 0.05%> (w/v),

- Methylene Blue, if present, has a concentration within the range of 0.001% (w/v) to 0.1% (w/v), preferably within the range of 0.01% (w/v) to 0.1% (w/v),

- Crystal Violet, if present, has a concentration within the range of 0.001 % (w/v) to 0.1 % (w/v), preferably within the range of 0.01% (w/v) to 0.05%> (w/v),

- Methyl Violet, if present, has a concentration within the range of 0.001% (w/v) to 0.1% (w/v), preferably within the range of 0.01% (w/v) to 0.05%> (w/v),

- Ethyl Violet, if present, has a concentration within the range of 0.001% (w/v) to 0.1% (w/v), preferably within the range of 0.01% (w/v) to 0.05%> (w/v),

- Evans Blue, if present, has a concentration within the range of 0.001% (w/v) to 0.4% (w/v), preferably within the range of 0.0025%) (w/v) to 0.1 % (w/v),

- Indocyanine Green, if present, has a concentration within the range of 0.001% (w/v) to 0.4% (w/v), preferably within the range of 0.0025% (w/v) to 0.1% (w/v),

- Methyl Blue, if present, has a concentration within the range of 0.001% (w/v) to 0.4% (w/v), preferably within the range of 0.0025%) (w/v) to 0.1 % (w/v), and,

- Trypan Blue, if present, has a concentration within the range of 0.001% (w/v) to 0.25% (w/v), preferably within the range of 0.01% (w/v) to 0.15% (w/v). Even more preferably, in a composition according to the invention:

- Janus Green, if present, has a concentration within the range of about 0.005% (w/v) to about 0.01% (w/v), - Methyl Green, if present, has a concentration within the range of about 0.01% (w/v) to about 0.05% (w/v),

- Methylene Blue, if present, has a concentration within the range of about 0.01% (w/v) to about 0.1% (w/v),

- Crystal Violet, if present, has a concentration within the range of about 0.01% (w/v) to about 0.05% (w/v),

- Methyl Violet, if present, has a concentration within the range of about 0.01% (w/v) to about 0.05% (w/v),

- Ethyl Violet, if present, has a concentration within the range of about 0.01% (w/v) to about 0.05% (w/v),

- Evans Blue, if present, has a concentration within the range of about 0.025% (w/v) to about 0.1% (w/v),

- Indocyanine Green, if present, has a concentration within the range of about 0.025% (w/v) to about 0.1% (w/v),

- Methyl Blue, if present, has a concentration within the range of about 0.025% (w/v) to about 0.1%) (w/v), and,

- Trypan Blue, if present, has a concentration within the range of about 0.01% (w/v) to about 0.25% (w/v). Even more preferably, in a composition according to the invention:

- Janus Green, if present, has a concentration within the range of 0.005% (w/v) to 0.01% (w/v),

- Methyl Green, if present, has a concentration within the range of 0.01% (w/v) to 0.05% (w/v),

- Methylene Blue, if present, has a concentration within the range of 0.01 % (w/v) to 0.1 % (w/v),

- Crystal Violet, if present, has a concentration within the range of 0.01% (w/v) to 0.05% (w/v),

- Methyl Violet, if present, has a concentration within the range of 0.01% (w/v) to 0.05% (w/v),

- Ethyl Violet, if present, has a concentration within the range of 0.01% (w/v) to 0.05% (w/v), - Evans Blue, if present, has a concentration within the range of 0.025% (w/v) to 0.1% (w/v),

- Indocyanine Green, if present, has a concentration within the range of 0.025% (w/v) to 0.1% (w/v),

- Methyl Blue, if present, has a concentration within the range of 0.025% (w/v) to 0.1% (w/v), and,

- Trypan Blue, if present, has a concentration within the range of 0.01% (w/v) to 0.25% (w/v). A more preferred further dye is Trypan Blue. When the composition according to the invention comprises Trypan Blue as a further dye, the concentrations are most preferably the following: the compound according to the invention is within the range of about 0.005%) (w/v) to about 0.5%> (w/v) and Trypan Blue is within the range of about 0.005%) (w/v) to about 0.5%) (w/v), more preferably the compound according to the invention is within the range of about 0.01% (w/v) to about 0.5%> (w/v) and Trypan Blue is within the range of about 0.01% (w/v) to about 0.5%> (w/v), more preferably the compound according to the invention is within the range of about 0.025%) (w/v) to about 0.1 % (w/v) and Trypan Blue is within the range of about 0.05%> (w/v) to about 0.15% (w/v). Even more preferably, the compound according to the invention is within the range of 0.005% (w/v) to 0.5% (w/v) and Trypan Blue is within the range of 0.005% (w/v) to 0.5% (w/v), more preferably the compound according to the invention is within the range of 0.01% (w/v) to 0.5%) (w/v) and Trypan Blue is within the range of 0.01% (w/v) to 0.5%> (w/v), more preferably the compound according to the invention is within the range of 0.025% (w/v) to 0.1%) (w/v) and Trypan Blue is within the range of 0.05%> (w/v) to 0.15% (w/v). The purity of a compound according to the invention and other dyes is preferably as high as possible, preferably of pharmaceutical grade, although the dyes herein are not necessarily limited thereto. Preferably, the purity of a dye herein is at least 70%>, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or most preferably at least 99% pure. Examples of pharmaceutical acceptable salts include but are not limited to, salts formed with organic bases (such as glucosamine, galactosamine, mannosamine, meglumine, trimethylamine, choline, procaine, triethanolamine, diethanolamine, and ethanolamine), inorganic bases (such as ammonia, alkaline metals, and alkaline earth metals), organic acids (such as para-toluene sulfonic acid, methane sulfonic acid, formic acid, trifluoro acetic acid, and maleic acid), inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid), basis amino acids (such as lysine, arginine, histidine and ornithine), halogen ions (such as F and CI ions) and intramolecular salts.

Ophthalmic dye compositions are typically prepared in commercially available phosphate-buffered saline solution (PBS). Accordingly, the compositions according to the invention preferably comprise PBS or are PBS based. When a composition according to the invention comprises an agent that increases the density and/or viscosity of the composition, such as specified here below, PBS may be replaced (in part) by the stock solution of the agent and/or may be replaced in part by water to adjust its osmolarity to the desired physiological range, such as specified here below. Alternatively, the composition according to the invention is a non-aqueous liquid and is prepared on the basis of a perfluorocarbon liquid as defined elsewhere herein.

The composition according to the invention can conveniently be used in ophthalmic manipulations on a subject such as ophthalmic surgery, preferably as an ophthalmic adjuvant. Examples of diseases and conditions of the eye that require ophthalmic manipulations such as ophthalmic surgery are, but are not limited to, vitreo-retinal disease such as macular hole, retinal detachment due to hymoyopathic macular hole, epiretinal membrane, proliferative diabetic retinopathy, diabetic macular edema, proliferative vitreoretinopathy, specific cataracts such as hypermature cataract, congenital cataract, and split thickness corneal transplantation. The subject is preferably a mammal, more preferably a human.

The composition according to the invention can be in any form, such as a mixture of solids and a liquid, preferably an aqueous liquid or a perfluorocarbon liquid. The compound according to Formula I is preferably soluble in water or a in watery composition such as a buffer. Alternatively or in combination, the compound according to Formula I is soluble in a perfluorocarbon liquid. Preferably, at least 0.1% (w/v) can be dissolved in water, in a watery composition such as a buffer or in a perfluorocarbon liquid, more preferably at least 0.1 % (w/v), even more preferably at least 1% (w/v) and most preferably at least 5% (w/v) in water, in a watery composition such as a buffer, or in a perfluorocarbon liquid. All components may be present in a single composition or may be present in different compositions that are mixed before use; all (dye) components may be present in a solid mixture that is dissolved before use; for preparation it may conveniently be dissolved in intraocular cleaning solution, intraocular rinsing solution, physiological saline or a balanced salt solution. The composition according to the invention may comprise or may be mixed with a pharmaceutically acceptable excipient and/or carrier and/or a drug known to the persons skilled in the art. After preparation, the composition according to the invention may be sterilized, e.g. by filtration or autoclaving. Preferably, the pH of the solution is within the range of pH 7.0 to pH 7.6, more preferably in the range of pH 7.1 to pH 7.5, more preferably in the range of pH 7.2 to pH 7.5, more preferably in the range of pH 7.3 to pH 7.5 even more preferably the pH is physiological, i.e. neutral, i.e. about pH 7.4, most preferably the pH is 7.4.

The person skilled in the art will comprehend that the composition according to the invention is not limited to a composition comprising a compound according to the invention and a single further dye; the composition may comprise another further dye or further dyes either selected from the dyes listed herein or other dyes.

Perfluorocarbon liquids (PCFLs) can be used in ophthalmic surgery. The physical properties of PFCLs including high specific gravity, moderate surface tension, low viscosity, and optical clarity and transparency make them convenient intraoperative tools for ophthalmic surgery.

Accordingly, preferably, a composition according to the invention further comprises a perfluorocarbon liquid (PCFL), preferably a perfluorocarbon liquid selected from the group consisting of perfluorooctane, perfluoroperhydrophenanthrene, perfluorodecalin, perfluorotributylamide and perfluorooctylbromide, more preferably perfluorooctane or perfluorodecalin. The person skilled in the art will comprehend that a PCFL for use in ophthalmic surgery should be as pure as possibly, preferably at least 95%, 96%, 97%, 98%, 99% or 100% pure. The person skilled in the art will comprehend that a composition according to the invention comprising a PCFL and a dye according to the invention will not be a watery composition since a PCFL and water hardly mix. Accordingly, the concentration of a PCFL or the total concentration of PCFL in a composition according to the invention is preferably at least 95%, 96%, 97%, 98%, 99% or 100%

Preferably, a composition according to the invention further comprises an agent that that increases the density and/or viscosity of the composition, such that the density and/or viscosity of the composition is increased compared to the density and/or viscosity of H₂0 when measured under identical conditions. Such agent is preferably selected from the group consisting of heavy water, diglycerol, triglycerol, a Gadolinium complex, a sugar, a sugar alcohol, a polymer and a polysaccharide, more preferably such agent is selected from the group consisting of diglycerol, triglycerol and a Gadolinium complex.

Preferably, in a composition according to the invention the total concentration of the diglycerol and/or triglycerol, if present, is within the range of about 0.1% to about 25% (v/v), preferably within the range of about 2% to about 20% (v/v), more preferably within the range of about 3% to about 6%> (v/v). The physiologically normal osmotic pressure is around 300 mosmol/kg, and deviations above about ± 50 mosmol/kg from the normal value may eventually lead to cellular damage. Accordingly, the osmotic pressure of the composition according to the invention is preferably within the range of about 250 mosmol/kg to about 350 mosmol/kg, more preferably from about 275 mosmol/kg to about 325 mosmol/kg, more preferably from about 285 mosmol/kg to about 315 mosmol/kg, more preferably from about 290 mosmol/kg to about 310 mosmol/kg, more preferably from about 295 mosmol/kg to about 305 mosmol/kg and most preferably is about 300 mosmol/kg. More preferably, the osmotic pressure of the composition according to the invention is preferably within the range of 250 mosmol/kg to 350 mosmol/kg, more preferably from 275 mosmol/kg to 325 mosmol/kg, more preferably from 285 mosmol/kg to 315 mosmol/kg, more preferably from 290 mosmol/kg to 310 mosmol/kg, more preferably from 295 mosmol/kg to 305 mosmol/kg and most preferably is 300 mosmol/kg.

Gadolinium complexes such as those used in magnetic resonance imaging (MRI) substantially enhance the staining of dyes in ophthalmic staining from barely discernable to evident and strong. This phenomenon allows for a reduction in dye concentration which is associated with a reduction in risk for the patient. In addition, it was demonstrated that gadolinium complexes such as those used in MRI increase the density of composition suitable for staining an ophthalmic structure such that excellent sinking is observed. For the same increase in density, the gadolinium complexes contribute less to osmotic pressure than the sugars, sugar alcohols and PEG that are routinely used in the art.

Preferably, in a composition according to the invention comprising a Gadolinium complex, the concentration of the Gadolinium complex is within the range of about 0.01 M to about 0.1 M, preferably within the range of about 0.01 M to about 0.05 M, more preferably in the range of about 0.02 M to about 0.04 M. More preferably, in such composition, the concentration of the Gadolinium complex is within the range of 0.01 M to 0.1 M, preferably within the range of 0.01 M to 0.05 M, more preferably in the range of 0.02 M to 0.04 M.

Preferably, the gadolinium complex is a complex with limited or no toxicity such as gadopentetate (CAS number 80529-93-7, gadoterate (CAS number 72573-82-1, gadodiamide (CAS number 122795-43-1, gadoteridol (CAS number 120066-54-8, gadoversetamide (CAS number 131069-91-5, gadobenate (CAS number 113662-23-0, gadobutrol (CAS number 138071-82-6, gadoxetate (CAS number 135326-11-3, gadofosveset (CAS number 193901-90-5, preferably gadopentetate, gadobutrol and gadoterate, more preferably gadopentetate.

A compositions according to the invention can conveniently be provided in a kit.

Accordingly, the invention provides for a kit of parts comprising a composition according to the invention, wherein the kit comprises the compounds within a single composition or comprises the compounds in multiple compositions. The kit may further comprise containers, instructions for use and the like.

A composition according to the invention can, as described earlier herein, conveniently be used, preferably as a surgical adjuvant, in ophthalmic surgeries defined previously herein.

Accordingly, there is provided the use of a composition according to the invention or a kit of parts according to the invention, as a surgical adjuvant, preferably in ophthalmic surgery

In addition, there is provided a method of treatment comprising ophthalmic surgery comprising staining of an ophthalmic structure using a composition or kit according to the invention.

In addition, there is provided the use of a composition or kit according to the invention for the preparation of a medicament, preferably a medicament for ophthalmic surgery, said surgery preferably comprising staining of an ophthalmic structure.

In addition, there is provided the use of a composition or a kit according to the invention for use as a medicament, preferably for use in a method for ophthalmic surgery, preferably comprising staining of an ophthalmic structure. In addition, there is provided a method for staining an ophthalmic structure, preferably an ophthalmic membrane using a composition or kit according to the invention.

In the embodiments of the invention, the ophthalmic structure is preferably an ophthalmic membrane, more preferably the internal limiting membrane (ILM) and/or the epiretinal membrane (ERM) and/or the anterior cavity. Other preferred ophthalmic structures are selected from the group consisting of the anterior lens capsule, the conjunctiva, the anterior segment, Descemet's membrane and endothelium, and the anterior vitreous. Preferred surgical procedures are selected from the group consisting of cataract surgery, refractive surgery, corneal surgery, vitreo -retinal surgery, and conjunctival surgery, deep lamellar keratoplasty and keratoplasty.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less about 10% of the value (a range from 9 to 11).

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

FIGURE LEGENDS

Figure 1. Retinas stained with various dye compositions (see examples for further details).

lA: 0.1% Dye l

IB: 0.05% Dye 1 and 0.075% Trypan Blue

1C: 0.05% Dye 1 and 0.15% Trypan Blue

ID: 0.1% Dye 1 and 0.15% Trypan Blue

IE: 0.1% Dye 2

IF: 0.1% Dye 3

1G: 0.15% Trypan Blue

Figure 2. Absorbance spectrum of a retina stained with Dye 1 (0.1%) (see examples for further details).

Figure 3. Toxicity data (see examples for further details)

Percent survival ± Standard Deviation of ARPE cells exposed for 30 minutes to Trypan Blue 0.15% (TB 0.15), Dye 1 0.1% (Dyel 0.1), Trypan Blue 0.15% and Dye 1 0.1% (TB 0.15 Dyel 0.1), Janus Green 0.1%>, Methyl Green 0.1%>. The cells were exposed in dark, washed with PBS, and where indicated exposed to intense light for 15 minutes, and assayed either immediately after exposure or following a recovery period of 24 hours. The survival of cells exposed to PBS only was set to 100%. Asterisks indicate survival values significantly lower (p < 0.05) than Trypan Blue 0.15%.

EXAMPLES

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Preparation of stain solutions

Stock solutions of the dyes were prepared by dissolving the appropriate amount of the dye in commercially available phosphate-buffered saline solution (PBS). For a stock solution of 0.3%, 30 mg are dissolved in 10 mL PBS. This stock solution is either mixed with a stock solution of the other dye, or with PBS, in appropriate volumes. As an example, a solution of Dye 1 of 0.1% and of TB of 0.1% is prepared by mixing 1 mL of the stock solution of Dye 1 (having a concentration of 0.3%), 1 mL of the stock solution of TB (having a concentration of 0.3%), and 1 mL PBS.

For a dye with good sinking behavior, part of the PBS is replaced by a stock solution of diglycerol (or any other desired agent to obtain a density higher than that of PBS). A 15% stock solution of diglycerol is prepared by adding 85 mL of PBS to 15 g of diglycerol. For other agents, PBS is replaced in part by water to adjust its osmolarity to the physiological range of 300 mosmol/1. The expert in the field is familiar with such adjustments.

As an example, a solution containing 0.1 % dye 1, 0.1 % TB, and 5% diglycerol is prepared from equal volumes of the three stock solutions. If a lower concentration of dye 1 of 0.05%) is desired, 500 of the stock solution of dye 1 is taken, and then an additional 0.5 mL of PBS is added. Staining ability

The staining ability of exemplary compounds according to the invention on the membrana interna limitans (internal limiting membrane, ILM) was tested in pig eyes obtained from the local slaughterhouse. These were used within the first 6 hours after the enucleation. The eyes were kept on ice until use.

A compound according to Formula 1 with R' '=R' "=R' ' ' ' equal to H and R=R' equal to (CH₂)4S03H (depicted herein as "Dye 1") as Na salt was used in various concentrations and combinations (0.1% (w/v) Dyel; 0.05% (w/v) Dyel and 0.075% (w/v) Trypan Blue; 0.05%) (w/v) Dyel and 0.015%) (w/v) Trypan Blue, and as a control 0.15% (w/v) Trypan Blue. All solutions contained 5% diglycerol, in order to represent as closely as possible the actual clinical situation. A compound according to Formula 1 with R"=R"'=R""=R^V=R^VI equal to H and R=R' equal to (CH₂)₅C0₂H) (depicted herein as "Dye 2") as Na salt was used in 0.1% (w/v). A compound according to Formula 1 with R"=R"'=R""=R^V=R^VI equal to H and R = (CH₂)₄S0₃H and R' = (CH₂)₅C0₂H) (depicted herein as "Dye 3") as Na salt was used in 0.1% (w/v).

Following removal of the anterior segment and the vitreous, the retina was rinsed three times with phosphate buffered saline (PBS), and a section of the retina was enclosed with a glass tube of 1 cm diameter. The volume outside the glass tube was filled with PBS, and 500 mL of the dye solution was pipetted into the glass tube. After 30 seconds, the glass tube was removed, and the eye cup was rinsed with PBS until the rinsing liquid was colorless. A 10-mm punch of the retina was taken, photographed, and the red channel of the photo was evaluated by densitometry. The stained area had an average density of 121 (on a scale from black to white from 0 to 255), whereas the non-stained area had an average density of 225. The results are depicted in Figures 1A-1G and in Table 1.

In comparison, the staining which can be achieved with trypan blue in the red channel (which gives the highest contrast to the surgeon because of the color of the fundus) is much smaller.

Table 1: Staining results of the compound according to the invention (Dye 1) and Trypan Blue (TB)

The improvement of compounds according to the invention with or without Trypan Blue is evident. Spectrum of Dye 1 on retina

A punch of 4 mm in diameter was taken from a retina stained with Dye 1 (0.1% (w/v) in PBS, and an absorption spectrum was prepared using a spectrophotometer. The results are depicted in Figure 2. The absorption peak around 650 nm is clearly discernible.

Toxicity

Toxicity was evaluated with the WST-1 assay [7, 8] using ARPE-19 cells cultivated in a 96-well plate. Prior to the assay, the cells were exposed to Dye 1 (0.1 % Dye 1 in PBS; 0.1% Dye 1 + 0.15% TB in PBS) for 30 minutes at 37°C, and the dye solution was rinsed off with PBS. The WST-1 reagent was added either directly to the cells, or after a 15- minute exposure to a 300-W LED panel at a distance of 75 mm from the 96-well plate. The same experiment was repeated with a delay of 24 hours between exposure and addition of the WST-1 reagent. As a control, PBS was used under same conditions as for Dye. The results are depicted in Figure. The percentage survival was normalized against the control of PBS alone; i.e. survival of the cells exposed to the PBS control was set at 100%.

From Figure 3, it is evident that Dye 1 and its combination with TB at 0.15% is not toxic to ARPE-19 cells after 30 minutes exposure to the dye. Other green dyes, such as Janus Green or Methyl Green, are toxic to ARPE-19 cells either already in the dark, or after exposure to light.

Summary

We have demonstrated that a new class of dyes for ocular surgery has been found, which offers the following advantages over previously described dyes: higher extinction coefficient; higher staining ability; staining in green; even higher staining ability in combination with other dyes.

The dye is stable in solution, and it shows no toxicity against cells even after exposure to the dye for 30 minutes and exposure to high- intensity light for 15 minutes. REFERENCE LIST

1. Jhanji V, Chan E, Das S, Zhang H, Vajpayee RB (2011) Trypan blue dye for anterior segment surgeries. Eye (Lond) 25: 1113-1120

2. Semeraro F, Morescalchi F, Duse S, Gambicorti E, Russo A, Costagliola C (2015) Current Trends about Inner Limiting Membrane Peeling in Surgery for Epiretinal

Membranes. J Ophthalmol 2015: Article ID 671905

3. Yamamoto N, Ozaki N, Murakami K (2004) Double Visualization Using Triamcinolone Acetonide and Trypan Blue during Stage 3 Macular Hole Surgery. Ophthalmologica 218: 297-305

4. Veckeneer M, Mohr A, Alharthi E, Azad R, Bashshur ZF, Bertelli E, Bejjani RA, Bouassida B, Bourla D, Crespo IC, Fahed C, Fayyad F, Mura M, Nawrocki J, Rivett K, Scharioth GB, Shkvorchenko DO, Szurman P, Van Wijck H, Wong IY, Wong DS, Frank J, Oellerich S, Bruinsma M, Melles GR (2013) Novel 'heavy' dyes for retinal membrane staining during macular surgery: multicenter clinical assessment. Acta Ophthalmol 10.111 l/aos.12208

5. Henrich PB, Priglinger SG, Haritoglou C, Schumann RG, Strauss RW, Schneider U, Josifova T, Cattin PC (2013) Quantification of contrast recognizability in sequential epiretinal membrane removal and internal limiting membrane peeling in Trypan Blue- assisted macular surgery. Retina 33: 818-824

6. Awad D, Schrader I, Bartok M, Mohr A, Gabel D (2011) Comparative toxicology of trypan blue, brilliant blue G, and their combination together with polyethylene glycol on human pigment epithelial cells. Invest Ophthalmol Vis Sci 52: 4085-4090

7. Awad, D., I. Schrader, M. Bartok, N. Sudumbrekar, A. Mohr and D. Gabel (2013). "Brilliant Blue G as protective agent against trypan blue toxicity in human retinal pigment epithelial cells in vitro." Graefes Arch Clin Exp Ophthalmol 251 : 1735-1740.

8. Gerding, H. (2016). "Acid Violet 17: a New Dye for Chromo vitrectomy?" Klin Monbl Augenheilkd 233: 460-464.

9. Mohr, A., M. Bruinsma, S. Oellerich, H. Frank, D. Gabel and G. R. Melles (2013). "Dyes for eyes: hydrodynamics, biocompatibility and efficacy of 'heavy' (dual) dyes for chromo vitrectomy." Ophthalmologica 230 Suppl 2: 51-58.

10. Rodrigues, E. B., F. M. Penha, E. de Paula Fiod Costa, M. Maia, E. Dib, M. J. Moraes, C. H. Meyer, O. J. Magalhaes, G. B. Melo, V. Stefano, A. B. Dias and M. E. Farah (2010). "Ability of new vital dyes to stain intraocular membranes and tissues in ocular surgery." Am J Ophthalmol 149: 265-277.

. Steel, D. H. W., A. A. Karimi and K. White (2015). "An evaluation of two heavier- than- water internal limiting membrane-specific dyes during macular hole surgery." Graefes Arch Clin Exp Ophthalmol: 1-7.

. Veckeneer, M., A. Mohr, E. Alharthi, R. Azad, Z. F. Bashshur, E. Bertelli, R. A. Bejjani, B. Bouassida, D. Bourla, I. C. Crespo, C. Fahed, F. Fayyad, M. Mura, J. Nawrocki, K. Rivett, G. B. Scharioth, D. O. Shkvorchenko, P. Szurman, H. Van Wijck, I. Y. Wong, D. S. H. Wong, J. Frank, S. Oellerich, M. Bruinsma and G. R. J. Melles (2014). "Novel 'heavy' dyes for retinal membrane staining during macular surgery: multicenter clinical assessment." Acta Ophthalmologica 92: 339-344.

Claims

1. A composition suitable for staining an ophthalmic structure comprising a compound according to Formula I:

, or a pharmaceutically acceptable salt or hydrate thereof, wherein:

b. R' is selected from the group consisting of: H; CH₃; (CH₂)_nH, wherein n is an integer from 1 - 6; (CH₂)_nA, wherein n is an integer from 1 - 6 and A is selected from C0₂H, S0₃H, OH, NH₂, and a functional derivative of C0₂H, such as an ester,an amide or a hydrazide;

c. R" is selected from the group consisting of: H and S0₃H;

d. R" ' is selected from the group consisting of: H and S0₃H;

e. R"" is selected from the group consisting of: H; (CH₂)_nH, wherein n is an integer from 1 - 6; Br; CI; I; (CH₂)_nCHO and (CH₂)_nC0₂H, wherein n is an integer from 0 - 6; and a functional derivative of C0₂H, such as an ester or an amide or a hydrazide;

h. wherein each of the double bonds can be a Z or and E stereoisomer.

2. A composition according to claim 1, wherein:

a. R = CH₃ and R' = (CH₂)₅C0₂H and R" = R' " = R" " = R^v = R^VI = H; b. R = (CH₂)₄C0₂H and R' = (CH₂)₅C0₂H and R" = R'" = R"" = R^v = R^VI = H; or, c. R = R' = (CH₂)₄S0₃H and R" = R" ' = R" " = R^V=R^VI= H.

A composition according to claim 1 or 2, wherein the concentration of the compound according to Formula I, or a pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.005% (w/v) to about 1% (w/v).

A composition according to claim 1 or 2, wherein the concentration of the compound according to Formula I, or a pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.025% (w/v) to about 0.1 %> (w/v), more preferably within the range of about 0.05%> (w/v) to about 0.1 %> (w/v).

A composition according to any one of claims 1 - 4, further comprising a dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue and their respective pharmaceutically acceptable salts and hydrates thereof.

A composition according to any one of claims 1 - 5, wherein the concentration of the dye selected from the group consisting of Trypan Blue, Janus Green, Methyl Green, Methylene Blue, Crystal Violet, Methyl Violet, Ethyl Violet, Evans Blue, Methyl Blue or a respective pharmaceutically acceptable salt or hydrate thereof, is within the range of about 0.005%) to about 0.5%> (w/v).

7. A composition according to any one of claims 1 - 6, further comprising a perfluorocarbon liquid, preferably a perfluorocarbon liquid selected from the group consisting of perfluorooctane, perfluoroperhydrophenanthrene, perfluorodecalin, perfluorotributylamide and perfluorooctylbromide, more preferably perfluorooctane or perfluorodecalin.

8. A composition according to any one of claims 1 - 6, further comprising an agent that increases the density and/or viscosity of the composition, such that the density and/or viscosity of the composition is increased compared to the density and/or viscosity of H₂0.

9. A composition according to claim 8, wherein the agent is selected from the group consisting of: heavy water, diglycerol, triglycerol, a Gadolinium complex, a sugar, a sugar alcohol, a polymer and a polysaccharide.

10. A composition according to claim 8, wherein the agent is selected from the group consisting of: diglycerol, triglycerol and a Gadolinium complex.

11. A composition according to claim 10, wherein the total concentration of the diglycerol and/or triglycerol, if present, is within the range of about 0.1% to about 25% (v/v), preferably within the range of about 2% to about 20%> (v/v), more preferably within the range of about 3% to about 6% (v/v), or wherein the concentration of a Gadolinium complex is within the range of about 0.01 M to about 0.1 M, preferably within the range of about 0.01 M to about 0.05 M, more preferably in the range of about 0.02 M to about 0.04 M.

12. A kit of parts comprising a composition according to any of claims 1 - 11, wherein the kit comprises the compounds within a single composition or comprises the compounds in multiple compositions.

13. A composition according to any one of claims 1 - 11, or a kit of parts according to claim 12, for use as a medicament, preferably for use in a method of ophthalmic surgery.

14. A composition for use according to claim 13, wherein the ophthalmic surgery comprises staining of an ophthalmic structure.

15. A composition for use according to claim 12, 13 or 14, wherein the ophthalmic structure is the internal limiting membrane (ILM) and/or the epiretinal membrane (ERM) and/or the anterior cavity.

16. Use of a composition according to any one of claims 1 - 11, or a kit of parts according to claim 12, as a surgical adjuvant, preferably in ophthalmic surgery.