OPHTHALMIC DYE

Abstract

The present invention relates to the field of medicine, specifically to ophthalmic surgery, more specifically to a novel ophthalmic dye for surgery which does not stain living cells.

Claims

1. A compound represented by structure (A), or a pharmaceutically acceptable salt or hydrate thereof, ##STR00003## wherein X is O or NH; wherein R.sup.1 and R.sup.2 are each independently a C.sub.1-8 hydrocarbon or a substituted C.sub.1-8 hydrocarbon; wherein R.sup.3 is selected from the group consisting of hydrogen, methyl, a substituted methyl, ethyl and a substituted ethyl; and wherein at least one of R.sup.1 and R.sup.2 is a C.sub.3-8 hydrocarbon or a substituted C.sub.3-8 hydrocarbon and/or wherein at least one of R.sup.1 and R.sup.2 is methyl or a substituted methyl.

2. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are each independently a C.sub.1-8 hydrocarbon, and wherein R.sup.3 is selected from the group consisting of hydrogen, methyl and ethyl.

3. The compound according to claim 1, wherein C.sub.1-8 hydrocarbon is selected from the group consisting of C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 alkenyl, C.sub.2-8 alkynyl, phenyl, and benzyl; and/or wherein C.sub.3-8 hydrocarbon is selected from the group consisting of C.sub.3-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.3-8 alkenyl, C.sub.3-8 alkynyl, phenyl, and benzyl.

4. The compound according to claim 1, wherein C.sub.1-8 hydrocarbon is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, phenyl, and benzyl, preferably from the group consisting of methyl, ethyl, n-propyl, i-propyl, phenyl, and benzyl; wherein C.sub.3-8 hydrocarbon is selected from the group consisting of n-propyl, i-propyl, n-butyl, phenyl, and benzyl, preferably from the group consisting of n-propyl, i-propyl, phenyl, and benzyl.

5. The compound according to claim 1, wherein R.sup.3 is H.

6. The compound according to claim 1, wherein X is O.

7. The compound according to claim 1, wherein R.sup.2 is identical for each instance.

8. The compound according to claim 1, wherein X is NH, R.sup.1 is phenyl, each R.sup.2 is ethyl, and R.sup.3 is H; or wherein X is O, R.sup.1 is benzyl, each R.sup.2 is ethyl, and R.sup.3 is H; or wherein X is O, R.sup.1 is methyl, each R.sup.2 is ethyl, and R.sup.3 is H; or wherein X is O, R.sup.1 is n-propyl, each R.sup.2 is ethyl, and R.sup.3 is H.

9. A composition comprising a compound according to claim 1, and a pharmaceutically acceptable excipient.

10. The composition according to claim 9, wherein said composition further comprises diglycerol and/or a dye, preferably wherein said dye is trypan blue.

11-15. (canceled)

16. A method for ophthalmic surgery comprising staining an ophthalmic structure with the compound of claim 1.

17. The method according to claim 16, wherein said staining is obtained by contacting the compound according to claim 1 with a vitreoretinal interface of a subject.

18. The method according to claim 16, wherein said compound is does not stain living cells.

19. The method according to claim 16, wherein said ophthalmic structure is the internal limiting membrane (ILM) and/or the epiretinal membrane (ERM) and/or within the anterior cavity.

Description

FIG. LEGENDS

[0119] FIG. 1. Correlation of staining of human ILM (data from [8]) and PVDF filter (own data). BB: Bromophenol Blue; BBG: Brilliant Blue G; EB: Evans Blue; FG: Fast Green; ICG: Indocyanine Green; LGSF: Light Green SF; PB: Patent Blue; TB: Trypan Blue

[0120] FIG. 2. Staining of PVDF filter by BBG and by three of the dyes according to the invention. The concentrations of the dyes are given in % (weight/volume).

[0121] FIG. 3. Photograph with stained cells pointed out by arrows (a), photograph with 485 nm/535 nm for calcein (b), photograph with 530 nm/620 nm for propidium iodide (c).

EXAMPLES

[0122] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Example 1

Preparation of Compounds (1) and (2)

[0123] N-Methyl-m-toluidine (17.65 g) was reacted with benzyl bromide (18 mL) in 150 mL acetonitrile and 30 g K.sub.2CO.sub.3for 24 h. The reaction solution was freed from the solids by filtration and mixed with 200 mL ethyl acetate. Water (100 mL) was added, and the organic phase was separated and washed with water. The solvent was removed by evaporation, yielding 27.93 g of N-benzyl-N-methyl-m-toluidine.

[0124] The product was dissolved in 120 mL concentrated sulfuric acid, and 17 mL fuming sulfuric acid (60% SO.sub.3) was added at 0° C. The mixture was heated to 100° C. overnight, and then poured on ice. After neutralization with NaOH, solids were removed by filtration, and water was removed by lyophilization. The residue was extracted with 200 mL methanol to yield 3-((methyl(m-tolyl)amino)methyl)benzenesulfonic acid.

[0125] The sulfonic acid (9.54 g) was condensed with p-chlorobenzaldehyde (2.34 g) in a mixture of water (180 mL) and ethanol (60 mL), and the pH was adjusted to 0.7 with concentrated HCl. The reaction was heated to 120° C. for 3 days.

[0126] After cooling, 1.07 g K.sub.2Cr.sub.2O.sub.7 (dissolved in 10 mL of water) was added to the reaction flask, and then 1.79 g oxalic acid (dissolved in 20 mL of water). After 1 h, 25 g of Na.sub.2SO.sub.4 was added. The aqueous layer was mixed with 200 mL butanol, the butanol phase was separated, and the solvent was removed by rotary evaporation.

[0127] A part (0.60 g) of the green dye obtained was used without further purification for condensation with 4-iosopropoxyaniline (500 μL) in 20 mL butanol and refluxed for 24 h. After cooling, the solution was poured into 200 mL diethyl ether, the solid was recovered by centrifugation, and subjected to column chromatography on silica gel with a gradient of dichloromethane and methanol. The dye-containing fractions were pooled.

[0128] The synthesis described in this example yields compound (1). The compound (2) was obtained in the same way, using p-anisidine in the last step.

Example 2

Preparation of Compounds (3) to (8)

[0129] In a round-bottom flask with stirrer and reflux condensor, 3.0 g (0.01 mol) 3-((ethyl(m-tolyl)amino)methyl)benzenesulfonic acid and 4-chlorobenzaldehyde (0.005 mol, 0.7 g) were 40 dissolved in a mixture of 50 mL water and 20 mL ethanol. The pH value was adjusted to pH=1.7 with HCl. The reaction mixture was refluxed for 72 hours. The solvent was removed by rotary evaporation, and the residue was dissolved in 30 mL acetonitrile.

[0130] Ce(IV)(NH.sub.4).sub.2(NO.sub.3).sub.6 (2.3 g, dissolved in 10 mL of a KCl/HCl solution adjusted to pH =1.8) was 5 added under stirring. After 1 hour, the dye was extracted into 100 mL 1-butanol and the solvent was removed by rotary evaporation.

[0131] The raw product was dissolved in 50 mL 1-butanol in a round-bottom flask equipped with stirrer and reflux condensor. 4-Isopropoxyaniline (0.01 mol =1.35 mL) was added, and the solution was refluxed for 18 hours. After cooling to room temperature, the mixture was poured into 500 mL diethylether, and the precipitate was collected by centrifugation. It was chromatographed on silica gel with a linear gradient of dichloromethane and methanol. The fractions containing the blue dye were pooled.

[0132] 15 The synthesis described in this example yields compound (3). The compounds (4), (5), (6), (7), and (8) were obtained in the same way, using p-propyloxyaniline, p-benzyloxyaniline, p-anisidine, p-phenoxyaniline, and 4-aminodiphenylamine, respectively.

Example 3

Assessment of Staining of Dead Cells and/or of Living Cells

[0133] ARPE19 cells (a human retinal pigment epithelial cell line) were grown in 96-well plates. In half of the wells, cell death was induced by fixation with 70% ethanol for 5 minutes.

[0134] Assessing staining of living cells and of dead cells with dyes was carried out with solutions of the dyes (all in a concentration 0.1% weight per volume, except where indicated differently) in PBS (ICG was dissolved in water at 0.5% and diluted to 0.1% with 5% glucose solution). The duration of staining of the cells was 15 minutes. After the incubation, the wells were washed with PBS. A mixture of DMSO (90%) and PBS (10%) was used to lyse the cells and solubilize the dyes.

[0135] The dye content in the ARPE19 cells as measured by spectrophotometry was used as a measure for (visual) staining. A complete spectrum from 400 to 1000 nm was recorded. The absorbance from cells only was subtracted, and the minimum absorbance was set to 0. The maximum absorbance for dead cells (A.sub.dead) and for living cells (A.sub.living) was recorded, and interpreted as measures for the staining of dead and living cells, respectively.

[0136] Absorbances below 0.07 were considered too weak to assume that staining can be visualized. Hence, dyes for which A.sub.living<0.07 can be considered as dyes which do not stain living cells.

TABLE-US-00002 TABLE 2 Staining of living cells and dead cells by dyes represented by structure (A) Compound X R.sup.1 R.sup.2 R.sup.3 A.sub.living A.sub.dead (1) O iPr Me H 0.0283 0.3481 (2) O Bn Me H 0.0253 0.2454 (3) O iPr Et H 0.0440 0.6387 (4) O nPr Et H 0.0485 0.8084 (5) O Bn Et H 0.0327 0.6734 (6) O Me Et H 0.0603 0.8755 (6)* O Me Et H 0.0635 0.3630 (7)* O Ph Et H 0.0216 0.1976 (8) NH Ph Et H 0.0629 0.8411 BBG O Et Et H 0.0328 0.5651 MBBG O Et Et Me 0.0355 0.6876 EBBG O Et Et Et 0.0724 0.9066 PBBG O Et Et Pr 0.1331 0.7369 BBBG O Et Et Bu 0.1491 0.7941 *Concentration of the dye: 0.025%

TABLE-US-00003 TABLE 3 Staining of living cells and of dead cells by various dyes Compound A.sub.living A.sub.dead Acid Violet 0.0184 0.2077 Aniline Blue 0.0080 0.0057 BBR1 0.0273 0.1467 Brilliant Green 0.1088 0.0568 Bromophenol Blue 0.0059 0.0357 Crystal Violet 0.5797 0.4365 Eosin 0.0362 0.2358 ICG 0.0703 1.1751 Light Green SF 0.0065 0.0080 Malachite Green 0.0058 0.0068 Methyl Green 0.0489 0.0750 Methyl Violet 1.2503 0.8900 Patent Blue 1 0.0155 0.0113 Trypan Blue 0.0135 0.0847

[0137] All compounds in Table 2 may be represented by Structure (A), and may be referred to as BBG derivatives herein. Compounds (1)-(8) are compounds according to the invention. BBG, EBBG, PBBG and BBBG are compounds known in the art.

[0138] Clinically used BBG does not stain livings cells (A.sub.living=0.0328). The staining of dead cells can be increased by substituting R.sup.3 by longer alkyl groups in EBBG (R.sup.3=Et, A.sub.dead=0.9066), PBBG (R.sup.3=Pr, A.sub.dead=0.7369) and BBBG (R.sup.3=0.7941, A.sub.dead=0.7941). However, in the search for a BBG derivative which have an increased ability to stain the ILM, this substitution pattern comes at a cost. The staining of living cells increases with increasing size of the R.sup.3 group (A.sub.living=0.0724 for EBBG; 0.1331 for PBBG; 0.1491 for BBBG). Hence, the art suggests that increasing the number of carbon atoms comprised in R.sup.1, R.sup.2 or R.sup.3 does not yield compounds which do no stain living cells.

[0139] The current invention unexpectedly provides a new set of compounds, exemplified but not limited 10 to (1)-(8), wherein the number of which compounds have increased ability for staining the ILM and which do not stain living cells. Indeed, for all (1)-(8) A.sub.living<0.07. The structural difference between the BBG derivatives known in the art (BBG, EBBG, PBBG, BBBG) and the compounds according to the inventions is the variation of R.sup.1 and R.sup.2 relative to BBG in (1)-(8) instead of the variation in R.sup.3 in BBG, EBBG, PBBG, BBBG.

[0140] As explained, the art seems to suggest that increasing the number of carbon atoms in R.sup.1, R.sup.2 and R.sup.3 would lead to an increased staining of living cells. Interestingly, the compounds according to the invention wherein one of R.sup.1 and R.sup.2 has more than two carbon atoms and the other one has exactly two carbon atoms ((3), (4), (5), (7), (8)), have a high staining ability. In other words, the compounds wherein R.sup.1 or R.sup.2 has been elongated with respect to the ethyl group in BBG, show a high staining but do not stain living cells. This set of compounds is a preferred subset of the compounds according to the invention.

[0141] All non-BBG dyes tested and listed in Table 3 show either staining of living cells with A.sub.living>0.07 (Brilliant Green, Crystal Violet, ICG, Methyl Violet), or are characterized by a relatively low ability of staining ophthalmic structures.

[0142] In conclusion, using the dye content in the ARPE19 cells as measured by spectrophotometry as do not stain living cells.

Example 4

Staining by Compound (6)

[0143] An experiment was carried out with ARPE-19 cells and staining with compound (6). The aim of the experiment was to confirm that compound (6) does not stain living cells.

[0144] A three-step staining procedure was used: first the dye was introduced, then calcein-acetoxymethyl ester (calcein-AM), then propidium iodide (PI). Calcein-AM (which is non-fluorescent) is able to penetrate cell membranes and is hydrolyzed by intracellular esterases to calcein, which is fluorescent and which cannot escape through the intact cell membrane. Cells fluorescent for calcein-AM are therefore intact and living cells. Dead cells (cells with a compromised cell membrane) might still hydrolyze calcein-AM, but they are not able to retain the calcein. Cells with compromised cell membranes can take up propidium iodide, a dye which intercalates into DNA and turns fluorescent upon binding.

[0145] Cells were grown in 48-well plates. Growth medium was removed and a solution of 20% ethanol was added for 5 minutes in order to induce cell death in some cells. The ethanol solutions were removed. Staining with compound (6) (0.025%) in PBS was carried out at room temperature for 15 minutes. The dye was washed off with PBS. The cells were then exposed to calcein-acetoxymethyl ester (calcein-AM) in PBS for 15 minutes at room temperature. After a wash with PBS, the cells were then exposed to PI for 5 minutes, and microphotographs were taken with a black-and-white camera, using a halogen lamp for illumination for the picture showing the stain (FIG. 3(a)), and with a xenon lamp and appropriate filter combinations for the two fluorescent dyes (FIGS. 3(a) and 3(b)).

[0146] The picture with the stain was overlayed with arrows pointing toward the stained cells, and the overlay was then (with maintained x-y coordinates) overlayed on the fluorescent pictures.

[0147] From FIG. 3 it clearly follows that compound (6) is does not stain living cells.

Example 5

Staining Ability

[0148] A PVDF filter has been found to represent the best non-physiological model of the ILM in patients. Correlation between staining of the filter and observed staining of the ILM in patients (data from (Rodrigues, Penha et al. 2010)) show a good correlation (see FIG. 1).

[0149] A 96-well filter (MSBVN1210 MultiScreen BV Filter Plate, Merck-Millipore, Darmstadt, Germany) was used as ILM model. The filter material is hydrophilic polyvinylidene difluoride (PVDF). A Collagen Cell Carrier membrane (CCC) (Viscofan, Weinheim, Germany) was used as model for the ERM.

[0150] Punches of the membrane (5 mm diameter) were fixed to the filter with a hypodermic needle and soaked in PBS for 30 minutes. The PBS was removed by vacuum filtration, and 50 μL of the dye solution was pipetted into the filter well. After 30 s, the dye was removed by vacuum filtration, and membrane and filter were washed three times with PBS. The membrane was removed and put into a 96-well plate for spectrum recording, using a Tecan Spark 20M (Tecan Group Ltd., Männedorf, Switzerland) plate reader.

[0151] The filter staining of the compounds (6) and (8) according to the invention are shown in FIG. 2 in comparison to that of BBG. Much stronger staining is observed for compound (6) than for BBG for equal concentrations of 0.025%, and even stronger staining is observed with compound (8) at 0.005%. The intensity of the grey values was measured. For BBG the mean intensity was 201, for compound (6) it was 175, for compound (8) it was 197. On the scale used, 255 is white and 0 is black.

Example 6

Toxicity

[0152] The dyes were tested for toxicity as described in the literature [9] at concentrations which stain the filter at an intensity comparable to or surpassing the staining achieved with BBG at 0.025% (the concentration used clinically for this dye). The results are listed in Table 4.

TABLE-US-00004 TABLE 4 The survival of the cells after exposure to dyes is given in percent Compound Concentration (%) Survival (%) BBG 0.025 99 (5) 0.025 81 (6) 0.025 103 (7) 0.025 81 (8) 0.025 79 BBG 0.025 99 EBBG 0.025 93 PBBG 0.005 66 BBBG 0.005 67

REFERENCE LIST

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