DYE MOLECULE AND DYE PREPARATIONS, IN PARTICULAR FOR USE IN SURGICAL METHODS OF OPHTHALMIC SURGERY AND FOR DYEING PROTEINS

Abstract

A dye molecule having the structure (I): where R.sub.1 is an SO.sub.3.sup. group bound to a hydrogen H atom or to another atom or to an ammonium NH4 group or to a lysine salt or to an arginine salt or to a different monovalent cation, and R.sub.2 is an SO.sub.3.sup. group, in particular for making dye preparations for use in methods for the treatment of the bodies of humans or animals, for example for dyeing the internal limiting membrane ILM and/or epiretinal membranes EPRM in a surgical method which involves subsequent removal respectively of the ILM and/or of the EPRM; as well as a dye preparation having a density higher than 1.01 g/cm.sup.3 and comprising at least one dye and at least one agent for increasing its density which is a polymer with the empirical formula (C.sub.12H.sub.22O.sub.11.C.sub.3H.sub.5ClO).sub.n, and structure (II).

Claims

1. A dye molecule having the structure: ##STR00003## where: R.sub.1 is constituted of an SO.sub.3.sup. group bound with an ionic bond to a hydrogen H atom or to another atom or to an ammonium NH.sub.4 group or to a lysine salt or to an arginine salt or to a different monovalent cation; and R.sub.2 is constituted of an SO.sub.3.sup. group.

2. A dye preparation comprising at least one first dye with the dye molecule according to claim 1, or a pharmaceutically acceptable salt of the dye molecule or a hydrate of the dye molecule.

3. The dye preparation according to claim 2, characterised in that the first dye is present in a quantity, by weight relative to the total volume of the preparation (w/v), of between 0.0001% and 0.5%.

4. The dye preparation according to claim 2, also comprising a second dye which is different to the first dye.

5. The dye preparation according to claim 4, wherein the second dye is trypan blue.

6. The dye preparation according to claim 2, characterised in that it has a density of between 1.01 g/cm.sup.3 and 1.5 g/cm.sup.3 and in that it also comprises at least one agent for increasing the density.

7. The dye preparation according to claim 6, characterised in that the at least one agent for increasing the density is selected from the group consisting of: heavy water D.sub.2O, monosaccharides, disaccharides, polysaccharides, neutral polymers such as polyethers, polyvinyl alcohol, polyesters, polyacrylic acid copolymers, mannitol and polyvinyl pyrrolidone.

8. The dye preparation according to claim 6, characterised in that the agent for increasing its density is a polymer with the empirical formula (C.sub.12H.sub.22O.sub.11.C.sub.3H.sub.5ClO)n, and the structure ##STR00004##

9. A dye preparation having a density greater than 1.01 g/cm.sup.3 and comprising at least one dye and at least one agent for increasing its density that is a polymer with the empirical formula (C.sub.12H.sub.22O.sub.11.C.sub.3H.sub.5ClO)x, and the structure ##STR00005##

10. The dye preparation according to claim 9, comprising at least one dye selected from the group consisting of triphenylmethane dyes, azo dyes, cyanine dyes, natural dyes and/or mixtures of them.

11. The dye preparation according to claim 10, wherein the dye is Brilliant Blue G (BBG) or a structural analogue of it.

12. The dye preparation according to claim 10, wherein the dye is selected from the group consisting of BBG, a pharmaceutically acceptable salt of BBG, a hydrate of BBG and a methylate of BBG.

13. The dye preparation according to claim 8, wherein the agent for increasing its density is present in a quantity, by weight relative to the total volume of the preparation (w/v), of between 0.001% and 20%.

14. The dye preparation according to claim 2, characterised in that it also comprises a phosphate buffered matrix.

15. The dye preparation according to claim 2, characterised in that it is also liquid at least in the temperature range between 0 C. and 50 C.

16-32. (canceled)

33. A method for dyeing a protein, comprising the following steps: providing a dye preparation according to claim 2; applying an effective amount of the dye preparation to the protein.

34. A method for making a tissue more visible, comprising the following steps: providing a dye preparation according to claim 2; applying an effective amount of the dye preparation to the tissue and so dyeing proteins which belong to the tissue.

35. A method for the treatment of a human or animal body, comprising the following steps: providing a dye preparation according to claim 2; applying an effective amount of the dye preparation to the human or animal body.

36. The method according to claim 35 wherein the method is a vitreoretinal surgery method.

37. The method according to claim 36 wherein the vitreoretinal surgery method comprises the following steps: applying the effective amount of the dye preparation to an internal limiting membrane ILM and/or epiretinal membranes EPRM of an eye; removing respectively the ILM and/or the EPRM.

38. The method according to claim 36 wherein the step of providing the dye preparation comprises providing a dye preparation comprising both the first dye and a second dye which is different to the first dye; and wherein the vitreoretinal surgery method comprises the following steps: applying an effective amount of the dye preparation to an internal limiting membrane ILM and epiretinal membranes EPRM of an eye, with the first dye dyeing at least the ILM and the second dye dyeing the epiretinal membranes EPRM, and subsequently removing the ILM and the EPRM.

39. A method for the treatment of a human or animal body, comprising the following steps: providing a dye preparation according to claim 9; applying an effective amount of the dye preparation to the human or animal body.

40. The method according to claim 39 wherein the method is a vitreoretinal surgery method.

41. The method according to claim 40 wherein the vitreoretinal surgery method comprises the following steps: applying an effective amount of the dye preparation to an internal limiting membrane ILM and/or epiretinal membranes EPRM of an eye; removing respectively the ILM and/or the EPRM.

Description

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0025] Regarding the dye molecule, in the context of this invention, a family of it was provided that is represented by the following structure (I):

##STR00001##

where R.sub.2 is constituted of an SO.sub.3.sup. group (which forms an inner salt with the nearby positively charged nitrogen N atom) and R.sub.1 is constituted of an SO.sub.3.sup. group bound with an ionic bond to a hydrogen H atom or to another atom (for example, sodium Na) or to an ammonium NH.sub.4 group or to a lysine salt or to an arginine salt or to a different monovalent cation.

[0026] As can be seen, the molecule disclosed according to this invention differs from a molecule of BBG (it should be noticed that in the context of this invention, that name is also intended to indicate any product identified by synonyms, such as Coomassie brilliant blue, Acid Blue 90, C. I. 42655 and Brilliant Blue G 250, all uniquely identified by CAS number: 6104-58-1) due to the presence of a methyl group rather than a hydrogen atom bound to the disubstituted amino nitrogen atom.

[0027] Synthesis of the new molecule disclosed may be achieved by means of a methylation reaction to the disubstituted amino nitrogen of a starting product constituted of Brilliant Blue G250. In particular, the reaction is particularly advantageous starting with commercial BBG which, as said, usually has a level of purity less than or equal to 90% and is contaminated with BBR. In fact, at the end of the methylation reaction and of the subsequent purification, the contamination with BBR has been eliminated. Generally speaking, the synthesis method involves making the starting product react in one or more successive steps with sodium hydroxide and methyl iodide, if necessary varying their proportions to one another each time.

Example

[0028] The following is a description of a preferred production example. Obviously, the quantities indicated could be varied, preserving the proportions between the various substances, it being understood that the process described leads to the production of approximately 1.6 g of molecule disclosed, and the production of significantly larger quantities could require some modifications to the process for its industrial optimisation.

Materials and Methods

Reagents

[0029]

TABLE-US-00001 Quantity for a batch Reagents Manufacturer of approx. 1.6 grams Brilliant Blue G-pure (BBG) (Sigma Aldrich, 2 g B0770) RP grade methanol (Sigma Aldrich) 50 ml Sodium hydroxide (Sigma Aldrich) 0.20 g Methyl iodide (Sigma Aldrich) 2.20 ml HPLC grade acetonitrile (Sigma Aldrich) 4 l Deionised water 10 l UV grade trifluoroacetic acid (Sigma Aldrich) 40 ml HPLC grade methanol (Sigma Aldrich) 4 l

[0030] As regards the starting BBG, in the tests carried out it was a product with 91% purity (Analysis certificate B0770, Sigma Aldrich, batch SLBJ8621V). Confirming that, FIG. 1 shows its mass spectrum which indicates at least two significant peaks.

Materials

[0031]

TABLE-US-00002 Quantity for a batch Materials of approx. 1.6 grams 250 ml glass flask 1 5 ml pipettes 2 500 ml, 1 l freeze-drying flasks 5 16*160 mm glass test tubes washed 300 in a laboratory washing machine 200 ul vials for HPLC autosampler 600

Instruments

[0032]

TABLE-US-00003 Instruments Manufacturer Medium Pressure Liquid Chromatography REVELERIS C18 WP 40 g column Grace Freeze Dryer Freeze Dryer Modulyo Edwards HPLC Shimadzu LC10 Magnetic stirrer Ika Rotary evaporator Buchi

Description of Reaction

[0033] 0.12 g of sodium hydroxide and 0.92 ml of methyl iodide are added to 1.7 g of BBG dissolved in methanol-water 1:1 v/v. The reaction is allowed to run for around two days at ambient temperature. The reaction mixture can then be checked using HPLC to highlight how the starting product is still present. The results of the HPLC analysis for the tests carried out are shown in FIG. 3 and highlight three peaks that correspond, from left to right, the first to the solvent used in the reaction, the second to the molecule of BBG and the third to the new molecule disclosed. At this point, another 0.92 ml of methyl iodide and 0.08 g of sodium hydroxide are added. After 7 days, another 0.36 ml of methyl iodide and 0.08 g of sodium hydroxide are added and the reaction is allowed to finish for another two days.

[0034] The reaction mixture is then concentrated in small volumes and the unrefined mixture is purified by means of Medium Pressure Liquid Chromatography (MPLC) using a REVELERIS C18 WP 40 g column (Grace) eluted with a mixture of water (A) and 90% v/v acetonitrile in water (B) both containing 0.05% v/v of trifluoroacetic acid.

[0035] After purification, the fractions containing the desired product are put together, concentrated in small volumes and freeze-dried. The solid dissolved in sodium hydroxide 0.01 is then purified using the same column with a mixture of water (A)methanol (B). MPLC conditions: flow rate 20 ml/min, detection 254 nm, gradient 40% (B) for 5 min, 40-60% (B) in 40 min, 60-95% (B) in 2 min, 95% (B) 2 min, 95-40% (B) 2 min.

[0036] FIG. 4 shows the result of the HPLC check on the purified and freeze-dried end product obtained in the tests carried out, and shows the presence of a single peak corresponding to the molecule disclosed (in the form of sodium salt). Confirming that, FIG. 2 shows the ESI-MS mass spectrum of the purified end product.

[0037] Also in the tests carried out, the purity of the end product was approximately 96.6% as calculated starting with the HPLC spectra, whilst the yield of the reaction was around 60% unrefined product (100 g of BBG-250 therefore yielded approximately 60 g of the molecule disclosed, in the form of sodium salt).

[0038] As indicated, this invention also relates to a dye preparation comprising at least one first dye whose molecule is the new dye molecule disclosed, or a pharmaceutically acceptable salt of it or a hydrate of it.

[0039] Preferably, the dye preparation is an aqueous solution and/or has a phosphate buffered matrix. Moreover, it is liquid at least in the temperature range between 0 C. and 50 C.

[0040] Moreover, at least for the uses in the context of methods for treatment of the bodies of humans or animals, the preparation is also advantageously sterile and biocompatible.

[0041] Advantageously, the first dye is present in a quantity, by weight relative to the total volume of the preparation (w/v), of between 0.0001% and 0.5%, preferably between 0.015% and 0.05%.

[0042] Moreover, in some preferred embodiments the dye preparation has a density of between 1.01 g/cm.sup.3 and 1.5 g/cm.sup.3 thanks to the additional presence of at least one agent for increasing its density.

[0043] The at least one agent for increasing the density may be selected according to requirements. For example, it may be selected in the group consisting of: heavy water D.sub.2O, monosaccharides, disaccharides, polysaccharides, and neutral polymers; amongst the neutral polymers in particular it may be selected from polyethers, polyvinyl alcohol, polyesters, polyacrylic acid copolymers, mannitol and polyvinyl pyrrolidone.

[0044] However, in a particularly preferred embodiment the agent for increasing the density is a polymer with the empirical formula (C12H22O11.C3H5ClO)n, and structure

##STR00002##

[0045] That agent is commercially known as Ficoll or Polysucrose (CAS number: 26873-85-8), and is advantageously present in a quantity, by weight relative to the total volume of the preparation (w/v), of between 0.001% and 20%, preferably between 0.1% and 10%. Hereinafter, when reference is made to Ficoll, the intention is to indicate that agent.

[0046] In a particularly preferred formulation the preparation comprises 0.05% (w/v) of the molecule disclosed and 4% (w/v) of Ficoll in a phosphate buffer.

[0047] Depending on the applications for which it is intended, the dye preparation may also comprise other substances, and in particular it may also comprise at least one second dye that is different to the first dye. In a preferred embodiment in which the dye preparation is intended to be used in the context of methods for treatment of the bodies of humans or animals, the second dye is advantageously trypan blue.

[0048] As regards the other properties of the dye preparation, such as dynamic viscosity, pH, osmolality, etc., in each case the expert in the field will be able to adapt them to the use of the dye preparation. For example, in the case of use of the dye preparation in the bodies of humans or animals, if necessary they can be adjusted in such a way that they are as close as possible to the physiological conditions.

[0049] As regards the possible uses of the dye preparation, one of those for which this invention is specifically intended is use in methods for treatment of the bodies of humans or animals.

[0050] Advantageously, in particular, the dye preparation is intended for use in a surgical method of vitreoretinal surgery.

[0051] Even more advantageously, the dye preparation is usable for dyeing the internal limiting membrane ILM and/or epiretinal membranes in a surgical method which involves subsequent removal of the ILM and/or of the EPRM. In fact, it has been seen that the dye molecule disclosed is able to selectively dye at least the ILM in such a way that during the step of detaching the ILM (peeling) and the EPRM, a clearly visible difference is created between the dyed ILM and the undyed underlying structures. To guarantee that all possible EPRM are also dyed, the dye preparation may also comprise a second dye, able to selectively dye them (such as trypan blue).

[0052] A further preferred use of the dye preparation disclosed is in a method in which it dyes proteins, for example to make a tissue to which the proteins belong more visible.

Experimental Data

[0053] The following are some experimental data obtained following tests and analyses carried out by the Applicant.

Cytotoxicity Analysis

[0054] The Applicant carried out various tests which highlighted how dye preparations according to this invention are not cytotoxic.

[0055] The following, by way of example, are the results obtained by the Eurofins

[0056] Biolab S.r.l. laboratories in Vimodrone (Milan province), Italy, for three different batches of a dye preparation comprising 0.05% (w/v) of a molecule made in accordance with this invention (with the group R.sub.1 constituted of an SO.sub.3.sup. group bound to a sodium cation), 4% (w/v) Ficoll in a phosphate buffer, which show how none of the three samples were cytotoxic. It should be noticed how the concentration used is much higher than that (0.03% w/v) at which BBG is declared to be cytotoxic.

TABLE-US-00004 degree of cellular degeneration % reduction in viability degree % cyto- cyto- value toxicity value toxicity batch sterilisation measured limit measured limit 1 autoclave 1.6 >30 0 >2 121.1 C. 20 2 autoclave 0.42 >30 0 >2 121.1 C. 20 3 autoclave 2.77 >30 0 >2 121.1 C. 20

Affinity Tests

[0057] These are tests carried out by the Applicant to compare the molecule disclosed and commercial BBG-250, in terms of affinity with proteins and protein chains. As in the previous case, the molecule produced according to this invention used in the tests is a molecule obtained with the method described in the Example, and in which the group R.sub.1 is constituted of an SO.sub.3.sup. group bound to a sodium cation.

[0058] In contrast, as regards the commercial BBG-250, given that the purity of said compound was approximately 91% (compared with the declared 80%), before carrying out the tests it was purified to approximately 98%.

[0059] In particular, tests were carried out on four different dye preparations, water-based in a phosphate buffer: for each molecule one comprising Ficoll (at 4% w/v) and one comprising heavy water D.sub.2O (at 13% v/v). In all of the dye preparations tested, the phosphate buffer comprised: [0060] Sodium phosphate monobasic monohydrate NaH2PO4 H2O: 0.266 mg/ml; [0061] Sodium phosphate dibasic anhydrous Na2HPO4: 1.51 mg/ml; [0062] Sodium Chloride NaCl: 8.2 mg/ml.

[0063] Affinity was determined by means of CD (circular dichroism) spectroscopy. The CD spectra were measured in the 800-400 nm region in the presence of increasing quantities of egg white albumin which, as is known, for analyses of this type is commonly considered to be representative of proteins in general.

[0064] It should be noticed that in the conditions used (400-800 nm) the albumin does not absorb and therefore does not give rise to any dichroic signal, whilst the dyes have a high level of absorption, but do not show any dichroic signal. In contrast, the interaction of the albumin with the dye molecule (BBG-250 or disclosed herein) induces a chirality in the dye that can be measured by means of CD. Consequently, titrating the dye with the albumin it is possible to determine its apparent binding constant or association constant, by means of a non-linear regression of the titration curve.

[0065] The CD spectroscopy data relative to a dye preparation comprising the molecule disclosed and Ficoll are shown in FIG. 5 where the number 1 indicates the dichroic spectrum of the dye preparation in the absence of protein, whilst the other lines relate to the same preparation in the presence of increasing quantities of albumin (according to the arrow).

[0066] As already indicated, using a non-linear regression of the intensity of the dichroic signal expressed in A (A.sub.L-A.sub.R) depending on the concentration of egg white albumin, the values of the association constant K.sub.a are determined. FIG. 6 shows the trend of the regression concerning the dye preparation comprising the molecule disclosed and Ficoll. It should be noticed that in the calculation of the association constant K.sub.a the different concentrations of the two dyes was taken into account.

[0067] The results obtained are as follows: [0068] Dye preparation with BBG-250 in Ficoll 4% in a phosphate buffer (sterilised): K.sub.a approximately 60,000 M.sup.1; [0069] Dye preparation with the molecule disclosed in Ficoll 4% in a phosphate buffer (sterilised): K.sub.a approximately 300,000 M.sup.1; [0070] Dye preparation with BBG-250 in a phosphate buffer (1.22 mg/ml in D.sub.2O) (sterilised): K.sub.a approximately 38,000 M.sup.1; [0071] Dye preparation with the molecule disclosed in a phosphate buffer (0.58 mg/ml in D.sub.2O) (sterilised): K.sub.a approximately 180,000 M.sup.1.

[0072] Therefore, as can be seen, irrespective of the agent for increasing the density used, the molecule disclosed surprisingly showed an affinity for the albumin equal to 4.7-5 times that of commercial BBG-250.

[0073] Moreover, it was possible to establish that, for both of the dye molecules, the presence of Ficoll promoted (by 1.66-1.58 times) the association of the dye with the albumin compared with used in heavy water. Although the reason for this latter result is not currently known for sure, the theory of the Applicant is that the Ficoll, in addition to the role of agent for increasing the density, may also fulfil the role of crowding agent (hereinafter referred to as CA) and promote the interaction of the dyes with the egg albumin by means of excluded volume and spatial factors.

[0074] In fact, recently, Ficoll, together with PEG and dextran, has been studied and used as a CA, that is to say, as a filling/crowding agent, to mimic the conditions of the intracellular environment which is characterised by an extremely crowded environment, with a limited quantity of free water and an almost total absence of space. Many studies on CA (see, for example, What Macromolecular Crowding Can Do to a Protein, Irina M. Kuznetsova 1,2, Konstantin K. Turoverov 1,2 and Vladimir N. Uversky 1,3,4,5) have in fact shown how macromolecular crowding created by the CA could affect the structure of the proteins, the folding, the form, shape, stability, the bond with the small molecules, enzymatic activity, protein-protein interactions, protein-nucleic acid interactions, and pathological aggregation.

[0075] The main mechanism of CA is to act for excluded volume, which corresponds to spatial occupation and, therefore, to movement of the other molecules into the remaining free spaces. Locally this may result in an increase in concentration or in steric molecular changes of solutes. Therefore, this could also occur in the case in question.

[0076] In light of the experimental results just discussed concerning the surprising advantages than can be obtained using Ficoll as the agent for increasing the density, to conclude, this invention also relates to a dye preparation for use in a surgical method of ophthalmic surgery having a density higher than 1.01 g/cm.sup.3 and comprising Ficoll as an agent for increasing its density. That preparation may be used for all of the uses indicated above with reference to the new dye molecule disclosed.

[0077] Advantageously, this dye preparation may comprise at least one dye selected from the group consisting of: triphenylmethane dyes, azo dyes, cyanine dyes, natural dyes and/or mixtures of them, but preferably it will comprise BBG or a structural analogue of it.

[0078] Amongst the latter, those preferred are the pharmaceutically acceptable salts of BBG, BBG hydrates and BBG methylates.

[0079] From an operating viewpoint, in the case of use of a dyed preparation which uses Ficoll to obtain a density higher than 1.01 g/cm.sup.3, for dyeing the ILM, it should be remembered that in these surgeries following removal of the vitreous by means of the vitrectomy, the dye preparation is injected into the posterior chamber. Thanks to its high density it is deposited on the ILM with which it remains in contact only for several seconds before being removed by means of the usual abundant washing of the posterior chamber with BSS. However, despite just several seconds of contact, the dye preparation dyes the ILM.

[0080] It should be noticed, however, that in these conditions of use (therefore even in the case of other ophthalmic surgeries performed under similar conditions), any Ficoll used as a thickener is not able to act as a CA, since the dye preparation is removed immediately after dyeing the membrane and is diluted in the posterior chamber with BSS.

[0081] Therefore, limited to the use for intraoperative dyeing in ophthalmic surgery methods, using Ficoll gives only performance similar to that obtainable, the density of the dye preparation being equal, with normal agents for increasing the density, such as heavy water.

[0082] This invention brings important advantages.

[0083] Regarding the new dye molecule provided, first the great advantage was achieved of that molecule proving able to dye better than commercial BBG not just the ILM (circumstance declared by all of the surgeons involved in the experimental tests), but also the proteins and protein chains (see the results of the tests shown above), the consequence being that it is possible to use less dye to achieve the same results.

[0084] Moreover, it is not cytotoxic at concentrations significantly higher than those currently permitted for commercial BBG.

[0085] In contrast, as regards the use of Ficoll as an agent for increasing the density, as an alternative to those used in the sector until now, it was possible to establish that its use allows in some applications results similar to those obtainable until now (as in the case of ophthalmic surgery), whilst in other applications it allows the use of less dye to achieve the same results.

[0086] Finally, it should be noticed that this invention is relatively easy to produce and that even the cost linked to implementing the invention is not very high.

[0087] The invention described above may be modified and adapted in several ways without thereby departing from the scope of the inventive concept.

[0088] All details of the invention may be substituted with other technically equivalent elements and the materials used, as well as the shapes and dimensions of the various components, may vary according to requirements.