BIOLOGICAL STRUCTURES

Field of the Invention

The present invention refers to pharmaceutical compositions comprising delphinidin, alone or combined with other dyes, namely brilliant blue (G or FCF) and trypan blue, to a process for producing those pharmaceutical compositions and to a method for staining biological membranes and structures using those pharmaceutical compositions.

The present invention facilitates the identification of biomembranes and their structures, including different types of ocular membranes and tissues, during surgical procedures such as chromovitrectomy. It also protects cells and tissues from damage induced by surgical light.

Therefore, this invention is in the technical domain of medical and pharmaceutical industry and related ones.

Background of the Invention

Surgery frequently requires the visualization, manipulation and removal of fragile biological membranes. These structures are often semi-transparent and thin and, therefore, their adequate identification and manipulation is difficult, which makes the surgical procedure complex and impacts on the recovery time. The use of vital dyes for staining human tissues during surgery has been established for more than 30 years. However, in ophthalmology, the use of this tool dates back to the thirties. Indeed, Sorsby in 1939 and Gifford in 1940 began the injection of Kiton-fast-green V, Xylene-Fast-green B and fluorescein intravenously. Kutschera, in 1969, began injecting patent blue intra-vitreally to stain retinal tissue and assess retinal breaks in retinal detachments.

The onset of the pars plana vitrectomy surgical procedure allowed the treatment of serious retinal diseases such as diabetic retinopathy, macular hole and retinal detachment. The procedure consists in removing the intraocular vitreous gel and pre-retinal membranes, followed by the restoration of the ocular volume, for instance, with balanced salt solution (BSS).

The removal of pre-retinal membranes, specifically, constitutes a technically difficult surgical step because of the thin and semi-transparent nature of the tissues. In fact, several studies have shown retinal complications caused by the inaccurate removal of these structures.

The use of stains in chromovitrectomy was first described by Burk et al. in 2000 and it was a major breakthrough in vitreoretinal surgery: the vital dye indocyanine green (ICG) was shown to exhibit high affinity for the internal limiting membrane (ILM) and the previously complex task of removing the ILM and epiretinal membranes became much easier. This highly improved anatomic and functional surgery outcomes. Nevertheless, clinical trials soon raised safety concerns demonstrating postoperative complications such as visual field and retinal pigment epithelium changes related to the use of ICG. In vitro and in vivo studies have shown a dose-dependent toxicity of ICG in retinal cells. Such toxicity has resulted into an active search for less toxic compounds. Two candidate compounds are trypan blue and patent blue.

However, the acceptance of these dyes has been limited due to low chemical affinity for retinal membranes and no precise safety indicators .

The ideal dye would then exhibit high affinity to retinal membranes and reduced or no retinal toxicity. In the search for these putative pharmaceutical compositions, the present invention proposes the use of a natural dye, delphinidin, as a new alternative for use in chromovitrectomy or other procedures where it is necessary or helpful to guide a health care professional by defining layers or boundaries.

Delphinidin or 2-(3,4,5-Trihydroxyphenyl)chromenylium-3,5,7- triol is a natural anthocyanin, produced by plants such as cranberries and blueberries (Vaccinium), blackcurrants (Ribes), violets (Viola), delphinium (Delphinium), some types of grapes and eggplant (Solanum) being responsible for the blue and blue red colour of flowers and fruits. This plant pigment also shows strong antioxidant activity and due to its pH sensitivity can additionally be used as a natural pH indicator, changing from red in acidic solution to blue in basic solution. Delphinidin has a diphenylpropane-based polyphenolic ring structure that carries a positive charge in its central ring. Anti-angiogenic and anticancer properties have been attributed to this pigment. It has also been shown to protect keratocytes and mouse skin from UVB-Mediated oxidative stress and apoptosis and to have anti-inflammatory properties in an in vitro model of psoriasis.

Document EP0410749 A2 refers to the use of delphinidin to treat injury or harmful agent-induced hyperpermeability of ciliary body blood vessels, known regulators of ocular pressure and of the blood-water barrier, as well as of the production of aqueous humour. However, said document does not disclose compositions, or methods for staining biological membranes and related structures nor the protection of tissues and cells exposed to surgical light during surgery.

Document WO2013167632 A1 discloses a viscoelastic device comprising a viscoelastic polymer covalently bound to at least one dye, being the said dye for example an anthocyanin, that is used to fill the anterior chamber after phacoemulsification, a cataract surgery in which the lens is emulsified with ultrasounds and aspirated from the eye. Aspirated fluids are then replaced with BSS irrigation to maintain the anterior chamber. This viscoelastic device is used in order to protect the endothelium and to easily detect or indicate normal or abnormal pH values in the ocular tissue. This document does not disclose compositions, methods for staining biological membranes and related structures to facilitate their identification, manipulation and removal during surgical procedures nor does it disclose the protection of tissues and cells exposed to light during surgical procedures. A recent scientific paper (Chuang L., Wu A., Wang N., Chen K., Liu, L., Hwang, Y., Yeung, L., Wu W., Lai C. The intraocular staining potential of anthocyanins and their retinal biocompatibility: a preclinical study. Cutaneous and Optical Toxicology 2018, 4:1-8) has shown preclinically the potential of delphinidin as an intraocular stain. Several staining suspensions comprising anthocyanins, namely delphinidin, are herein disclosed. Each anthocyanin was suspended in 10 ml of 5% glucose in water solvent with resulting anthocyanin concentration of 1 mg/ml for improving the contrast of the ILM and remaining retinal tissues during chromovitrectomy procedures. Results show that delphinidin was used with success to stain pig eye lenses and intravitreal and subretinal injection in rats promoted no morphologic nor functional toxicity. A clinical trial has shown that oral delphinidin improves glucose metabolism in pre-diabetic individuals, is well tolerated and generates no adverse effects. However, the concentration of the staining solutions based on the studied anthocyanins had to be substantially higher, since each formulation comprises only one dye. Moreover, despite staining solutions comprising other known dyes are mentioned, such as ICG, these dyes are referred to as non-usable mainly due to their toxicity. There is no mention or hint of staining solutions comprising a combination of at least one anthocyanin and another dye to reduce the toxicity of said solutions, to increase the contrast between the different tissues of the eye during intraocular interventions or to protect bio structures from damage caused by exposure to surgical light.

Therefore, there is a need to develop staining pharmaceutical compositions that are not only able to stain biological tissues but are also able to do it selectively in order to produce a contrast between close related tissues and allow accurate identification, manipulation and removal of biological membranes and structures. This improves the safety of surgical procedures and promotes better recovery periods. There is also a need to protect bio structures from damage caused by exposure to surgical light.

In this sense, the present invention proposes pharmaceutical compositions that can be used not only for staining biological membranes, in particular ocular membranes, but also for protecting intraocular structures from light exposure by topical application or injection into the eye.

Summary of the Invention

The present invention refers to pharmaceutical compositions comprising delphinidin, alone or combined with other dyes, such as brilliant blue (G or FCF) and/ or trypan blue, to a process for producing the said compositions and to a method for staining biological membranes and structures to improve their identification and processing during surgical procedures, such as chromovitrectomy. It also discloses the protection of tissues from damage induced by surgical light.

Therefore, it is a first object of the present invention to provide pharmaceutical compositions comprising delphinidin alone or in combination with brilliant blue (G or FCF) and/ or trypan blue for staining biological membranes and cell structures, according to claim 1. These pharmaceutical compositions selectively dye the biological structure of interest whereas the surrounding structures are not dyed or are not dyed to the same extent. Furthermore, they also stain biological structures at a concentration that is physiologically and toxicologically acceptable and therefore, the lowest amount of dye that provides a visible differentiation between the selected biological membranes and the surrounding tissues can be used. Despite these dyes show little or no toxicity, any remaining pharmaceutical composition in the surgical field is removed briefly after the procedure, further reducing the possibility of adverse effects.

It is another object of the present invention to provide a process for producing staining pharmaceutical compositions based on delphinidin, according to claim 10.

It is yet another object of the present invention to provide a method for staining biological membranes and structures by using pharmaceutical compositions based on delphinidin, according to claim 6.

This method allows dyeing the biological structure of interest selectively whereas the surrounding structures are not dyed or are not dyed to the same extent resulting in a more accurate identification of the desired structure and its easier surgical manipulation/ removal, reducing the risk of damaging the surrounding tissues and consequently reducing the recovery time.

This process can be used during surgical procedures or in any other procedures that require staining of biological material, improving the contrast between the different types of biological tissues.

By using the pharmaceutical compositions and methods of the present invention it is possible to increase the protection of tissues and cells from damage due to light present in the surgical field during surgical procedures.

Furthermore, by applying the present invention it is also possible to protect intra-ocular structures by topical application or by injection into the eye of one or more of the said pharmaceutical compositions that include the natural substance delphinidin, with low toxicity risk for the patient.

Detailed Description of the Invention

The present invention refers to pharmaceutical compositions comprising delphinidin, alone or combined with other dyes, namely brilliant blue (G or FCF) and/ or trypan blue, to a process for producing said pharmaceutical compositions and to a method for staining biological membranes and their structures.

The present invention allows improved identification of biological materials and their structures, including different types of ocular membranes and tissues, during surgical procedures such as chromovitrectomy. It also relates to the use of delphinidin comprising pharmaceutical compositions that protect tissues and cells from surgical light-induced damage.

In the scope of the present invention intraocular membranes referred herein are particularly the anterior and posterior capsule, epiretinal membrane, ILM, vitreous and posterior hyaloid.

1. Dyes of the compositions of the invention

The first aspect of the present invention relates to pharmaceutical compositions comprising delphinidin, alone or in combination with brilliant blue (G or FCF) and/ or trypan blue dyes and pharmaceutically acceptable vehicles.

Staining pharmaceutical compositions according to the present invention comprise delphinidin or 2-(3,4,5-trihydroxyphenyl) chromenylium-3,5,7-triol (formula 1), as a main dyeing agent.

Delphinidin, is a safe and an effective natural dye for staining biological tissues and structures that can be used in surgical procedures, such as chromovitrectomy, allowing an easy identification of biological membranes and structures by the ophthalmic surgeon, an effective performance during surgery and, remarkably, a decrease of iatrogenic retinal complications derived from the surgical procedure.

Formula 1 Comparing with other anthocyanins, delphinidin is the one exhibiting the best staining properties with no toxicity. It has also shown the ability to protect skin from UV radiation.

Delphinidin as a dying agent can be used in combination with other dyes. In the scope of the present invention, the pharmaceutical compositions comprise delphinidin in combination with a dye selected from the group including ingredients or extracts of brilliant blue (G or FCF) and/ or trypan blue.

Brilliant Blue FCF is an azo and triarylmethane synthetic dye (Formula 2) that is commonly used as food colorant due to its poor absorption in the gastrointestinal tract. It is added to drug formulations as an inactive substance.

Formula 2

Brilliant Blue G is a triphenylmethane dye commonly used for staining proteins (Formula 3) by interacting electrostatically but non-covalently with the amino and carboxyl groups of proteins. Its colour varies in function of the acidity of the solution due to the different charged states of the dye molecule, in particular at a value pH less than 0 all three nitrogen atoms carry a positive charge conferring to the dye a red color, whilst at pH values above 2, the two sulfonic acid groups have extremely low pf _a and are negatively charged conferring to the dye a bright blue color.

Formula 3

Trypan blue, also known as diamine blue and Niagara blue (Formula 4), is a blue azo synthetic dye that is used to selectively dye dead tissues or cells. In ophthalmic cataract surgery it is used to stain the anterior capsule in the presence of a mature cataract, to aid in visualization, before creating the continuous curvilinear capsulorhexis.

Formula 4 2. Formulations of the invention

Typically, the pharmaceutical compositions of the present invention comprise delphinidin as the main dye in concentrations of 0.01 to 0.1%, preferably of 0.05 to 0.5%, more preferably of 0.55 to 0.65%, even more preferably in a concentration of 0.06%.

Brilliant blue (G or FCF) can be present in the pharmaceutical compositions of the present invention in an amount of 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055%.

Trypan blue can be present in the pharmaceutical compositions of the present invention in an amount of 0.001 to 0.2% of the total composition, preferably of 0.04 to 0.15%.

These dyes can be advantageously combined with delphinidin to produce pharmaceutical compositions for staining biological material, namely biological membranes and/or structures, particularly ocular membranes.

Therefore, in the scope of the present invention, pharmaceutical compositions comprising delphinidin as main dye, for staining biological material can be produced in one of the following formulations : a) Delphinidin + brilliant blue (G or FCF); b) Delphinidin + brilliant blue (G or FCF) + trypan blue; c) Delphinidin + trypan blue; d) Delphinidin.

Acceptable pharmaceutical vehicles suitable to be included in delphinidin pharmaceutical compositions of the present invention can be, for example acetic acid, benzyl alcohol, borax, boric acid, BSS, calcium chloride, carbomer 934, carbopol, chondroitin sulphate, citric acid, dextran sodium polysorbate, glycocholic acid, hyaluronic acid, magnesium chloride, metaphosphoric acid, methylcellulose and derivatives, phenylphosphate, phosphate buffer, polyethylene glycol, polyvinyl alcohol, potassium chloride, potassium phosphate, propylene glycol, purified water, sodium acetate, sodium chloride, sodium citrate, sodium edetate, sodium phosphate, sodium phthalate and/or tweens.

Each one of the above-mentioned synthetic dyes has its staining specificity and accordingly, when used in combination with delphinidin, the biological structure of interest is selectively stained and the surrounding structures are not or are not to the same extent. This allows accurate identification of the structure and surgical manipulation/ removal, reducing the risk of damaging the surrounding tissues and consequently reducing the time of recovery and iatrogenic complications.

Moreover, they show little or no toxicity and any remaining residues that are left in the surgical field are removed briefly after the procedure, further reducing the possibility of adverse effects. In addition, they also stain biological structures at a concentration that is physiologically and toxicologically acceptable .

In short, benefits of using pharmaceutical compositions of the present invention comprising delphinidin, a natural dye, alone or in combination with one or both of these synthetic dyes include: clear and easy identification of the desired membrane and/ or biological structure to manipulate and/ or remove during eye surgery; prevention of adverse effects caused by inaccurate (insufficient or excessive) removal of the desired biological structure; reduction of surgery time and reduction of the post-operative period with consequent reduction of health care costs; increased safety during the procedure and later phases; possible antioxidant/ anti-inflammatory effects associated with the use of delphinidin in the surgery or other medical procedure; protection of bio structures from damage caused by exposure to surgical light.

The compositions of the present invention can be presented in the form of a solution, a dispersion, a suspension or an emulsion.

3. Process for producing the pharmaceutical compositions

The overall process of pharmaceutical composition preparation includes diluting the selected dyes in a pharmaceutical vehicle to achieve the final concentrations of:

- Delphinidin: 0.01 to 0.1%, preferably 0.05 to 0.5%; more preferably 0.55 to 0.65%;

- Brilliant blue (G or FCF): 0.025 to 0.05%; preferably 0.045 to 0.055%, and/or

- Trypan blue: 0.001 to 0.2%, preferably 0.04 to 0.15%. A pharmaceutical acceptable vehicle is added to the above compositions. The said compounds are already listed in the previous section.

In addition, the dye may be formulated as a solution, suspension, dispersion or emulsion by the known techniques in the art.

All the steps are conducted under the required conditions for manufacturing, handling and processing pharmaceutical quality materials.

4. Method for staining biological material

The method for staining biological material, such as biological tissue, membranes or other structures of the eye includes the application of a pharmaceutical composition according to the present invention, topically or by injection. This application can be done during a surgical procedure or any other procedure that requires staining of a biological material and/ or structure.

Examples

Example 1. A pharmaceutical composition with delphinidin

The packaging material and raw materials are received and the raw materials are weighed. Handling and/ or filtration takes place in an ISO 8 class room. In-process quality control is performed before septic or aseptic filling. Should the filling be aseptic, it follows the flow: Filling in the ISO 5 class room > Sealing in ISO 7 class room > Sterilization in the final sterilization room. Should the filling be septic, it follows the flow: Sterilizing Filtration in ISO 5 class room > Filling in ISO 5 class room > Sealing in ISO 7 class room. Packaging and quality control of the finished product then follows.

Example 2 . A pharmaceutical composition with delphinidin and brilliant blue FCF

All the steps were performed as described in Example 1 with exception of the amounts of dyes that were the following: Delphinidin and brilliant blue were weighted in separate and diluted together in a pharmaceutical vehicle to achieve amounts of delphinidin 0.06% and brilliant blue of 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055% of the final composition .

Example 3. A pharmaceutical composition with delphinidin and trypan blue

Similarly, to the described in example 2, delphinidin and trypan blue were weighted in separate and diluted together to achieve amounts of delphinidin 0.06% and trypan blue of 0.001 to 0.2% of the total composition, preferably of 0.04 to 0.15% of the final composition . Example 4. A pharmaceutical composition with delphinidin, brilliant blue FCF and trypan blue

In this example, all the described steps of the previous examples were followed and a pharmaceutical composition having delphinidin 0.06%, brilliant blue FCF of 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055% and trypan blue of 0.001 to 0.2% of the total composition, preferably of 0.04 to 0.15% of the final composition was achieved.

Example 5. Pharmaceutical compositions with different concentrations of delphinidin alone or in combination with brilliant blue and/or trypan blue

Different pharmaceutical compositions were obtained, by the process described in any of the previous examples, with different concentrations of the 3 dyes of interest.

Example 6. Efficacy of delphinidin in staining the lens capsule and ILM - Comparative example (COMP)

Lens capsule and ILM were stained with a delphinidin solution according to the method described by Chuang et al.

Lenses and retinas were obtained from fresh pig eyes purchased from a slaughterhouse, thirty minutes before staining at room temperature. A delphinidin solution was prepared at a concentration of 1 mg/ml in a 5% glucose solution in water and twenty-four lenses were soaked in the dye solution for 1 minute. The lenses were rinsed with BSS to remove the excess dye and examined under a surgical microscope by the same cataract surgeon. Regarding ILM staining, the vitreous of the eye globe was removed from the surface of retina, stained and examined similarly to the lenses. Both the lens capsule and ILM were stained, exhibiting a blue color.

Example 7. Delphinidin safety and neuroprotective effects in vitro - Comparative example (COMP)

The viability ARPE19 and RGC5 cell cultures was determined by the method described by Chuang et al. after incubation with delphinidin. Cell viability was determined using the reagent WST- 1 (Roche Diagnostics GmbH, Penzberg, Germany), a tetrazolium salt that is cleaved by mitochondrial dehydrogenase, present in metabolically active cells. Such reaction generates a coloured formazan dye, detected in a microplate reader that measures absorbance at 450nm. At a concentration of 1 mg/ml, delphinidin was non-toxic with similar survival rates as controls treated with 5% glucose. Additionally, after H2O2 treatment, delphinidin significantly increased the survival rate of the ARPE19 cells compared with that of the control cells, indicating a neuroprotective effect.

Example 8. Delphinidin injection in rats, histology and retinal function evaluation - Comparative example (COMP)

Anesthetized rats were injected with a solution of 0.15 ml/kg of an equal volume of 2% lidocaine (Xylocaine; Astra, Sweden) and 50 mg/ml ketamine (Ketalar; Parke-Davis, USA), according to the method described in Chuang et al. After anaesthesia, the pupils were dilated and the eyes were protruded. A 1.5-cm/33-gauge Hamilton blunt-tip syringe needle was inserted into the vitreous under a surgical microscope, injecting 5 mΐ of dye solution. The control eye was injected with BSS. Regarding the subretinal injection, a 1.5-cm/33-gauge blunt-tip syringe (Hamilton, Reno, NV) was inserted tangentially toward the posterior pole of the eye, and delphinidin was injected. The control eye was injected with BSS.

For histologic examination, the eyeballs were collected from six rats, 1 and 3 months after delphinidin injection, and fixed with 4% paraformaldehyde at 4°C for 24 hours. The fixed tissues were embedded in paraffin, sectioned at 5 pm and stained with haematoxylin and eosin. The histology of the delphinidin-treated and control eyes did not show major anatomical signs of toxicity. Apoptotic cell death evaluation by TUNEL essay performed 1 month after intravitreal injection showed similar staining among the groups, with no differences in cell death compared with the control group.

Corneal electroretinograms (ERGs) were obtained from the injected rats a month after the intravitreal injections of delphinidin or BSS. ERGs were recorded with an ERG instrument (UTAS-E 300; LKC Technologies, Gaithersburg, MD, USA). Briefly, rats were maintained in the dark for 1 hour before recording the ERGs and all procedures were performed in a dark room subsequently. Animals were placed on a heating pad at 35-36°C and, after anaesthesia, the eyes were dilated and protruded. The Ag/AgCl recording electrode was attached to the cornea, the reference electrode to the shaven skin of the head and the ground electrode to the rat's ear. The light stimulus was a light flash of 100 ms at a distance of 30 cm from both eyes. Responses were then recorded. ERG data from the rats were normal, showing no reductions in function at baseline or one month after the intravitreal injection of delphinidin.