Pharmaceutical compositions for staining membranes and other biological structures using a natural or synthetic CBD-based vital dye isolated and / or associated with other DYES for vitreoretinal surgery

Abstract

The present invention describes the use of a pharmaceutical compositions for dyeing or staining membranes and other biological structures using a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, as a promising approach during surgical or medical procedures, such as vitreoretinal. The use of such compounds for the preparation of dyeing or staining compositions in order to facilitate their identification and biomolecular membrane composition, results in the visualization of different types of ocular membranes and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light. The present invention describes the entirety process, since the production of the composition purpose along with formulation and studies conducted, until the purport method for staining biological membranes and structures to improve their adequate identification and manipulation, responding to the challenged presented by the complexity of the surgical procedure mainly chromovitrectomy.

Description

[0001] DESCRIPTION

[0002] PHARMACEUTICAL COMPOSITIONS FOR STAINING MEMBRANES AND OTHER BIOLOGICAL STRUCTURES USING A NATURAL OR SYNTHETIC CBD-BASED VITAL

[0003] DYE ISOLATED AND / OR ASSOCIATED WITH OTHER DYES FOR VITREORETINAL

[0004] SURGERY

[0005] FIELD OF INNOVATION

[0006] The present invention describes the use of a pharmaceutical compositions for staining membranes and other biological structures using a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely Brilliant Blue G and / or Trypan Blue, as a promising approach in surgery, such as vitreoretinal. Those pharmaceutical compositions are to be used as a practice for staining biological membranes and structures in surgery, in order to facilitate their identification during surgical procedures such as vitreoretinal surgery, resulting in the visualization of different types of ocular membranes, structures and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light.

[0007] The use of one and / or more natural or synthetic vital dyes, isolated and / or associated with other dyes, namely Brilliant Blue G and / or Trypan Blue among others, could emerge as an innovation in the medical field of ophthalmology by introducing a natural or synthetic CBD- based isolated and / or associated with other natural or synthetic dyes into the pharmaceutical formulation being capable of dye or stain living cells or tissues, both in vitro and in vivo, leading to target-delivery of the purposed dye, as it will have affinity to the receptors of the endocannabinoid system present in the biomolecular membranes and structures of the human eye. Therefore, this invention is in the technical domain of medical and pharmaceutical industry, and related ones.

[0008] Figure 1. Schematic representation of the pharmaceutical composition delivery along with Anti-inflammatory Sinking Antioxidant effect.

[0009] 1 - Natural or synthetic CBD-based vital dye

[0010] 2- Other dyes, including but not limited Brilliant Blue G and / orTrypan Blue

[0011] 3- ASA effect - Anti-inflammatory Sinking Antioxidant effect in stain delivery and surgical light protection

[0012] 4- Vitreous stain

[0013] 5- Epiretinal membrane stain

[0014] 6- Internal limiting membrane stain

[0015] BACKGROUND As our understanding of ocular anatomy and pathology has expanded, the quest for refined surgical techniques and cutting-edge technologies to enhance visual outcomes and patient safety was increase over the years. The evolution of ophthalmic surgery reflects a continuous pursuit to break down precision challenges, safety, and outcomes for a variety of eye-related conditions, such as cataract and vitreoretinal surgery (Abrams et al., 1978; Brod, 2009; Ribeiro et al., 2022; Rodrigues et al., 2009).

[0016] In recent years, a notable surge in innovation has reshaped the landscape of surgery by advancements in imaging technologies and minimally invasive procedures, namely in ophthalmic field (Abrams et al., 1978; Brod, 2009; Ribeiro et al., 2022; Rodrigues et al., 2009). These innovations not only strive to address conventional challenges in ocular surgery but also open newfrontiers in treating complex eye disorders with unique precision.

[0017] Since biological membranes and structures are often semi-transparent and thin, their adequate identification and manipulation is challenging, which makes the surgical procedures complex and may impact on the recovery time due to inadequate practice (Abrams et al., 1978; Dhaliwal et al., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0018] The incorporation of dyes in surgery procedures namely in ophthalmic field plays a key role as a therapeutical assistance representing one of the most efficient, objective, non- invasive, and directly visible means for biological membranes and structures identification, facilitating the delicate and precise manipulation and removal of fragile biological membranes and structures, which in most cases are thin and semi-transparent (Farah et al., 2009; Rodrigues et al., 2009). A comprehensive understanding of the layered composition and structural intricacies of both the internal limiting membrane and epiretinal membrane along with other biological membranes and structures is fundamental for unravelling their roles in physiology and pathology, and it can also advance surgical outcomes, enabling surgeons to navigate complex anatomical sites with heightened accuracy and reduced invasiveness (Abrams et al., 1978; Dhaliwal et al., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0019] Dyes are chemical compounds that bind to various substances in nature to induce colour. Vital dyes emerged recently as an important and effective surgical adjuvants to enhance visualization of biological membranes and structures, since these compounds have the ability to intravital and supravital dyeing or staining of living tissues or cells in a living organism and living cells or tissues freshly removed from the body. Vital dyes have been used in cataract and vitreoretinal surgery, as well as in corneal, glaucoma, orbit, strabismus, and conjunctival surgery (Abrams et al., 1978; Dhaliwal et al., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0020] Vitreoretinal surgery, also known as Pars plana vitrectomy, refers to a group of advanced, highly delicate procedures that is performed in the part of your eye where the vitreous and retina are located. The vitreous is a substance filling the cavity between the lens of your eye and your retina (Abrams et al., 1978; Ribeiro et al., 2022; Thompson, 2006).

[0021] The purpose of vitreoretinal surgery is to restore, preserve and improve vision for a wide range of conditions, therefore understanding of vitreoretinal diseases has evolved with the development of those new diagnostic technologies (Abrams et al., 1978; Ribeiro et al., 2022; Thompson, 2006). Likewise, the use of vital dyes for surgical approaches have also evolved overtheyears, contributingforthe advances of Robert Machemerfirst vitreoretinal surgery in 1970. Initially considered an unsafe and ineffective procedure, it has become highly technologic, safe, and predictable in most cases due to recent targeted drug delivery strategies and technologies for enhancing the possible visualization of biological membranes and structures (Ribeiro et al., 2022). Chromovitrectomy, raised as a groundbreaking technique in vitreoretinal surgery changing the way surgeons approach the delicate vitreous and retinal tissue (Farah et al., 2009). 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 and stability, for instance, with balanced salt solution (BSS) (Abrams et al., 1978; Brod, 2009; Ribeiro et al., 2022; Samuel et al., 2003; Thompson, 2006).

[0022] The ocular membranes and tissues stained with vital dyes during vitreoretinal surgery, also known as chromovitrectomy, allows the visualization of the transparent tissues in the vitreoretinal interface: the internal limiting membrane (ILM) a transparent structure that defines the boundary between the retina and the vitreous and is the surface upon which the epiretinal membrane (ERM), a cellular tissue found on the inner surface of the retina developed by several factors, including aging, inflammation, or trauma, addressing the challenges in vital dye for staining human tissues during surgery with a particular focus on their efficacy within the ocular membranes, provides a full view of the balance between therapeutic effectiveness and surgical interventions. This technique represents one of the most efficient, objective, non-invasive, and directly visible means for biological membranes and structures identification, facilitating the delicate and precise manipulation and removal of fragile biological membranes and structures, which in most cases are thin and semi-transparent, with the required precision (Abrams et al., 1978; Aiello et al., 2020; Delyfer et al., 2020; Dhaliwal et al., 2022; Farah et al., 2009; Guber et al., 2019; Mantelli et al., 2013; Ribeiro et al., 2022; Rodrigues et al., 2009).

[0023] Although, the first use of chromovitrectomy in pars plana vitrectomy surgery was reported by Abrams et al in 1978 (Abrams et al., 1978), showing that fluorescein intravenously staining improves the visualization of the vitreous, this procedure has only become more extensively over the last few years (Abrams et al., 1978; Dhaliwal et al., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0024] Early studies embraced vital dyes such as fluorescein intravenously and ICG in chromovitrectomy, establishing that ICG presented affinityforthe ILM and ERM. This highly improved anatomic and functional surgery outcomes (Farah et al., 2009; Iriyama et al., 2004; Rodrigues et al., 2009).

[0025] BurkS. etal. (Burk et al., 2000) first described this technique in 2000, by using the vital dye indocyanine green (ICG), which has a high affinity for the ILM. This breakthrough simplifies the complex task of removing the biological membranes and tissues, significantly improving surgical outcomes and give respond to the eye diseases that are affecting more and more people worldwide every year. This innovative method utilizes vital dyes to selectively stain specific structures within the eye, enhancing visualization and precision during surgical procedures, since the vitreous and retina exhibit a transparent and intricately layered nature and several studies have shown retinal complications caused by the inaccurate removal of these structures (Abrams et al., 1978; Dhaliwal etal., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0026] Nevertheless, clinical trials soon raised safety concerns demonstrating potential risks and limitations, including retinal toxicity, difficulty in dye removal, and the need for precise dosage control, and postoperative complications such as visual field and retinal pigment epithelium alterations, by creating ocular instability and oxidative stress together with the surgical light induced damage. In vitro and in vivo studies have shown a dose-dependent toxicity of ICG in retinal cells, resulted into an active search for less toxic compounds (Gandorfer et al., 2008; RODRIGUES et al., 2007). Studies also shown that although fluorescein intravenously and ICG presented to be effective in the intraocular membranes identification, those vital dyes still lacked specificity (I riya ma etal., 2004; MAIA et al., 2004).

[0027] Hence, the prolonged dye exposure, in addition to the extended time olzlcoaxial microscope light application, could have contributed to the transient cellular toxicity and oxidative stress observed in vivo (Bacsal and Chee, 2006). Given the increase oGfeaGfety concerns due to postoperative complications, subsequent studies have shown that ICG that ICG is toxic to retinal cells in a dose-dependent manner I3)r cells and layered tissues. Therein re, researchers began investigating less toxic vital dyes (Bacsal and Chee, 2006; Dhaliwal et al., 2022; Farah et al., 2009; Narayanan et al., 2006; Rodrigues et al., 2009; Soni et al., 2022).

[0028] Nowadays, chromovitrectomy performed with Brilliant Blue G (BBG) (Enaida and Ishibashi, 2008), Trypan Blue (TB) (Aguilera Teba et al., 2003), and Triamcinolone Acetonide (Bakri, 2008) have emerged to offlfer heightened precision due to the Facilitated visualization and removal 00 pre- retina I membranes as a result 00 their different aHBnities to intraocular collagen and cellular elements present with the tissue barriers.

[0029] The TB provides a contrast between the stained capsule and the underlying lens, while crystalline steroid Triamcinolone Acetonide is the primary line I3)r vitreous identification and BBG shown to be best stains I3)r the ILM. However, recent studies shown that the use 00 vital dyes in biological membranes and structures dyeing or staining has been limited due to their low chemical aHBnity and lack 00 precise saGfety indicators presented during trials, therein re achieving optimal staining while avoiding adverse effects remains a challenge (Dhaliwal et al., 2022; Farah et al., 2009; Rodrigues et al., 2009).

[0030] Since cannabinoid molecules have affinity to the receptors o0the endocannabinoid system present in the biomolecular membranes and structures o0the human eye, the staining 00 ocular membranes will be enhanced by increasing the target delivery 00 the staining molecules Trypan Blue, along with CBD's anti-inflammatory a nd antioxidant properties that could provide cellular protection during the procedure.

[0031] Studies have shown that TP and BBG are more effective Gbr optimal coloration, particularly at low concentrations, however, their delivery to membranes and structures is oGten compromised and less effective, resulting in insufficient contrast between the target intraocular membranes and the surrounding structures, which could complicate the surgical procedure. Therein re, natural or synthetic CBD-based vital dye is a potential solution Gbr stabilising these dyes in structures. The pharmaceutically acceptable vehicles currently used, could achieve the creation of larger molecules, being a carrier system that allows controlled release of the innovative formulation and have the potentialto improve effectiveness. Altering the molecularweight of an ocular dye will improve its adherence to retinal membranes during vitrectomy by utilizing the molecular sinking effect. For instance, having the patient in a supine position (lying face up) during the dye delivery can also optimize dye contact with the ocular surface. A higher viscosity formulation may address the issue of current dyes being too liquid for optimal adherence during surgery along the target-delivery provided by the natural or synthetic CBD-based vital dye isolated and / or associated with other dyes formulation.

[0032] The endocannabinoid system (ECS) is an active and complex cell signalling system mediated by the endocannabinoids lipidic molecules such as endocannabinoids 2- arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide or AEA) that bind to the G protein-coupled cannabinoid receptors (CB1 R and CB2R) (Lu and Mackie, 2016; Schwitzer et al., 2016).

[0033] While the CB1 R are primarily found in central nervous system (CNS), particularly in cortex, basal ganglia, hippocampus, and cerebellum, outnumbering many of the other receptor types on the brain, CB2R can also be observed in the peripheral organs especially is primarily present the immune tissues and playa key role in the immune functioning control and maintenance. Studies shown that it can also modulate intestinal inflammation, contraction, and pain in inflammatory bowel conditions (Bradshaw and Walker, 2005; Lu and Mackie, 2016; Schwitzer et al., 2016).

[0034] In response to specific endogenous or exogenous signals, the ECS can act like a neuromodulatory system in the development CNS by influencing neuronal synaptic plasticity and transmission, and regulation of several physiological functions, for instance body temperature, movement and motor coordination, learning and memory, addictive- like behaviour and pain modulation, inflammatory and immune responses and other basic body needs such as cardiovascular function, sleep and visual processing by the optical nerve (Bradshaw and Walker, 2005; McCormick et al., 2019; Schwitzer et al., 2016),

[0035] In addition to the endogenous activation of the cannabinoid system, the cannabinoid receptors are the primary target of exogenous cannabinoids, such as A-9-tetra- hydrocannabinol (THC) and cannabidiol (CBD) present in Cannabis, highly interact with the cannabinoid receptors CB1 R and CB2R (Saraiva et al., 2023). Cannabis is a plant that belongs to the family Cannabaceae and can be separated it into three subspecies defined by their phytocannabinoid content: subspecies Cannabis indica, with relatively high amounts of the psychoactive constituent THC, subspecies Cannabis sativa, with low amounts of THC and an intermediate subspecies Cannabis ruderalis Janisch (Saraiva et al., 2023; Tahir et al., 2021 ).

[0036] Cannabis sativa contains a wide range of bioactive compounds including phenols, flavonoids and anthraquinones. The THC and CBD are the most studied ones, due to their therapeutic and biological properties and recent association with neuromodulatory systems when interacting with ECS leading to neuronal synaptic communication and affect biological functions (Zantut et al., 2020).

[0037] Although THC and CBD interact with the ECS, their molecular structures and functions within the ECS differ significantly. While, THC has a cyclic ring structure and a side chain with a phenol group (OH) and an alkene group, contributing to its ability to bind to CB1 R in the CNS; CBD has a similar cyclic structure but lacks the side chain found in THC being considered non-psychoactive. CBD contains a phenol group (OH) associated with various therapeutic properties, including anti-inflammatory and anxiolytic effects, however the underlying biomolecular mechanisms still remain unclear (Ibeas Bih et al., 2015; Saraiva et al., 2023; Watkins, 2019; Zantut et al., 2020; Zhornitsky and Potvin, 2012).

[0038] CBD has a low binding affinity for CB1 receptors, mainly interacting with CB2R, which are predominantly found in peripheral tissues and the immune system contributing to antiinflammatory and immunomodulatory process, but also found in the retinal glia, more specifically in the Muller cells (Borowska-Fielding et al., 2018; Bouskila et al., 2016). In addition to binding to CB1 R and CB2R of the endocannabinoid system, as mentioned before, there is evidence that CBD activates 5-HT1A serotonergic and TRPV1-2 vanilloid receptors, antagonizes alpha-1 adrenergic and p-opioid receptors(lbeas Bih et al., 2015; Zhornitsky and Potvin, 2012).

[0039] Although the exact mechanism and magnitude of effects of CBD are not fully understood, CBD has been shown to have analgesic (Petersen et al., 2023), anticonvulsant, muscle relaxant (Isenmann et al., 2021 ), neuroprotective (Fernandez- Ruiz et al., 2013), antioxidant (Atalay et al., 2019), and anti-psychotic activity (Waldo Zuardi et al., 2012).

[0040] Moreover, Borowska-Fielding J et al. (Borowska-Fielding et al., 2018) demonstrated that cb2 deletion altered retinal visual processing and the cannabinoid-related enzymatic profile in murine models, since CBD modulates the ECS by influencing the activity of enzymes responsible for endocannabinoid breakdown, indirectly affecting cannabinoid receptors and other non-cannabinoid receptors (Borowska-Fielding et al., 2018; Bouskila et al., 2021 ; Watkins, 2019; Zantut et al., 2020).

[0041] Upon critical revision of the current literature related to this matter, cannabidiol, or CBD (C21H30O2), is one of at least 85 active cannabinoids identified within the Cannabis plant. As mentioned above, although the exact mechanism and magnitude of effects of CBD are not fully understood, CBD has been shown to have analgesic (Petersen et al., 2023), anticonvulsant, muscle relaxant (Isenmann et al., 2021 ), neuroprotective(Fernandez-Ruiz et al., 2013), antioxidant (Atalay et al., 2019), and anti-psychotic activity (Waldo Zuardi et al., 2012). In addition to binding to CB1 R and CB2R of the endocannabinoid system, as mentioned before, there is evidence that CBD activates 5-HT1 A serotonergic and TRPV1-2 vanilloid receptors, antagonizes alpha-1 adrenergic and p-opioid receptors (Ibeas Bih etal., 2015; Zhornitsky and Potvin, 2012).

[0042] Although CBD is a colourless molecular, after degradation in the membrane cells, it may appear purple due to the increase in the concentration of the photochemically unstable oxidation product CBD-hydroxyquinone (HU-331 ). In the presence of oxygen, water and / or exposed to natural light / dark cycles, this hydroxyquinol rapidly reacts to form a multitude of violet / purple -coloured cannabinoid by-products (Thomson et al., 2023).

[0043] Previous studies support evidence that HU-331 displays a potential anticancer therapeutic activity without some of the known adverse events associated with traditional anticancer agent, exhibiting to be much less cardiotoxic than chemotherapy drug doxorubicin (Kogan et al., 2007b, 2007a; Trac et al., 2021). For evaluation of the antiangiogenic action of HU- 331 was found to be strongly antiangiogenic, significantly inhibiting angiogenesis at low concentrations inducing apoptosis of vascular endothelial cells without changing the expression of pro- and antiangiogenic cytokines and their receptors(Kogan et al., 2006).

[0044] However, more studies will have to be conducted in this regard, as sufficient information about this cellular metabolic pathway and photochemical process is not yet available, and since release of reactive oxygen species such as HU-331 anions and superoxide may occur but could be controlled due to CBD’s antioxidant behaviour giving possible stability to the natural vital dye, protection of the retinal membrane and immune system activation (Forrester and Xu, 2012; Mecha et al., 2012; Thomson et al., 2023).

[0045] As explained above there are many benefits associated with the use of natural dyes, including: easy identification of eye membrane and biological layers to identify and remove; prevention of side effects caused by incomplete removal of membranes and biological structures; reducing the time of surgery with reduction in medical and hospital fees; increased safety because the proposed dye is a natural dye, alone or in combination with other dyes and all the alternatives on the market today have demonstrated toxicity; and possible antioxidant and antiinflammation effect associated with the cannabinoid molecules along with the targeted delivery strategy for other dyes such as BBG and / orTB.

[0046] In this context, the present invention describes the use of a new technique to achieve the coloration of the ocular tissue using pharmaceutical compositions for dyeing or staining membranes and other biological structures using a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, as a promising approach in vitreoretinal surgery. Those pharmaceutical compositions are to be used as a practice for dyeing or staining biological membranes and structures, in order to facilitate their identification and biomolecular membrane composition during surgical procedures such as vitreoretinal surgery, resulting in the visualization of different types of ocular membranes and tissues alongwith the cellularand tissue protection from damage induced by the exposition to surgical light. The antioxidative and anti-inflammatory effects of a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes will be also expected.

[0047] The ASA effect - Anti-inflammatory Sinking Antioxidant effect - could be an innovative pharmaceutical mechanism of action for the purposed stain, exhibiting antioxidative and anti-inflammatory properties. This dye could be either naturally derived or synthetically produced and could be used alone or in combination with other dyes.

[0048] Since the use of one and / or more natural or synthetic CBD-based vital dyes, isolated and / or associated with other dyes, namely BBG and / or TB, could emerge as an innovation in the medical field of ophthalmology by introducing a natural or synthetic CBD-based isolated and / or associated with other dyes into the pharmaceutical formulation being capable of dye or stain living cells or tissues, both in vitro and on the surface of the eye while to providing a protective effect to intraocular tissues, particularly against surgical light and damage.

[0049] This narrative provides a compelling context for an innovative material, showcasing the development of techniques and the continuous pursuit of advance and improvement. The text underscores the pursuit of a promising approach in vitreoretinal surgery, this invention is in the technical domain of medical and pharmaceutical industry and related ones, therefore the current literature related to this matter will be summarised and critical analyse in order to better understand and support the natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB novel pharmaceutical formulation and its use in the human eye. This sets the stage for the introduction of the inventive material in the subsequent sections.

[0050] SUMMARY OF THE INVENTION

[0051] The present invention describes the use of a pharmaceutical compositions for dyeing or staining membranes and other biological structures using a natural or synthetic CBD- based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, as a promising approach during surgical or medical procedures, such as vitreoretinal. The use of such compounds for the preparation of dyeing or staining compositions in order to facilitate their identification and biomolecular membrane composition, results in the visualization of different types of ocular membranes and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light. The present invention describes the entirety process, since the production of the composition purpose along with formulation and studies conducted, until the purport method for staining biological membranes and structures to improve their adequate identification and manipulation, responding to the challenged presented by the complexity of the surgical procedure mainly chromovitrectomy.

[0052] In accordance with the present invention, a novel method is proposed for the protection of tissues and cells exposed during surgical procedures, since cannabinoid-based vital dye provides a defensive effect against the unstable toxic free radicals induced by phototoxic agents and oxidative environment created by ocular instability and oxidative stress, shielding the retina from light-induced damage, known as phototoxicity. Free radicals are charged molecules that are highly reactive and can cause damage to the biological membranes and tissues in the body. Thus, antioxidants agents donate an electron to a free radical, neutralizing it and stopping the chain reaction. Ongoing research worldwide investigates the protective effect of CBD antioxidants properties. Studies suggest that antioxidants may manage or prevent the development of various oxidant linked diseases.

[0053] This method involves the administration of a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, leading to the protection of intraocular tissues and cells through the procedure application of these dyes into the eye. These dyes, which include a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / orTB, offer a significant advancement in the field of surgical and medical procedures by providing an effective means of protecting vulnerable tissues and cells.

[0054] It also discloses the protection of tissues from damage induced by surgical light, since, as mentioned above, although the exact mechanism and magnitude of effects of CBD are not fully understood, CBD has been shown to have analgesic(Petersen et al., 2023), anticonvulsant, muscle relaxant (Isenmann et al., 2021), neuroprotective (Fernandez-Ruiz et al., 2013), antioxidant (Atalay et al., 2019), and anti-psychotic activity (Waldo Zuardi et al., 2012). In addition to binding to CB1 R and CB2R of the endocannabinoid system, there is evidence that CBD activates 5-HT1A serotonergic and TRPV1-2 vanilloid receptors, antagonizes alpha-1 adrenergic and p-opioid receptors (Ibeas Bih et al., 2015; Zhornitsky and Potvin, 2012).

[0055] Since the use of one and / or more natural or synthetic vital dyes, isolated and / or associated with other dyes, namely BBG and / or TB, emerge as an innovation in the medical field of ophthalmology by introducing a natural or synthetic CBD-based isolated and / or associated with other natural or synthetic dyes into the pharmaceutical formulation being capable of dye or stain living cells or tissues, both in vitro and in vivo. Therefore, this invention is in the technical domain of medical and pharmaceutical industry, and related ones.

[0056] The present invention introduces a novel technique for staining intraocular structures and membranes, aiming to enhance visualization, precision, and safety during surgical procedures. This technique involves the use a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, to achieve optimal staining of the vitreous and retinal tissue.

[0057] These compositions selectively stain the biological structure of interest, staining the target biological membranes and structures at a concentration that is physiologically and toxicologically acceptable, allowing for the use of the minimum concentration of dye that provides visible differentiation between the selected biological membranes and the surrounding. Despite the low or non-existent toxicity of these dyes, any remaining pharmaceutical composition in the surgical and medical field is promptly removed after the procedure, further reducing the possibility of post-process adverse effects.

[0058] The invention also offers a method for staining biological membranes and structures with these pharmaceutical compositions. This method allows for the selective staining of the biological structure of interest, leavin the surroundingstructures undyed or less dyed. This leads to a more precise identification of the desired biological membranes and structures and facilitates its surgical manipulation or removal, reducing the risk of damaging the surrounding tissues and, consequently, reducing the recovery time. The pharmaceutical compositions and methods described here can improve the contrast between different types of biological tissues during surgical or other procedures that require staining of biological material, along with the increased protection to tissues and cells from light damage in the surgical field. In addition, the present invention allows for the protection of intraocular structures through topical application or injection of pharmaceutical compositions containing the natural substance a natural or synthetic CBD-based vital dye . This method poses a low risk of toxicity for the patient.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] The primary objective of this project is to explore an innovative technique for the coloration of various ocular tissues. This technique employs a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, along with / or without BBG and / or TB and / or other natural or synthetic dyes, to achieve optimal staining of ocular membranes and structures, presenting a promising approach for a wide range of ocular procedures, including but not limited to vitreoretinal surgery.

[0061] A comprehensive review and critical analysis of the existing literature was undertaken to gain a deeper understanding of the application method of this technique across various ocular procedures currently in practice are ineffective and unsafe for patients due to the challenges mentioned above. This project aims to contribute significantly to the surgical and medical field by providing a more target-delivery of the purposed dye, as it will have affinity to the receptors of the endocannabinoid system present in the biomolecular membranes and structures of the human eye and potentially safer alternative for tissue staining in surgeries, along with shield tissues and cells from surgical light-induced damage due the natural or synthetic CBD-based molecular anti-inflammatory and antioxidant properties that could provide cellular protection over an extended period post-operation.

[0062] 1. DYES OF THE COMPOSITIONS OF THE INVENTION

[0063] The present invention describes the use of a pharmaceutical compositions for dyeing or staining membranes and other biological structures using a natural or synthetic CBD- based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, as a promising approach in surgery, such as vitreoretinal. Those pharmaceutical compositions are to be used as a practice for dyeing or staining biological membranes and structures in surgery, in order to facilitate their identification and biomolecular membrane composition during surgical procedures such as vitreoretinal surgery, resulting in the visualization of different types of ocular membranes and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light.

[0064] The first aspect of the present invention relates to pharmaceutical compositions comprising a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, namely BBG and / or TB dyes and pharmaceutically acceptable vehicles.

[0065] Staining pharmaceutical compositions according to the present invention comprise a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, as a main dyeing and / or delivery agent (please see Formula 1 below). It also ensure protection tissues and cells from surgical light-induced damage due the natural or synthetic CBD- based molecular anti-inflammatory and antioxidant properties during and after surgical or medical procedures.

[0066] CBD, is a safe and an effective natural molecule for staining biological tissues and structures that can be used in surgical procedures, such as chromovitrectomy, and it not only provides a clear differentiation between the target and surrounding structures by providing an adequate identification and manipulation of the biological membranes and structures for the ophthalmic surgeon but also minimizes iatrogenic retinal complications associated with the surgical or medical procedure. This innovative approach, therefore, offers a saferand more effective method for ocular surgeries, potentially revolutionizingthe field of ophthalmology and significantly improving surgical outcomes and patient safety.

[0067] Formula 1 . Although CBD is a colourless molecular, after degradation in the membrane cells, it may appear purple due to the increase in the concentration of the photochemically unstable oxidation product CBD-hydroxyquinone (HU-331 , Formula 2). In the presence of oxygen, water and / or exposed to natural light / dark cycles, this hydroxyquinol rapidly reacts to form a multitude of violet / purple -coloured cannabinoid by-products (Thomson et al., 2023).

[0068] Formula 2.

[0069] Previous studies support evidence that HU-331 displays a potential anticancer therapeutic activity without some of the known adverse events associated with traditional anticancer agent, exhibiting to be much less cardiotoxic than chemotherapy drug doxorubicin (Kogan et al., 2007b, 2007a; Trac et al., 2021). For evaluation of the antiangiogenic action of HU- 331 was found to be strongly antiangiogenic, significantly inhibiting angiogenesis at low concentrations inducing apoptosis of vascular endothelial cells without changing the expression of pro- and antiangiogenic cytokines and their receptors(Kogan et al., 2006).

[0070] BBG (refer to Formula 3.) is a specialized stain that selectively targets the internal biological membranes and structures within the eye. This unique property enhances the visibility of the ILM during surgical procedures, thereby facilitating its removal.

[0071] Formula 3.

[0072]

[0073] BBG is a triphenylmethane dye that interacts electrostatically with the amino and carboxyl groups of proteins, allowing for protein staining. The colour of the dye changes depending on the acidity of the solution, due to the charge states of the dye molecule. While at pH levels below 0, the dye appears red because of the positively charged nitrogen atoms, at pH levels above 2, the negatively charged sulfonic acid groups produce a bright blue colour. This pH-responsive behaviour makes it useful in various scientific applications. This unique combination of properties makes it a valuable component pharmaceutical and medical field. Despite its prevalent use, the underlying mechanism that enables its selective staining of the ILM remains a subject of ongoing research.

[0074] Trypan Blue, also referred to as Diamine Blue or Niagara Blue (please referto Formula 4), is a synthetic azo dye that selectively colours biological membranes and structures. It is particularly useful in ophthalmic cataract surgery, where it stains the anterior capsule in the presence of a mature cataract, enhancing visualisation prior to a continuous curvilinear capsulorhexis, an important step in of modern phacoemulsification, which has crucial influence on the surgical process and prognosis.

[0075] Formula 4.

[0076]

[0077] TB is used as a visual aid during ophthalmic surgical vitrectomy procedures to stain the epiretinal membranes and facilitate their removal. TB ophthalmic drops are utilised in posterior eye surgery to selectively stain membranes, such as Epiretinal Membranes and Internal Limiting Membranes, to improve surgical outcomes.

[0078] Those pharmaceutical compositions are to be used as a practice for dyeing or staining biological membranes and structures in surgery, in order to facilitate their identification and biomolecular membrane composition during surgical procedures such as vitreoretinal surgery, resulting in the visualization of different types of ocular membranes and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light.

[0079] 2. FORMULATIONS OF THE INVENTION

[0080] The present invention describes the use of a pharmaceutical compositions for dyeing or staining membranes and other biological structures using a natural or synthetic CBD- based vital dye isolated and / or associated with other dyes, namely BBG and / or TB, as a promising approach in surgery, such as vitreoretinal. Those pharmaceutical compositions are to be used as a practice for dyeing or staining biological membranes and structures in surgery, in order to facilitate their identification and biomolecular membrane composition during surgical procedures such as vitreoretinal surgery, resulting in the visualization of different types of ocular membranes and tissues along with the cellular and tissue protection from damage induced by the exposition to surgical light.

[0081] Within this framework of thought, the present invention proposes a pharmaceutical formulation with the following composition:

[0082] • Composition of Cannabinoid-based vital dye This formulation contains the main active ingredient (Cannabinoids) mainly CBD- based stain responsible for staining biological tissues and structures during surgical procedures that can be either natural or synthetic, which can be either isolated and / or associated with other dyes, including but not limited to BBG and / or TB.

[0083] Cannabinoids could also have neuroprotective and plasticity-mediating properties, along with anti-inflammatory and antioxidant properties that could provide ocular protection during surgery.

[0084] • Composition of natural or synthetic CBD-based vital dye and / or associated with other dyes, namely BBG and / or TB

[0085] This formulation contains the main active ingredient (Cannabinoids) mainly CBD- based stain responsible for staining biological tissues and structures during surgical procedures that can be either natural or synthetic, and / or associated with other dyes, namely BBG and / or TB.

[0086] This formulation has the capacity to combine the CBD-based vital dye either natural or synthetic and / or with additional dyes, which contributes to the staining effect since it can act as a target delivery mediator of the others known to be dyeing agents currently used for intraocular membranes, as mentioned above, and will provide anti-inflammatory and antioxidant biomolecular mechanisms that could provide cellular protection due to the ocular instability / damage and oxidative stress induced by surgical light.

[0087] The pharmaceutically acceptable vehicles currently used, could achieve the creation of larger molecules, being a carrier system that allows controlled release of the innovative formulation and have the potential to improve effectiveness. Altering the molecular weight of an ocular dye will improve its adherence to retinal membranes during vitrectomy by utilizing the molecular sinking effect. For instance, having the patient in a supine position (lying face up) during the dye delivery can also optimize dye contact with the ocular surface, due to the higher viscosity formulation for optimal adherence during surgery along the target-delivery provided by the natural or synthetic CBD-based vital dye isolated and / or associated with other dyes formulation.

[0088] Composition of natural or synthetic CBD-based vital dye and / or associated with other dyes, namely BBG and / or TB, include acceptable pharmaceutical vehicles including but not limited to 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 its 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, among others.

[0089] Within this framework oESthought, the pharmaceutical composition o0the present invention comprise a natural or synthetic CBD-based vital dye isolated and / or associated with other dyes, as the main dye in concentrations ranging ETom 0.001 to 10%, preferably o00.005 to 0.5%, even more definitely in the optimal concentration oE?l0.05%.

[0090] Brilliant blue (G or FCF) can be present in the pharmaceutical composition oESthe present invention in a concentration of?l0.025 to 0.05% o0the total composition, preferably 0.045 to 0.055%.

[0091] TB can be present in the pharmaceutical composition 00 the present invention in a concentration o00.001 to 5% ofthe total composition, preferably of?l0.04 to 0.2%.

[0092] These compositions selectively stain the biological structure 00 interest, leaving the surrounding structures undyed or less dyed, staining the target biological membranes and structures at a concentration that is physiologically and toxicologically acceptable, allowing ie>r the use 00 the minimum concentration 00 dye that provides visible differentiation between the selected biological membranes and the surrounding. Despite the low or non-existent toxicity oESthese dyes, any remaining pharmaceutical composition in the surgical and medical Held is promptly removed aESer the procedure, Elirther reducing the possibility oESpost-process adverse effects.

[0093] The compositions described in this invention can be formulated as solutions, dispersions, suspensions, or emulsions, providing a wide range oESapplications, which contain a natural or synthetic CBD-based vital dye, either alone or in combination with other dyes, namely BBG and / or TB.

[0094] As explained above there are many benefits associated with the use oH natural dyes, including: easy identification oESeye membrane and biological layers to identify and remove; prevention of side effects caused by incomplete removal of membranes and biological structures; reducing the time of surgery with reduction in medical and hospital fees; increased safety because the proposed dye is a natural dye, alone or in combination with other dyes and all the alternatives on the market today have demonstrated toxicity; and possible antioxidant and antiinflammation effect associated with the cannabinoid molecules along with the targeted delivery strategy for BBG and / or TB.

[0095] 3. PROCESS FOR PRODUCING THE PHARMACEUTICAL COMPOSITIONS

[0096] The overall process of pharmaceutical purposed composition preparation includes diluting the selected dyes with or without pharmaceutical vehicle to achieve the homogeneous final concentrations of:

[0097] • Natural or synthetic CBD-based vital dye as the main dye: ranging from 0.001 to 10%, preferably of 0.005 to 0.5%, even more definitely in the optimal concentration of 0.05%.

[0098] • Brilliant blue (G or FCF): 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055%.

[0099] • Trypan blue: 0.001 to 5% of the total composition, preferably of 0.04 to 0.2%.

[0100] • A pharmaceutical acceptable vehicle is added to the above compositions. The referred compounds are already listed in the previous sections.

[0101] In addition, the dye can be prepared as a solution, suspension, dispersion, or emulsion using established methods in the field. All procedures are carried out under the necessary conditions for the production, management, and processing of pharmaceutical-grade materials. The mixture is then sterilized and packaged into suitable containers for storage and transportation.

[0102] 4. METHOD FOR STAINING BIOLOGICAL MATERIAL

[0103] 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.

[0104] Example 1.

[0105] A pharmaceutical composition with a natural or synthetic CBD-based vital dye

[0106] 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.

[0107] Example 2 .

[0108] A pharmaceutical composition with a natural or synthetic CBD-based vital dye and brilliant blue B

[0109] All the steps were performed as described in Example 1 with exception of the composition is the following: A natural or synthetic CBD-based vital dye and brilliant blue were weighted in separate and diluted together / or without a pharmaceutical vehicle to achieve amounts of a natural or synthetic CBD-based vital dye ranging from 0.001 to 10%, preferably of 0.005 to 0.5%, even more definitely in the optimal concentration of 0.05% and brilliant blue of 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055% to obtain final homogeneous composition.

[0110] Example 3.

[0111] A pharmaceutical composition with a natural or synthetic CBD-based vital dye and trypan blue

[0112] Similarly, to the described in example 2, a natural or synthetic CBD-based vital dye and trypan blue were weighted in separate and diluted together / or without a pharmaceutical vehicle to achieve amounts of a natural or synthetic CBD-based vital dye ranging from 0.001 to 10%, preferably of 0.005 to 0.5%, even more definitely in the optimal concentration of 0.05% and trypan blue of 0.001 to 5% of the total composition, preferably of 0.04 to 0.2% to obtain final homogeneous composition.

[0113] Example 4.

[0114] A pharmaceutical composition with a natural or synthetic CBD-based vital dye, brilliant blue FCF and trypan blue

[0115] In this example, all the described steps of the previous examples were followed and a pharmaceutical composition having a natural or synthetic CBD-based vital dye ranging from 0.001 to 10%, preferably of 0.005 to 0.5%, even more definitely in the optimal concentration of 0.05%, brilliant blue of 0.025 to 0.05% of the total composition, preferably 0.045 to 0.055% and trypan blue of 0.001 to 5% of the total composition, preferably of 0.04 to 0.2% to obtain final homogeneous composition.

[0116] Example 5. Pharmaceutical compositions with different concentrations of a natural or synthetic CBD-based vital dye alone or in combination with Indocyanine Green

[0117] Using the process described on the previous examples, countless pharmaceutical compositions containing different concentrations of the three dyes involved can be obtained.

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Claims

CLAIMS1. A pharmaceutical composition for staining and protection of biological structures comprising a CBD-based vital dye with a concentration of 0.001 to 10%.

2. A pharmaceutical composition for staining and protection of biological structures according to claims 1 comprising a CBD-based vital dye with a concentration of 0.5%.

3. A pharmaceutical composition for staining and protection of biological structures according to claims 1 to 2 additionally comprising other dying agents, namely one or a combination of brilliant blue G and trypan blue.

4. A pharmaceutical composition for staining and protection of biological structures according to claim 3 where the concentration of the brilliant blue G is of 0.025 to 0.05% of the total composition .

6. A pharmaceutical composition for staining and protection of biological structures according to claim 4 where the concentration of the trypan blue is of 0.001 to 5% of the total composition.

7. A pharmaceutical composition for staining and protection of biological structures according to claims 6 where the concentration of the trypan blue is of 0.05%.

8. A pharmaceutical composition for staining and protection of biological structures according to any of the previous claims additionally comprising a pharmaceutical acceptable vehicle.

9. A pharmaceutical composition for staining and protection of biological structures according to claim 8 where the pharmaceutical acceptable vehicle is one of 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, propyleneglycol, purified water, sodium acetate, sodium chloride , sodium citrate , sodium edetate , sodium phosphate , sodium phthalate or tweens .10 . A pharmaceutical composition for staining and protection of biological structures according to any of the previous claims wherein said pharmaceutical composition can be formulated as a solution, dispersion, suspension or emulsion, depending on the application .11 . A method for staining biological membranes and structures comprising the usage of the pharmaceutical formulation as described claims 1 to 10 .12 . A method for staining biological membranes and structures according to claim 13 wherein the pharmaceutical composition is applied topically or by inj ection to the biological material to be stained and protected .13 . A method for staining biological membranes and structures according to claims 11 and 12 for use in eye surgery and other medical interventions .14 . A method for staining biological membranes and structures according to claims 11 to 13 for use in chromovitrectomy .15 . A process for producing a pharmaceutical composition, as described in claims 1 to 10 , comprising the steps of diluting CBD- based vital dye in a pharmaceutical vehicle .16 . A process for producing a pharmaceutical composition according to claim 15 further comprising the addition of a second contrasting agent , namely brilliant blue G or trypan blue .

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