BR102020015496A2 - PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF EYE DISEASES ASSOCIATED WITH THROMBUS AND CLOTH FORMATION IN RETINAL VEINS AND USE - Google Patents

Abstract

pharmaceutical composition for treating eye diseases associated with thrombus and clot formation in retinal veins and use. the present technology relates to a pharmaceutical composition containing leuc-a for the treatment of retinal vein occlusion. the fibrinolytic enzyme, leuc-a, can be administered in the form of an aqueous solution by intravitreal injection, as a new alternative for the treatment of retinal vein occlusion (vr).

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF EYE DISEASES ASSOCIATED WITH THROMBUS AND CLOTH FORMATION IN RETINAL VEINS AND USE

[001] The present technology relates to a pharmaceutical composition containing Leuc-a for the treatment of retinal vein occlusion. The fibrinolytic enzyme, Leuc-a, can be administered in the form of an aqueous solution by intravitreal injection, as a new alternative for the treatment of retinal vein occlusion (RVO).

[002] The technology described here belongs to the field of ophthalmology and aims to provide a new and effective therapeutic option for the treatment of retinal vein occlusion, due to its ability to degrade fibrin clots.

[003] Degenerative diseases of the retina are the leading cause of blindness in the world and for which there is currently no cure (Petit, L.; Punzo, C. Gene Therapy Approaches For The Treatment Of Retinal Disorders. Discovery Medicine, v. 22, no. 121, p. 221-229, 2016). Associated with degenerative diseases are other disorders that are equally harmful to vision and constitute an important public health problem, such as retinal vascular obstructions.

[004] OVRs are a heterogeneous group of chronic disorders that present impaired venous return from the retinal circulation (IP, M.; Hendrick, A. Retinal vein occlusion review. Asia-Pacific Journal of Ophthalmology, v. 7, n. 1, p. 40-45, 2018). They can be subdivided into branched retinal vein occlusion (RVO), which occurs when a branch of the central vein is obstructed, and central retinal vein occlusion (RCO), when the outflow of the central vein is impaired, the latter being the most severe case, since the entire retina is affected (Cehofski, L. J.; et al. Review: Proteomics in retinal artery occlusion, retinal vein occlusion, diabetic retinopathy and acquired macular disorders. International Journal of Molecular Sciences, v. 18, No. 5, p. 907, 2017).

[005] Currently, the vast majority of treatments are aimed at complications resulting from OVR, such as ischemia, neovascularization, macular edema and glaucoma, however, few advances have been made to reverse retinal vascular obstruction. Among the most used treatments are laser photocoagulation, anti-VEGF (Vascular Endothelial Growth Factor) therapy, steroid implants and vitrectomy (Jonas, J. B. et al. Retinal vein occlusions. Developments in Ophthalmology, v. 58, p. 139- 167, 2017.; Kida, T. Mystery of Retinal Vein Occlusion: Vasoactivity of the Vein and Possible Involvement of Endothelin-1. BioMed Research International, v. 2017, 2017; Pierru, A. et al. Occlusions veineuses rétiniennes ଝ Retinal vein occlusions. Journal de Gynecologie Obstetrique et Biologie de la Reproduction, 2017. )

[006] The eye is an organ that presents many anatomical and physiological barriers designed to keep the systemic circulation separate from the ocular tissues, such as the cornea and anterior segment barriers, conjunctival plasma clearance mechanisms, nasolacrimal drainage, the choroid-membrane complex of Bruch and the blood-retinal barrier (HRB). Thus, the delivery of drugs to specific intraocular targets in therapeutic concentrations is limited, especially with regard to tissues of the posterior segment of the eye, such as the retina, in such a way that the treatment of diseases that affect this region is limited. becomes a challenge (Huang, D. et al. Overcoming ocular drug delivery barriers through the use of physical forces. Advanced Drug Delivery Reviews, 2017; Lalu, L. et al. Novel nanosystems for the treatment of ocular inflammation: Current paradigms and future research directions. Journal of Controlled Release, 2017; Tsai, C. et al. Ocular Drug Delivery: Role of Degradable Polymeric Nanocarriers for Ophthalmic Application. Molecular Sciences, v. 19, p. 1-20, 2018).

[007] The injection of drugs directly into the vitreous cavity presents itself as an alternative to reach the necessary concentration in the retinal tissue, since, when injected into the vitreous humor, the drug diffuses in several directions and crosses the inner membrane. limiting factor for the retina (Ogura, Y. Drug delivery to the posterior segments of the eye. Advanced Drug Delivery Reviews, v. 52, p. 1-3, 2001; Penha, F. M. et al. Retinal and Ocular Toxicity in Ocular Application of Drugs and Chemicals - Part II: Retinal Toxicity of Current and New Drugs, Ophtalmic Research, v. 44, pp. 205-224, 2010; Tsai, C. et al. Ocular Drug Delivery: Role of Degradable Polymeric Nanocarriers for Ophthalmic Application. Molecular Sciences, v. 19, p. 1-20, 2018).

[008] Snake venoms are a rich source of biochemically active molecules that have aroused the interest of researchers in understanding their mechanism of action in some physiological systems for the development of new therapeutic agents directed to specific protein/receptor targets such as hemostasis/thrombosis and vasculature proteins, to arrive at a pharmacological application. The genus Bothrops is made up of several species of snakes distributed throughout South and Central America, and in Brazil the species Bothrops leucurus (B. leucurus), responsible for the highest rates of snakebite envenoming, is found in the northeast and part of Southeast, mainly in the states of Bahia and Espírito Santo.

[009] These venoms are a complex of biologically active proteins and peptides that, when injected into the victim, disrupt normal physiological and biochemical processes, causing local and systemic injuries such as pain, edema, hemorrhage, tissue necrosis and disorders in hemostasis and blood clotting.

[010] Among these compounds, metalloproteinases are predominant and conserved proteins in the venoms of viperidae snakes, for example Bothrops, and some elapids, for example. Cobra sp. Some of these molecules are interesting models for the development of new drugs for therapeutic use, such as hemostatic disorders and thrombosis. The purification and characterization of these substances demonstrated the interest in the important role associated with the vascular system, especially the mechanisms that coordinate the coagulation cascade, hemostasis and platelet function (Sanchez, E.F., et al. A novel fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (Barnett's pitviper) snake venom with anti-platelet properties. Biochimica et Biophysica Acta, v. 1860, n. 3, p.542-556, 2016; De Souza, R. A. et al. Unraveling the distinctive features of hemorrhagic and non-hemorrhagic snake venom metalloproteinases using molecular simulations. Journal of Computer-Aided Molecular Design, v. 30, n. 1, p. 69-83, 2016; Gomes, M. S. R. et al. Purification and functional characterization of a new metalloproteinsa (BleucMP) from Bothrops leucurus sanke venom. Comparative Biochemistry and Physiology, Part C, v. 153, n. 3, p. 290-300, 2011; Higuchi, D. A. et al. Toxicon Leucurogin , a new recombinant disintegrin cloned from Bothrops leucu rus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth. Toxicon, v. 58, no. 1, p. 123-129, 2011).

[011] Snake venom metalloproteinases (SVMPs) are zinc-dependent proteases divided into three classes (P-I, P-II and P-III) based on their structural organization. Class P-I SVMPs are formed by the metalloprotease (M) domain, which contains zinc in the catalytic center; whereas the P-II contain the disintegrin domain (D) with the Arg-Gly-Asp sequence (fibrinogen receptor) in addition to the M domain, and the P-III class has the cysteine-rich domain (C) at the C-terminus. . Some of them, particularly those of class III, have the ability to induce severe hemorrhage, through the hydrolysis of proteins present in the basement membrane of the extracellular matrix (eg collagen IV, laminin), in addition to affecting platelet aggregation by acting on membrane receptors. of platelets. SVMPs are insensitive to the action of plasma serine proteinase inhibitors (eg anti-thrombin). Class I SVMPs (~23-kDa) are rapidly inhibited and cleared from circulation by the main plasma protease inhibitor, a2-macroglobulin (Sanchez, E. F. et al. Direct fibrinolytic snake venom metalloproteinases affecting hemostasis: Structural, biochemical features and therapeutics potential. Toxins, v. 9, n. 12, 2017; Escalante, T. et al. Role of collagens and perlecan in microvascular stability: Exploring the mechanism of capillary vessel damage by snake venom metalloproteinases. PLoS ONE, v. 6, n . 12, 2011). Some of these P-I enzymes (without hemorrhagic activity) are interesting models for the development of new therapeutic agents in medicine, particularly in the area of hemostasis/thrombosis.

[012] Among the non-hemorrhagic metalloproteinases purified from B. leucurus venom is Leuc-a, a 23 kDa α-fibrinogenase of the P-I class. It is composed of 202 amino acid residues, being able to dissolve fibrin clots through hydrolysis of fibrin/fibrinogen alpha chains, in addition to affecting platelet aggregation. (Bello, C. A., et al. Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed pit viper) snake venom. Biochimie, v. 88, n. 2, p. 189-200, 2006). Furthermore, leuk-a does not exert proteolytic activity on capillary basement membrane and extracellular matrix (ECM) proteins (Ferreira, R. N. et al. Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a , a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venom. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, v. 65, n. 8, p. 798-801, 2009; Gremski, L. H. et al. Cytotoxic , thrombolytic and edematogenic activities of leucurolysin-a , a metalloproteinase from Bothrops leucurus snake venom. Toxicology in Vitro, v. 50, p. 120-134, 2007; Bello, C. A., et al. Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed) jararaca) snake venom. Biochimie, v. 88, n. 2, p. 189-200, 2006).

[013] Considering that the treatment of venous thrombosis is mainly through the use of thrombolytic agents derived from tissue plasminogen activator (t-PA) and that, despite demonstrating effectiveness in dissolving the thrombus, bring with them the inevitable risk of bleeding , it is necessary to search for new alternatives that are equally effective, but with greater vascular efficacy and safety. Thus, Leuc-a, whose fibrinolytic/thrombolytic activity has already been elucidated, appears as a potential alternative for the treatment of retinal vein occlusions. However, despite being promising, the elucidation of the biological activity of peptides, proteins/toxins extracted from snake venoms with therapeutic potential and/or as models for the development of new therapeutic agents for the treatment of eye diseases and complications is still very recent, so that very few works can be found in the literature reporting this use.

[014] Figure 1 represents the cell viability curve in the ARPE-19 line after treatment with a product containing Leuc-a for 48 hours, at the different concentrations tested, by the Sulfarrhodamine B method.

[015] In Figure 2 are the photographs of the cells of the ARPE-19 lineage stained with Sulfarrhodamine B, and the figures from A to G represent the different concentrations tested of the product containing Leuc-a, , being (A) control; (B) Leuc-a 12.5 µg/ml; (C) Leuc-a 25 µg/ml; (D) Leuc-a 50 µg/ml; (E) Leuc-a 100 µg/ml; (F) Leuc-a 200 μg/mL and (G) Leuc-a 500 μg/mL.

[016] In Figure 3 are the fundus images of Wistar rats, after intravitreal administration of different concentrations of the product containing Leuc-a in the pre-injection, 7 and 14 days after the injections.

[017] Figure 4 represents the graph of the variation of intraocular pressure in Wistar rats after intravitreal administration of different concentrations of the product containing Leuc-a in the pre-injection, 7 and 14 days after injections.

[018] Figure 5 represents the electroretinogram curve in the scotopic condition, 7 and 14 days after intravitreal injections of different concentrations of the product containing Leuc-a in eyes of Wistar rats, being (A) vehicle; (B) Leuc-a 12.5 µg/ml; (C) Leuc-a 25 µg/ml; (D) Leuc-a 50 μg/mL and (E) Leuc-a 100 μg/mL.

[019] Figure 6 shows the measurements of the amplitudes of waves a and b in steps 6 and 11 of the different concentrations tested, in the pre-injection periods, 7 and 14 days after the injections, being (A) the amplitude of wave a in step 11 ; (B) the amplitude of wave b in step 11 and (C) the a/b ratio in step 11. Statistical difference in relation to pre-injection: *p<0.05 **p<0.01 ***p<0.001

[020] Figure 7 represents the amplitude curves of waves a and b due to the log of light intensity at the different concentrations tested, in the pre-injection periods, 7 and 14 days after injections, , being (A) vehicle; (B) Leuc-a 12.5 µg/ml; (C) Leuc-a 25 µg/ml; (D) Leuc-a 50 μg/mL and (E) Leuc-a 100 μg/mL.

[021] In Figure 8 are shown the optical microscopy images of the retina of Wistar rats, stained by hematoxylin-eosin, at the time of 14 days after the intraviral injection of the product containing Leuc-a, being (A) vehicle; (B) Leuc-a 12.5 µg/ml; (C) Leuc-a 25 µg/ml; (B) Leuc-a 50 μg/mL and (B) Leuc-a 100 μg/mL.

DETAILED DESCRIPTION OF THE INVENTION

[022] The present technology concerns a pharmaceutical composition for the treatment of eye diseases associated with the formation of thrombi and clots in retinal veins containing Leuc-a, a protein purified from the venom of the Bothrops leucurus snake.

[023] The pharmaceutical composition is characterized by being prepared in the form of an aqueous solution for intravitreal injection.

[024] The pharmaceutical composition containing Leuc-a is characterized by comprising enzyme concentration between 10 to 50 μg/mL.

[025] The composition proposed in the present technology is characterized by being administered by intravitreal injection.

[026] The composition containing purified Leuc-a can be used to treat eye diseases associated with thrombus and clot formation in retinal veins.

[027] The present technology can be better understood through the following non-limiting examples of the technology.

Example 1: In vitro evaluation of the cell viability of the product containing Leuc-a on ARPE-19 cells

[028] In these assays, the cytotoxicity of the product containing Leuc-a was determined by cell viability by the sulfarrhodamine B (SRB) colorimetric assay, described by Skehan (1990), at concentrations of 12.5, 25, 50, 100, 200 and 500 µg/mL.

[029] ARPE-19 cells were incubated in 96-well plates at 37°C and 5% CO2 for 24 h. After this period, the prepared treatments were added in triplicate and incubated for 48 hours.

[030] After fixing the cells with 10% trichloroacetic acid (TCA) for 1 hour at 4°C, the plates were washed and dried at room temperature and stained with 100 μL of the 0.057% (w/v) SRB solution , for 30 minutes. Then, the wells were washed with 1% acetic acid solution (v/v), dried and photographed using a camera attached to an optical microscope.

[031] Next, 100 μL of 10 mM tris base solution were added to each well to solubilize the dye bound to cellular proteins. Absorbance values were measured using a plate spectrophotometer.

[032] The results were expressed as the mean of the percentage of cell viability ± standard deviation. Data were evaluated using one-way ANOVA analysis of variance with Tukey's post-test, adopting a significance level of α<0.05.

[033] From the photographs obtained and the dose-response curve, maximum cell viability of approximately 75% was obtained against this cell line.

Example 2: Evaluation of the toxicity and safety of the product containing Leuc-a in vivo.

[034] The toxicity of the product containing purified Leuc-a on the retina was evaluated after intravitreal injection into the eyes of adult male Wistar rats. The animals were divided into four treatment groups (12.5, 25, 50 and 100 μg/mL).

[035] The animals of all groups received an intravitreal injection of 5 μL of sterile PBS pH 7.4 (vehicle) in the left eyes, while in the right eyes 5 μL of the product containing Leuc-a were injected at the doses presented.

[036] After intraperitoneal anesthesia of the animals with a solution containing 10 mg/kg xylazine and 90 mg/kg ketamine, the eyes were also anesthetized with 0.1% phenylephrine hydrochloride eye drops, followed by the injections themselves using an insulin syringe. .

[037] The eyes of the treated animals were clinically evaluated by ocular inspection and binocular indirect ophthalmoscopy at pre-injection, 7 and 14 days after injections. The appearance of the retina was evaluated by analyzing the fundus of the eyes of the animals before and after the intravitreal injections of PBS (control) or Leuc-a, and it was not possible to observe hemorrhages, retinal detachment, ischemic process or other ocular alteration indicative of retinal toxicity for doses up to 50 μg/mL.

[038] Intraocular pressure (IOP) was also evaluated, using a veterinary tonometer, in the pre-injection periods, 7 and 14 days after the injections. The IOP was calculated based on the average of 3 readings for each eye, and the difference in values between the evaluated periods was calculated by subtracting the IOP value obtained on days 7 and 14 from the value obtained before the injections. Statistical analysis of the data presented confirmed that the IOP remained stable throughout the experiment.

[039] The data were evaluated by two-way ANOVA followed by the Bonferroni post-test using the GraphPad Prism®5 program and adopting a significance level of α<0.05.

[040] Functional analyzes of the retina by electroretinogram (ERG) were performed before the intravitreal injections, as a control and 7 and 14 days after the injections, according to the protocol established by the ISCEV (International Society for Clinical Electrophysiology of Vision). For this, the animals were dark-adapted for 12 h, anesthetized via intraperitoneal injection of a solution containing xylazine 10 mg/kg of ketamine 90 mg/kg. The eyes were anesthetized with 0.1% phenylephrine hydrochloride eye drops, and the pupils were dilated with 1% tropicamide eye drops 15 min before the start of the examination. The animals were positioned on a styrofoam support adapted to the Ganzfeld dome.

[041] ERG responses were acquired simultaneously in both eyes using a lens-type bipolar electrode over the cornea; Needle-type electrodes were fixed subcutaneously on the skin, in the frontal region above the eyes, and the ground electrode was fixed subcutaneously on the animal's back. The eyes were stimulated using a Ganzfeld LED stimulator.

[042] For scotopic-type examinations, flashes of white light (6500 K) lasting 4 ms were conducted in 11 steps, with steps 6 and 11 being the main ones: the light intensity of 0.01 cd.s.m-2 for evaluation of the function of the rods and the light intensity of 3.0 cd.s.m-2 for the evaluation of the function of the cones and rods.

[043] Responses were amplified (filtering: 0.3-300 Hz) and stored for off-line analysis. Data were evaluated from comparisons between the amplitudes of waves a and b, under the mentioned light stimulus intensities. Comparisons were performed both for the concentrations of Leuc-a applied and for the time interval of the exams.

[044] Data were evaluated by two-way ANOVA (α<0.05) with Bonferroni post-test, in order to compare the changes resulting from the days and concentrations evaluated, with the control eyes as a reference ( vehicle).

[045] In a first analysis of steps 6 and 11, it was observed that for doses of 12.5, 25 and 50 μg/mL there was practically no difference in amplitude or implicit time shift for waves a and b in relation to the curves of the vehicle.

[046] After verifying the statistical difference in wave amplitudes between the tested doses and the vehicle, only the dose of 100 μg/mL showed a significant reduction in the amplitude of the wave a in the period of 7 days; in addition, it is possible to notice that the statistical difference found for the 7-day period is repeated for the 14-day period with respect to wave b amplitude, that is, the reduction in wave b amplitude can be attributed to the loss of function due to the toxicity presented by Leuc-a at that dose.

[047] Furthermore, the toxicity of the product containing Leuc-a was evaluated by measuring the maximum amplitude of the wave (Vmax), which indicates the saturation of the photoreceptors. While for the vehicle there was practically no change in Vmax throughout the study, for doses of 12.5, 25 and 50 μg/mL, there was a reduction in Vmax after 7 days, which was reestablished after 14 days; at the dose of 100 μg/mL, there was a very sharp reduction in Vmax after 7 days, which was maintained up to 14 days, mainly in the b wave, which provides further evidence of retinal cell dysfunction after administration of this dose.

[048] For histological evaluation, at the end of the last ERG the animals were euthanized with an overdose of anesthetic and the eyes were prepared for analysis. The eyeballs were enucleated and fixed in Davidson's solution (95% alcohol, formaldehyde, glacial acetic acid and distilled water) for 24 h for fixation. Then, the eyes were transferred to a 70% alcohol solution, where they remained for another 24 h for dehydration. After these two steps, the eyes were sectioned transversally to the optic nerve with the aid of a scalpel. The samples were then embedded in paraffin and the micrometric sections were placed on slides and stained with hematoxylin-eosin before being taken to an optical microscope for analysis.

[049] As in the results obtained by the ERG exam, the similarity of the architecture and morphology of the retina of the eyes that received doses of 12.5, 25 and 50 pg/mL in relation to those that received PBS (vehicle ). The cell layers appear to be preserved and organized, with no evidence of misalignment, decrease in thickness or in the number of cells. In addition, no signs of necrosis, fibrosis or the presence of inflammatory infiltrate were found.

[050] For the dose of 100 μg/mL, however, the histological findings have already been shown to be close to the high toxicity presented by the ERG, in such a way that it is possible to perceive the disorganization of the cell layers and a more evident thickening of the outer nuclear layer.

Claims (6)

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF EYE DISEASES ASSOCIATED WITH THE FORMATION OF THROMBS AND CLOTS IN RETINAL VEINS, characterized by containing the enzyme Leuc-A. PHARMACEUTICAL COMPOSITION, according to claim 1, characterized in that the enzyme is a non-hemorrhagic metalloproteinase purified from B. leucurus venom. PHARMACEUTICAL COMPOSITION, according to claims 1 and 2, characterized in that it is prepared in the form of an aqueous solution. PHARMACEUTICAL COMPOSITION, according to claims 1 to 3, characterized in that the enzyme concentration is between 10 to 50 μg/mL. PHARMACEUTICAL COMPOSITION, according to claims 1 to 4, characterized in that it is administered by injection. USE of the composition defined in claims 1 to 5, characterized in that it is for the treatment of eye diseases associated with the formation of thrombus and clots in retinal veins.

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