Intravitreal lupeol: A new potential therapeutic strategy for noninfectious uveitis.

Lupeol is a pentacyclic triterpene with known anti-inflammatory effects. However, its role in the treatment of noninfectious uveitis has not been explored. This work investigated anti-inflammatory activity of lupeol in ocular tissues with in vitro and in vivo models. First, we evaluated the effect of lupeol (100 µM) on inflammatory response induced by lipopolysaccharide (LPS) in retinal pigment epithelium cells (ARPE-19) by measuring levels of released interleukins (IL-6 and IL-8). Then, we investigated the anti-inflammatory action of intravitreal lupeol in a rodent model of panuveitis induced by Mycobacterium bovis Calmette-Guérin Bacillus (BCG). Rats were submitted to electroretinography and clinical analyses on days 3, 7, and 15 after uveitis induction. In addition, histopathological analysis, and indirect quantification of myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) in the posterior segment were performed. Treatment with lupeol (100 µM) significantly decreased IL-6 and IL-8 levels in comparison to untreated LPS-activated ARPE-19 cells. This reduction was similar to that detected in ARPE-19 cells treated with dexamethasone. The results of the in vivo assay demonstrated that intravitreal lupeol is able to modulate inflammation in the anterior and posterior segment of the rat eyes, indicating that it should be further investigated as a novel potential candidate for management of uveitis.

Anti-angiogenic activity and safety of intraocular application of triterpenes.

PURPOSE: Assessment of the anti-angiogenic activity and the safety of ophthalmic use of four pentacyclic triterpenes (friedelin, friedelinol, lupenone, and lupeol). METHODS: Triterpenes cytotoxicity (5-640 µmol L-1) was examined in ARPE-19 cells by sulforhodamine B colorimetric method, and the anti-angiogenic activity (50-1000 µmol L-1) was evaluated in the chorioallantoic membrane model. Full-field electroretinography and histological analysis were performed to evaluate intraocular effects of these four triterpenes (at 100 or 500 µmol L-1) in eyes of Wistar rats, for 15 days. RESULTS: In the cytotoxicity assay, friedelin and friedelinol were not able to drastically reduce cell growth. A dose-dependent response was observed in groups exposed to lupeol or lupenone. During the chorioallantoic membrane assay, friedelinol at 500 µmol L-1 reduced the vascularity in 26%; lupeol and lupenone showed promising anti-angiogenic activity, reducing three parameters: vascularized area (> 30%), number of junctions (> 20%), and vessel length (> 15%). According to the electroretinographic and histologic findings, triterpenes at 100 µmol L-1 or lupenone at 500 µmol L-1 did not induce any transient or permanent disturbance in retinal structure or functioning. CONCLUSIONS: Triterpenes at 100 µmol L-1 or lupenone at 500 µmol L-1 were considered safe for potential ophthalmic use.

Intravitreal ketamine promotes neuroprotection in rat eyes after experimental ischemia.

Retinal ischemia, one of the most common cause of visual loss, is associated with blood flow inadequacy and subsequent tissue injury. In this setting, some treatments that can counteract glutamate increase, arouse interest in ischemic pathogenesis. Ketamine, a potent N-methyl-d-aspartate (NMDA) receptor antagonist, provides a neuroprotective pathway via decreasing the excitotoxicity triggered by excess glutamatergic. Thus, the goal of this study was to evaluate the safety of intravitreal use of ketamine and their potential protective effects on retinal cells in retinal ischemia/reperfusion model. Initially, ketamine toxicity was evaluated by cytotoxicity assay and Hen's egg chorioallantoic membrane (HET-CAM) method. Afterward, some ketamine concentrations were tested in rat's eyes to verify the safety of the intravitreal use. To investigate the neuroprotective effect on retinal, a single intravitreal injection of ketamine in concentrations of 0.059 mmol.L-1 and 0.118 mmol.L-1 was performed one day before the retinal injury by ischemia/reperfusion model. After 7 and 15 days, the retina activity was evaluated by electroretinogram (ERG) records and, lastly, by morphological analyzes. Cytotoxicity assay reveals that the maximum ketamine concentration that could reach retinal pigmented epithelium cells is 0.353 mmol.L-1. HET-CAM assay showed that concentrations above 0.237 mmol.L-1 are irritants to the eye. Thus, Ketamine in concentrations of 0.0237 mmol.L-1, 0.118 mmol.L-1, and 0.059 mmol.L-1 were selected for in vivo toxicity test. ERG records reveal a tendency of b-wave amplitude to decrease as the luminous intensity increased, in the group receiving ketamine at 0.237 mmol.L-1. Therefore, ketamine in concentrations at 0.059 mmol.L-1 and 0.118 mmol.L-1 were chosen for the following tests. In the ischemia retinal degeneration model, pretreatment with ketamine was capable to promote a recovery of retinal electrophysiological function minimizing the ischemic effects. In histological analysis, the groups that received intravitreal ketamine showed a number of retinal cells significantly higher than the vehicle group. In TUNEL assay a reduction on TUNEL-positive cells was observed in all the layers for both concentrations which allow to affirm that ketamine contributes to reducing cell death in the retina. Transmission electron microscopy (TEM) reaffirms this finding. Ketamine intravitreal pretreatment showed reduced ultrastructural changes. Our findings demonstrate that ketamine is safe for intravitreal use in doses up to 0.118 mmol.L-1. They seem to be particularly efficient to protect the retina from ischemic injury.

Vancomycin-loaded N,N-dodecyl,methyl-polyethylenimine nanoparticles coated with hyaluronic acid to treat bacterial endophthalmitis: Development, characterization, and ocular biocompatibility.

Vancomycin-loaded N,N-dodecyl,methyl-polyethylenimine nanoparticles coated with hyaluronic acid (VCM-DMPEI nanoparticles/HA) were synthesized as an adjuvant for the treatment of bacterial endophthalmitis. The nanoparticles were formulated by experimental statistical design, thoroughly characterized, and evaluated in terms of bactericidal activity and both in vitro and in vivo ocular biocompatibility. The VCM-DMPEI nanoparticles/HA were 154 ± 3 nm in diameter with a 0.197 ± 0.020 polydispersity index; had a + 26.4 ± 3.3 mV zeta potential; exhibited a 93% VCM encapsulation efficiency; and released 58% of the encapsulated VCM over 96 h. VCM and DMPEI exhibited a synergistic bactericidal effect. The VCM-DMPEI nanoparticles/HA were neither toxic to ARPE-19 cells nor irritating to the chorioallantoic membrane. Moreover, the VCM-DMPEI nanoparticles/HA did not induce modifications in retinal functions, as determined by electroretinography, and in the morphology of the ocular tissues. In conclusion, the VCM-DMPEI nanoparticles/HA may be a useful therapeutic adjuvant to treat bacterial endophthalmitis.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage.

BACKGROUND: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. METHODS: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. RESULTS: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. CONCLUSIONS: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

PLGA- corosolic acid implants for potential application in ocular neovascularization diseases

Angiogenesis is the formation of new blood vessels from preexisting vasculature. Uncontrolled angiogenesis is associated with progression of several ocular pathologies, such as diabetic retinopathy and macular degeneration. Thus, the inhibition of this process consists in an interesting therapeutic target. Corosolic acid (CA) is a natural derivative of ursolic acid, found in many medicinal herbs and exhibits numerous biological properties, including the antiangiogenic activity. The present study reports the production of CA-loaded poly d,l-lactidecoglycolide acid (PLGA) devices by melt technique. HPLC-UV method was developed and validated to evaluate the uniformity and the release profile of the developed systems. The devices were also characterized by Fourier transform infrared spectroscopy, thermal analysis, and scanning electron morphology. It was studied the antiangiogenic activity of the CA-polymer system, using an in vivo model, the chorioallantoic membrane assay (CAM). CA was dispersed uniformly in the polymer matrix and no chemical interaction between the components of the formulation was verified. The implants presented a sustained release of the drug, which was confirmed by the morphological study and demonstrated an antiangiogenic activity. Therefore, the developed delivery system is a promising therapeutic tool for the treatment of ocular diseases associated with neovascularization or others related to the angiogenic process.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage

PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.(AU)

Corosolic acid: antiangiogenic activity and safety of intravitreal injection in rats eyes.

PURPOSE: Investigate the potential application of corosolic acid (CA) in the treatment of diseases causing retinal neovascularization. METHODS: CA cytotoxicity effect was evaluated in ARPE-19 cells by sulforhodamine B colorimetric method, and antiangiogenic activity was studied using chorioallantoic membrane (CAM) assay. An amount of 0.01 mL of CA formulations at 5, 10 and 25 µM was injected in the right eyes of Wistar rats, and the contralateral eyes received the vehicle to verify the safety of ophthalmic use. Electroretinography (ERG) was performed before, 7 and 15 days after CA administration. Animals were killed on the 15th day, and the histological analysis of retina was carried out under light microscopy. RESULTS: CA did not present cytotoxicity at concentrations below 35.5 µM after 48 h of treatment. The antiangiogenic activity was confirmed by CAM assay, since CA (range from 5 to 25 µM) induced a significant reduction in vascularity without any signs of toxicity. ERG recordings and histological evaluation did not show any signs of retinal toxicity. CONCLUSIONS: CA was effective in reducing vascularity in a CAM model and was found to be safe for potential ophthalmic use.

Intravitreal injection of the synthetic peptide LyeTx I b, derived from a spider toxin, into the rabbit eye is safe and prevents neovascularization in a chorio-allantoic membrane model.

BACKGROUND: The great diversity of molecules found in spider venoms include amino acids, polyamines, proteins and peptides, among others. Some of these compounds can interact with different neuronal receptors and ion channels including those present in the ocular system. To study potential toxicity and safety of intravitreal injection in rabbits of LyeTx I b, a synthetic peptide derived from the toxin LyeTx I found in venom from the spider Lycosa eritrognatha and to evaluate the angiogenic activity on a CAM model. METHODS: ARPE-19 cells were treated with LyeTx I b (0.36; 0.54; 0.72; 2.89; 4.34 or 9.06 µM). In this study, New Zealand rabbits were used. LyeTx I b (2.89 µM) labeled with FITC dissolved in PBS, or only PBS, were injected into vitreous humor. Electroretinogram (ERG) was recorded 1 day before injection and at 7, 14 and 28 days post-injection. Clinical examination of the retina was conducted through tonometer and eye fundus after ERG. Eyes were enucleated and retinas were prepared for histology in order to assess retinal structure. CAMs were exposed to LyeTx I b (0.54; 0.72; 2.17 or 2.89 µM). RESULTS: ARPE-19 cells exposed to LyeTx I b showed cell viability at the same levels of the control. The fluorescence of LyeTx I b labeled with FITC indicated its retinal localization. Our findings indicate ERG responses from rats injected in the eye with LyeTx I b were very similar to the corresponding responses of those animals injected only with vehicle. Clinical examination found no alterations of intraocular pressure or retinal integrity. No histological damage in retinal layers was observed. CAM presented reduced neovascularization when exposed to LyeTx I b. CONCLUSIONS: Intravitreal injection of LyeTx I b is safe for use in the rabbit eye and prevents neovascularization in the CAM model, at Bevacizumab levels. These findings support intravitreal LyeTx I b as a good candidate to develop future alternative treatment for the retina in neovascularization diseases.

Intravitreal injection of the synthetic peptide LyeTx I b, derived from a spider toxin, into the rabbit eye is safe and prevents neovascularization in a chorioallantoic membrane model

The great diversity of molecules found in spider venoms include amino acids, polyamines, proteins and peptides, among others. Some of these compounds can interact with different neuronal receptors and ion channels including those present in the ocular system. To study potential toxicity and safety of intravitreal injection in rabbits of LyeTx I b, a synthetic peptide derived from the toxin LyeTx I found in venom from the spider Lycosa eritrognatha and to evaluate the angiogenic activity on a CAM model. Methods: ARPE-19 cells were treated with LyeTx I b (0.36; 0.54; 0.72; 2.89; 4.34 or 9.06 µM). In this study, New Zealand rabbits were used. LyeTx I b (2.89 µM) labeled with FITC dissolved in PBS, or only PBS, were injected into vitreous humor. Electroretinogram (ERG) was recorded 1 day before injection and at 7,14 and 28 days post-injection. Clinical examination of the retina was conducted through tonometer and eye fundus after ERG. Eyes were enucleated and retinas were prepared for histology in order to assess retinal structure. CAMs were exposed to LyeTx I b (0.54; 0.72; 2.17 or 2.89 µM). Results: ARPE-19 cells exposed to LyeTx I b showed cell viability at the same levels of the control. The fluorescence of LyeTx I b labeled with FITC indicated its retinal localization. Our findings indicate ERG responses from rats injected in the eye with LyeTx I b were very similar to the corresponding responses of those animals injected only with vehicle. Clinical examination found no alterations of intraocular pressure or retinal integrity. No histological damage in retinal layers was observed. CAM presented reduced neovascularization when exposed to LyeTx I b. Conclusions: Intravitreal injection of LyeTx I b is safe for use in the rabbit eye and prevents neovascularization in the CAM model, at Bevacizumab levels. These findings support intravitreal LyeTx l b as a good candidate to develop future alternative treatment for the retina in neovascularization diseases.(AU)

Development and validation of alternative methods by non-aqueous acid-base titration and derivative ultraviolet spectrophotometry for quantification of sildenafil in raw material and tablets

Sildenafil citrate (SILC) is a potent phosphodiesterase-5 inhibitor used for erectile dysfunction and pulmonary hypertension. This study shows two simple, fast and alternative analytical methods for SILC determination by non-aqueous titration and by derivative ultraviolet spectrophotometry (DUS) in active pharmaceutical ingredient and/or dosage forms. The quantitation method of SILC active pharmaceutical ingredient by non-aqueous acid-base titration was developed using methanol as solvent and 0.1 mol/L of perchloric acid in acetic acid as titrant. The endpoint was potentiometrically detected. The non-aqueous titration method shows satisfactory repeatability and intermediate precision (RSD 0.70-1.09%). The neutralization reaction occurred in the stoichiometric ratio 1:1 in methanol. The determination of SILC active pharmaceutical ingredient or dosage forms by DUS was developed in the linear range from 10 to 40 µg/mL, in 0.01 mol/L HCl, using the first order zero-peak method at λ 256 nm. The DUS method shows selectivity toward tablets excipients, appropriate linearity (R2 0.9996), trueness (recovery range 98.86-99.30%), repeatability and intermediate precision in three concentration levels (RSD 1.17-1.28%; 1.29-1.71%, respectively). Therefore, the methods developed are excellent alternatives to sophisticated instrumental methods and can be easily applied in any pharmaceutical laboratory routine due to simple and fast executions.