Corneal Permeability and Uptake of Twenty-Five Drugs: Species Comparison and Quantitative Structure-Permeability Relationships.

The purpose of this study was to determine corneal permeability and uptake in rabbit, porcine, and bovine corneas for twenty-five drugs using an N-in-1 (cassette) approach and relate these parameters to drug physicochemical properties and tissue thickness through quantitative structure permeability relationships (QSPRs). A twenty-five-drug cassette containing ß-blockers, NSAIDs, and corticosteroids in solution at a micro-dose was exposed to the epithelial side of rabbit, porcine, or bovine corneas mounted in a diffusion chamber, and the corneal drug permeability and tissue uptake were monitored using an LC-MS/MS method. Data obtained were used to construct and evaluate over 46,000 quantitative structure-permeability (QSPR) models using multiple linear regression, and the best-fit models were cross-validated by Y-randomization. Drug permeability was generally higher in rabbit cornea and comparable between bovine and porcine corneas. Permeability differences between species could be explained in part by differences in corneal thickness. Corneal uptake between species correlated with a slope close to 1, indicating generally similar drug uptake per unit weight of tissue. A high correlation was observed between bovine, porcine, and rabbit corneas for permeability and between bovine and porcine corneas for uptake (R2 ≥ 0.94). MLR models indicated that drug characteristics such as lipophilicity (LogD), heteroatom ratio (HR), nitrogen ratio (NR), hydrogen bond acceptors (HBA), rotatable bonds (RB), index of refraction (IR), and tissue thickness (TT) are of great influence on drug permeability and uptake. When data for all species along with thickness as a parameter was used in MLR, the best fit equation for permeability was Log (% transport/cm2·s) = 0.441 LogD - 8.29 IR + 8.357 NR - 0.279 HBA - 3.833 TT + 10.432 (R2 = 0.826), and the best-fit equation for uptake was Log (%/g) = 0.387 LogD + 4.442 HR + 0.105 RB - 0.303 HBA - 2.235 TT + 1.422 (R2 = 0.750). Thus, it is feasible to explain corneal drug delivery in three species using a single equation.

Protective effect of cinnamon on diabetic cardiomyopathy in nicotinamide-streptozotocin induced diabetic rat model.

There is an increase in the incidence and prevalence of type-2 diabetes and obesity which leads to the structural and functional changes in myocardium leading to a lethal complication called diabetic cardiomyopathy (DCM). In the present study, we investigated the preventive effect of cinnamon (3% of Cinnamomum zeylanicum bark powder in AIN-93 diet for 3 months) feeding on DCM and the concerned mechanisms in a rodent model. Experimental diabetes was induced by a single intraperitoneal injection of 40 mg/kg b.w streptozotocin (STZ), 15 min after the ip administration of 60 mg/kg b.w of nicotinamide (NA) in Wistar-NIN (WNIN) male rats. The oxidative stress parameters were investigated by assessing superoxide dismutase (SOD), glutathione-s-transferase (GST) enzyme activity, protein carbonyls and malondialdehyde (MDA) levels. The histopathology of myocardium was analyzed by H&E and Masson's trichrome staining, and scanning electron microscopy. The changes in diabetic rat heart involved the altered left ventricular parietal pericardium, structural changes in myocardial cells, enhanced oxidative stress. Masson's trichrome and H&E staining have shown increased fibrosis, and perinuclear vacuolization in NA-STZ induced diabetic rat myocardium. Cinnamon feeding prevented the oxidative stress and myocardial alterations in the heart of diabetic rats. Taken together, these results suggest that cinnamon can effectively prevent the metabolic and structural changes in NA-STZ induced diabetic cardiomyopathy.

Noninvasive monitoring of suprachoroidal, subretinal, and intravitreal implants using confocal scanning laser ophthalmoscope (cSLO) and optical coherence tomography (OCT).

To address the need for noninvasive monitoring of injectable preformed drug delivery implants in the eye, we developed noninvasive methods to monitor such implants in different locations within the eye. Cylindrical polymeric poly(lactide-co-glycolide) or metal implants were injected into isolated bovine eyes at suprachoroidal, subretinal, and intravitreal locations and imaged noninvasively using the cSLO and OCT modes of a Heidelberg Spectralis HRA + OCT instrument after adjusting for the corneal curvature. Length and diameter of implants were obtained using cSLO images for all three locations, and the volume was calculated. Additionally, implant volume for suprachoroidal and subretinal location was estimated by integrating the cross-sectional bleb area over the implant length in multiple OCT images or using the maximum thickness of the implant based on thickness map along with length in cSLO image. Simultaneous cSLO and OCT imaging identified implants in different regions of the eye. Image-based measurements of implant dimensions mostly correlated well with the values prior to injection using blade micrometer. The accuracy (82-112%) and precision (1-19%) for noninvasive measurement of length was better than the diameter (accuracy 69-130%; precision 3-38%) using cSLO image for both types of implants. The accuracy for the measurement of volume of both types of implants from all three intraocular locations was better with cSLO imaging (42-152%) compared to those obtained using OCT cross-sectional bleb area integration (117-556%) or cSLO and thickness map (32-279%) methods. Suprachoroidal, subretinal, and intravitreal implants can be monitored for length, diameter, and volume using cSLO and OCT imaging. Such measurements may be useful in noninvasively monitoring implant degradation and drug release in the eye.

Extraocular, periocular, and intraocular routes for sustained drug delivery for glaucoma.

Although once daily anti-glaucoma drug therapy is a current clinical reality, most therapies require multiple dosing and there is an unmet need to develop convenient, safe, and effective sustained release drug delivery systems for long-term treatment to improve patient adherence and outcomes. One of the first sustained release drug delivery systems was approved for the reduction of intraocular pressure in glaucoma patients. It is a polymeric reservoir-type insert delivery system, Ocusert™, placed under the eyelid and on the ocular surface for zero-order drug release over one week. The insert, marketed in two strengths, released pilocarpine on the eye surface. While many clinicians appreciated this drug product, it was eventually discontinued. No similar sustained release non-invasive drug delivery system has made it to the market to date for treating glaucoma. Drug delivery systems under development include punctal plugs, ring-type systems, contact lenses, implants, microspheres, nanospheres, gels, and other depot systems placed in the extraocular, periocular, or intraocular regions including intracameral, supraciliary, and intravitreal spaces. This article discusses the advantages and disadvantages of the various routes of administration and delivery systems for sustained glaucoma therapy. It also provides the reader with some examples and discussion of drug delivery systems that could potentially be applied for glaucoma treatment. Interestingly, one intracamerally injected implant, Durysta™, was approved recently for sustained intraocular pressure reduction. However, long-term acceptance of such devices has yet to be established. The ultimate success of the delivery system will depend on efficacy relative to eye drop dosing, safety, reimbursement options, and patient acceptance. Cautious development efforts are warranted considering prior failed approaches for sustained glaucoma drug delivery. Neuroprotective approaches for glaucoma therapy including cell, gene, protein, and drug-combination therapies, mostly administered intravitreally, are also rapidly progressing towards assessment in humans.

Noninvasive Monitoring of Choroid-Retina Autofluorescence and Intravitreal Nanoparticle Disposition in Royal College of Surgeon Rats of Different Ages and Retinal Thinning.

Purpose: To determine the baseline choroid-retina fluorescence signal in Royal College of Surgeon (RCS) rats of various ages with different degrees of retinal degeneration and assess the persistence of intravitreal nanoparticles. Methods: In RCS rats of age 6, 12, and 20 weeks and Sprague Dawley (SD) rats of age 6 and 20 weeks, baseline eye tissue fluorescence and retinal thickness were recorded noninvasively using fluorophotometry and optical coherence tomography (OCT), respectively. Further, 20-nm carboxylate-modified fluorescent particles were injected intravitreally in the above groups of rats, and the depth-wise fluorescence signal was monitored over 7 days using fluorophotometry and confocal laser scanning ophthalmoscopy (cSLO). Additionally, 200 nm particles of the same material were injected intravitreally into about 7-week-old RCS rats and the fluorescence signal was monitored up to 35 days using fluorophotometry. Results: Reduction in retinal thickness and an increase in choroid-retina and lens baseline fluorescence was observed with increasing age of RCS and SD rats. The 20 nm particles persisted in the vitreous of animals from all age groups for at least 7 days postadministration, irrespective of the differences in retinal thickness. cSLO confirmed nanoparticle persistence in the eye. The fluorescence signal from 200 nm particles persisted for 35 days in the vitreous humor. Conclusions: Choroid-retina and lens autofluorescence monitored using fluorophotometry increase with age. Intravitreally injected nanoparticles can be monitored noninvasively in rats using fluorophotometry and cSLO imaging. Both 20 and 200 nm particles persist in the back of the eye tissues, for several days following intravitreal injection.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0⁻24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Targeted delivery of YSA-functionalized and non-functionalized polymeric nanoparticles to injured pulmonary vasculature.

Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.

Vanillic Acid Ameliorates Cationic Bovine Serum Albumin Induced Immune Complex Glomerulonephritis in BALB/c Mice.

Preclinical Research Vanillic acid (VA) is a dihydroxybenzoic acid derivative widely used as a flavoring agent. It has chemopreventive effects on experimentally-induced carcinogenesis and in ulcerative colitis. The object of the present study was to investigate the effects of VA, alone and in combination with methylprednisolone (MP), on cationic bovine serum albumin (cBSA induced immune-complex glomerulonephritis in female BALB/c mice. Pre-immunization was carried out with cBSA in BALB/c mice and repeated (cBSA, 13 mg/kg, 3 times/week, i.v.) for 6 weeks to induce glomerulonephritis which was confirmed by the presence of severe proteinuria. The effect of VA (50, 100, and 200 mg/kg, p.o.) and its combination with MP (12.5 mg/kg, p.o.) was assessed in the nephrotic disease model. Treatment with VA decreased inflammatory nephrotic injury as evidenced by decreased proteinuria, serum creatinine, blood urea nitrogen, serum IgG1 and TNF-α levels. Co-administration of VA with MP showed an improvement in the immunohistochemistry of glomerular nephrin and podocin. The present results indicate that VA has a nephroprotective effect in the management of autoimmune nephritis. Drug Dev Res 77 : 171-179, 2016. © 2016 Wiley Periodicals, Inc.

Evaluation of neonatal streptozotocin induced diabetic rat model for the development of cataract.

Type 2 diabetes (T2D) generally follows prediabetes (PD) conditions such as impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT). Although studies reported an association of IGT or IFG with cataract, the experimental basis for PD associated cataract is not known. Hence, we evaluated neonatal streptozotocin (nSTZ) induced rat model to study PD associated cataractogenesis by injecting STZ to two-day old rats. While majority (70%) of nSTZ injected pups developed IGT (nSTZ-PD) by two months but not cataract even after seven months, remaining (30%) nSTZ rats developed hyperglycemia (nSTZ-D) by two months and mature cataract by seven months. Lens biochemical analysis indicated increased oxidative stress as indicated by increased SOD activity, lipid peroxidation, and protein carbonyl levels in nSTZ-D cataractous lens. There was also increased polyol pathway as assessed by aldose reductase activity and sorbitol levels. Though nSTZ-PD animals have not shown any signs of lenticular opacity, insolubilization of proteins along with enhanced polyol pathway was observed in the lens. Further there was increased oxidative stress in lens of IGT animals. These results suggest that oxidative stress along with increased polyol pathway might play a role in IGT-associated lens abnormalities. In conclusion, nSTZ-PD rat model could aid to investigate IGT-associated lens abnormalities.

Efficacy of biodegradable curcumin nanoparticles in delaying cataract in diabetic rat model.

Curcumin, the active principle present in the yellow spice turmeric, has been shown to exhibit various pharmacological actions such as antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic activities. Previously we have reported that dietary curcumin delays diabetes-induced cataract in rats. However, low peroral bioavailability is a major limiting factor for the success of clinical utilization of curcumin. In this study, we have administered curcumin encapsulated nanoparticles in streptozotocin (STZ) induced diabetic cataract model. Oral administration of 2 mg/day nanocurcumin was significantly more effective than curcumin in delaying diabetic cataracts in rats. The significant delay in progression of diabetic cataract by nanocurcumin is attributed to its ability to intervene the biochemical pathways of disease progression such as protein insolubilization, polyol pathway, protein glycation, crystallin distribution and oxidative stress. The enhanced performance of nanocurcumin can be attributed probably to its improved oral bioavailability. Together, the results of the present study demonstrate the potential of nanocurcumin in managing diabetic cataract.

Insulin resistance mediated biochemical alterations in eye lens of neonatal streptozotocin-induced diabetic rat.

Cataract, the leading cause of blindness worldwide, is associated with many risk factors including diabetes. Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) states are associated with pre-diabetes and insulin resistance. This condition subsequently leads to the development of type-2 diabetes. Epidemiological studies indicated that not only diabetes but IGT/IFG will also lead to the development of microvascular disorders and cataract. However, there are no studies on the mechanism of insulin resistance induced changes in the eye lens. In the present study, IGT/IFG-induced changes in lens using neonatal-streptozotocin (nSTZ) rat model have been investigated. Though, nSTZ rats showed the signs of IGT and insulin resistance starting from two months old, they did not develop cataract even at the age of 8-months. However, biochemical analysis indicates a three-fold increase in sorbitol levels in nSTZ lens upon prolonged (6-months) IGT and insulin resistance. Also there was an increase in lipid peroxidation and alterations in antioxidant enzymes. Results of this study showed that activation of polyol pathway and increased oxidative stress, commonly associated with long-term complications of diabetes, have been observed in eye lens due to prolonged IGT and insulin resistance which may lead to cataract.

Temperature-dependent coaggregation of eye lens αB- and ß-crystallins.

Crystallin is essential not only for the maintenance of eye lens transparency, but also in the biology of other tissues. Eye lens α-crystallin exists as a heteropolymer composed of two homologous subunits, αA and αB. Despite the critical role of α-crystallin in many tissues, little is known regarding structural and functional significance of the two subunits. Herein, we describe a unique feature of αB-crystallin. At high temperatures (>70°C) not only αB-crystallin aggregates but also enhances the aggregation of other lens proteins. Intriguingly, αB-crystallin-mediated coaggregation at and above 70°C involves ß- but not γ-crystallin. Further, αA-crystallin, but not a mutant (F71L) αA-crystallin, prevented aggregation of αB-crystallin and also reduced coaggregation of αB- and ß-crystallin. These studies explain the rationale for the existence of α-crystallin heteropolymer with αA subunit as a major partner that is vital for lens transparency and provide insights into αB-crystallin-induced coaggregation which may have a bearing in some pathological conditions where αB-crystallin is overexpressed.

Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats.

PURPOSE: Advanced glycation end products (AGE) are associated in the development of several pathophysiologies including diabetic cataract. Earlier we have reported that some common dietary agents have antiglycating activity and ginger (Zingiber officinalis) was one of the few prominent agents that effectively prevented AGE formation in vitro. In this study we investigated the potential of ginger to prevent diabetic cataract in rats. METHODS: Diabetes was induced in Wistar-NIN rats by intraperitoneal injection of streptozotocin (35 mg/kg bodyweight) and the control rats received vehicle alone. While a set of diabetic animals received AIN-93 diet, another set received either 0.5 or 3% ginger in their diet for a period of two months. Cataract progression was monitored by slit-lamp biomicroscope. At the end of two months, the animals were sacrificed to evaluate non-enzymatic glycation and osmotic stress in the eye lens. RESULTS: Slit-lamp examination revealed that feeding of ginger not only delayed the onset but also the progression of cataract in rats. Molecular analyses indicated that feeding of ginger significantly inhibited the formation of various AGE products including carboxymethyl lysine in the eye lens. In addition, ginger also countered hyperglycemia-induced osmotic stress in the lens. CONCLUSIONS: The results indicated that ginger was effective against the development of diabetic cataract in rats mainly through its antiglycating potential and to a lesser extent by inhibition of the polyol pathway. Thus, ingredients of dietary sources, such as ginger, may be explored for the prevention or delay of diabetic complications.