Vitreous Concentrations Research Articles

A Mechanistic and Translational Pharmacokinetic-Pharmacodynamic Model of Abicipar Pegol and Vascular Endothelial Growth Factor Inhibition.

Abicipar pegol (abicipar) is a novel DARPin therapeutic and highly potent vascular endothelial growth factor (VEGF) inhibitor intended for the treatment of neovascular age-related macular degeneration (nAMD). Here we develop a translational pharmacokinetic/pharmacodynamic (PK/PD) model for abicipar to guide dosing regimens in the clinic. The model incorporated abicipar-VEGF binding kinetics, VEGF expression levels, and VEGF turnover rates to describe the ocular and systemic PK data collected from the vitreous, aqueous humor (AH), choroid, retina, and serum of rabbits after a 1-mg abicipar intravitreal (IVT) dose. The model was translated to humans using human-specific mechanistic parameters and refitted to human serum and AH concentrations from patients with diabetic macular edema and nAMD. The model was then used to simulate 8-, 12- (quarterly), and 16-week dosing intervals in the clinic. Simulations of 2 mg abicipar IVT at 8-week or quarterly dosing in humans indicates minimum steady-state vitreal concentrations are maintained above both in vitro IC50 and in vivo human IC50 values. The model predicted virtually complete VEGF inhibition for the 8-week and quarterly dosing schedule during the 52-week treatment period. In the 16-week schedule, clinically significant VEGF inhibition was maintained during the 52-week period. The model quantitatively described abicipar-VEGF target engagement leading to rapid reduction of VEGF and a long duration of VEGF inhibition demonstrating the clinical feasibility of up to a 16-week dosing interval. Abicipar is predicted to reduce IVT dosing compared with other anti-VEGF therapies with the potential to lessen patient treatment burden. SIGNIFICANCE STATEMENT: Current anti-VEGF treatments for neovascular age-related macular degeneration require frequent (monthly) intravitreal injections and monitoring, which increases patient burden. We developed a mechanistic pharmakinetic/pharmadynamic model to describe the interaction between abicipar (a novel VEGF inhibitor) and VEGF to evaluate the duration of action. The model demonstrates extended abicipar-VEGF target engagement leading to clinical feasibility of up to a 16-week dosing interval. Our model predicted that abicipar 8-week and quarterly dosing schedules maintain virtually complete VEGF inhibition during the 52-week period.

The distribution and redistribution of carfentanil in post mortem samples

This article compares 290 post mortem case reports that were positive for carfentanil. All the cases were submitted to, and analyzed by, the toxicology department of the Office of the Chief Medical Examiner, Edmonton, Alberta, Canada. This study highlights the varied distribution of carfentanil in the body after death as a result of misadventure, i.e., these are accidental drug overdose cases.Post mortem samples were collected from more than one anatomical site and analysed for carfentanil using a validated liquid chromatography-tandem mass spectrometry method. Ante-mortem samples were available in 15 of these cases and were also analysed.Post mortem mean blood carfentanil concentrations were found to be 0.362 μg/L (femoral), 0.442 μg/L (iliac), 0.484 μg/L (cardiac) and 0.692 μg/L (subclavian). The mean vitreous humor carfentanil concentration was 0.238 μg/L; the mean urine carfentanil concentration was found to be 0.697 μg/L.Little difference was found between ligated and ‘blindstick’ femoral blood carfentanil concentrations. Whilst carfentanil can readily be detected in both vitreous humor and urine samples neither were found to correlate with blood concentrations, limiting their use in interpretation.This study demonstrates the importance of multi-site sample collection and subsequent analysis for a thorough post mortem toxicological investigation. The study also highlights the risks and limitations associated with the interpretation of post mortem analytical results concerning carfentanil.

Angiogenic and inflammatory biomarker levels in aqueous humor and vitreous of neovascular glaucoma and proliferative diabetic retinopathy.

To explore the relationships between the aqueous and vitreous levels of vascular endothelial growth factor-A (VEGF-A), interleukin-8 (IL-8), placental growth factor (PlGF) and erythropoietin (EPO) in proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). Aqueous and vitreous samples were obtained from patients with PDR and NVG during surgery. Aqueous and vitreous concentrations of VEGF-A, IL-8, PlGF and EPO were measured via enzyme-linked immunosorbent assay. No correlation between the aqueous and vitreous levels of VEGF-A, IL-8, PlGF or EPO was found in both the PDR and the NVG eyes. Aqueous VEGF-A was significantly higher in the NVG group (317.55 ± 36.25pg/ml, n = 15) than that in the PDR group (256.23 ± 46.11pg/ml, n = 17, P < 0.001). The level of VEGF-A in aqueous (317.55 ± 36.25pg/ml, n = 15) was significantly higher than that in vitreous (224.74 ± 60.32pg/ml, n = 15, P < 0.001) in NVG patients. The level of IL-8 in aqueous (76.55 ± 10.88pg/ml, n = 17) was significantly higher than that in vitreous (63.55 ± 10.74pg/ml, n = 17, P = 0.001) in PDR patients. The level of EPO in aqueous (18.62 ± 2.87mIU/ml, n = 15) was significantly higher than that in vitreous (15.97 ± 3.11mIU/ml, n = 15, P = 0.022) in NVG patients. The ratio of aqueous versus vitreous for VEGF-A was significantly higher in the NVG group (1.475 ± 0.289, n = 15) than that in the PDR group (0.996 ± 0.227, n = 17, P < 0.001). Aqueous levels of VEGF-A, IL-8, PlGF and EPO do not correlate with vitreous levels of those proteins. The relationship between protein levels in aqueous humor and vitreous might be dependent on different disease status or protein types investigated.

Safety and Bioavailability of Complete and Half-Dose Intravitreal Ziv-Aflibercept in an Experimental Model: Contralateral Eye Study.

To evaluate the safety and bioavailability of complete and half-dose of intravitreal ziv-aflibercept (IVZ) in an experimental model. Thirty-two eyes of 16 male rabbits received one IVZ injection under anesthesia and the operating microscope. All right and left eyes received 1,250 μg/0.05 mL and 625 μg/0.05 mL of ziv-aflibercept, respectively. Then, rabbits were randomly allocated to four groups (four rabbits in each group). The rabbits were euthanized at predesignated intervals (at 24, 168, 336, and 720 hours), and the eyes were enucleated. Indirect ophthalmoscopy, vitreous sampling, and electrophysiological recordings were obtained before euthanization. Histological examination was performed after enucleation. Vitreous samples were evaluated by enzyme-linked immunosorbent assay to measure the concentration of aflibercept. No serious drug-related ocular inflammation and toxicity or systemic adverse events were identified. Electroretinogram findings showed no significant difference to the baseline measurements. Remaining vitreal concentrations of ziv-aflibercept injection for the 625 μg/mL group were 416 μg/mL, 349 μg/mL, 124 μg/mL, 41.2 μg/mL, and 18.1 μg/mL (± 10 μg/mL) and for the 1,250 μg/mL group were 833 μg/mL, 737 μg/mL, 284 μg/mL, 87.3 μg/mL, and 38.2 μg/mL (± 10 μg/mL), at zero, 24, 168, 336, and 720 hours after injection, respectively. The vitreous concentration of aflibercept was analyzed by one-compartment model. The area under curve from time 0 to the end point (AUC last) was 147,637 hours × μg/mL for the complete dose group (1,250 μg/0.05mL) and 68,498 hours × μg/mL for the half-dose group (625 μg/0.05 mL). The assessed vitreous half-life of ziv-aflibercept was 113 hours in both groups. IVZ proved to be safe and well tolerated, even in the complete dose group. It seems to be a cost-effective therapeutic option for the treatment of retinal vascular diseases. However, the long-term safety and efficacy of intravitreal ziv-aflibercept remain unknown. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:785-790.].

VITREOUS PROSTAGLANDIN E2 CHANGES AFTER TOPICAL ADMINISTRATION OF DICLOFENAC 0.1%, INDOMETHACIN 0.5%, NEPAFENAC 0.3%, AND BROMFENAC 0.09.

To evaluate the vitreous concentration of different nonsteroidal anti-inflammatory drugs (NSAIDs) after topical administration and the related prostaglandin E2 (PGE2) levels in patients undergoing pars plana vitrectomy. A prospective, randomized, investigator-masked study was performed. One hundred four patients scheduled for a pars plana vitrectomy for an epiretinal membrane or a macular hole were randomized to receive topical diclofenac 0.1%, indomethacin 0.5%, nepafenac 0.3%, bromfenac 0.09%, or placebo 3 days before surgery. At the beginning of surgery, a sample of undiluted vitreous was collected in each patient to assess NSAIDs concentration and PGE2 levels. The median vitreous concentrations were 203.35 (interquartile range 146.54-264.18) pg/mL for diclofenac, 243.45 (interquartile range 156.96-365.37) pg/mL for nepafenac, 438.21 pg/mL (interquartile range, 282.52-645.87) for its active metabolite amfenac, 350.14 (interquartile range, 290.88-481.95) pg/mL for indomethacin, and 274.59 (245.43-358.25) pg/mL for bromfenac. Vitreous PGE2 levels were significantly lower for all the NSAIDs groups compared with the control group (P < 0.001). A statistically significant higher vitreous PGE2 level was found in the diclofenac group compared with the other NSAIDs groups (P < 0.05). Topical NSAIDs achieve sufficient vitreous concentration to decrease vitreous PGE2 levels compared with the control group. The different efficacy in reducing PGE2 concentration may affect the management of posterior segment inflammation.

Increased interleukin-26 expression in proliferative diabetic retinopathy.

To detect the possible role of interleukin (IL)-26 in diabetic retinopathy (DR) patients. Subjects were divided into diabetes without retinopathy (DWR) group (n=20), non-proliferative diabetic retinopathy (NPDR) group (n=20), proliferative diabetic retinopathy (PDR) group (n=20) and normal control group (n=20). The protein expression of IL-26 in the serum and vitreous fluid were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA change of IL-26 in peripheral blood mononuclear cells (PBMCs) was assessed by real-time polymerase chain reaction. The serum expression of IL-26 in PDR group was significantly elevated compared with the normal control group, DWR group and NPDR group. The vitreous fluid concentration of IL-26 in PDR patients (without anti-VEGF therapy) was also higher compared to normal controls. However, no obvious significance was found concerning the expression of IL-26 in vitreous fluid between PDR after anti-VEGF therapy and normal controls. In PDR group, the mRNA level of IL-26 significantly increased compared with the normal controls and DWR patients in the PBMCs. Protein and mRNA expression of IL-26 are increased in serum, vitreous fluid and PBMCs in PDR patients, suggesting that IL-26 may be associated with the pathogenesis of PDR.

Investigations Into Bioenergetic Neuroprotection of Cone Photoreceptors: Relevance to Retinitis Pigmentosa.

Recent studies suggest cone degeneration in retinitis pigmentosa (RP) may result from intracellular energy depletion. We tested the hypothesis that cones die when depleted of energy by examining the effect of two bioenergetic, nutraceutical agents on cone survival. The study had three specific aims: firstly, we, studied the neuroprotective efficacies of glucose and creatine in an in vitro model of RP. Next, we utilized a well-characterized mouse model of RP to examine whether surviving cones, devoid of their inner segments, continue to express genes vital for glucose, and creatine utilization. Finally, we analyzed the neuroprotective properties of glucose and creatine on cone photoreceptors in a mouse model of RP. Two different bioenergy-based therapies were tested in rd1 mice: repeated local delivery of glucose and systemic creatine. Optomotor responses were tested and cone density was quantified on retinal wholemounts. The results showed that glucose supplementation increased survival of cones in culture subjected to mitochondrial stress or oxidative insult. Despite losing their inner segments, surviving cones in the rd1 retina continued to express the various glycolytic enzymes. Following a single subconjunctival injection, the mean vitreous glucose concentration was significantly elevated at 1 and 8 h, but not at 16 h after injection; however, daily subconjunctival injection of glucose neither enhanced spatial visual performance nor slowed cone cell degeneration in rd1 mice relative to isotonic saline. Creatine dose-dependently increased survival of cones in culture subjected to mitochondrial dysfunction, but not to oxidative stress. Despite the loss of their mitochondrial-rich inner segments, cone somas and axonal terminals in the rd1 retina were strongly positive for both the mitochondrial and cytosolic forms of creatine kinase at each time point examined. Creatine-fed rd1 mice displayed enhanced optomotor responses compared to mice fed normal chow. Moreover, cone density was significantly greater in creatine-treated mice compared to controls. The overall results of this study provide tentative support for the hypothesis that creatine supplementation may delay secondary degeneration of cones in individuals with RP.

Human vitreous concentrations of citicoline following topical application of citicoline 2% ophthalmic solution.

PurposeTo evaluate the presence and concentration of citicoline and its metabolites (choline, cytidine and uridine) in the vitreous body in human eyes after topical application of an ophthalmic solution of citicoline 2%, in vivo.MethodsTwenty-one subjects affected by epiretinal membrane with surgical indication for pars-plana vitrectomy underwent treatment with 1 drop 3 times/day of a solution of citicoline 2%, 0.2% high molecular weight hyaluronic acid and 0.01% benzalkonium chloride (OMK1, Omikron Italia s.r.l., Rome, Italy) 14 days before surgery and 2 hours prior to surgery. Five additional patients served as controls and received an OMK1 vehicle solution without citicoline. The vitreous samples were taken at the beginning of the pars-plana vitrectomy and analyzed for qualitative/quantitative determination of vitreous concentration of citicoline and its metabolites by means of high performance liquid chromatography.ResultsThe overall mean concentration of citicoline in patients treated with citicoline 2% solution was 406.72 ± 52.99 μg/mL, while the mean concentration of choline, cytidine and uridine was 180.88 ± 41.49 μg/mL, 44.45 ± 10.19 μg/mL and 330.41 ± 75.8 μg/mL, respectively. The concentration of citicoline in phakic eyes (n = 13, 366.61 ± 129.61 μg/mL) was lower compared to that found in pseudophakic eyes (n = 8, 435.89 ± 131.42 μg/mL) and the difference was not statistically significant. The concentration of citicoline in the control eyes was 45.66 ± 26.36 μg/mL, while the concentration of choline, cytidine and uridine were 17.21 ± 9.93 μg/mL, 6.24 ± 3.6 μg/mL and 172.80 ± 99.76 μg/mL, respectively.ConclusionCiticoline can reach the human vitreous in high concentration when administered in ophthalmic solution. This evidence contributes to the build-up of the pyramid of the evidences required for determining the role of citicoline administered in ophthalmic formulation in retinal and optic nerve neurodegenerative diseases.

Gender differences in albumin and ascorbic acid in the vitreous antioxidant system

Ascorbic acid is present at high concentrations in the vitreous and plays a central role in vitreous redox chemistry. Albumin is the main protein in the vitreous with antioxidant properties and occurs in different oxidation states, which can be used as redox indicators, but have not been studied in the vitreous. This study, therefore, addressed the vitreous redox state of cysteine-34 of albumin in relation to the ascorbic acid content, which has been suggested to exert a main function in detoxifying reactive oxygen in the vitreous.A total of 58 vitreous samples obtained from patients undergoing vitrectomy were analyzed for (i) human mercaptalbumin (HMA), the reduced thiol form; (ii) human non-mercaptalbumin1 (HNA1), a reversible oxidative modification with a disulfide at cysteine-34; and (iii) human non-mercaptalbumin2 (HNA2), a non-reversibly (highly) oxidized form of albumin; as well as (iv) ascorbic acid concentrations, to study possible relations. In addition, blood samples were taken to compare albumin redox state between plasma and the vitreous.Vitreous albumin showed greater variability in the redox state of cysteine-34 and a shift to the oxidized fractions compared to plasma albumin (P < 0.001). A strong positive relation was observed between the vitreous ascorbic acid concentrations and the reversibly oxidized form, HNA1 (P < 0.001), and a negative relation with the reduced form, HMA. Positive relations between ascorbic acid and HNA1 in the vitreous were stronger in men than in women. In contrast to HMA and HNA1, there was a distinct gender difference noted for the irreversibly oxidized form, HNA2. While males showed a positive relation between the vitreous ascorbic acid concentrations and HNA2, there was no correlation found with HNA2 in females.Our results support the view that ascorbic acid, by decreasing either directly or indirectly the concentrations of molecular oxygen, generates hydrogen peroxide, and that thiols, including HMA, are acting as antioxidants. This study for the first time provides evidence that vitreous albumin can be used as a marker molecule for the appearance of reactive oxygen species in the vitreous of patients undergoing vitrectomy. Moreover, it can be shown that there are gender differences in vitreous ascorbic acid and albumin concentrations as well as in oxidation state of vitreous albumin.

Acidic Drug Concentrations in Postmortem Vitreous Humor and Peripheral Blood.

Vitreous humor is a potential alternative matrix for postmortem toxicology drug screens when peripheral blood is unavailable. It is easily and reliably collected and may not suffer from the same postmortem redistribution as seen in blood. Here, we compared the concentrations of 7 acidic drugs (acetaminophen, ibuprofen, naproxen, salicylic acid, carbamazepine, phenobarbital and phenytoin) in peripheral blood and vitreous fluid collected in 89 autopsy cases. Analysis was done by high-performance liquid chromatography with diode-array detection. Overall, we found that vitreous drug concentrations were significantly lower than peripheral blood with median vitreous to peripheral blood (V/PB) ratios ranging from 0.0 to 0.6 (mean, 0.1-0.6). The correlations between the concentrations of over-the-counter analgesics in peripheral blood versus vitreous fluid were poor, with acetaminophen exhibiting the best linearity (R2=0.72). The antiepileptic drugs (carbamazepine, phenytoin and phenobarbital) exhibited good correlations between peripheral blood and vitreous humor, with all exhibiting an R2≥0.95. Overall, we have demonstrated the potential of vitreous fluid as an alternative matrix for the detection of select acidic drugs.

Changes in Aqueous and Vitreous Inflammatory Cytokine Levels in Retinal Vein Occlusion: A Systematic Review and Meta-analysis.

Evidence suggests that inflammatory cytokines not only play a role in the pathogenesis of retinal vein occlusion (RVO) but also may be useful as biomarkers to predict disease severity and response to treatment. We aimed to quantitatively summarize data on inflammatory cytokines associated with RVO. A systematic search of peer-reviewed English-language articles was performed without year limitation up to August 19, 2019. Studies were included if they provided data on aqueous or vitreous cytokine concentrations in patients with RVO. Data were extracted from 116 studies that encompassed 3242 study eyes with RVO and 1402 control eyes. Effect sizes were generated as standardized mean differences (SMDs) of cytokine concentrations between patients with RVO vs controls. Among the 4644 eyes in 116 studies, aqueous and vitreous concentrations (SMD, 95% CI, and P value) of interleukin (IL)-6 (aqueous: 1.23, 0.65 to 1.81, P < .001 vitreous: 0.70, 0.49 to 0.90, P < .001), IL-8 (aqueous: 1.11, 0.73 to 1.49, P < .001; vitreous: 1.19, 0.73 to 1.65, P < .001), monocyte chemoattractant protein 1(aqueous: 1.22, 0.72 to 1.72, P < .001; vitreous 1.42, 0.92 to 1.91, P < .001), vascular endothelial growth factor (VEGF) (aqueous: 1.52, 1.09 to 1.94, P < .001; vitreous: 0.99, 0.78 to 1.21, P < .001) were significantly higher in patients with RVO than in healthy controls. Only aqueous concentrations of IL-10 (0.81, 0.45 to 1.18, P < .001), angiopoietin 4 (1.96, 0.92 to 3.00, P < .001), and platelet-derived growth factor (PDGF)-AA (0.82, 0.35 to 1.30, P < .001) were significantly higher in patients with RVO than in healthy controls. Only the vitreous concentration of soluble intercellular adhesion molecule-1 (sICAM-1) (1.23, 0.83 to 1.63, P < .001) was significantly higher in patients with RVO. No differences, failed sensitivity analyses, or insufficient data were found between patients with RVO and healthy controls for the concentrations of the remaining cytokines. Several cytokines in addition to VEGF have the potential to be useful biomarkers and therapeutic targets in RVO.

Characterisation of the inflammatory cytokine and growth factor profile in a rabbit model of proliferative vitreoretinopathy

To clarify the mechanisms and their temporal relationship in the development of proliferative vitreoretinopathy (PVR), we measured vitreous levels of pro-inflammatory cytokines and growth factors in a rabbit model of PVR. PVR was surgically induced in 11 rabbit eyes by vitrectomy, retinotomy, cryotherapy and injection of platelet-rich plasma at baseline. Severity of PVR was assessed on dilated fundal examination with indirect binocular ophthalmoscopy and graded based on the revised experimental PVR classification. Severe PVR was defined as stage 5 or worse. Vitreous concentrations of interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 beta (IL-1 β), tumor necrosis factor beta (TNF-β), granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), C reactive protein; (CRP), placental growth factor (PlGF), platelet derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2) at weeks 2, 3 and 4 were compared to baseline and correlations between the cytokines with PVR severity were assessed. Four weeks after PVR induction, 5 eyes (45.5%) had developed severe PVR. IL-8 was raised at 2 weeks post PVR induction (1.46 ± 0.48 pg/ml vs 0.53 ± 0.25 pg/ml, p = 0.04) and remained significantly elevated at week 4 (2.6 ± 3.1 pg/ml, p = 0.03). CRP was significantly raised at week 4 (34.8 ± 12.0 pg/ml vs 13.0 ± 13.1 pg/ml, p < 0.001). Among the growth factors, PDGF-BB was the earliest to show significantly elevated levels, at 3 weeks (50.4 ± 19.0 pg/ml vs 6.2 ± 10.1 pg/ml) and remained elevated at week 4 (p = 0.002), while PlGF (11.2 ± 7.7 pg/ml vs 5.3 ± 3.8 pg/ml, p = 0.002) and Ang2 (13617.0 ± 8170.2 pg/ml vs 38593.8 ± 8313.4, p = 0.02) were significantly raised at week 4. IFN-γ (p = 0.03), PDGF-BB (p = 0.02) and VEGF (p = 0.02) were significantly associated with PVR severity. We demonstrated that inflammatory cytokines IL-6, -8, elevation post PVR induction is followed by elevated levels of fibroproliferative growth factors, Ang2, PlGF, VEGF and PDGF-BB in the development of PVR. These findings will guide future studies targeting appropriate therapeutic strategies for the treatment of PVR.

Effects of 7-Methylxanthine on Deprivation Myopia and Retinal Dopamine Release in Chickens

Introduction: Intake of 7-methylxanthine (7-MX), an adenosine receptor (AR) antagonist, has been shown to inhibit school myopia in children and deprivation myopia in rhesus monkeys, but the underlying mechanisms are not known. Also retinal dopamine seems to be involved in the control of eye growth, and in the brain, ARs and dopamine receptors interact widely by heteromerization. We have studied whether 7-MX can inhibit deprivation myopia also in chickens and whether inhibition may involve the retinal dopamine system. Methods: 7-MX was applied by either tube-feeding (100 µg/g body weight, twice a day) or intravitreal injection (12.5 µg, every other day). Forty-eight 2-week-old chicks wore unilateral diffusers and were randomly assigned to either the tube-feeding group (involving 7-MX, vehicle [xanthan gum], or no feeding, for 13 days) or the intravitreal injection group (involving 7-MX, vehicle, or DMSO, for 8 days). Refractions (REs), ocular biometry (AL, VCD), and scleral and choroidal thickness (ChT) were measured before and after treatment. Dopamine and dihydroxyphenylacetic acid (DOPAC) content were determined in retina and vitreous by HPLC at the end of the experiments. Results: No matter how 7-MX was applied, it did not inhibit deprivation myopia in chicks. No significant differences were observed in RE, VCD, AL, and scleral fibrous layer thickness. Feeding 7-MX produced more choroidal thinning in the open contralateral eye compared to control eyes in the vehicle-fed group (–40 ± 14 vs. –1 ± 7 µm, unpaired t test, p < 0.05). DOPAC and dopamine concentration in vitreous and DOPAC concentration in retina did not change with 7-MX. Vitreal dopamine content was significantly decreased in deprived eyes in the groups fed with the vehicle xanthan gum (paired t test, p < 0.01) but not in 7-MX-treated eyes, perhaps indicating a small effect of 7-MX on dopamine. Conclusions: In our study, 7-MX had no effect on DM in chicks and only minor effects on ChT and retinal dopamine. It remains unclear whether 7-MX inhibits myopia through a retinal mechanism or whether it acts directly on choroid and sclera. In the latter case, the finding that myopia is suppressed in mammals but not birds might be explained by differences in scleral structure.

Vitreous potassium concentration as a predictor of postmortem interval in severe burn deaths at Alexandreia mortuary

ABSTRACT Background: Postmortem interval (PMI) estimation is an important goal in forensic medicine. After death, many physiochemical changes occur in a regular sequence and thanatochemistry is the chemistry of death. They can be used to arrive at an approximate time of death. Aim: to estimate the time since death using a scoring method for three postmortem changes; hypostasis, rigidity and corneal turbidity in sever burn cases. Also, to evaluate the use of thanatochemistry; potassium (K+) in vitreous humor (VH) and serum in determination of (PMI) and compare the accuracy of thanatochemistry and the scoring method for postmortem changes in estimation of PMI. Methods: The study was conducted on 100 adult autopsy cases of known postmortem interval presented to the mortuary of Alexandria from May 2017 to May 2018, 50 burn cases where autopsy confirmed the cause of death as severe burns and another 50 control cases. The development of postmortem rigidity, hypostasis and corneal turbidity was assessed and numerically scored. The potassium (K+) levels in vitreous humor (VH) and serum were measured. The data were statistically analyzed and linear regression analysis was used to obtain equations for calculation of PMI .Results: All the studied variables in the present study were significantly correlated with PMI; highest correlation coefficient in control and burn groups was for K+ level in VH, followed by corneal turbidity and serum K+ then rigidity in burn groups, but in control groups the highest correlation coefficient was for K+ level in VH and serum, followed by rigidity then corneal turbidity and lastly hypostasis. All equations obtained from the present study can predict PMI but with different levels of accuracy. Conclusion: The most accurate equation in control cases was that concerning with all the five studied variables. Furthermore, the scoring method for the physical postmortem changes was proved to be more valuable in PMI estimation than thanatochemistry. No much difference between accuracy of equations in burn cases was that concerning with all the five studied variables.

The efficacy of systemic and intravitreal infliximab treatments in an endotoxin-induced uveitis model

Purpose: To compare the efficacy of systemic and intravitreal infliximab treatments in an experimental endotoxin-induced uveitis (EIU) model.Methods: Twenty-eight white New Zealand rabbits were equally divided into 4 groups. Group 1 received an intravitreal injection of 0.1 cc saline, group 2 received an intravitreal injection of 2 µg/0.1 cc lipopolysaccharide (LPS), group 3 received an intravitreal injection of 2 µg/0.1 cc LPS and 2 mg/0.1 cc infliximab, and group 4 received intravitreal injection of 2 µg/0.1 cc LPS and intravenous injection of 5 mg/kg infliximab. Clinical, biochemical (aqueous and vitreous humour protein levels and TNF-α concentrations), and histopathological evaluations were performed.Results: The clinical examination score was lower in group 4 than in group 2 (p = 0.006); but there was no significant difference between groups 2 and 3 (Bonferroni correction, p = 0.016). No statistically significant difference was found among groups 2, 3, and 4 for aqueous humour protein levels (p > 0.05). Significantly higher aqueous humour concentrations of TNF-α was measured in group 3 comparing to both group 1 and 4 (p = 0.003 and p = 0.002, respectively). No significant difference was found in vitreous protein levels or TNF-α concentrations among all study groups (Bonferroni correction, p = 0.026 and p = 0.101, respectively). Histopathological evaluation of the uveal tissue and anterior chamber reaction revealed the highest inflammation in group 3 (p < 0.001). In group 4, histopathological evaluation of uveal tissue was lower than in groups 2 and 3 (p < 0.001 and p = 0.001, respectively); whereas there was no difference in anterior chamber inflammation between groups 2 and 4 (p = 1.00).Conclusion: Intravitreal 2 mg/0.1 cc infliximab injection exacerbated inflammation in an EIU model; whereas systemic infliximab treatment at a dose of 5 mg/kg suppressed inflammation effectively and rapidly.

Safety of anti-VEGF treatments in a diabetic rat model and retinal cell culture.

PurposeTo analyze the safety of different concentrations of anti-VEGF on retinal cells.MethodsNon-diabetic and streptozotocin (STZ)-induced diabetic rats received intravitreal rat anti-VEGF injections that had final vitreous concentrations of 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL. Rats were also injected with the clinical dose of ranibizumab. TUNEL assay was performed on sectioned eyes to evaluate apoptotic cells. In vitro, rat retinal cell cultures were exposed to 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL of ranibizumab for 48 and 72 hrs. Cellular metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, necrosis by lactate dehydrogenase (LDH), and apoptosis by cell death enzyme-linked immunosorbent assay (ELISA).ResultsDiabetic rats had a significant increase (p<0.03) in apoptotic cell death at half the clinical dose, at the clinical dose, and at double the clinical dose. In vitro, MTT showed a significant decrease (p<0.04) in cellular metabolic activity at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. LDH showed a significant increase (p<0.04) in necrosis at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. ELISA showed a significant increase (p<0.04) in apoptosis at half the clinical dose, at the clinical dose, and double the clinical dose, compared to control at 48 and 72 hrs.ConclusionsAnti-VEGF treatment may be potentially detrimental to the retina by decreasing cellular metabolic activity and increasing cytotoxicity of retinal cells. The results provide a cautionary note to monitor both the retina and optic nerve status in patients undergoing frequent injections.

586-P: Vitreous and Serum Measurements of Retinol Binding Protein 3 in the Clinical Evaluation of Diabetic Retinopathy

The Joslin 50-Year Medalist Study discovered that elevated photoreceptor-secreted Retinol Binding Protein 3 (RBP3) in the retina and vitreous of individuals with extreme duration of type 1 diabetes is associated with long-term protection from advanced diabetic retinopathy (DR), independent of glycemic control. This represents the first neuroretinal protein identified with potential protective activity against the toxic effects of hyperglycemia on the retinal vasculature, a major cause of DR onset and worsening. We extended these findings by correlating vitreous and serum RBP3 concentrations and examining associated clinical characteristics. We developed a novel RBP3 ELISA assay with high sensitivity (0.1-1 nM), specificity (no detection of immunoglobulin G, albumin, or RBP4), and intra-and inter-assay correlations (p&amp;lt;0.001 and p=0.003, respectively). Vascular endothelial growth factor (VEGF) concentrations were measured by ELISA. A total of 101 individuals with type 1 diabetes with mean±SD diabetes duration 44.6±21.0 years, A1c 7.6±1.2%, age 73.5±8.9 years, with 46.5% female, were evaluated. Vitreous RBP3 concentration correlated with vitreous VEGF concentration, age, diabetes duration, cardiovascular disease, and DR severity (p&amp;lt;0.05, all). Mean serum RBP3 concentration was 1000-fold less than vitreous, but correlated with vitreous RBP3 concentration and DR severity (p&amp;lt;0.05, all), even after adjusting for age and A1c. There was a strong association between lower serum RBP3 concentration and increasing age and diabetes duration (p&amp;lt;0.01 and p&amp;lt;0.05, respectively). Vitreous (p=0.01), but not plasma VEGF concentrations were positively associated with DR severity. The fact that serum RBP3 and not plasma VEGF concentrations correlate with DR severity, suggests potential for the systemic measurement of RBP3 in the clinical evaluation of DR and its therapeutic outcomes. Disclosure W. Fickweiler: None. H. Yokomizo: None. K. Park: None. S.M. Paniagua: None. I. Wu: None. D.M. Pober: None. V. Bahnam: None. A. Clermont: Consultant; Self; KalVista Pharmaceuticals, Inc. L.P. Aiello: Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Decision Resources Group, KalVista Pharmaceuticals, Inc., Mingsight, Retinal Solutions. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J.K. Sun: Research Support; Self; Adaptive Sensory Technology, Boston Micromachines Corporation, Genentech, Inc., KalVista Pharmaceuticals, Inc., Optovue, Incorporated. Other Relationship; Self; Genentech, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc. G.L. King: Research Support; Self; Sanofi. Funding National Eye Institute (R01EY026080-01); National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK094333-01); JDRF (17-2013-310); Thomas J. Beatson, Jr. Foundation Inc.; Dianne Nunnally Hoppes Scholarship Fund

Effectiveness of prophylactic intravitreal bevacizumab injection to proliferative diabetic retinopathy patients with elevated preoperative intraocular VEGF in preventing complications after vitrectomy.

PurposeThis study aimed to elucidate the effects of intravitreal bevacizumab (IVB) injections for the prevention of post-vitrectomy complications in proliferative diabetic retinopathy (PDR) patients with elevated vitreous vascular endothelial growth factor (VEGF) concentration.DesignProspective case series.MethodsThirty-three patients (42 eyes) with PDR who underwent primary vitrectomy in the Department of Ophthalmology, Tokyo Medical University Hospital were studied. We measured VEGF concentrations in vitreous humor collected at the time of vitrectomy using ELISA. IVB injections were performed after vitrectomy in patients with vitreous VEGF levels exceeding 1,000 pg/mL. New bleeding occurring within 1 month of vitrectomy was defined as early vitreous hemorrhage (VH).Main outcome measureThe incidence of complications after vitrectomy including postoperative early VH.ResultsIVB injections were administered to 11 eyes (26%) with vitreous VEGF concentrations exceeding 1,000 pg/mL. None of the 11 eyes that received an IVB injection developed early VH. Among 31 eyes (74%) with vitreous VEGF concentrations lower than 1,000 pg/mL, two eyes (6%) developed early VH after vitrectomy.ConclusionsProphylactic IVB injections administered to patients with elevated preoperative intraocular VEGF concentrations were effective in preventing post-vitrectomy early VH.

597-P: Retinol Binding Protein 3 in Diabetic Retinopathy: Mechanisms of Protective Activity

The Joslin Medalist study reported that after having type 1 diabetes (T1D) for &amp;gt; 50 years, over 35% of Medalists exhibit no to mild diabetic retinopathy (DR) independent of glycemic control, suggesting presence of endogenous protective factors against DR. Proteomic analysis of Medalist retina and vitreous found that Retinol Binding Protein 3 (RBP3), secreted by photoreceptors, is elevated in individuals protected from DR. This finding was confirmed by RBP3 ELISA of replicate vitreous samples from Medalists and other T1D and T2D individuals. In contrast, vitreous VEGF concentrations increased with DR severity. Thus, elevated ratios of intravitreous RBP3/VEGF correlated with lack of progression to advanced DR. Intervention and prevention studies overexpressing RBP3 in retina/photoreceptors of nondiabetic and diabetic animals inhibited VEGF actions and normalized diabetes-induced retinal capillary permeability, neuro-retinal dysfunctions, expressions of VEGF and IL-6, and formation of acellular capillaries. Mechanistically, physiological levels of recombinant RBP3 (10-100nM), but not RBP4 or FGF, inhibited the increased in VEGFR2 tyrosine phosphorylation, hyperglycemia-induced expression of pro-inflammatory cytokines (VEGF and IL-6), and migration of retinal endothelial cells and Müller cells. RBP3, but not RBP4, physically bound to VEGFR2 in cross linkage studies. At physiological levels, RBP3 significantly inhibited glucose uptake into retinal endothelial and Müller cells, and subsequently decreased expression of VEGF and IL-6. These results suggest novel actions for RBP3 to slow glucose uptake in retinal cells and inhibit VEGF actions, thereby preventing, delaying and even arresting diabetes-induced neuro- and vascular retinal dysfunction, inflammatory abnormalities and DR pathologies. These findings strongly support therapeutic potential of RBP3 in DR. Disclosure H. Yokomizo: None. K. Park: None. A. Clermont: Consultant; Self; KalVista Pharmaceuticals, Inc. Y. Maeda: Research Support; Self; Abbott, Arkray, Inc. Speaker's Bureau; Self; Abbott, Arkray, Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Terumo Medical Corporation. W. Fickweiler: None. A. Ishikado: Employee; Self; SunStar Inc. Employee; Spouse/Partner; SunStar Inc. Q. Li: None. C. Wang: None. D.M. Pober: None. I. Wu: None. H.A. Keenan: Employee; Self; Sanofi Genzyme. L.P. Aiello: Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Decision Resources Group, KalVista Pharmaceuticals, Inc., Mingsight, Retinal Solutions. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J.K. Sun: Research Support; Self; Adaptive Sensory Technology, Boston Micromachines Corporation, Genentech, Inc., KalVista Pharmaceuticals, Inc., Optovue, Incorporated. Other Relationship; Self; Genentech, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc. G.L. King: Research Support; Self; Sanofi. Funding National Eye Institute; National Institute of Diabetes and Digestive and Kidney Diseases; JDRF; Thomas J. Beatson, Jr. Foundation Inc.; Dianne Nunnally Hoppes Scholarship Fund

Elevation of the vitreous body concentrations of oxidative stress-responsive apoptosis-inducing protein (ORAIP) in proliferative diabetic retinopathy.

Oxidative stress has been implicated in the pathogenesis of various disorders, including diabetic retinopathy (DR). Oxidative stress-responsive apoptosis-inducing protein (ORAIP; a tyrosine-sulfated secreted form of eukaryotic translation initiation factor 5A [eIF5A]) is a recently discovered pro-apoptotic ligand that is secreted from cells in response to oxidative stress and induces apoptosis in an autocrine fashion. This study aimed to determine if ORAIP plays a role in DR. To investigate the role of ORAIP in DR, we analyzed the levels of ORAIP in the vitreous body and their relationship with the extent of proliferative diabetic retinopathy (PDR). Enzyme-linked immunosorbent assay was used to quantify the levels of ORAIP, vascular endothelial growth factor (VEGF), C-C motif chemokine ligand 2 (CCL2), interleukin-6 (IL-6), and IL-8 in the vitreous body of 40 eyes from 28 patients with PDR and 11 patients with non-PDR (NPDR). We also analyzed the expression of ORAIP in insoluble proliferative tissues from vitreous body samples by immunofluorescent staining. The vitreous body concentration of ORAIP was significantly (P = 0.0433) higher in the PDR group (52.26 ± 8.68 [mean ± SE] ng/mL, n = 29) than in the NPDR group (28.21 ± 7.30ng/mL, n = 11). However, there were no significant correlations between the concentration of ORAIP and those of VEGF, IL-6, CCL2, or IL-8. ORAIP expression was observed in the insoluble proliferative tissues in vitreous body samples of most patients in the PDR group, whereas almost no expression of ORAIP was observed in patients in the NPDR group. Our findings strongly suggest that ORAIP plays a role in oxidative stress-induced retinal injury and may be a sensitive diagnostic marker and a promising therapeutic target for oxidative stress-induced cytotoxicity.