Subconjunctival Administration Research Articles

Controlled release of dexamethasone sodium phosphate with biodegradable nanoparticles for preventing experimental corneal neovascularization

Corneal neovascularization (CNV) leads to the loss of corneal transparency and vision impairment, and can ultimately cause blindness. Topical corticosteroids are the first line treatment for suppressing CNV, but poor ocular bioavailability and rapid clearance of eye drops makes frequent administration necessary. Patient compliance with frequent eye drop application regimens is poor. We developed biodegradable nanoparticles (NP) loaded with dexamethasone sodium phosphate (DSP) using zinc ion bridging, DSP-Zn-NP, with dense coatings of poly(ethylene glycol) (PEG). DSP-Zn-NP were safe and capable of providing sustained delivery of DSP to the front of the eye following subconjunctival (SCT) administration in rats. We reported that a single SCT administration of DSP-Zn-NP prevented suture-induced CNV in rats for two weeks. In contrast, the eyes receiving SCT administration of either saline or DSP solution developed extensive CNV in less than 1 week. SCT administration of DSP-Zn-NP could be an effective strategy in preventing and treating CNV.

Co-delivery of metformin and levofloxacin hydrochloride using biodegradable thermosensitive hydrogel for the treatment of corneal neovascularization

Corneal neovascularization (CNV) is one of the major causes of severe disorders in ocular surface. Subconjunctival administration provides a localized and effective delivery of anti-angiogenic agents to inhibit neovascularization. In the present study, the ABA triblock copolymer of poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol)-block-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) was used as a sustained drug delivery carrier for metformin (MET) and levofloxacin hydrochloride (LFH). Both drugs and PLGA-PEG-PLGA copolymers could be easily dissolved in water at low or room temperature and the mixed solution could form a drug-loaded thermosensitive hydrogel in terms of body temperature response. The in vitro release investigation displayed a sustained release of MET and LFH from the formulation for one month. The in vivo efficacy of subconjunctival injection of the MET + LFH loaded thermosensitive hydrogel in inhibiting CNV was evaluated on a mouse model of corneal alkali burn. Compared with the single administration of MET or LFH loaded thermosensitive hydrogel, the MET + LFH loaded thermosensitive hydrogel remarkably inhibited the formation of CNV. The sustained release of MET and an antibiotic (LFH) provides synergistic therapeutic outcome. As a result, the co-delivery of MET and LFH using PLGA-PEG-PLGA thermosensitive hydrogel by subconjunctival injection has great potential for ocular anti-angiogenic therapy.

Personalized Nano Delivery Strategy in Treating Uveitis

Uveitis is an inflammatory disease causes the damage of eye tissues and leads to the loss of vision once kept untreated. Early detection and identification of the disease are necessary for the prevention of the progressing inflammation and tissue damage. Existing treatment comprise of the administration of oral steroids, immunosuppressant, biological and adjuvant therapy. Even though topical, intravitreal and subconjunctival administration routes have been chosen for the drug administration, they fail to achieve the drug concentration to elicit a better therapeutic action due to the presence of ocular barriers such as tear, cornea conjunctiva, sclera, choroid membrane, retina, and blood-retinal barrier. Design of delivery systems that can prolong the precorneal habitation time increase the drug release as well as reduce the side effects such as methods to achieve prolonged can promise effective ocular drug delivery. Delivery systems such as hydrogels, liposomes, micro carrier systems, vitreous implants, medicated lenses, nanotechnology approaches such as nanocrystals, nanoemulsions, nanosuspensions, dendrimers, microneedles etc. exist as current research and the future research perspectives to achieve effective ocular drug delivery.

Development of Targeted siRNA Nanocomplexes to Prevent Fibrosis in Experimental Glaucoma Filtration Surgery.

RNAi induced by double-stranded small interfering RNA (siRNA) molecules has attracted great attention as a naturally occurring approach to silence gene expression with high specificity. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and, thus, represents a promising target to prevent fibrosis. A major hurdle to implementing siRNA therapies is the method of delivery, and we have, thus, optimized lipid-peptide-siRNA (LPR) nanoparticles containing MRTF-B siRNAs as a targeted approach to prevent conjunctival fibrosis. We tested 15 LPR nanoparticle formulations with different lipid compositions, surface charges, and targeting or non-targeting peptides in human conjunctival fibroblasts. In vitro, the LPR formulation of the DOTMA/DOPE lipid with the targeting peptide Y (LYR) was the most efficient in MRTF-B gene silencing and non-cytotoxic compared to the non-targeting formulation. In vivo, subconjunctival administration of LYR nanoparticles containing MRTF-B siRNAs doubled bleb survival in a pre-clinical rabbit model of glaucoma filtration surgery. Furthermore, MRTF-B LYR nanoparticles reduced the MRTF-B mRNA by 29.6% in rabbit conjunctival tissues, which led to significantly decreased conjunctival scarring with no adverse side effects. LYR-mediated delivery of siRNA shows promising results to increase bleb survival and to prevent conjunctival fibrosis after glaucoma filtration surgery.

Role of IL-18 induced Amphiregulin expression on virus induced ocular lesions.

This report deals with the possible mechanism by which IL-18 can contribute to the control and resolution of inflammatory lesions in the cornea caused by herpes simplex virus infection. Our results demonstrate that the expression of the IL-18R by both regulatory T cells (Treg) and effector T cells was a pivotal event that influenced lesion pathogenesis. The engagement of IL-18R on Treg with its cytokine ligand resulted in Amphiregulin expression a molecule associated with tissue repair. In support of this scheme of events, lesion severity became more severe in animals unable to express the IL-18R because of gene knockout and was reduced in severity when IL-18 was overexpressed in the cornea. These changes in lesion severity correlated with the frequency and number of both Treg and Teff that expressed Amphiregulin. Additional experiments indicated that IL-12 and IL-18 acted synergistically to enhance Amphiregulin expression in Treg, an event partly dependent on P38 MAPK activity. Finally, sub-conjunctival administration of Amphiregulin resulted in resolution of both developing and developed lesions. Thus, overall our results imply that IL-18 may participate in controlling the severity of SK and contribute to tissue repair by converting both Treg and effector T cells into those that produce Amphiregulin.

Serotype survey of AAV gene delivery via subconjunctival injection in mice.

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

Functional and Structural Effects of Erythropoietin Subconjunctival Administration in Glaucomatous Animals

Purpose: The present study aimed to assess functional and structural benefits of erythropoietin (EPO) when administered subconjunctivally in the retina of glaucomatous rats using electroretinography (ERG) and retinal thickness (RT) measurements. Methods: Glaucoma was experimentally induced in 26 Wistar Hannover albino rats. Animals were divided into 2 groups of 13 animals each: a treated group receiving a unique subconjunctival injection of 1,000 IU of EPO and a control group receiving a saline solution. In each group, 7 animals were used for retinal function evaluation (ERG) and 6 animals were used for retinal structural evaluation (histology). RT was measured, dorsally and ventrally, at 500 μm (RT1) and at 1,500 μm (RT2) from the optic nerve. Results: Retinal function evaluation: for both scotopic and photopic conditions, ERG wave amplitudes increased in the treated group. This increase was statistically significant (p < 0.05) in photopic conditions. Structural evaluation: for both locations RT1 and RT2, the retinas were significantly (p < 0.05) thicker in the treated group. Conclusion: Subconjunctival EPO administration showed beneficial effects both on retinal structure and on retinal function in induced glaucoma in albino rats. This neuroprotective effect should be applied in other animal species.

The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy.

Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long–term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.

Новая модель транзиторной ишемии глаза кроликов и количественная оценка степени ишемии методом офтальмоплетизмографии

Purpose: to create a model of transient eye ischemia in order to study the effectiveness of drugs affecting the intensity of ocular blood flow in ischemic conditions, and to develop a method of quantifying the extent of ischemia. Material and methods. The study involved 72 Chinchilla rabbits. For the first time, the vasoconstrictive drug Phenylephrine 10 mg/ml was used to create an experimental model of ocular ischemia. The degree of ischemia was quantitatively assessed by the method of ophthalmoplethysmography, adapted for animals, while the degree of hypoxia was measured by enzymatic amperometric determination of lactate concentration in the intraocular fluid. Results. The effect of phenylephrine on the development of eye ischemia was found to be dose-dependent. After subconjunctival administration of phenylephrine, the maximum decrease in the minute volume of blood flow occurs in the interval from 60 min to 120 min. The time of permissible repeated injection of phenylephrine for the prolongation of ischemia development period was established at the 90th minute after the first injection, while the period of development of transient ischemia of the rabbit's eye is longer - 240 min. An increased content of lactate in the intraocular fluid after an injection of phenylephrine was noted, which points to the development of hypoxia due to transient ischemia of eye tissues. Conclusion. The proposed experimental model of transient rabbit ischemia developed by us may be used to study the efficiency of drugs affecting the intensity of ocular blood flow. It provides the possibility to control the dose of the ischemic factor and to avoid side effects. For citation: Chesnokova N.B., Grigoryev A.V., Mustafayeva K.N., Beznos O.V. A new model of transient ischemia of rabbit eyes and a quantitative assessment of ischemia degree using ophthalmoplethysmography. Russian ophthalmological journal. 2018; 11 (2): 35-40. doi: 10.21516/2072-0076-2018-11-2-35-40 (In Russian).

Freeze-dried matrices for ocular administration of bevacizumab: a comparison between subconjunctival and intravitreal administration in rabbits.

This work was aimed to tune solid matrices for bevacizumab (BVZ) subconjunctival or intravitreal administration in order to prolong drug release, to reduce the number of applications and consequently the side effects. Matrices, with sizes suitable for intravitreal or subconjunctival administration, based on hydroxypropylmethyl cellulose (HPMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and polyacrylic acid (PAA) were obtained by freeze-drying of polymeric dispersions either in phosphate buffer solution or water and were sterilized by gamma rays. The matrices were characterized from the technological point of view and evaluated for in vitro release of dextran and BVZ. In vivo evaluation of BVZ release in ocular humours was finally carried out on rabbits. The obtained matrices showed solvent sorption time ranging from a few seconds for PAA to 46min for HPMC, with shorter times when prepared in buffer solution. The hydration times were up to 5.5-fold higher after sterilization. HPMC and PVA matrices showed a slowdown of the release rate of both dextran and BVZ, but HPMC was selected for following in vivo studies also in consideration of its higher viscosity after rehydration of the matrix. HPMC matrix was well tolerated by the rabbit eye when intravitreally and subconjunctivally administered. The different treatment produced the same effect in terms of drug concentration in aqueous and vitreous humour up to 12weeks after administration. The results of this study support the possible use of lyophilized matrices as a BVZ delivery system to the posterior segment of the eye.

Ocular tissue distribution and pharmacokinetic study of a small 13kDa domain antibody after intravitreal, subconjuctival and eye drop administration in rabbits

Domain antibodies (dAb's) comprise the smallest functional unit of human IgG and can be targeted to a range of different soluble cytokine and receptor targets in the eye. In particular their small size may offer advantage for ocular tissue penetration and distribution. To investigate this we used a 13kDa tool molecule to undertake a preliminary short term ocular tissue distribution and pharmacokinetic study in the rabbit eye. The dAb was administered by the intravitreal or subconjunctival route or, as topical eye drops for up to five days and dAb concentrations measured in vitreous, aqueous, conjunctiva, choroid-RPE, retina, iris, sclera, and ciliary body. The observed elimination half-live of the dAb (~3 days) in vitreous showed a similar elimination rate to that of a much larger (∼50kDa) Fab fragment whilst the half-life following subconjunctival administration was ∼24 h and, after eye drop dosing the dAb was detectable in aqueous and conjunctiva. These preliminary data show that the intravitreal half-life of dAb's are similar to much larger antibody fragments, offering the potential to deliver significantly more drug to target on a molar basis with a single intravitreal injection potentially enabling dosing frequencies of once a month or less. Subconjunctival injection may provide short duration therapeutic levels of dAb to the anterior and posterior chamber whilst topical eye drop delivery of dAbs may be useful in front-of-eye disease. These data indicate that small domain antibodies may have utility in ophthalmology. Further studies are warranted.

Effects of subconjunctival administration of anti-high mobility group box 1 on dry eye in a mouse model of Sjӧgren's syndrome.

PurposeExtracellular high mobility group box 1 (HMGB1) acts as a damage associated molecular pattern molecule through the Toll-like receptor to promote autoreactive B cell activation, which may be involved in the pathogenesis of Sjӧgren’s syndrome. The aim of this study was to investigate the effect of subconjunctival administration of anti-HMGB1 on dry eye in a mouse model of Sjӧgren’s syndrome.MethodsTen weeks-old NOD.B10.H2b mice were subconjunctivally injected with 0.02 to 2 μg of anti-HMGB1 antibodies or PBS twice a week for two consecutive weeks. Tear volume and corneal staining scores were measured and compared between before- and after-treatment. Goblet cell density was counted in PAS stained forniceal conjunctiva and inflammatory foci score (>50 cells/focus) was measured in extraorbital glands. Flow cytometry was performed to evaluate the changes in BrdU+ cells, IL-17-, IL-10-, or IFNγ-secreting cells, functional B cells, and IL-22 secreting innate lymphoid cells (ILC3s) in cervical lymph nodes. The level of IL-22 in intraorbital glands was measured by ELISA.ResultsInjection of 2 μg or 0.02 μg anti-HMGB1 attenuated corneal epithelial erosions and increased tear secretion (p<0.05). Goblet cell density was increased in 0.2 μg and 2 μg anti-HMGB1-treated-mice with marginal significance. The inflammatory foci score, and the number of BrdU+ cells, IL-17-, IL-10-, IFNγ-secreting cells, and functional B cells did not significantly change following anti-HMGB1 treatment. Surprisingly, the percentage of ILC3s was significantly increased in the draining lymph nodes (p<0.05), and the expression of IL-22 was significantly increased in the intraorbital glands (p<0.05) after administration of 2 μg anti-HMGB1.ConclusionThis study shows that subconjunctival administration of anti-HMGB1 attenuates clinical manifestations of dry eye. The improvement of dry eye may involve an increase of ILC3s, rather than modulation of B or plasma cells, as shown using a mouse model of Sjӧgren’s syndrome.

Efficacy of Subconjunctival Bevacizumab Injections before and after Surgical Excision in Preventing Pterygium Recurrence.

Purpose To evaluate the efficacy of subconjunctival bevacizumab injections, before and after surgical excision with bare sclera technique, in preventing postoperative pterygium recurrence. Material and Methods 83 eyes of 83 patients affected with primary pterygia underwent surgical excision. 42 eyes received two subconjunctival bevacizumab injections, at the dosage of 2.5 mg/0.1 ml, one week prior surgery and one week after intervention. Recurrence rate was evaluated among the two groups. Moreover, modifications of pterygium size and grade one week after the first injection were evaluated. Results At 6 months after surgery, the recurrence rate was 7.14% in the bevacizumab group and 24.39% in the control group. Significant changes of pterygium size and grade were reported after the first injection. No important complications related to bevacizumab subconjunctival injections were registered. Conclusions The application of subconjunctival bevacizumab injections, at the dosage of 2.5 mg/0.1 ml, before and after surgical pterygium excision, may be useful in preventing lesion recurrence after bare scleral procedures. Furthermore, bevacizumab subconjunctival administration is well tolerated and may represent a safer alternative if compared with other surgical techniques and adjunctive drugs. This trial is retrospectively registered with ISRCTN Registry on 18 April 2017, TRN: ISRCTN11424742.

Comparison of different doses of subconjunctival sunitinib with bevacizumab in the treatment of corneal neovascularization in experimental rats.

Background:To compare the efficacy of subconjunctival administration of bevacizumab and different doses of sunitinib malate in reducing corneal neovascularization (CNV).Materials and Methods:In this experimental study, central corneal cauterization was created in the right eye of fifty male Sprague–Dawley rats. On day 1 (1 week after cauterization), rats were randomly assigned into five treatment groups. Group control (n = 10) received subconjunctival injection of 0.02 ml of base saline solution. Group 1 (n = 10) received 0.02 ml of bevacizumab (25 mg/ml). Group 2, 3, and 4 (n = 10 for each group) were treated with 0.02 ml of sunitinib malate (10, 20, and 50 μg/ml, respectively). On days 1, 7, and 14, digital photographs of the cornea were taken, and the area of CNV was measured.Results:During the 2-week follow-up, CNV area in treatment groups was less than in control group (P < 0.05). On day 7, corneal avascular area was highest in Group 3 at 63%. On day 14, the area of CNV in Groups 2 and 3 was less than in Group 1 (P = 0.031 and 0.011, respectively), but the difference between Groups 2 and 3 was not statistically significant (P = 0.552). The decreased CNV area on day 14 in Group 4 was significant in comparison to bevacizumab, but it was not significant on day 7 (P = 0.25 on day 7 and 0.002 on day 14).Conclusion:Subconjunctival sunitinib malate is more effective than bevacizumab in regressing CNV. This effect is more prominent on day 14.

Сравнительное исследование фармакокинетики рекомбинантной проурокиназы при субконъюнктивальном и инстилляционном методах введения

Purpose: to compare the dynamics of recombinant prourokinase (RPU) in humor of the anterior chamber of the eye with regard to administration technique (instillations vs. subconjunctival injection) using fluorescent spectrophotometry. Material and methods. 62 samples of anterior chamber humor were obtained from 62 patients (62 eyes) who underwent cataract surgery, with rigid pupil due exfoliation syndrome or after an earlier acute iridocyclitis. The patients were divided into two groups. Group 1 received the medication by forced instillations of RPU solution with the concentration of 330 IU per 1 ml of 0.9 % sodium chloride continuously for 10 minutes, the rate being 30 drops per minute. Group 2 (31 patients, 31 eyes) received a subconjunctival injection of 0.5 ml of 0.9 % sodium chloride solution that contained 5,000 IU of RPU. For each sample, the time elapsed from the moment of RPU administration to the moment of humor sampling was noted. RPU concentration in the samples was determined by fluorescent spectrophotometry using the HITACHI 650-60 device (Japan). Results. Inforced instillations, RPU is detected in the humor of the anterior chamber starting from the 5th minute after the instillation, with rapid growth of concentration, which attains the level of 3100 IU/ml in 30 to 60 minutes. Then, however, the concentration drops, with the half-life period being 160 to 180 minutes. In subconjunctival injections RPU is detected after the same time span after the procedure; however, its concentration grows for 120 to 160 minutes, reaching the level of 2500 IU/ml, and only then begins to fall gradually, the half-life period being as long as 320-360 minutes. Conclusions. RPU may be used for the treatment of fibrinoid syndrome with practically the same efficacy by subconjunctival injections or by force instillation. This is explained by the fact that the enzyme is found in anterior chamber humor in sufficient concentrations in either case. The pharmacokinetics of therapeutic concentration of the medicine in the humor of the anterior chamber envisages a single subconjunctival administration and permits two instillations in the 24-hour period Russian ophthalmological journal. 2017; 10 (2): 18-22. (in Russian). doi: 10.21516/2072-0076-2017-10-2-18-22.

Effects of topical and subconjunctival use of bevacizumab on corneal neovascularization in rabbits’ eyes

To evaluate and compare the effects of topical application and subconjunctival injection of bevacizumab on corneal neovascularization (CNV) in rabbits' eyes after chemical burning of the cornea. The animals were randomly distributed into four groups of five animals. In one group, the drug was instilled, while in another, it was administered by subconjunctival injection. The two procedures using bevacizumab were compared with instillation and subconjunctival injection of saline solution (S). Neovascularization was evaluated according to the size of the invasion area of new blood vessels and through computerized analysis of this area. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for two-by-two comparison of the groups, to assess the external examination of CNV. Analysis of variance was used to assess the area of CNV. P<0.05 was considered statistically significant. Assessing both the external examination and the invasion area of neovessels on the 5th and 10th days, there was a clear difference between the groups. The group to which saline solution had been applied showed higher scores for CNV, as well as increases in the invasion area of neovessels. Two-by-two comparison of groups revealed no significant differences. However, an analysis of the factors involved (injection vs. instillation and bevacizumab vs. saline solution) showed that injection did not differ from instillation, but that bevacizumab differed from saline solution. Bevacizumab showed an inhibitory effect on CNV in rabbits' eyes after chemical burning of the cornea. There was no difference between the topical or subconjunctival administration of bevacizumab in the inhibition of CNV.

Subconjunctival antimicrobial poloxamer gel for treatment of corneal ulceration in stranded California sea lions (Zalophus californianus).

Corneal ulcers are commonly encountered in pinnipeds. Prolonged oral antibiotics and topical ophthalmic solutions may not be practical to administer, and novel treatment techniques are desired. Thermodynamic gels are a potential solution because they hold antimicrobials at the site of injection, slowly releasing drug. This study investigated the clinical efficacy of antibiotic-impregnated poloxamer gel in management of corneal ulceration. Twenty-six California sea lions undergoing rehabilitation at The Marine Mammal Center. A poloxamer gel mixed with 2% enrofloxacin was subconjunctivally injected in the treatment group. Control animals received oral doxycycline. Systemic anti-inflammatories and analgesics were administered as needed. Corneal examinations under general anesthesia were repeated weekly, and included sampling for bacterial culture and corneal cytology, collection of high-quality corneal images, and treatment administration until the ulcers were healed. There was no gross or histologic evidence of a localized tissue reaction to the gel administration in the conjunctiva, and no evidence of systemic reaction to therapy in animals that died due to unrelated causes during the study period (n = 17). In animals that experienced a superficial corneal ulcer involving only epithelium or superficial stroma (n = 12), all lesions resolved completely, in both treatment and control groups. Of those animals with deeper or more complex ulcers involving keratomalacia or descemetoceles (n = 15), four demonstrated complete lesion resolution (all four received gel treatment). This study demonstrates that subconjunctival antibiotic poloxamer gel administration is a safe and effective alternative therapeutic option to traditional treatments for superficial corneal ulceration in pinnipeds.

Sustained Delivery of Timolol Maleate for Over 90 Days by Subconjunctival Injection.

Medical treatment of glaucoma relies on intraocular pressure (IOP)-lowering medications, typically administered daily by the patient. While these medications are effective when applied correctly, patient adherence is a major obstacle in glaucoma treatment. We have developed a sustained-release formulation of timolol maleate that can be injected subconjunctivally to avoid patient noncompliance. A biodegradable microsphere formulation for timolol maleate was injected subconjunctivally in normal rabbits. We measured timolol levels in tears, aqueous humor, vitreous humor, and serum of study rabbits. Furthermore, IOP profiles were recorded longitudinally. Tissue compatibility and side effects were evaluated using histochemistry. The microsphere formulation led to measureable amounts of timolol in the aqueous humor and the tear film for up to 90 days. Timolol was not detectable in the serum at any time. A significant reduction of IOP was observed in treated eyes. Clinically, the subconjunctival administration of the microspheres was well tolerated with no signs of inflammation or infection. The absence of local inflammation was confirmed by histology. A single subconjunctival administration of timolol microspheres achieved delivery and IOP reduction in rabbits for up to 90 days without local or systemic inflammation or toxicity. This approach has the potential to improve the management of glaucoma in patient populations, who are challenged to adhere to a regimen of daily eye drops.

The Combination of PlGF Inhibition and MMC as a Novel Anti-Scarring Strategy for Glaucoma Filtration Surgery.

The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients. In the trabeculectomy mouse model, intracameral injection(s) of the PlGF inhibitor (5D11D4) were compared to MMC or aflibercept and to the combination of both compounds. Treatment outcome was studied by bleb investigation and by Sirius Red staining. The effect of subconjunctival PlGF administration and topical 5D11D4 on IOP was investigated in normotensive mice and was compared to topical administration of latanoprost, the gold standard for IOP-lowering. Combination of MMC and 5D11D4 was able to significantly improve surgical outcome compared to monotherapy of MMC or 5D11D4 (n = 20; P < 0.001). Compared to combined treatment of MMC with aflibercept, the simultaneous administration of MMC and 5D11D4 was equally efficacious in improving surgical outcome (n = 15; P = 0.88). In normotensive mice, 5D11D4 was able to significantly reduce the IOP-elevation induced by PlGF (n = 10; P < 0.05), whereas no effect of 5D11D4 was seen in naive mice, which was in contrast to latanoprost. The current data suggest that application of MMC together with PlGF inhibition may have complementary effects in the improvement of surgical outcome and is equally efficacious as the combined treatment of MMC and aflibercept. Inhibition of PlGF also might open alternative perspectives as IOP-lowering strategy for glaucoma patients with increased aqueous PlGF levels.

Ocular Erythropoietin Penetration after Subconjunctival Administration in Glaucomatous Rats

Purpose: The present study aimed to determine whether the subconjunctival administration of recombinant human erythropoietin (rHuEPO) reached the retina in glaucoma conditions. After subconjunctival rHuEPO administration, in a rat glaucoma model, erythropoietin (EPO) distribution in the rat's retina was studied by immunohistochemistry. Methods: Female Wistar Hannover albino rats (n = 15) were divided into 2 groups, control (n = 3) and treated (n = 12). The animals' unilateral glaucoma was induced by coagulation of episcleral veins, under general anaesthesia. After vein coagulation, 1,000 IU of rHuEPO were administered by the subconjunctival route to the treated group (n = 12). The control group (n = 3) received only a subconjunctival saline injection. The contralateral eye of each animal remained untouched. Treated group animals were euthanized at different time points, i.e. days 1, 3, 7 and 14. Bilateral enucleation was performed, and EPO distribution in the rat's retina was assessed by immunohistochemistry. Results: Glaucoma was confirmed by results of repeated intraocular pressure measurements over the experimental period. In the test group, EPO was identified in different neuroretinal cells, showing a stronger immunostaining signal during the first 2 time points in the retinal ganglion cell (RGC) layer. EPO protein was still present on day 14 after the subconjunctival injection. EPO was not detected in any of the control eyes or in any contralateral eye of the treated group. Conclusion: When administered subconjunctivally to glaucomatous eyes, rHuEPO reached the RGC layer and was still present at least 14 days after administration. The subconjunctival route was shown to be a promising alternative for ocular EPO delivery in glaucomatous conditions in a rat animal model.