Nedosiran population pharmacokinetic and pharmacodynamic modelling and simulation to guide clinical development and dose selection in patients with primary hyperoxaluria type 1.

AIM: To characterize pharmacokinetic and pharmacodynamic profiles of nedosiran in patients with primary hyperoxaluria type 1 (PH1), identify influential covariates and confirm therapeutic doses. METHODS: A population pharmacokinetic (PK)/pharmacodynamic (PD) (POP-PKPD) model was developed to characterize the concentration-time course of nedosiran and the corresponding effect on 24-h urinary oxalate (Uox). Simulations of dosing to achieve clinically meaningful reduction in Uox in children, adolescents and adults with PH1 were performed. RESULTS: Analyses included PK data from 143 healthy participants and PH1/PH2 patients, and PD data from 46 PH1 patients. Nedosiran PK was described by a two-compartment model with dual n-transit absorption and parallel linear and nonlinear elimination. The relationship between nedosiran exposure and Uox was described by an indirect response model. Body weight, estimated glomerular filtration rate (eGFR) and disease status were identified as influential covariates for the POP-PK model. The simulation results supported a weight-banded dosing regimen of nedosiran sodium in adolescents and adults (≥12 years) with PH1 of 170 mg (weight ≥50 kg) and 136 mg (weight <50 kg), in children (6-11 years) with PH1 of 3.5 mg/kg, and no dose adjustments for PH1 patients with relatively preserved kidney function (eGFR ≥ 30 mL/min/1.73m2). Following the proposed dosing regimens, the simulated median fold-changes in PK AUC0-τ,ss were acceptable (≤1.51 fold-change) and ~71% of PH1 patients across all age groups achieved near-normal Uox (<0.6 mmol) by week 52. CONCLUSIONS: The final POP-PKPD model characterizes observed nedosiran PK and Uox data. Simulations support nedosiran dosing regimens in PH1 patients aged ≥6 years with relatively preserved kidney function.

Safety, Pharmacokinetics, and Exposure-Response Modeling of Nedosiran in Participants With Severe Chronic Kidney Disease.

Nedosiran is an investigational RNA-interference therapeutic in development for primary hyperoxaluria (PH). Because nedosiran undergoes renal clearance, we assessed its pharmacokinetic profile in non-PH participants with normal kidney function and Stages 4/5 chronic kidney disease (CKD), the latter with/without dialysis. Nedosiran exposure-response modeling in patients with PH Subtype 1 (PH1) with different renal function level was performed to recommend a nedosiran dose for this subpatient population. In this open-label, single-dose, Phase 1 study, 24 participants with estimated glomerular filtration rate <30 mL/min/1.73 m2 (CKD Stages 4/5; on hemodialysis [Groups 1a, 1b] and not on hemodialysis [Group 2]) and 10 participants with normal kidney function (estimated glomerular filtration rate ≥90 mL/min/1.73 m2 ; Group 3) received a single dose of subcutaneous nedosiran sodium 170 mg. Group 1a received nedosiran 8 hours before beginning hemodialysis, Group 1b received nedosiran 2 hours after completing hemodialysis; Group 2 was not on hemodialysis. Nedosiran population pharmacokinetic-pharmacodynamic analyses were conducted using pooled data from this study and 4 others. Nedosiran pharmacokinetic exposure in non-PH participants with CKD Stages 4/5 was approximately 2-fold higher versus participants with normal kidney function. Hemodialysis timing relative to nedosiran administration had no clinically significant impact on pharmacokinetics (Group 1a vs 1b). Nedosiran was well tolerated. Modeling indicated that in patients with PH1 with CKD Stages 4/5, lower nedosiran doses provide similar exposure and potential reduction in 24-hour urinary oxalate to standard nedosiran doses in patients with PH1 with normal kidney function or CKD Stages 2/3. Nedosiran dosage reductions are recommended in patients with PH1 with CKD Stages 4/5; further adjustments are unnecessary if dialysis is started.

A sensitive LC-MS/MS assay to quantitate free payload Aur0101 from ADC PYX-201 in rat and monkey plasma.

Aim: Aur0101 is a cytotoxic and small-molecule microtubule depolymerizing agent, and is the payload conjugated to antibody-drug conjugate PYX-201. Developing and validating a sensitive bioanalytical method to quantitate Aur0101 was novel and crucial in preclinical PYX-201 studies. Materials & methods: Reference standard Aur0101 and its stable isotope labelled internal standard Aur0101-d8 were used in this LC-MS/MS method. Results: This sensitive assay was validated at a lower limit of quantitation of 15 pg/ml and successfully applied to support preclinical rat and monkey toxicology studies. Preclinical plasma toxicokinetic parameters were presented. Conclusion: A sensitive and robust LC-MS/MS assay was validated for Aur0101 in rat and monkey plasma.

Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies.

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 - 10,000 ng/mL and 250 - 6000 ng/mL in rat and monkey K2EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix.

Quantification of antibody-drug conjugate PYX-201 in rat and monkey plasma via ELISA and its application in preclinical studies.

Aim: PYX-201 is a novel antibody-drug conjugate targeting the extra domain B splice variant of fibronectin in the tumor microenvironment. Accurate quantification of PYX-201 is critical for PYX-201 pharmacokinetics profiling in preclinical studies. Materials & methods: ELISA was performed using reference standard PYX-201, mouse monoclonal anti-monomethyl auristatin E antibody, mouse IgG1, mouse monoclonal anti-human IgG horseradish peroxidase and donkey anti-human IgG horseradish peroxidase. Results: This assay was validated at 50.0-10,000 ng/ml in rat dipotassium EDTA plasma and 250-10,000 ng/ml in monkey dipotassium EDTA plasma. Conclusion: This is the first time for a PYX-201 bioanalytical assay in any matrix to be reported.

PHYOX2: a pivotal randomized study of nedosiran in primary hyperoxaluria type 1 or 2.

Nedosiran is an investigational RNA interference agent designed to inhibit expression of hepatic lactate dehydrogenase, the enzyme thought responsible for the terminal step of oxalate synthesis. Oxalate overproduction is the hallmark of all genetic subtypes of primary hyperoxaluria (PH). In this double-blind, placebo-controlled study, we randomly assigned (2:1) 35 participants with PH1 (n = 29) or PH2 (n = 6) with eGFR ≥30 mL/min/1.73 m2 to subcutaneous nedosiran or placebo once monthly for 6 months. The area under the curve (AUC) of percent reduction from baseline in 24-hour urinary oxalate (Uox) excretion (primary endpoint), between day 90-180, was significantly greater with nedosiran vs placebo (least squares mean [SE], +3507 [788] vs -1664 [1190], respectively; difference, 5172; 95% CI 2929-7414; P < 0.001). A greater proportion of participants receiving nedosiran vs placebo achieved normal or near-normal (<0.60 mmol/24 hours; <1.3 × ULN) Uox excretion on ≥2 consecutive visits starting at day 90 (50% vs 0; P = 0.002); this effect was mirrored in the nedosiran-treated PH1 subgroup (64.7% vs 0; P < 0.001). The PH1 subgroup maintained a sustained Uox reduction while on nedosiran, whereas no consistent effect was seen in the PH2 subgroup. Nedosiran-treated participants with PH1 also showed a significant reduction in plasma oxalate versus placebo (P = 0.017). Nedosiran was generally safe and well tolerated. In the nedosiran arm, the incidence of injection-site reactions was 9% (all mild and self-limiting). In conclusion, participants with PH1 receiving nedosiran had clinically meaningful reductions in Uox, the mediator of kidney damage in PH.

Safety, pharmacodynamics, and exposure-response modeling results from a first-in-human phase 1 study of nedosiran (PHYOX1) in primary hyperoxaluria.

Primary hyperoxaluria (PH) is a family of ultra-rare autosomal recessive inherited disorders of hepatic glyoxylate metabolism characterized by oxalate overproduction. Nedosiran is an RNA interference agent that inhibits hepatic lactate dehydrogenase, the enzyme responsible for the common, final step of oxalate production in all three genetic subtypes of PH. Here, we assessed in a two-part, randomized, single-ascending-dose, phase 1 study (PHYOX1) the safety, pharmacokinetics, pharmacodynamics, and exposure-response of subcutaneous nedosiran in 25 healthy participants (Group A) and 18 patients with PH1 or PH2 (Group B). Group A received nedosiran (0.3, 1.5, 3.0, 6.0, then 12.0 mg/kg) or placebo, and Group B received open-label nedosiran (1.5, 3.0, or 6.0 mg/kg). No significant safety concerns were identified. Injection site reactions (four or more hours post dose) occurred in 13.3% of participants in Group A and 27.8% of participants in Group B. Mean maximum reduction in 24-hour urinary oxalate excretion from baseline to day 57 (end of study) across Group B dose cohorts was 55% (range: 22%-100%) after single-dose nedosiran, with 33% participants reaching normal 24-hour urinary oxalate excretion. Based on the available modeling and simulation data, a fixed monthly dose of nedosiran 160 mg (free acid; equivalent to 170 mg sodium salt) in adults was associated with the highest proportion of simulated individuals achieving normal or near-normal 24-hour urinary oxalate excretion and fewest fluctuations in urinary oxalate response. Thus, single-dose nedosiran demonstrated acceptable safety and evidence of a pharmacodynamic effect in both PH1 and PH2 subpopulations consistent with its mechanism of action.

Nanomedicines for back of the eye drug delivery, gene delivery, and imaging.

Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology. Nanomedicines, products of nanotechnology, having at least one dimension in the nanoscale include nanoparticles, micelles, nanotubes, and dendrimers, with and without targeting ligands. Nanomedicines are making a significant impact in the fields of ocular drug delivery, gene delivery, and imaging, the focus of this review. Key applications of nanotechnology discussed in this review include a) bioadhesive nanomedicines; b) functionalized nanomedicines that enhance target recognition and/or cell entry; c) nanomedicines capable of controlled release of the payload; d) nanomedicines capable of enhancing gene transfection and duration of transfection; f) nanomedicines responsive to stimuli including light, heat, ultrasound, electrical signals, pH, and oxidative stress; g) diversely sized and colored nanoparticles for imaging, and h) nanowires for retinal prostheses. Additionally, nanofabricated delivery systems including implants, films, microparticles, and nanoparticles are described. Although the above nanomedicines may be administered by various routes including topical, intravitreal, intravenous, transscleral, suprachoroidal, and subretinal routes, each nanomedicine should be tailored for the disease, drug, and site of administration. In addition to the nature of materials used in nanomedicine design, depending on the site of nanomedicine administration, clearance and toxicity are expected to differ.

In vitro transport and partitioning of AL-4940, active metabolite of angiostatic agent anecortave acetate, in ocular tissues of the posterior segment.

PURPOSE: The purpose of this study was to evaluate partitioning into and transport across posterior segment tissues (sclera, retinal pigment epithelium (RPE)-choroid) of AL-4940, the active metabolite of angiostatic cortisene anecortave acetate (AL-3789). METHODS: Transport of [(14)C]-AL-4940 was measured through RPE-choroid-sclera (RCS) and sclera, excised from Dutch Belted pigmented rabbits' eyes, in the directions of scleral to vitreal (S-->V) and vitreal to scleral (V-->S) for 3 h at 37 degrees C using Ussing chambers. Tissue integrity was monitored by transepithelial electrical resistance (TEER), potential difference (PD), and biochemical assay (LDH). Partitioning in RPE-choroid and sclera was determined separately for both [(14)C]-AL-4940 and [(14)C]-AL-3789. Mathematical analysis for bilaminate membranes used partitioning and transport data to derive diffusion coefficients for 2 tissue layers sclera and RPE-choroid. RESULTS: Partitioning of drug in tissue was comparable for both [(14)C]-AL-4940 and [(14)C]-AL-3789. Partition coefficients of drug in tissue were 2.2 for sclera and about 4 for RPE-choroid. Permeability through sclera alone was about 3 x 10(-5) cm/s and about 1 x 10(-5) cm/s through the RCS tissue, irrespective of the direction of transport (S-->V) or (V-->S). Results from bioelectrical and biochemical evaluation of tissue with modified LDH assay provided evidence that the RCS tissue preparation remained viable during the period of transport study. CONCLUSIONS: The thin RPE-choroid layer contributes significantly to resistance to drug transport, and diffusivity in this layer is 10 times less than in sclera. This experimental scheme is proposed as an important component for the development of a general ocular physiologically based pharmacokinetic model.

Delivery of celecoxib for treating diseases of the eye: influence of pigment and diabetes.

IMPORTANCE OF THE FIELD: Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are two major causes of blindness. In these disorders, growth factors such as vascular endothelial growth factor (VEGF) are upregulated, leading to either enhanced vascular permeability or proliferation of endothelium. While corticosteroid therapies available at present suffer from side effects including cataracts and elevated intraocular pressure, anti-VEGF antibody therapies require frequent intravitreal injections, a procedure that can potentially lead to retinal detachment or endophthalmitis. Thus, there is a need to develop safe, sustained release therapeutic approaches for treating AMD and DR. AREAS COVERED IN THIS REVIEW: This review discusses the pharmacological basis for using celecoxib, an anti-inflammatory drug capable of selectively inhibiting cycloxygenase 2, in treating AMD and DR. In addition, this article discusses the safety, delivery advantage and efficacy of celecoxib by transscleral retinal delivery, a periocular delivery approach that is less invasive to the globe compared with intravitreal injections. WHAT THE READER WILL GAIN: The reader will gain insights into the development of a pharmacological agent and a sustained release delivery system for treating DR and AMD. Further, the reader will gain insights into the influence of eye physiology including pigmentation and disease states such as DR on retinal drug delivery. TAKE HOME MESSAGE: Transscleral sustained delivery of anti-inflammatory agents is a viable option for treating retinal disorders.

Effect of diabetes on transscleral delivery of celecoxib.

PURPOSE: To investigate the effects of diabetes on transscleral retinal delivery of celecoxib in albino and pigmented rats. METHODS: Albino (Sprague Dawley-SD) and pigmented (Brown Norway-BN) rats were made diabetic by a single intraperitoneal injection of streptozotocin (60 mg/kg) following 24 h of fasting and diabetes was confirmed (blood glucose>250 mg/dL). Two months after diabetes induction, the integrity of blood-retinal-barrier in control versus diabetic rats from both strains was compared by using FITC-dextran leakage assay. Fifty microliter suspension of celecoxib (3 mg/rat) was injected periocularly in both the strains in one eye, 2 months following diabetes induction. The animals were euthanized at the end of 0.25, 0.5, 1, 2, 3, 4, 8, and 12 h post-dosing and celecoxib levels in ocular tissues and plasma were estimated using a HPLC assay. RESULTS: Diabetes (2-month duration) resulted in 2.4 and 3.5 fold higher blood-retinal barrier leakage in diabetic SD and BN rats, respectively, compared to controls. The area under tissue celecoxib concentration versus time curves (AUC) for sclera, cornea, and lens were not significantly different between control and diabetic animals. However, retinal and vitreal AUCs of celecoxib in treated eyes were approximately 1.5-fold and 2-fold higher in diabetic SD and BN rats, respectively, as compared to the controls. CONCLUSIONS: Transscleral retinal and vitreal delivery of celecoxib is significantly higher in diabetic animals of both strains. The increase in retinal delivery of celecoxib due to diabetes is higher in pigmented rats compared to albino rats. Higher delivery of celecoxib in diabetic animals compared to control animals can be attributed to the disruption of blood-retinal barrier due to diabetes.

Effect of circulation on the disposition and ocular tissue distribution of 20 nm nanoparticles after periocular administration.

PURPOSE: Our previous studies indicated that while 20 nm particles are rapidly cleared from the periocular space of the rat following posterior subconjunctival injection, 200 nm particles persisted for at least two months. To understand faster clearance of 20 nm particles, the purpose of this study was to determine transscleral permeability and in vivo disposition in the presence and absence of circulation. Further, it was the purpose of this study to simulate sustained retinal drug delivery after periocular administration of rapidly cleared and slowly cleared nanoparticles. METHODS: The permeability of 20 and 200 nm particles over 24 h was examined across isolated bovine sclera and sclera-choroid-RPE with or without a surfactant (Tween 20, 0.1% w/v) added to the preparation. The in vivo disposition of nanoparticles was performed using Sprague Dawley rats. The rats, either dead or alive, were administered with 400 microg of the nanoparticles in the periocular space, and the particle disposition in the eye tissues was assessed 6 h later. To evaluate the role of the reticulo-endothelial system and lymphatic circulation, isolated liver, spleen, and cervical, axillary, and mesenteric lymph nodes were analyzed using confocal microscopy. Mathematical simulations with Berkeley Madonna were used to evaluate the effect of nanoparticle size on retinal drug levels following periocular administration. Celecoxib was used as the model drug and the finalized pharmacokinetic model from a previous study was used with some modifications for the simulation. RESULTS: Transport of 20 nm particles across sclera in the presence and absence of the surfactant were 0.1%+/-0.07% and 0.46%+/-0.06%, respectively. These particles did not permeate across the sclera-choroid-RPE in 24 h. There was no quantifiable transport for 200 nm particles across the sclera or the sclera-choroid-RPE. In live animals, the 20 nm particles were undetectable in any of the ocular tissues except in the sclera-choroid following periocular administration; however, in dead animals, the particle concentrations in the sclera-choroid were 19 fold higher than those in live animals, and particles were detectable in the retina as well as vitreous. The retention of 20 nm particles at the site of administration was two fold higher in the dead animals. In live animals, the particles were clearly detectable in the spleen and to a very low extent in the liver as well. The particles were also detected in the cervical, axillary, and mesenteric lymph nodes of the live animals. Simulations with two particles (20 nm and 200 nm) with different clearance rates demonstrated that the retinal drug levels were affected by particle clearance. Larger nanoparticles sustained retinal drug delivery better than smaller nanoparticles. With an increase in drug release rate from the particles, these differences diminish. CONCLUSIONS: The 20 nm particles are transported across the sclera to a minor degree; however, there is no significant transport across the sclera-choroid-RPE. Periocular circulation (blood and lymphatic) plays an important role in the clearance of the 20 nm particles. The higher particle levels in the ocular tissues in the post-mortem studies indicate a dynamic physiologic barrier to the entry of particles into the ocular tissues after periocular administration. The particle size of the delivery system can play an important role in the observed retinal drug levels after periocular administration. Slow release nanoparticles with low clearance by blood and lymphatic circulations are suitable for prolonged transscleral drug delivery to the back of the eye.

Modeling of corneal and retinal pharmacokinetics after periocular drug administration.

PURPOSE: To develop pharmacokinetics models to describe the disposition of small lipophilic molecules in the cornea and retina after periocular (subconjunctival or posterior subconjunctival) administration. METHODS: Compartmental pharmacokinetics analysis was performed on the corneal and retinal data obtained after periocular administration of 3 mg of celecoxib (a selective COX-2 inhibitor) to Brown Norway (BN) rats. Berkeley Madonna, a differential and difference equation-based modeling software, was used for the pharmacokinetics modeling. The data were fit to different compartment models with first-order input and disposition, and the best fit was selected on the basis of coefficient of regression and Akaike information criteria (AIC). The models were validated by using the celecoxib data from a prior study in Sprague-Dawley (SD) rats. The corneal model was also fit to the corneal data for prednisolone at a dose of 2.61 mg in albino rabbits, and the model was validated at two other doses of prednisolone (0.261 and 26.1 mg) in these rabbits. Model simulations were performed with the finalized model to understand the effect of formulation on corneal and retinal pharmacokinetics after periocular administration. RESULTS: Celecoxib kinetics in the BN rat cornea can be described by a two-compartment (periocular space and cornea, with a dissolution step for periocular formulation) model, with parallel elimination from the cornea and the periocular space. The inclusion of a distribution compartment or a dissolution step for celecoxib suspension did not lead to an overall improvement in the corneal data fit compared with the two-compartment model. The more important parameter for enhanced fit and explaining the apparent lack of an increase phase in the corneal levels is the inclusion of the initial leak-back of the dose from the periocular space into the precorneal area. The predicted celecoxib concentrations from this model also showed very good correlation (r = 0.99) with the observed values in the SD rat corneas. Similar pharmacokinetics models explain drug delivery to the cornea in rat and rabbit animal models. Retinal pharmacokinetics after periocular drug administration can be explained with a four-compartment (periocular space, choroid-containing transfer compartment, retina, and distribution compartment) model with elimination from the periocular space, retina, and choroid compartment. Inclusion of a dissolution-release step before the drug is available for absorption or elimination better explains retinal t(max). Good fits were obtained in both the BN (r = 0.99) and SD (r = 0.99) rats for retinal celecoxib using the same model; however, the parameter estimates differed. CONCLUSIONS: Corneal and retinal pharmacokinetics of small lipophilic molecules after periocular administration can be described by compartment models. The modeling analysis shows that (1) leak-back from the site of administration most likely contributes to the apparent lack of an increase phase in corneal concentrations; (2) elimination via the conjunctival or periocular blood and lymphatic systems contributes significantly to drug clearance after periocular injection; (3) corneal pharmacokinetics of small lipophilic molecules can be explained by using similar models in rats and rabbits; and (4) although there are differences in some retinal pharmacokinetics parameters between the pigmented and nonpigmented rats, the physiological basis of these differences has yet to be ascertained.

Effect of eye pigmentation on transscleral drug delivery.

PURPOSE: To determine the influence of eye pigmentation on transscleral retinal delivery of celecoxib. METHODS: Melanin content in ocular tissues of both the strains was determined by sodium hydroxide solubilization METHOD: The affinity of celecoxib to synthetic and natural melanin was estimated by co-incubating celecoxib and melanin in isotonic phosphate-buffered saline. The binding affinity (k) and the maximum binding (r(max)) for celecoxib to both natural and synthetic melanin were estimated. Suspension of celecoxib (3 mg/rat) was injected periocularly into one eye of Sprague-Dawley (SD, albino) and Brown Norway (BN, pigmented) rats. The animals were euthanatized at the end of 0.25, 0.5, 1, 2, 3, 4, 8, or 12 hours after the drug was administered, and celecoxib levels in ocular tissues (sclera, choroid-RPE, retina, vitreous, lens, and cornea) were estimated with an HPLC assay. In addition, celecoxib-poly(lactide) microparticles (750 microg drug/rat) were administered periocularly in SD and BN rats, and celecoxib levels in these eye tissues were assessed on day 8, to determine the effectiveness of the sustained release system. RESULTS: The r(max) and k for celecoxib's binding to natural melanin were (3.92 +/- 0.06) x 10(-7) moles/mg of melanin and (0.08 +/- 0.01) x 10(6) M(-1), respectively. The affinity and the extent of celecoxib's binding to natural melanin were not significantly different from those observed with synthetic melanin. The concentrations of melanin in choroid-RPE, sclera, and retina of BN rats were 200 +/- 30, 12 +/- 4, and 3 +/- 0.2 mug/mg tissue, respectively. Melanin was not detectable in the vitreous, lens, and cornea of BN rats. In SD rats, melanin was not detected in all tissues assessed except in the choroid-RPE, wherein melanin-like activity was 100-fold less than in BN rats. The area under the curve (AUC) for tissue concentration versus time profiles for animals administered with celecoxib suspension was not significantly different between the two strains for sclera, cornea, and lens. However, the retinal (P = 0.001) and vitreal (P = 0.001) AUCs of celecoxib in the treated eyes were approximately 1.5-fold higher in SD rats than in BN rats. Further, the choroid-RPE AUC in the treated and untreated eyes, respectively, were 1.5-fold (P = 0.001) and 2-fold (P = 0.0001) higher in BN rats than in SD rats. With celecoxib-poly(lactide) microparticles, choroid-RPE, retina, and vitreous concentrations on day 8 exhibited similar trends in differences between the two strains, with the differences being greater than those recorded for the celecoxib suspension. CONCLUSIONS: Transscleral retinal and vitreal drug delivery of lipophilic celecoxib is significantly lower in pigmented rats than in albino rats. This difference may be attributable to significant binding of celecoxib to melanin and its accumulation/retention in the melanin-rich choroid-RPE of pigmented rats. The hindrance of retinal and vitreal drug delivery by the choroid-RPE in pigmented rats is also true of sustained-release microparticle systems.

Celecoxib inhibits proliferation of retinal pigment epithelial and choroid-retinal endothelial cells by a cyclooxygenase-2-independent mechanism.

Age-related macular degeneration (ARMD) is a leading cause of blindness. The major reason for severe vision loss in ARMD is choroidal neovascularization due to an elevation in the expression of angiogenic factors such as vascular endothelial growth factor (VEGF). Drugs with anti-VEGF and antiproliferative activities can be beneficial for the treatment of this disorder. We have previously demonstrated that celecoxib [a selective cyclooxygenase (Cox)-2 inhibitor] inhibits VEGF expression in retinal pigment epithelial cells. In this study, we investigated the antiproliferative effects of celecoxib in adult retinal pigment epithelial (ARPE-19) and choroidal endothelial (RF/6A) cells. The results indicate that celecoxib 1) causes a dose-dependent antiproliferative effect in ARPE-19 and RF/6A cells (IC(50) of 23 and 13 microM, respectively); 2) leads to a G(2)-M phase cell cycle arrest in these cell types; and 3) inhibits VEGF-induced proliferation of RF/6A cells (IC(50) of 20 microM). In addition, 4) the concentrations of celecoxib required for antiproliferative effects are lower than those required for the cytotoxicity. These effects of celecoxib are by mechanisms independent of its Cox-2 inhibitory activity because rofecoxib (another Cox-2 inhibitor) had no effects on the proliferation or cell cycle distribution of the two cell types, and flurbiprofen (an inhibitor of Cox-1 and Cox-2) had weak antiproliferative effects on ARPE-19 cells, with IC(50) of 90 microM. In summary, celecoxib has potent antiproliferative effects in RF/6A and ARPE-19 cells; thus, it can be a potential new treatment in proliferative disorders of the choroid-retina such as choroidal neovascularization in age-related macular degeneration.

Single periocular injection of celecoxib-PLGA microparticles inhibits diabetes-induced elevations in retinal PGE2, VEGF, and vascular leakage.

PURPOSE: To determine whether celecoxib inhibits VEGF secretion from ARPE-19 cells and to investigate further the safety and effectiveness of periocular celecoxib-poly (lactide-co-glycolide; PLGA) microparticles in inhibiting elevations in retinal PGE(2), VEGF, and blood-tissue barrier leakage at the end of 60 days in a streptozotocin diabetic rat model. METHODS: VEGF mRNA and protein expression in ARPE-19 cells was evaluated in the presence of 0 to 10 microM celecoxib, and cytotoxicity of celecoxib on ARPE-19 and RF6A cells was evaluated over a 0- to 100-microM concentration range. Celecoxib-PLGA microparticles were prepared by a modified solvent evaporation technique, sterilized by 25 kGy of gamma-irradiation, and characterized for size, zeta potential, drug loading, and in vitro release. Normal and streptozotocin-diabetic male Sprague-Dawley rats were divided into five groups: normal, diabetic, diabetic+placebo, normal+celecoxib, and diabetic+celecoxib. Phosphate-buffered saline (PBS) containing celecoxib-PLGA microparticles, placebo PLGA microparticles, or plain PBS in one eye was injected into the posterior subconjunctival (periocular) space in rats under anesthesia. Sixty days after administration, the animals were killed, and retinal PGE2 secretion, VEGF protein, and blood-retinal barrier leakage were estimated. Blood cell counts, blood chemistry and histology were used to assess the safety of the microparticulate system. RESULTS: Celecoxib (up to 25 microM) did not cause significant cytotoxicity in ARPE-19 or RF6A cells. Nanomolar concentrations of celecoxib reduced VEGF mRNA and VEGF protein secretion. Celecoxib-PLGA microparticles (diameter: 1140 +/- 15 nm), containing 14.93% +/- 0.21% of celecoxib sustained in vitro drug release and in vivo drug levels in the retina for 60 days. Diabetes elevated PGE2 secretion, VEGF protein, the vitreous-plasma protein ratio, and blood-retinal barrier leakage by 3-, 1.7-, 3.1-, and 2.7-fold, and celecoxib-PLGA microparticles significantly reduced these elevations by 40%, 50%, 40%, and 50%, respectively. Neither the placebo-treated eyes nor the contralateral eyes in celecoxib-PLGA microparticle-treated rats showed significant effects. Celecoxib-PLGA or placebo-PLGA particles had no effect on the body weight or blood sugar level of rats. The celecoxib-PLGA microparticles did not cause any changes in blood cell counts or chemistry and caused no histopathological damage to the retina or periocular tissues. CONCLUSIONS: Nanomolar concentrations of celecoxib can inhibit VEGF mRNA and protein expression from ARPE-19 cells. Periocular celecoxib microparticles are useful sustained drug delivery systems for inhibiting diabetes-induced elevations in PGE2, VEGF, and blood-retinal barrier leakage. The periocular celecoxib-PLGA microparticles are safe and do not cause any damage to the retina.

Size-dependent disposition of nanoparticles and microparticles following subconjunctival administration.

The purpose of this study was to determine the retention and ocular distribution of subconjunctivally administered nanoparticles and microparticles. Fluorescent polystyrene particles (carboxylate modified, negatively charged) of various sizes (20 nm, 200 nm and 2 microm; Fluospheres, dose 400 microg) were administered to male Sprague-Dawley rats by subconjunctival injection under anaesthesia. The disposition of the particles in the periocular and ocular tissues was studied for up to 60 days by quantifying the particle amounts using liquid extraction followed by spectrofluorimetric analysis. The effect of dose on the particle disposition was investigated with a 40-microg dose of the particles. The effect of an increase in surface hydrophobicity was evaluated for the 20 and 200 nm particles at 1 day post administration. Following periocular administration, penetration into the ocular tissues was negligible for the carboxylate-modified microparticles as well as nanoparticles. Almost the entire dose of the 200 nm and 2 microm particles was retained in the periocular tissue at 60 days post-administration. The 20 nm particles disappeared rapidly from the periocular tissue with 15 and 8% of administered dose remaining after 1 and 7 days, respectively. The 20 nm particles could not be detected in the periocular tissue at 60-days post-administration. An increase in the surface hydrophobicity did not affect the periocular retention of 200 nm particles but elevated that of the 20 nm particles, at the end of day 1. It was concluded that subconjunctivally administered 200 nm and larger particles can be almost completely retained at the site of administration for at least two months. Periocular administration of particulate systems of this size would likely be useful as sustained drug delivery systems.

Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas.

Up-regulation of cyclooxygenase-2 occurs in retinal cells during the early onset of diabetic retinopathy. Under these conditions, prostaglandin production is elevated, which in turn leads to an increased expression of vascular endothelial growth factor (VEGF)--a growth factor implicated in vascular leakage and neovascularization. In this ex vivo study, we tested whether cyclooxygenase-1 or cyclooxygenase-2 is responsible for diabetes-induced secretion of prostaglandin E2 from isolated rat retinas. Celecoxib, a selective cyclooxygenase-2 inhibitor, significantly inhibited prostaglandin E2 secretion, whereas SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole], a selective cyclooxygenase-1 inhibitor, had no inhibitory effect. These results suggests that the enzymatic activity of cyclooxygenase-2, but not cyclooxygenase-1, results in prostaglandin E2 secretion under diabetic conditions.