Tolerability Assessment of Formulation pH in New Zealand White Rabbits Following Intravitreal Administration.

Development of intravitreal drugs presents several challenges due to the delicate ocular environment and volume constraints of what can be safely administered in the eye. Formulation development of intravitreally administered drugs may necessitate the use of nonphysiological pH in order to accommodate manufacturing processes or achieve favorable drug properties. Clinical and nonclinical data show that intravitreal drugs formulated in the pH 5.5 to 7.4 range are well tolerated. The aim of this study was to provide ocular toxicity data for formulations in the pH 4.0 to 5.5 range following intravitreal administration in New Zealand White rabbits. This range was evaluated as part of formulation development for an intravitreal drug that necessitated the use of pH outside the available tolerability data for formulations. Toxicity was assessed by ophthalmic examinations, intraocular pressure (IOP) measurement, clinical observations, body weights, and microscopic analysis of ocular tissue. Histidine chloride pH 5.0 to 5.5 and acetate chloride pH 4.0 to 5.0 solutions were well tolerated, and no test article-related ocular inflammation, IOP changes, or gross or microscopic findings were observed in any eye. The data presented here add to the knowledge of pH ranges that can be explored for intravitreal drug formulation development.

Nonclinical Safety Assessment of FHTR2163, An Antigen-Binding Fragment Against HTRA1 for the Treatment of Geographic Atrophy.

FHTR2163 is an antigen-binding fragment of a humanized immunoglobulin G1 monoclonal antibody directed against high-temperature requirement A serine peptidase 1 (HTRA1) that is being developed as a potential intravitreal (ITV) treatment for patients with geographic atrophy (GA), an advanced form of dry age-related macular degeneration. The nonclinical toxicology program was designed to assess the safety and tolerability of HTRA1 inhibition following ITV administration of FHTR2163 to support ITV administration in patients with GA. FHTR2163 was well tolerated in a single-dose ITV-administered 8-day toxicity study in cynomolgus monkeys following a 50 µL high (>700 mOsm/kg) osmolality formulation up to 12.5 mg/eye; however, 100 µL (2× 50 µL injections) of a high-osmolality formulation resulted in transient retinal detachment. Repeat-dose ITV administration every 2 weeks of FHTR2163 was well tolerated in 8- and 26-week studies with ITV injection of 100 µL (2× 50 µL) of iso-osmolar formulation up to 15 mg/eye, or 50 µL of the high-osmolality formulation up to 12.5 mg/eye. Observed transient and reversible ocular effects included inflammation and perivascular infiltrates, consistent with an immune response attributed to the administration of heterologous (humanized) protein. Overall, FHTR2163 was well tolerated, and the nonclinical package supported the continued clinical development of FHTR2163 in patients with GA.

Fab-Nanolipoprotein Conjugate Causes Vitreous Opacity and Cataracts Following a Single Intravitreal Administration in New Zealand White Rabbits.

One strategy employed to prolong the ocular half-life of large molecule therapeutics is via covalent attachment to a carrier, resulting in an increase in size thereby slowing their clearance from the eye. Rabbit antigen-binding fragment conjugated to nanolipoprotein (RabFab-NLP) is a novel conjugate intended to prolong ocular half-life through an increase in hydrodynamic radius compared to Fab alone (∼12 vs ∼3 nm). Nanolipoproteins are mimetics of endogenous high-density lipoproteins and consist of lipids and apolipoproteins (ApoE422k), both biologically derived materials. The objective of this study was to evaluate the ocular toxicity and toxicokinetics of RabFab-NLP after a single intravitreal administration in New Zealand White rabbits. Serum toxicokinetic data suggested a significant increase in ocular residence time of RabFab-NLP compared to RabFab alone. Ophthalmic examinations showed that RabFab-NLP caused vitreous and lens opacities as early as day 3 and day 8 postdose, respectively, which persisted for the entire study duration to day 30. The RabFab-NLP-related microscopic findings were present in the lens, vitreous cavity, and/or optic nerve head. Based on the observed ocular toxicity, a single intravitreal dose of 1.3 mg/eye RabFab-NLP was not tolerated and caused vitreous opacity and cataracts in rabbit eyes.

Intravitreal Administration of Acetyl Triethyl Citrate and Benzyl Benzoate Is Retinotoxic in Rabbits but Not in Cynomolgus Monkeys.

Sustained drug delivery formulations are developed to reduce dose frequency while maintaining efficacy of intravitreal (ITV) administered therapeutics. Available safety data for components novel to the eye's posterior segment may be limited, requiring preclinical assessments to identify potential toxicities. We evaluated the in vivo and in vitro safety of two solvents, acetyl triethyl citrate (ATEC) and benzyl benzoate (BB), as novel sustained delivery formulations for ITV administration. In vivo tolerability was assessed following ITV administration of ATEC and BB to rabbits and cynomolgus monkeys. In rabbits, ITV solvent administration resulted in moderate to severe retinal toxicity characterized by focal retinal necrosis and/or degeneration, sometimes accompanied by inflammation, with a clear association between the physical presence of the solvent and areas of retinal damage. In contrast, solvent administration in monkeys appeared well tolerated, producing no histologic abnormalities. Toxicity in primary human retinal pigment epithelial cells, characterized by cellular toxicity and mitochondrial injury, corroborated the retinal toxicity in rabbits. In conclusion, ITV solvent depots of ATEC or BB result in chemical and focal retinal toxicity in rabbits, but not monkeys. Additional investigation is needed to demonstrate a sufficient margin of safety prior to use of ATEC or BB in ITV drug products.

Nonclinical Toxicology and Biocompatibility Program Supporting Clinical Development and Registration of the Port Delivery System With Ranibizumab for Neovascular Age-Related Macular Degeneration.

The Port Delivery System with ranibizumab (PDS) is an investigational drug delivery system designed to provide continuous intravitreal release of ranibizumab for extended durations. The PDS consists of a permanent, surgically placed, refillable intraocular implant; a customized formulation of ranibizumab; and ancillary devices to support surgery and refill procedures. A toxicology program was conducted to evaluate the ocular toxicology and biocompatibility of the PDS to support its clinical development program and product registrational activities. PDS safety studies included a 6-month chronic toxicology evaluation in minipigs as well as evaluation of nonfunctional surrogate implants (comprised of the same implant materials but without ranibizumab) in rabbits. Biocompatibility of the implant and ancillary devices was evaluated in both in vitro and in vivo studies. Implants and extracts from implants and ancillary devices were nongenotoxic, noncytotoxic, nonsensitizing, and nonirritating. Ocular findings were comparable between implanted and sham-operated eyes, and no systemic toxicity was observed. The results of this nonclinical toxicology program demonstrated that the PDS was biocompatible and that intravitreal delivery of ranibizumab via the PDS did not introduce any new toxicology-related safety concerns relative to intravitreal injections, supporting ongoing PDS clinical development and product registrational evaluation.

EVALUATION OF SURGICAL FACTORS AFFECTING VITREOUS HEMORRHAGE FOLLOWING PORT DELIVERY SYSTEM WITH RANIBIZUMAB IMPLANT INSERTION IN A MINIPIG MODEL.

PURPOSE: To develop an animal model of vitreous hemorrhage (VH) to explore the impact of surgical parameters on VH associated with insertion of the Port Delivery System with ranibizumab (PDS) implant. METHODS: Ninety eyes from 45 treatment-naive male Yucatan minipigs received PDS implant insertion or a sham procedure. The effect of prophylactic pars plana hemostasis, scleral incision length, scleral cauterization, surgical blade type/size, and viscoelastic usage on postsurgical VH was investigated. RESULTS: Postsurgical VH was detected in 60.0% (54/90) of implanted eyes. A systematic effect on VH was only detected for pars plana hemostasis before the pars plana incision. The percentage of eyes with VH was 96.6% (28/29) among eyes that did not receive prophylactic pars plana hemostasis and 42.4% (24/58) among eyes that did. There was no VH in eyes that received laser ablation of the pars plana using overlapping 1,000-ms spots; pars plana cautery or diathermy was less effective. The majority of all VH cases (83.3% [45/54]) were of mild to moderate severity (involving ≤25% of the fundus). CONCLUSION: In this minipig surgical model of VH, scleral dissection followed by pars plana laser ablation before pars plana incision most effectively mitigated VH secondary to PDS implant insertion.

Lens Capsule Perforation Without Inflammation in 4 Rabbits From Intravitreal Injection Studies.

Historically, it was thought that lens protein was sequestered, and injury to the lens capsule causing release of lens material into the eye would always result in ocular inflammation. Currently, it is believed that lens antigens are recognized as self, subject to normal T-cell tolerance. Three different single-dose intravitreal injection/implantation studies of 4 different test materials, ranging from 4 to 6 weeks in length, were performed in New Zealand White rabbits. The test materials included polymer microspheres, polymer rods, a solvent, and a hydrogel. Intravitreal injection/implantation procedures were performed on day 1, and indirect ophthalmoscopy and slit-lamp biomicroscopy examinations were performed by board-certified veterinary ophthalmologists periodically throughout the course of each study. None of the affected animals received corticosteroids or other immunomodulatory agents during the course of the studies. Four rabbits had perforation of the posterior lens capsule during the injection/implantation procedure on day 1, visible on clinical ophthalmic examination as lens capsule alterations described as "lens hits" and/or incipient posterior cataracts. Findings on slit-lamp biomicroscopy examination were limited to vitreous cells in 2 of the animals, although not centered on the area of lens capsule disturbance. Histologically, there was no evidence of inflammation in association with extruded lens protein material in any of the affected eyes. These results indicate that iatrogenic damage to the lens capsule during aseptically performed intravitreal injections/implantations does not appear to induce inflammation in rabbits.

Characterization of the pH and Temperature in the Rabbit, Pig, and Monkey Eye: Key Parameters for the Development of Long-Acting Delivery Ocular Strategies.

Many long-acting delivery strategies for ocular indications rely on pH- and/or temperature-driven release of the therapeutic agent and degradation of the drug carrier. Yet, these physiological parameters are poorly characterized in ocular animal models. These strategies aim at reducing the frequency of dosing, which is of particular interest for the treatment of chronic disorders affecting the posterior segment of the eye, such as macular degeneration that warrants monthly or every other month intravitreal injections. We used anesthetized white New Zealand rabbits, Yucatan mini pigs, and cynomolgus monkeys to characterize pH and temperature in several vitreous locations and the central aqueous location. We also established post mortem pH changes in the vitreous. Our data showed regional and species differences, which need to be factored into strategies for developing biodegradable long-acting delivery systems.

Intravitreal administration of known phototoxicants in the rabbit fails to produce phototoxicity: implications for phototoxicity testing of intravitreally administered small molecule therapeutics.

CONTEXT: Intravitreal (ITV) dosing has become a clinically important route of administration for the treatment of uveitis, endophthalmitis, retinal vein occlusion, diabetic macular edema and age-related macular degeneration. Despite this, there are no validated non-clinical models of phototoxicity for ITV products. OBJECTIVE: The objective of this study was to develop an ITV rabbit model of phototoxicity for use in assessing the photosafety of small molecules therapeutics. MATERIALS AND METHODS: Dutch Belted rabbits were intravitreally injected bilaterally with four known phototoxicants: 8-methoxypsoralen, lomefloxacin, doxycycline and stannsoporfrin. Triescence(®), a non-phototoxic triamcinolone acetonide steroid formulation designed for ITV administration, was used as a negative control. One eye was then irradiated with solar-simulated ultraviolet radiation for 30 min, 1 h after dosing, while the other eye was occluded, serving as a non-irradiated control. RESULTS: Despite the direct administration of known phototoxicants into the vitreous, no evidence of ocular phototoxicity was observed in any dose group. Direct (non-phototoxic) retinal toxicity was observed in the doxycycline dose group only. CONCLUSION: These data suggest that the posterior segment of the rabbit eye is protected against phototoxicity by anatomical and/or physiological mechanisms, and is not a useful model for the assessment of phototoxicity of intravitreally administered molecules.

Determination of a No-Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Dutch Belted Rabbits.

Purpose: The purpose of this study was to characterize the inflammatory response and determine a no-observable effect level (NOEL) in rabbit eyes after endotoxin intravitreal (ITV) injection. Methods: Fifty-three naïve male Dutch Belted rabbits were treated with a single 50-µL ITV injection ranging from 0.01 to 0.75 endotoxin units/eye (EU/eye) and monitored for up to 42 days post treatment. Ophthalmic examination included slit-lamp biomicroscopy and indirect ophthalmoscopy. Laser flare photometry was performed in a subset of animals. On days 2, 8, 16, and 43, a subset of animals was necropsied and eyes processed for histopathological evaluation. Results: Intravitreal injection of endotoxin at ≥0.05 EU/eye resulted in a dose-related anterior segment inflammation response. No aqueous flare or cell response was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreal cell response was observed beginning on day 5, peaking on day 9, and decreasing starting on day 16 that persisted at trace to a level of 1+ on day 43. Microscopy findings of infiltrates of minimal mixed inflammatory cells in the vitreous and subconjunctiva and proteinaceous fluid in the anterior chamber and/or vitreous were observed in eyes given ≥0.1 EU/eye. Conclusions: We defined the NOEL for ITV endotoxin to be 0.01 EU/eye, suggesting that the vitreal cavity is more sensitive to the effects of endotoxin than the anterior segment and aqueous chamber. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.

Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods: In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results: Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 µm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions: These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates.

An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant.