A Novel Eye Drop Formulation for Potential Treatment of Neovascular Age-Related Macular Degeneration.

Purpose: Drug delivery to posterior ocular tissues via topical eye drop administration is arduous due to the unique anatomy and physiology of the eye. Therefore, treatments for posterior eye disease have to be administered via intravitreal injection or systemic route, both of which have their drawbacks. Herein, the objective of this work was to demonstrate that a specially designed eye drop formulation could effectively deliver small-molecule vascular endothelial growth factor (VEGF) inhibitor to posterior ocular tissues for antiangiogenic therapy. Methods: The unique eye drop formulation, termed ITRI AXN eye drops, was obtained from self-assembly of (2-hydroxypropyl)-ß-cyclodextrin with a VEGF tyrosine kinase inhibitor, a hydrophilic polymer, hypromellose, and a complex stabilizer, caffeine. In vivo ocular pharmacokinetics studies were performed with New Zealand White (NZW) rabbits and Non Human Primates (NHP). The antiangiogenesis effect was evaluated on the Long-Evans rat with laser-induced choroidal neovascularization and pigmented Dutch-Belted rabbits with VEGF-induced retinal neovascularization. Results: The successful drug transport from ocular surface to posterior ocular cavity was indicated by a drug biodistribution pattern in pharmacokinetic studies. Excellent drug exposure in the choroid and retina with the concentrations of 900- and 750-fold greater than drug IC50 0.5 hours post the eye drop administration (drug level: 0.8%) was observed on the NHP study. The obtained formulation also demonstrated a comparable antiangiogenic outcome with the intravitreal injection of anti-VEGF antibody on rat and rabbit disease models. Conclusions: Our eye drop formulation has demonstrated great promise in antiangiogenic therapy against retinal and choroidal neovascularization in animal models. The results suggest that the aim of this work can be successfully achieved by the novel eye drop formulation. Translational Relevance: The preclinical results provide evidence that ITRI AXN eye drops could effectively deliver therapeutics to the choroid and retina for antiangiogenic therapy.

The Intraocular Pressure Lowering Effect of a Dual Kinase Inhibitor (ITRI-E-(S)4046) in Ocular Hypertensive Animal Models.

Purpose: The purpose of this study was to develop a preclinical compound, ITRI-E-(S)4046, a dual synergistic inhibitor of myosin light chain kinase 4 (MYLK4) and Rho-related protein kinase (ROCK), for reducing intraocular pressure (IOP). Methods: ITRI-E-(S)4046 is an amino-pyrazole derivative with physical and chemical properties suitable for ophthalmic formulation. In vitro kinase inhibition was evaluated using the Kinase-Glo Luminescent Kinase Assays. A comprehensive kinase selectivity analysis of ITRI-E-(S)4046 was performed using the KINOMEscan assay from DiscoverRx. The IOP reduction and tolerability of ITRI-E-(S)4046 were assessed in ocular normotensive rabbits, ocular normotensive non-human primates, and ocular hypertensive rabbits. In vivo studies were conducted to assess drug concentrations in ocular tissue. The adverse ocular effects of rabbit eyes were evaluated following the OECD405 guidelines. Results: ITRI-E-(S)4046 showed highly selective kinase inhibitory activity against ROCK1/2, MYLK4, and mitogen-activated protein kinase kinase kinase 19 (MAP3K19), with high specificity against protein kinase A, G, and C families. In ocular normotensive rabbits and non-human primates, the mean IOP reductions of 0.1% ITRI-E-(S)4046 eye drops were 29.8% and 28.5%, respectively. In hypertonic saline-induced and magnetic beads-induced ocular hypertensive rabbits, the mean IOP reductions of ITRI-E-(S)4046 0.1% eye drops were 46.9% and 22.0%, respectively. ITRI-E-(S)4046 was well tolerated with only temporary and minor signs of hyperemia. Conclusions: ITRI-E-(S)4046 is a novel type of highly specific ROCK1/2 and MYLK4 inhibitor that can reduce IOP in normotensive and hypertensive animal models. It has the potential to become an effective and well-tolerated treatment for glaucoma.

Inhibition of Retinal Ganglion Cell Loss By a Novel ROCK Inhibitor (E212) in Ischemic Optic Nerve Injury Via Antioxidative and Anti-Inflammatory Actions.

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.

A Highly Selective Rho-Kinase Inhibitor (ITRI-E-212) Potentially Treats Glaucoma Upon Topical Administration With Low Incidence of Ocular Hyperemia.

Purpose: The purpose of this study was to investigate the IOP-lowering effects of the ITRI-E-212, a new Rho-associated protein kinase (ROCK) inhibitor. ITRI-E-212 improved fluid outflow through the trabecular meshwork and reduced IOP with transient and mild conjunctival hyperemia. ITRI-E-212 can potentially be developed into new antiglaucoma agents. Methods: ITRI-E-212 was selected from more than 200 amino-isoquinoline structures because of its adequate solubility and drug-loading percentage in eye drops. ITRI-E-212 has less than 50% inhibitory concentration (IC50) against ROCK2. The in vitro kinase inhibition was evaluated using the ADP-Glo kinase assay. A comprehensive analysis of the kinase inhibitor selectivity of ITRI-E-212 was performed using the KINOMEscan methodology. The IOP-lowering effect and tolerability of ITRI-E-212 were investigated in normotensive and ocular hypertensive rabbits. The pharmacokinetics study was performed in vivo in the aqueous humor (AH), and hyperemia was assessed. Results: ITRI-E-212 showed high in vitro inhibitory activity against ROCK2 and high specificity against AGC kinases. The mean IOP-lowering effect of ITRI-E-212 in normotensive and ocular hypertensive models was 24.9% and 28.6%, respectively; 1% ITRI-E-212 produced notable reductions in IOP that were sustained for at least 6 hours after each dose once per day. Only transient, mild hyperemia was observed. The compound extracted from the AH reached 78.4% ROCK2 kinase inhibition at 1 hour after dose administration and was sustained for 4 hours. Conclusions: ITRI-E-212 is a novel and highly specific ROCK2 inhibitor with the ability to lower IOP in animal models. It has favorable pharmacokinetic and ocular tolerability profiles with only minimal conjunctival hyperemia.

In situ chemically crosslinked injectable hydrogels for the subcutaneous delivery of trastuzumab to treat breast cancer.

Recently, novel approaches for the delivery of therapeutic antibodies have attracted much attention, especially sustained release formulations. However, sustained release formulations capable of carrying a high antibody load remain a challenge for practical use. In this study, a novel injectable hydrogel composed of maleimide-modified γ-polyglutamic acid (γ-PGA-MA) and thiol end-functionalized 4-arm poly(ethylene glycol) (4-arm PEG-SH) was developed for the subcutaneous delivery of trastuzumab. γ-PGA-MA and 4-arm PEG-SH formed a hydrogel through thiol-maleimide reactions, which had shear-thinning properties and reversible rheological behaviors. Moreover, a high content of trastuzumab (>100 mg/mL) could be loaded into this hydrogel, and trastuzumab demonstrated a sustained release over several weeks through electrostatic attraction. In addition, trastuzumab released from the hydrogel had adequate stability in terms of its structural integrity, binding bioactivity, and antiproliferative effect on BT-474 cells. Pharmacokinetic studies demonstrated that trastuzumab-loaded hydrogel (Her-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, and 10 mg/mL trastuzumab) and trastuzumab/Zn-loaded hydrogel (Her/Zn-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, 5 mM ZnCl2, and 10 mg/mL trastuzumab) could lower the maximum plasma concentration (Cmax) than the trastuzumab solution. Furthermore, Her/Zn-hydrogel-10 was better able to release trastuzumab in a controlled manner, which was ascribed to electrostatic attraction and formation of trastuzumab/Zn nanocomplexes. In a BT-474 xenograft tumor model, Her-hydrogel-10 had a similar tumor growth-inhibitory effect as that of the trastuzumab solution. By contrast, Her/Zn-hydrogel-10 exhibited a superior tumor growth-inhibitory capability due to the functionality of Zn. This study demonstrated that this hydrogel has potential as a carrier for the local and systemic delivery of proteins and antibodies. STATEMENT OF SIGNIFICANCE: Recently, novel sustained-release formulations of therapeutic antibodies have attracted much attention. However, these formulations should be able to carry a high antibody load owing to the required high dose, and these formulations remain a challenge for practical use. In this study, a novel injectable chemically cross-linked hydrogel was developed for the subcutaneous delivery of trastuzumab. This novel hydrogel possessed ideal characteristics of loading high content of trastuzumab (>100 mg/mL), sustained release of trastuzumab over several weeks, and maintaining adequate stability of trastuzumab. In vivo studies demonstrated that a trastuzumab-loaded hydrogel possessed the ability of controlled release of trastuzumab and maintained antitumor efficacy same as that of trastuzumab. These results implied that a γ-PGA-MA and 4-arm PEG-SH-based hydrogel has great potential in serving as a carrier for the local or systemic delivery of therapeutic proteins or antibodies.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors.

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.