Eyes on Topical Ocular Disposition: The Considered Design of a Lead Janus Kinase (JAK) Inhibitor That Utilizes a Unique Azetidin-3-Amino Bridging Scaffold to Attenuate Off-Target Kinase Activity, While Driving Potency and Aqueous Solubility.

An unmet medical need remains for patients suffering from dry eye disease (DED). A fast-acting, better-tolerated noncorticosteroid anti-inflammatory eye drop could improve patient outcomes and quality of life. Herein, we describe a small-molecule drug discovery effort to identify novel, potent, and water-soluble JAK inhibitors as immunomodulating agents for topical ocular disposition. A focused library of known 3-(4-(2-(arylamino)pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitriles was evaluated as a molecular starting point. Structure-activity relationships (SARs) revealed a ligand-efficient (LE) JAK inhibitor series, amenable to aqueous solubility. Subsequent in vitro analysis indicated the potential for off-target toxicity. A KINOMEscan selectivity profile of 5 substantiated the likelihood of widespread series affinity across the human kinome. An sp2-to-sp3 drug design strategy was undertaken to attenuate off-target kinase activity while driving JAK-STAT potency and aqueous solubility. Tactics to reduce aromatic character, increase fraction sp3 (Fsp3), and bolster molecular complexity led to the azetidin-3-amino bridging scaffold in 31.

Discovery and Preclinical Development of Novel Intraocular Pressure-Lowering Rho Kinase Inhibitor: Corticosteroid Conjugates.

Purpose: A new class of ocular steroids designed to mitigate steroid-induced intraocular pressure (IOP) elevation while maintaining anti-inflammatory activity was developed. Herein is described the discovery and preclinical characterization of ROCK'Ster compound 1. Methods: Codrugs consisting of a Rho kinase inhibitor (ROCKi) and a corticosteroid were synthesized. Compounds were initially screened in vitro for ROCKi activity and anti-inflammatory activity against the proinflammatory interleukin 23 and bacterial lipopolysaccharide (LPS) pathways. Selected compounds were then screened for solubility, chemical stability, and ex vivo corneal metabolism. Lead compound 1 was evaluated for IOP lowering in the Dutch Belted rabbit and for anti-inflammatory efficacy in both a postcataract surgery model and an allergic eye disease (AED) mouse model. Results: Several ROCK'Sters were found to be potent inhibitors of ROCK (Kis < 50 nM), have high anti-inflammatory activity in vitro (IC50s < 50 nM), display sufficient stability in topical ophthalmic formulations, and have a moderate rate of corneal metabolism. Compound 1 (0.1% and 0.25%, quater in die [QID]-4 times a day) demonstrated IOP-lowering capability without inducing hyperemia in our rabbit model. When compared with the marketed steroids, Durezol® and Pred Forte®, compound 1 (0.1%, 0.25%) demonstrated noninferiority in clinical scoring in a rabbit model of inflammation after surgery. In addition, anti-inflammatory outcomes were observed with compound 1 (0.1%) relative to Lotemax® or vehicle control in an AED mouse model. Conclusion: ROCK'Ster compound 1 is a novel compound suitable for topical ocular dosing that possesses IOP-lowering capability along with similar anti-inflammatory activity compared with marketed steroids.

Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma.

PURPOSE: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein. METHODS: The kinase inhibitory activity of netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a commercially available kinase assay kit. Disruption of actin stress fibers was measured in primary porcine TM cells and disruption of focal adhesions in transformed human TM (HTM) cells. Induction of fibrosis markers after exposure to transforming growth factor-ß2 (TGF-ß2) was conducted in primary HTM cells. Ocular hypotensive activity and tolerability of topical formulations were evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted rabbits. RESULTS: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 and 16 nM, respectively, and blocked the profibrotic effects of TGF-ß2 in HTM cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that were sustained for at least 24 h after once daily dosing, with transient, mild hyperemia observed as the only adverse effect. CONCLUSION: Netarsudil is a novel ROCK/NET inhibitor with high potency in biochemical and cell-based assays, an ability to produce large and durable IOP reductions in animal models, and favorable pharmacokinetic and ocular tolerability profiles.

Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma.

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK inhibitor, 29 (Ki=0.2nM) and demonstrated an effective and sustained IOP reduction for 24h after dosing. From these studies, netarsudil (a.k.a. AR-13324) was discovered and is currently in clinical trials for the treatment of glaucoma and ocular hypertension.

DeoxyArbutin and its derivatives inhibit tyrosinase activity and melanin synthesis without inducing reactive oxygen species or apoptosis.

Safety is a major concern in developing commercial skin-lightening agents. Here, we report the modulating effects of deoxyArbutin (dA) and its second-generation derivatives - deoxyFuran (dF), 2-fluorodeoxyArbutin (fdA), and thiodeoxyArbutin (tdA) - on tyrosinase, and consequently, on melanization. Results demonstrate that dA and its derivatives inhibit tyrosine hydroxylase and dopa oxidase activity of tyrosinase. The inhibition is dose-dependent, thereby inhibiting melanin synthesis in intact melanocytes, when used at concentrations that retain 95% viability of the treated cells in culture. Herein we demonstrate that dA, and its second-generation derivatives dF, fdA, and tdA, exhibit dose-dependent reductions in melanocyte cell number, primarily due to inhibition of proliferation rather than initiation of apoptosis as exemplified by hydroquinone (HQ), ie, cytostatic as opposed to cytotoxic. Human and murine melanocytes with functional mutations in either tyrosinase or tyrosinase-related protein 1 (Tyrp1) are less sensitive to the cytostatic effects of dA and its derivatives. Minimal amounts of reactive oxygen species (ROS) were generated upon treatment with dA and its derivatives, in contrast to a dramatic amount of ROS induced by HQ. This increase in ROS subsequently induced the expression of the endogenous antioxidant catalase in treated melanocytes. Treatment with exogenous antioxidants provided protection for melanocytes treated with HQ, but not dA and its derivatives, suggesting that HQ exerts more oxidative stress. These studies demonstrate that dA and its derivatives are relatively safe tyrosinase inhibitors for skin lightening or for ameliorating hyperpigmented lesions.

Comparative efficacy and safety of deoxyarbutin, a new tyrosinase-inhibiting agent.

Several tyrosinase inhibitors have been developed and utilized to ameliorate various cutaneous hyperpigmentary disorders and complexion discolorations. Deoxyarbutin (dA) (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), designed using quantitative structure-activity relationships (QSAR), demonstrates effective inhibition of mushroom tyrosinase and skin-lightening capability (1). However, its comparative safety, effectiveness, and reversibility to other known tyrosinase inhibitors in human melanocytes had not been determined. The effect of dA was assessed in cultured human skin cells, on xenographs, and with a clinical trial. Using cultured human melanocytes, the maximum concentration of dA that allowed 95% viability was fourfold greater than for hydroquinone (HQ), indicating that dA is less cytotoxic/cytostatic than HQ. The viability of cultured human keratinocytes and fibroblasts was also less compromised by increasing concentrations of dA as opposed to HQ. At the maximum concentration allowing normal cellular viability, dA effectively inhibited tyrosinase activity and melanin content in human melanocytes, whereas HQ was marginally inhibitory. Upon removal of dA, tyrosinase activity and melanin content was normalized within five days. Topical application of dA on human xenografts resulted in a gradual and visually apparent skin lightening effect during an eight-week period. In a clinical trial, dA facilitated fading of pre-tanned skin to a statistically significant greater extent than either HQ or no treatment. These results demonstrate that dA is a potentially safe, effective, and reversible tyrosinase inhibitor.

DeoxyArbutin: a novel reversible tyrosinase inhibitor with effective in vivo skin lightening potency.

Modulation of melanogenesis in the melanocytes can be achieved using chemicals that share structural homologies with the substrate tyrosine and as thus competitively inhibit the catalytic function of tyrosinase. We have developed a new tyrosinase inhibitor, deoxyArbutin (dA), based on this premise. DeoxyArbutin demonstrates effective inhibition of mushroom tyrosinase in vitro with a Ki that is 10-fold lower that hydroquinone (HQ) and 350-fold lower than arbutin. In a hairless, pigmented guinea pig model, dA demonstrated rapid and sustained skin lightening that was completely reversible within 8 weeks after halt in topical application. In contrast, HQ induced a short but unsustained skin lightening effect whereas kojic acid and arbutin exhibit no skin lightening effect. Results from a panel of safety tests supported the overall establishment of dA as an actionable molecule. In a human clinical trial, topical treatment of dA for 12 weeks resulted in a significant or slight reduction in overall skin lightness and improvement of solar lentigines in a population of light skin or dark skin individuals, respectively. These data demonstrate that dA has potential tyrosinase inhibitory activity that can result in skin lightening and may be used to ameliorate hyperpigmentary lesions.

Glutathione, S-substituted glutathiones, and leukotriene C4 as substrates for peptidylglycine alpha-amidating monooxygenase.

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.