Purpose:To evaluate the pharmacology and toxicology of SAF312, a transient receptor potential vanilloid 1 (TRPV1) antagonist.Methods:TRPV1 expression in human ocular tissues was evaluated with immunohistochemistry. Inhibition of calcium influx in Chinese hamster ovary (CHO) cells expressing human TRPV1 (hTRPV1) and selectivity of SAF312 were assessed by a fluorescent imaging plate reader assay. Ocular tissue and plasma pharmacokinetics (PK) were assessed following a single topical ocular dose of SAF312 (0.5%, 1.0%, 1.5%, 2.5%) in rabbits. Safety and tolerability of SAF312 were evaluated in rabbits and dogs. Effects of SAF312 on corneal wound healing after photorefractive keratectomy (PRK) surgery were assessed in rabbits.Results:TRPV1 expression was noted in human cornea and conjunctiva. SAF312 inhibited calcium influx in CHO-hTRPV1 cells induced by pH 5.5 (2-[N-morpholino] ethanesulfonic acid), N-arachidonoylethanolamine, capsaicin, and N-arachidonoyl dopamine, with IC50 values of 5, 10, 12, and 27 nM, respectively, and inhibition appeared noncompetitive. SAF312 demonstrated high selectivity for TRPV1 (>149-fold) over other TRP channels. PK analysis showed highest concentrations of SAF312 in cornea and conjunctiva. SAF312 was found to be safe and well tolerated in rabbits and dogs up to the highest feasible concentration of 2.5%. No delay in wound healing after PRK was observed.Conclusions:SAF312 is a potent, selective, and noncompetitive antagonist of hTRPV1 with an acceptable preclinical safety profile for use in future clinical trials.Translational Relevance:SAF312, which was safe and well tolerated without causing delay in wound healing after PRK in rabbits, may be a potential therapeutic agent for ocular surface pain.

EFEMP1 R345W is a dominant mutation causing Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML), a rare blinding disease with clinical pathology similar to age-related macular degeneration (AMD). Aged Efemp1 R345W/R345W knock-in mice (Efemp1ki/ki) develop microscopic deposits on the basal side of retinal pigment epithelial cells (RPE), an early feature in DHRD/ML and AMD. Here, we assessed the role of alternative complement pathway component factor B (FB) in the formation of these deposits. RNA-seq analysis of the posterior eyecups revealed increased unfolded protein response, decreased mitochondrial function in the neural retina (by 3 months of age) and increased inflammatory pathways in both neural retina and posterior eyecups (at 17 months of age) of Efemp1ki/ki mice compared with wild-type littermate controls. Proteomics analysis of eye lysates confirmed similar dysregulated pathways as detected by RNA-seq. Complement activation was increased in aged Efemp1ki/ki eyes with an approximately 2-fold elevation of complement breakdown products iC3b and Ba (P < 0.05). Deletion of the Cfb gene in female Efemp1ki/ki mice partially normalized the above dysregulated biological pathway changes and oral dosing of a small molecule FB inhibitor from 10 to 12 months of age reduced sub-RPE deposits by 65% (P = 0.029). In contrast, male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females, no elevation of ocular complement activation and no effect of FB inhibition on sub-RPE deposits. The effects of FB deletion or inhibition on Efemp1ki/ki mice supports systemic inhibition of the alternative complement pathway as a potential treatment of dry AMD and DHRD/ML.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

MGV354was being developed as a novel ocular therapy for lowering of intraocular pressure, a key modifiable risk factor for glaucoma.MGV354is an activator of soluble guanylate cyclase, an enzyme known to be involved in the regulation of IOP.MGV354has been shown to robustly lower IOP over 24 h after a single topical ocular drop in rabbit and monkey pharmacology models. However,MGV354failed to produce similar results in patients with ocular hypertension or open-angle glaucoma. With an objective of explaining the lack of efficacy in the clinic, we attempted to study whether human metabolism was significantly different from animal metabolism. The present study documents the investigation of metabolism ofMGV354in an effort to understand potential differences in biotransformation pathways ofMGV354in rabbits, monkeys, and humans. Overall twenty-six metabolites, formed via oxidative and conjugative pathways, were identifiedin vitroandin vivo.In vitrohepatic metabolism was qualitatively similar across species, with minor but distinct differences. There were no observable interspecies differences in the hepatic and ocular metabolism ofMGV354. Although ocular metabolism was not as extensive as hepatic, the results do not explain the lack of efficacy ofMGV354in clinical studies.

Inhibition of neprilysin (NEP) is widely studied as a therapeutic target for the treatment of hypertension, heart failure, and kidney disease. Sacubitril/valsartan (LCZ696) is a drug approved to reduce the risk of cardiovascular death in heart failure patients with reduced ejection fraction. LBQ657 is the active metabolite of sacubitril and an inhibitor of NEP. Previously, we have reported the crystal structure of NEP bound with LBQ657, whereby we noted the presence of a subsite in S1' that has not been explored before. We were also intrigued by the zinc coordination made by one of the carboxylic acids of LBQ657, leading us to explore alternative linkers to efficiently engage zinc for NEP inhibition. Structure-guided design culminated in the synthesis of selective, orally bioavailable, and subnanomolar inhibitors of NEP. A 17-fold boost in biochemical potency was observed upon addition of a chlorine atom that occupied the newly found subsite in S1'. We report herein the discovery and preclinical profiling of compound 13, which paved the path to our clinical candidate.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein.

Complement factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis, and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

Purpose The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

Purpose: To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma. Design: Double-masked, prospective, randomized and vehicle-controlled three-part multicenter study with 98 subjects (ClinicalTrials.gov NCT02743780). Methods: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2). Part 3 evaluated IOP-lowering efficacy of the MTD administered nightly for one week in 50 patients with minimum IOP of 24mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at Day 8 compared to baseline. Results: There was no difference in favor of MGV354 for IOP lowering; change from Baseline to Day 8 in mean diurnal IOP was -0.6 mmHg for MGV354-treated patients and -1.1 mmHg for Vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common AEs reported following MGV354 administration in all the three parts was conjunctival and ocular hyperemia. Conclusions: Overall, MGV354 0.1% demonstrated no statistically significant effect compared to Vehicle in lowering IOP based upon the study’s main outcome measure. MGV354 produced ocular hyperemia consistent with target engagement in the conjunctiva, but human glaucomatous trabecular meshwork may have levels of oxidized sGC that are too low to benefit from MGV354.

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.

PURPOSE:To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma. DESIGN:Double-masked, randomized, and vehicle-controlled study. METHODS:Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once-daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site. Part 3 was a multisite trial that evaluated intraocular pressure (IOP)-lowering efficacy of the MTD administered nightly for 1 week in 50 patients with minimum IOP of 24 mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at day 8 compared to baseline (ClinicalTrials.govNCT02743780). RESULTS:There was no difference in favor of MGV354 for IOP lowering; change from baseline to day 8 in mean diurnal IOP was -0.6 mm Hg for MGV354-treated patients and -1.1 mm Hg for vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common adverse events reported after MGV354 administration were conjunctival and ocular hyperemia. CONCLUSIONS:Overall, MGV354 0.1% demonstrated no statistically significant effect compared to vehicle in lowering IOP based on the study's main outcome measure. MGV354 produced ocular hyperemia consistent with its pharmacology.

Background: Patients with resistant hypertension have a higher risk of cardiovascular morbidity and mortality. The current treatment options for patients with resistant hypertension are limited by ...

Phototransduction is initiated when the absorption of light converts the 11-cis-retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-cis-retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the “visual or retinoid” cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-cis-retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-cis-6-membered ring (11-cis-6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-cis-6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro-205 in green cone opsin could prevent entry and binding of 11-cis-6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-cis-6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-cis-6mr-retinal. These results and our structural modeling indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-cis-6mr-retinal.

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is becoming an important technology to determine the distribution of drugs and their metabolites in the tissue of preclinical species after dosing. Interest in IMS is growing in the ophthalmology field, but little work to this point has been done to investigate ocular drug transit using this technology. Information on where and how a drug is distributing through the eye is important in understanding efficacy and whether it is reaching the desired target tissue. For this study, ocular distribution of brimonidine was investigated in rabbits following topical administration. Brimonidine has been shown to lower intraocular pressure and is approved to treat glaucoma, the second leading cause of blindness in the world. We have developed IMS methods to assess transit of topically administered brimonidine from the anterior to the posterior segment of rabbit eyes. Using IMS, brimonidine was detected in the cornea, aqueous humor, iris, and posterior segments of the eye. The distribution of brimonidine suggests that the route of transit following topical administration is mainly through the uvea-scleral route. This study demonstrates that IMS can be applied to assess ocular transit and distribution of topically administered drugs.

Herein we describe the discovery and characterization of a novel, piperidine-based inhibitor of cholesteryl ester transfer protein (CETP) with a core structure distinct from other reported CETP inhibitors. A versatile synthesis starting from 4-methoxypyridine enabled an efficient exploration of the SAR, giving a lead molecule with potent CETP inhibition in human plasma. The subsequent optimization focused on improvement of pharmacokinetics and mitigation of off-target liabilities, such as CYP inhibition, whose improvement correlated with increased lipophilic efficiency. The effort led to the identification of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics in rats and robust efficacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo. Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma cells nor in chronically cannulated rats. On the basis of its preclinical efficacy and safety profile, the compound was advanced into clinical trials.