Pharmacology of novel intraocular pressure-lowering targets that enhance conventional outflow facility: Pitfalls, promises and what lies ahead?

Intraocular pressure (IOP) lowering drugs that are approved for the treatment of glaucoma and ocular hypertension have limited activity on increasing aqueous humor movement through the trabecular meshwork and Schlemm's canal (TM/SC). The TM/SC complex is considered the conventional outflow pathway and is a primary site of increased resistance to aqueous humor outflow in glaucoma. Novel mechanisms that enhance conventional outflow have shown promise in IOP reduction via modulation of several pathways including Rho kinase, nitric oxide/soluble guanylate cyclase/cGMP, adenosine A1, prostaglandin EP4/cAMP, and potassium channels. The clinical translatability of these pharmacological modulators based on pre-clinical efficacy models is currently being explored. In addition, identification of pathways from GWAS and other studies involving transgenic rodent models with elevated/reduced IOP phenotypes have begun to yield additional insights into IOP regulation and serve as a source for the next generation of IOP lowering targets. Lastly, improvements in drug delivery technologies to enable sustained IOP reduction are also discussed

Application of Imaging Mass Spectrometry to Assess Ocular Drug Transit

MALDI imaging mass spectrometry (IMS) is becoming an important technology to determine the distribution of drugs and their metabolites in 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 monitor ocular transit and distribution of topically administered drugs

Understanding metabolism related differences in ocular efficacy of MGV354

MGV354 was being developed as a novel ocular therapy for lowering of intraocular pressure, a key modifiable risk factor for glaucoma. MGV354 is an activator of soluble guanylate cyclase, an enzyme known to be involved in the regulation of IOP. MGV354 has been shown to robustly lower IOP over 24 h after a single topical ocular drop in rabbit and monkey pharmacology models. However, MGV354 failed 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 of MGV354 in an effort to understand potential differences in biotransformation pathways of MGV354 in rabbits, monkeys, and humans. Overall twenty-six metabolites, formed via oxidative and conjugative pathways, were identified in vitro and in vivo. In vitro hepatic metabolism was qualitatively similar across species, with minor but distinct differences. There were no observable interspecies differences in the hepatic and ocular metabolism of MGV354. Although ocular metabolism was not as extensive as hepatic, the results do not explain the lack of efficacy of MGV354 in clinical studies

Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked retinal analogue.

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

Structure of neprilysin in complex with the active metabolite of sacubitril

Sacubitril is an ethyl ester prodrug of LBQ657, the active neprilysin (NEP) inhibitor, and is a component of LCZ696 (sacubitril/valsartan). We report herein the three-dimensional structure of LBQ657 in complex with human NEP at 2Å resolution. The X-ray structure unravels the binding mode of the compound spanning the S1, S1’ and S2’ sub-pockets of the active site, consistent with a competitive inhibition mode. An induced fit conformational change upon binding of the P1’-biphenyl moiety of the inhibitor suggests an explanation for its selectivity against structurally homologous zinc metallopeptidases

A phase I/II study to evaluate the safety, tolerability and early efficacy of MGV354 in healthy subjects and in patients with ocular hypertension or glaucoma

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

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

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 (G(t)) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C(11)=C(12) 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 G(t) 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 G(t) 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

The discovery of (S)-1-(6-(3-((4-(1-(cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1H-inden-4-yl)pyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic acid, an sGC activator specifically designed for topical ocular delivery as a therapy for glaucoma

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 intra ocular pressure over 24h after a single topical ocular drop, and has been selected for clinical evaluation

Complement factor B is critical for sub-RPE deposit formation in a model of DHRD/ML with features of age-related macular degeneration

EFEMP1 R345W is a protein misfolding-prone mutation causing Doyne honeycomb retinal dystrophy/Mallatia Leventinese (DHRD/ML), a rare blinding disease with similar clinical pathology to a common disease of age-related macular degeneration (AMD). Aged Efemp1R345W/R345W knock-in mice (Efemp1ki/ki mice) develop deposits on the basal side of retinal pigment epithelial cells, which is complement C3- dependent. We assessed alternative complement pathway component factor B (FB) in sub-RPE deposit formation in Efemp1ki/ki mice. RNA-seq analysis of Efemp1ki/ki mice comparing to Efemp1+/+ reveals increased unfolded protein response in posterior eye cups, decreased mitochondrial function in neural retina (both by 3-month-old), and increased inflammatory pathways in both tissues (at 17-month-old). Aged Efemp1ki/ki mice also exhibits elevated ocular complement protein activation with around two-fold (p<0.05) elevation of breakdown products iC3b and Ba by Western blot analysis. Proteomics analysis of eye lysate confirmed similar Efemp1 R345W-dysregulated pathways as detected by RNA-seq. Cfb deficiency partially normalizes these biological pathway changes in the eyes of female Efemp1ki/ki mice. Female Efemp1ki/ki mice dosed with a small molecule FB inhibitor from 10- to 12-month-old reduced sub-RPE deposits by 65% (p=0.051). Male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females and no elevation of ocular complement activation, therefore not affected by FB inhibitor. Sub-RPE deposits/drusen, seen in Efemp1ki/ki mice, are a common early clinical feature of DHRD/ML and AMD. The broader effect on Efemp1ki/ki mice by either Cfb deficiency or oral FB inhibition suggest that systemic inhibition of the alternative complement pathway is potentially an effective strategy for treatment of dry AMD and DHRD/ML

Structure-guided design of substituted biphenyl butanoic acid derivatives as Neprilysin inhibitors

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 sub-site 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 sub-site in S1’. We report herein the discov-ery and pre-clinical profiling of compound 13, which paved the path to our clinical candidate