Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity.

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

Remote functionalization of SCH 39166: discovery of potent and selective benzazepine dopamine D1 receptor antagonists.

A series of novel benzazepine derived dopamine D(1) antagonists have been discovered. These compounds are highly potent at D(1) and showed excellent selectivity over D(2) and D(4) receptors. SAR studies revealed that a variety of functional groups are tolerated on the D-ring of known tetracyclic benzazepine analog 2, SCH 39166, leading to compounds with nanomolar potency at D(1) and good selectivity over D(2)-like receptors.

Discovery of new SCH 39166 analogs as potent and selective dopamine D1 receptor antagonists.

A series of novel dopamine D(1) antagonists derived from functionalization of the D-ring of SCH 39166 were prepared. A number of these compounds displayed subnanomolar D(1) activity and more than 1000-fold selectivity over D(2). We found C-3 derivatization afforded compounds with superior overall profile in comparison to the C-2 and C-4 derivatization. A number of highly potent D(1) antagonists were discovered which have excellent selectivity over other dopamine receptors and improved PK profile compared to SCH 39166.

Pharmacological profile of the NOP agonist and cough suppressing agent SCH 486757 (8-[Bis(2-Chlorophenyl)Methyl]-3-(2-Pyrimidinyl)-8-Azabicyclo[3.2.1]Octan-3-Ol) in preclinical models.

We describe the pharmacological and pharmacokinetic profiles of SCH 486757, a nociceptin/orphanin FQ peptide (NOP) receptor agonist that has recently entered human clinical trials for cough. SCH 486757 selectively binds human NOP receptor (K(i)=4.6+/-0.61nM) over classical opioid receptors. In a guinea pig capsaicin cough model, SCH 486757 (0.01-1mg/kg) suppressed cough at 2, 4, and 6h post oral administration with a maximum efficacy occurring at 4h equivalent to codeine, hydrocodone, dextromethorphan and baclofen. The antitussive effects of SCH 486757 (3.0mg/kg, p.o.) was blocked by the NOP receptor antagonist J113397 (12mg/kg, i.p.) but not by naltrexone (10mg/kg, p.o.). SCH 486757 does not produce tolerance to its antitussive activity after a 5-day BID dosing regimen. After acute and chronic dosing paradigms, SCH 486757 (1mg/kg) inhibited capsaicin-evoked coughing by 46+/-9% and 40+/-11%, respectively. In a feline mechanically-evoked cough model, SCH 486757 produces a maximum inhibition of cough and expiratory abdominal electromyogram amplitude of 59 and 61%, respectively. SCH 486757 did not significantly affect inspiratory electromyogram amplitude. We examined the abuse potential of SCH 486757 (10mg/kg, p.o.) in a rat conditioned place preference procedure which is sensitive to classical drugs of abuse, such as amphetamine and morphine. SCH 486757 was without effect in this model. Finally, SCH 486757 displays a good oral pharmacokinetic profile in the guinea pig, rat and dog. We conclude that SCH 486757 has a favorable antitussive profile in preclinical animal models.

Effect of a novel NOP receptor agonist (SCH 225288) on guinea pig irritant-evoked, feline mechanically induced and canine infectious tracheobronchitis cough.

BACKGROUND: Previous studies have demonstrated that nociceptin/orphanin FQ (N/OFQ), the endogenous peptide ligand for the G-protein-coupled NOP receptor, inhibits cough in experimental models. SCH 225288 is a nonpeptide, orally active NOP agonist that may provide the foundation for the development of novel treatments for cough. METHODS: First we characterized the selectivity of SCH 225288 in human receptor binding assays. Afterwards, the antitussive activity of SCH 225288 was studied in three mechanistically distinct cough models. Specifically, we observed the cough-suppressant effect of SCH 225288 in a guinea pig capsaicin irritant-evoked cough model, a feline mechanically induced cough model and finally in a canine Bordetella bronchiseptica disease model. RESULTS: SCH 225288 selectively binds human NOP receptor (K(i) = 0.38 +/- 0.02 nmol/l) over classical opioid receptors (COR). In a guinea pig capsaicin cough model, SCH 225288 (0.1-1 mg/kg) suppressed cough at 2, 4, and 6 h after oral administration. The antitussive effect of SCH 225288 (3.0 mg/kg, p.o.) was blocked by the NOP antagonist J113397 (12 mg/kg, i.p.) but not by the classical opioid receptor (COR) antagonist, naltrexone (3.0 mg/kg, i.p.). In the anesthetized cat, we evaluated the effects of SCH 225288 given either intravenously or via the intravertebral artery against the increases in cough number and respiratory expiratory and inspiratory muscle (rectus abdominis and parasternal) electromyographic (EMG) activities due to perturbations of the intrathoracic trachea. SCH 225288 (0.03-3.0 mg/kg, i.v.) inhibited both cough number and abdominal EMG amplitudes. Similarly, SCH 225288 (0.001-0.3 mg/kg) administered intra-arterially also diminished cough number and abdominal EMG amplitudes. No significant effect of the drug was noted on parasternal EMG activity. Finally, we studied the antitussive actions of SCH 225288 (1.0 mg/kg) in a canine B. bronchiseptica disease model. In this model, dogs were challenged intranasally with B. bronchiseptica. Comparisons were made between a vehicle group, an SCH 225288 (1.0 mg/kg, p.o., q.d.) and a butorphanol (0.6 mg/kg, p.o., b.i.d.) group on the mean change in cough scores from baseline values and days 6-9 after B. bronchiseptica challenge. SCH 225288 (1.0 mg/kg, p.o., q.d.) displayed a positive antitussive tendency (p = 0.06) to inhibit B. bronchiseptica cough whereas butorphanol (0.6 mg/kg, p.o., b.i.d.) was devoid of antitussive activity. CONCLUSIONS: Taken together, the present data show that SCH 225288 is a potent and effective antitussive agent in animal models of cough. Furthermore, these findings indicate that NOP agonists represent a promising new therapeutic approach for the treatment of cough without the side effect liabilities associated with opioid antitussives.

Design and discovery of 1,3-benzodiazepines as novel dopamine antagonists.

A series of novel 1,3-benzodiazapine based D1 antagonists was designed according to the understanding of pharmacophore models derived from SCH 23390 (1b), a potent and selective D1 antagonist. The new design features an achiral cyclic-amidine that maintains desired basicity. Solid phase synthesis was developed for SAR development of the novel dopamine antagonists.

The discovery of tropane derivatives as nociceptin receptor ligands for the management of cough and anxiety.

The discovery of 1 as a high-affinity ligand for the nociceptin receptor has led to the synthesis of a series of tropane (8-methyl-8-azabicyclo[3.2.1]octane) derivatives as optimized ligands. These compounds exhibit high affinity for the nociceptin receptor, moderate to excellent selectivity over the opioid mu receptor, and behave as full agonists. In this Letter, we present the synthesis and highlight the structure-activity relationship of tropane derivatives culminating in the identification of 24 and 32 as potent and orally active antitussive and anxiolytic agents. The in vitro and in vivo activities, pharmacokinetic profile, and the hPXR activity, which predicts the potential 3A4 induction in human, are disclosed.

Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety.

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in the guinea pig models.

Synthesis and structure-activity relationships of N-substituted spiropiperidines as nociceptin receptor ligands: part 2.

A series of N-8 substituted analogs based upon the spiropiperidine core of the original lead compound 1 was synthesized. This lead has been elaborated to compounds to give compounds 2 and 3 (R=H) that exhibited high NOP binding affinity as well as selectivity against other known opioid receptors. These two series have been further functionalized at the amido nitrogen. The synthesis and structure-activity relationship (SAR) of these and related compounds are discussed.

The anxiolytic-like effects of the novel, orally active nociceptin opioid receptor agonist 8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510).

Orphanin FQ/nociceptin (OFQ/N) is the endogenously occurring peptide ligand for the nociceptin opioid receptor (NOP) that produces anxiolytic-like effects in mice and rats. The present study assessed the anxiolytic-like activity of 8-[bis(2-methylphenyl)-methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510), a novel potent piperidine NOP agonist (EC(50) = 12 nM) that binds with high affinity (K(i) = 0.3 nM) and functional selectivity (>50-fold over the mu-, kappa-, and delta-opioid receptors). The anxiolytic-like activity and side-effect profile of SCH 221510 were assessed in a variety of models and the benzodiazepine, chlordiazepoxide (CDP), was included for comparison. The effects of chronic dosing of SCH 221510 were also assessed. Furthermore, the specificity of the anxiolytic-like effect of SCH 221510 was investigated with the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and the opioid receptor antagonist naltrexone. Like CDP (1-30 mg/kg i.p.), SCH 221510 (1-30 mg/kg p.o.) produced anxiolytic-like effects in the elevated plus-maze (rat and gerbil), Vogel conflict (rat), conditioned lick suppression (rat), fear-potentiated startle (rat), and pup separation-induced vocalization (guinea pig) assays. In the Vogel conflict, the anxiolytic-like effect of SCH 221510 (10 mg/kg) was attenuated by J-113397 (3-10 mg/kg p.o.), but not naltrexone (3-30 mg/kg i.p.). Additionally, the anxiolytic-like effects of SCH 221510 did not change appreciably following 14-day b.i.d. dosing in rats (10 mg/kg). Furthermore, unlike CDP, SCH 221510 (3-30 mg/kg) produced anxiolytic-like activity at doses that did not disrupt overt behavior. Collectively, these data suggest that NOP agonists such as SCH 221510 may have an anxiolytic-like profile similar to benzodiazepines, with a reduced side-effect liability.

Protective effects of myricetin and related flavonols against A2E and light mediated-cell death in bovine retinal primary cell culture.

The present study was performed to investigate the effect of flavonols, namely myricetin and structurally related quercetin and kaempferol against A2E and blue light-induced photoreceptors death in primary retinal cell cultures. Primary retinal cell cultures were prepared from bovine retinas. Fourteen-day-old cultures were pretreated with different concentrations of myricetin, quercetin, kaempferol (1-40 microM) for 24 h, then treated with 30 microM of A2E or exposed to blue-actinic light for 20 h. Green nucleic acid stain assay was used to evaluate cell death. Photoreceptor and bipolar cells were immunolabeled with specific antibodies and were counted using automated microscope imaging and image-based cell counting software. Twenty hours exposure to blue light induced approximately 75% death of photoreceptors in bovine retinal cell cultures. Myricetin protected 100% of photoreceptors against blue-light-mediated damage with an EC(50) of 9+/-0.7 microM. Quercetin resulted in a maximum of 15% protection against light damage, and kaempferol was inactive. A2E induced photoreceptor and bipolar cell death in a concentration-dependent manner with EC(50) of 25 microM for photoreceptors and 31 microM for bipolar cells. Myricetin, quercetin and kaempferol protected against A2E-induced photoreceptors and bipolar cells death with EC(50) values of 2+/-0.3 microM, 2+/-0.3 microM, 5+/-0.09 microM and 0.8+/-0.07 microM, 0.44+/-0.06 microM, 1+/-0.4 microM, respectively. Caspase-3 inhibitor (Z-DEVD-fmk) protected 42% photoreceptors and 57% bipolar cells from A2E toxicity. In contrast, this inhibitor had no effect against light-induced photoreceptor damage. Despite the poor activity of quercetin and the inactivity of kaempferol against blue light, myricetin, quercetin and kaempferol exhibited approximately 100% protection against A2E toxicity. This suggests that light- and A2E-induced cell deaths are mediated through different pathways. These results suggest that myricetin functions as potent and effective neuroprotective agent for photoreceptor cells against A2E and light damage.

Synthesis and structure-activity relationships of 4-hydroxy-4-phenylpiperidines as nociceptin receptor ligands: Part 2.

A series of 4-[2-(aminomethyl)phenyl]-1-[bis(2-chlorophenyl)methyl]-4-hydroxypiperidine analogs has been identified as nociceptin receptor ligands. These compounds display high affinity and functional activity at the nociceptin receptor. The synthesis and structure-activity relationships at the C-4 phenyl and N-1 positions are described and the antitussive activity of a selected compound is reported.

Synthesis and structure-activity relationships of 4-hydroxy-4-phenylpiperidines as nociceptin receptor ligands: Part 1.

A series of 4-hydroxy-4-phenylpiperidines have been synthesized and bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships at the N-1 and C-4 are described.

Synthesis and structure-activity relationships of N-substituted spiropiperidines as nociceptin receptor ligands.

A series of N-substituted analogs based upon the spiropiperidine core of 1 was synthesized and exhibited high binding affinity to the nociceptin (NOP) receptor. The selectivities against other known opioid receptors were determined.

Minocycline protects photoreceptors from light and oxidative stress in primary bovine retinal cell culture.

PURPOSE: To determine whether minocycline, a compound known to protect the retina against light-induced damage in rodent models, and its structurally related analogues would protect photoreceptor cells in primary bovine retinal cell culture against light and oxidative stress. METHODS: Minocycline and its analogues were tested in primary retinal cell culture to see whether they would inhibit light or oxidative stress-induced cell death. Primary cell cultures composed of photoreceptors, bipolar cells, and glial cells were prepared from bovine retinas. The extent of cell death induced by light or oxidative stress was assessed by using Sytox Green (Invitrogen-Molecular Probes, Eugene, OR) a nucleic acid dye uptake assay. Differential protection of photoreceptor cells from stress were examined using immunocytochemistry. RESULTS: Minocycline and methacycline were cytoprotective against light- or oxidative stress-induced damage of bovine primary photoreceptors in culture with an EC(50) < 10 microM. In contrast, structurally related analogues such as demeclocycline, meclocycline, and doxycycline were phototoxic at >3 to >10 microM. Though demeclocycline was found to be phototoxic, it was cytoprotective (EC(50) = 5 microM) against oxidative stress in the absence of exposure to light. CONCLUSIONS: The protective action of minocycline against light-induced damage in the cell-based assays agrees with earlier reports in animal models and suggests that the in vitro assay using bovine primary retinal cell culture is a suitable model for evaluating compounds for retinal protection. Cellular protection or toxicity produced by structurally related compounds show that minor structural modifications can alter the function of minocycline and lead to potent retinal protective compounds.

Protective effect of crocin against blue light- and white light-mediated photoreceptor cell death in bovine and primate retinal primary cell culture.

PURPOSE: The present study was performed to investigate the effect of crocin on blue light- and white light-induced rod and cone death in primary retinal cell cultures. METHODS: Primary retinal cell cultures were prepared from primate and bovine retinas. Fifteen-day-old cultures were exposed to blue actinic light or to white fluorescent light for 24 hours. Cultures were treated by the addition of different concentrations of crocin for 24 hours before light exposure or for 8 hours after light exposure. Cultures kept in the dark were used as controls. Green nucleic acid stain assay was used to evaluate cell death. Rods and cones were immunolabeled with specific antibodies and counted. TUNEL labeling was used to detect fragmented DNA in fixed cells after light exposure. RESULTS: Primary retinal cell cultures contained a mixture of retinal cells enriched in photoreceptors, bipolar cells, and Müller cells. Twenty-four-hour exposure to blue and white light induced death in 70% to 80% of the photoreceptors in bovine and primate retinal cell cultures. Crocin protected the photoreceptors against blue light- or white light-mediated damage in a concentration-dependent manner with an EC50 of approximately 30 microM. TUNEL assays confirmed that crocin protected photoreceptors from light damage. CONCLUSIONS: These results show that blue and white light selectively induce rod and cone cell death in an in vitro model. Crocin protects retinal photoreceptors against light-induced cell death.

Modification of the clozapine structure by parallel synthesis.

A structure-activity study based on the core structure of clozapine 1b was accomplished by utilizing high-throughput synthesis. Several focused libraries were designed and synthesized to quickly develop SAR. The results indicate that by varying different regions of clozapine, both D(1)-selective and D(2)-selective compounds can be obtained.

2-(2-Furanyl)-7-phenyl[1,2,4]triazolo[1,5-c]pyrimidin-5-amine analogs as adenosine A2A antagonists: the successful reduction of hERG activity. Part 2.

The structure-activity relationship (SAR) exploration using 2-(2-furanyl)-7-phenyl[1,2,4]triazolo-[1,5-c]pyrimidin-5-amine (1) as a template led to the identification of a novel class of potent and selective adenosine A2A receptor (AR) antagonists. However, these compounds were found to be associated with significant hERG activity. This report discusses the strategy and outcome of an expanded SAR focused on addressing the hERG liability. As a result, compounds 21 and 24 possess excellent in vitro profiles, highly promising in vivo profiles, and acceptable levels of hERG channel inhibition.

2-(2-Furanyl)-7-phenyl[1,2,4]triazolo[1,5-c]pyrimidin-5-amine analogs: highly potent, orally active, adenosine A2A antagonists. Part 1.

The structure-activity relationship of this novel class of compounds based on 2-(2-furanyl)-7-phenyl[1,2,4]-triazolo[1,5-c]pyrimidin-5-amine, 1, and its analogs was evaluated for their in vitro and in vivo adenosine A(2A) receptor antagonism. Several compounds displayed oral activity at 3 mg/kg in a rat catalepsy model. Specifically, compound 8g displayed an excellent in vitro profile, as well as a highly promising in vivo profile.

6-(2-Furanyl)-9H-purin-2-amine derivatives as A2A adenosine antagonists.

Structure-activity relationships have been investigated through substitutions at the 9-position of the 2-amino-6-(2-furanyl) purine (5) to identify novel and selective A(2A) adenosine receptor antagonists. Several potent and selective antagonists were identified. In particular, compounds 20, 25, and 26 show very high affinity with excellent selectivity.