ABSTRACT
Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. Results: RG7774 was found to be a potent (EC50: 2.8 nM and Ki: 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.
ABSTRACT
Diabetic retinopathy (DR) is a microvascular disease of the retina and a serious complication of type I and type II diabetes mellitus. DR affects working-age populations and can cause permanent vision loss if left untreated. The standard of care for proliferative DR is inhibiting VEGF. However, the mechanisms that induce excessive VEGF production in the retina remain elusive, although some evidence links elevated VEGF in the diabetic retina with local and systemic TGFß1 upexpression. Here, we present evidence from animal models of disease suggesting that excessive TGFß1 production in the early DR is correlated with VEGF mRNA and protein production by senescent pericytes and other retinal cells. Collectively, these results confirm that TGFß1 is strongly implicated in the vascular complications of DR.
ABSTRACT
The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.
Subject(s)
Endothelial Cells/drug effects , Pluripotent Stem Cells/drug effects , Small Molecule Libraries/pharmacology , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Blood-Retinal Barrier/drug effects , Blood-Retinal Barrier/metabolism , Cell Differentiation , Cell Line , Cell Proliferation/drug effects , Claudin-5/genetics , Claudin-5/metabolism , Drug Evaluation, Preclinical , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Editing , Genome , Humans , Mice , Mice, Knockout , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Pyrazoles/pharmacology , Pyridines/pharmacology , Tight Junctions/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Chronic renal disease (CRD) accelerates the development of atherosclerosis. The potent protease cathepsin S cleaves elastin and generates bioactive elastin peptides, thus promoting vascular inflammation and calcification. We hypothesized that selective cathepsin S inhibition attenuates atherogenesis in hypercholesterolemic mice with CRD. CRD was induced by 5/6 nephrectomy in high-fat high-cholesterol fed apolipoprotein E-deficient mice. CRD mice received a diet admixed with 6.6 or 60 mg/kg of the potent and selective cathepsin S inhibitor RO5444101 or a control diet. CRD mice had significantly higher plasma levels of osteopontin, osteocalcin, and osteoprotegerin (204%, 148%, and 55%, respectively; P < 0.05), which were inhibited by RO5444101 (60%, 40%, and 36%, respectively; P < 0.05). Near-infrared fluorescence molecular imaging revealed a significant reduction in cathepsin activity in treated mice. RO5444101 decreased osteogenic activity. Histologic assessment in atherosclerotic plaque demonstrated that RO5444101 reduced immunoreactive cathepsin S (P < 0.05), elastin degradation (P = 0.01), plaque size (P = 0.01), macrophage accumulation (P < 0.01), growth differentiation factor-15 (P = 0.0001), and calcification (alkaline phosphatase activity, P < 0.01; osteocalcin, P < 0.05). Furthermore, cathepsin S inhibitor or siRNA significantly decreased expression of growth differentiation factor-15 and monocyte chemotactic protein-1 in a murine macrophage cell line and human primary macrophages. Systemic inhibition of cathepsin S attenuates the progression of atherosclerotic lesions in 5/6 nephrectomized mice, serving as a potential treatment for atherosclerosis in patients with CRD.
Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/pathology , Cathepsins/antagonists & inhibitors , Kidney Failure, Chronic/enzymology , Kidney Failure, Chronic/pathology , Animals , Arteries/enzymology , Arteries/pathology , Atherosclerosis/complications , Biomarkers/blood , Cathepsins/metabolism , Chemokine CCL2/metabolism , Growth Differentiation Factor 15/metabolism , Humans , Interferon-gamma/pharmacology , Kidney Failure, Chronic/blood , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Osteogenesis/drug effects , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Vascular Calcification/complications , Vascular Calcification/pathologyABSTRACT
RATIONALE: Rescuing adverse myocardial remodeling is an unmet clinical goal and, correspondingly, pharmacological means for its intended reversal are urgently needed. OBJECTIVES: To harness a newly-developed experimental model recapitulating progressive heart failure development for the discovery of new drugs capable of reversing adverse remodeling. METHODS AND RESULTS: A VEGF-based conditional transgenic system was employed in which an induced perfusion deficit and a resultant compromised cardiac function lead to progressive remodeling and eventually heart failure. Ability of candidate drugs administered at sequential remodeling stages to reverse hypertrophy, enlarged LV size and improve cardiac function was monitored. Arguing for clinical relevance of the experimental system, clinically-used drugs operating on the Renin-Angiotensin-Aldosterone-System (RAAS), namely, the ACE inhibitor Enalapril and the direct renin inhibitor Aliskerin fully reversed remodeling. Remodeling reversal by these drugs was not accompanied by neovascularization and reached a point-of-no-return. Similarly, the PPARγ agonist Pioglitazone was proven capable of reversing all aspects of cardiac remodeling without affecting the vasculature. Extending the arsenal of remodeling-reversing drugs to pathways other than RAAS, a specific inhibitor of 11ß-hydroxy-steroid dehydrogenase type 1 (11ß HSD1), a key enzyme required for generating active glucocorticoids, fully rescued myocardial hypertrophy. This was associated with mitigating the hypertrophy-associated gene signature, including reversing the myosin heavy chain isoform switch but in a pattern distinguishable from that associated with neovascularization-induced reversal. CONCLUSIONS: A system was developed suitable for identifying novel remodeling-reversing drugs operating in different pathways and for gaining insights into their mechanisms of action, exemplified here by uncoupling their vascular affects.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/antagonists & inhibitors , Cardiomegaly/drug therapy , Enzyme Inhibitors/pharmacology , Renin-Angiotensin System/drug effects , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Animals , Cardiomegaly/enzymology , Cardiomegaly/genetics , Drug Evaluation, Preclinical , Mice , Mice, Transgenic , Renin-Angiotensin System/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Starting from the weakly active dual CatS/K inhibitor 5, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50,000-fold vs CatK) cyclopentane derivative 22 by exploiting specific ligand-receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative 57 decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., 79). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound 79 reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED50 of 5 mg/kg.
Subject(s)
Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Animals , Cyclopentanes/chemistry , Cysteine Proteinase Inhibitors/pharmacokinetics , Humans , Mice , Proline/analogs & derivatives , Structure-Activity RelationshipABSTRACT
AIMS: mast cells have been shown to accumulate in the adventitia of human atherosclerotic plaques and were recently demonstrated by us to contribute to plaque progression and instability. In this study, we investigated whether selective inhibition of mast cell chymases would affect the lesion development and stability. METHODS AND RESULTS: the protease inhibitor RO5066852 appeared to be a potent inhibitor of chymase activity in vitro and ex vivo. With this inhibitor, we provide three lines of evidence that chymase inhibition can prevent many pro-atherogenic activities. First, oral administration of RO5066852 reduced spontaneous atherosclerosis in the thoracic aorta of apoE(-/-) mice. Second, chymase inhibition prevented the accelerated plaque progression observed in apoE(-/-) mice that were exposed to repetitive episodes of systemic mast cell activation. Furthermore, RO5066852 enhanced lesional collagen content and reduced necrotic core size. Third, RO5066852 treatment almost completely normalized the increased frequency and size of intraplaque haemorrhages observed in apoE(-/-) mice after acute perivascular mast cell activation in advanced atherosclerosis. CONCLUSION: our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.
Subject(s)
Apolipoproteins E/deficiency , Chymases/antagonists & inhibitors , Indoleacetic Acids/pharmacology , Mast Cells/drug effects , Mast Cells/enzymology , Naphthalenes/pharmacology , Plaque, Atherosclerotic/prevention & control , Animals , Apolipoproteins E/genetics , Chymases/genetics , Gene Expression , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Plaque, Atherosclerotic/drug therapy , Plaque, Atherosclerotic/enzymology , Plaque, Atherosclerotic/pathology , Protease Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, LDL/deficiency , Receptors, LDL/geneticsABSTRACT
Bark from Pinus brutia was extracted with supercritical fluid extraction (SFE), using CO(2), at various extraction conditions both at laboratory and at pilot scale. Optimized parameters were 200 bar, 60 degrees C, and 3% ethanol at a solvent/feed ratio of 30. Additionally, the pine bark was sonicated (1 h at 50 degrees C) by different solvents (n-hexane, dichloromethane, ethyl acetate, and ethanol) to investigate the correlation between the different extraction setups and to obtain information on SFE up-scaling possibilities. Analyzed by HPLC, 7.2% of (-)-catechin was extractable at laboratory scale, and 58.4% (800 bar) and 47.8% (200 bar), both with modifiers, at pilot scale. By sonication with ethanol, 46.8% of (-)-catechin and almost 100% of (-)-epicatechin and (-)-catechin gallate were extracted. Ethyl acetate extract revealed high correlations with the laboratory scale SFE (r = 0.98) and also pilot scale SFE runs at 200 (r = 0.99) and 800 bar (r = 0.98) without modifiers.