Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions.

mRNA LNPs can experience a decline in activity over short periods (ranging from weeks to months). As a result, they require frozen storage and transportation conditions to maintain their full functionality when utilized. Currently approved commercially available mRNA LNP vaccines also necessitate frozen storage and supply chain management. Overcoming this significant inconvenience in the future is crucial to reducing unnecessary costs and challenges associated with storage and transport. In this study, our objective was to illuminate the potential time frame for nonfrozen storage and transportation conditions of mRNA LNPs without compromising their activity. To achieve this goal, we conducted a stability assessment and an in vitro cell culture delivery study involving five mRNA LNPs. These LNPs were constructed by using a standard formulation similar to that employed in the three commercially available LNP formulations. Among these formulations, we selected five structurally diverse ionizable lipids─C12-200, CKK-E12, MC3, SM-102, and lipid 23─from the existing literature. We incorporated these lipids into a standard LNP formulation, keeping all other components identical. The LNPs, carrying mRNA payloads, were synthesized by using microfluidic mixing technology. We evaluated the shelf life stability of these LNPs over a span of 9 weeks at temperatures of 2-8, 25, and 40 °C, utilizing an array of analytical techniques. Our findings indicated minimal impact on the hydrodynamic diameter, zeta potential, encapsulation efficiency, and polydispersity of all LNPs across the various temperatures over the studied period. The RiboGreen assay analysis of LNPs showed consistent mRNA contents over several weeks at various nonfrozen storage temperatures, leading to the incorrect assumption of intact and functional LNPs. This misunderstanding was rectified by the significant differences observed in EGFP protein expression in an in vitro cell culture (using HEK293 cells) across the five LNPs. Specifically, only LNP 1 (C12-200) and LNP 4 (SM-102) exhibited high levels of EGFP expression at the start (T0), with over 90% of HEK293 cells transfected and mean fluorescence intensity (MFI) levels exceeding 1. Interestingly, LNP 1 (C12-200) maintained largely unchanged levels of in vitro activity over 11 weeks when stored at both 2-8 and 25 °C. In contrast, LNP 4 (SM-102) retained its functionality when stored at 2-8 °C over 11 weeks but experienced a gradual decline of in vitro activity when stored at room temperature over the same period. Importantly, we observed distinct LNP architectures for the five formulations through cryo-EM imaging. This highlights the necessity for a deeper comprehension of structure-activity relationships within these complex nanoparticle structures. Enhancing our understanding in this regard is vital for overcoming storage and stability limitations, ultimately facilitating the broader application of this technology beyond vaccines.

Effects of Cathepsin S Inhibition in the Age-Related Dry Eye Phenotype.

Purpose: Aged C57BL/6J (B6) mice have increased levels of cathepsin S, and aged cathepsin S (Ctss-/-) knockout mice are resistant to age-related dry eye. This study investigated the effects of cathepsin S inhibition on age-related dry eye disease. Methods: Female B6 mice aged 15.5 to 17 months were randomized to receive a medicated diet formulated by mixing the RO5461111 cathepsin S inhibitor or a standard diet for at least 12 weeks. Cornea mechanosensitivity was measured with a Cochet-Bonnet esthesiometer. Ocular draining lymph nodes and lacrimal glands (LGs) were excised and prepared for histology or assayed by flow cytometry to quantify infiltrating immune cells. The inflammatory foci (>50 cells) were counted under a 10× microscope lens and quantified using the focus score. Goblet cell density was investigated in periodic acid-Schiff stained sections. Ctss-/- mice were compared to age-matched wild-type mice. Results: Aged mice subjected to cathepsin S inhibition or Ctss-/- mice showed improved conjunctival goblet cell density and cornea mechanosensitivity. There was no change in total LG focus score in the diet or Ctss-/- mice, but there was a lower frequency of CD4+IFN-γ+ cell infiltration in the LGs. Furthermore, aged Ctss-/- LGs had an increase in T central memory, higher numbers of CD19+B220-, and fewer CD19+B220+ cells than wild-type LGs. Conclusions: Our results indicate that therapies aimed at decreasing cathepsin S can ameliorate age-related dry eye disease with a highly beneficial impact on the ocular surface. Further studies are needed to investigate the role of cathepsin S during aging.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.

Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Real-time measurements of vascular permeability in the mouse eye using vitreous fluorophotometry.

Breakdown of blood-retinal barrier integrity underpins pathological changes in numerous ocular diseases, including neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME). Whilst anti-vascular endothelial growth factor (VEGF) therapies have revolutionised disease treatment, novel therapies are still required to meet patients' unmet needs. To help develop new treatments, robust methods are needed to measure changes in vascular permeability in ocular tissues in animal models. We present here a method for detecting vascular permeability using fluorophotometry, which enables real-time measurements of fluorescent dye accumulation in different compartments of the mouse eye. We applied this method in several mouse models with different increased vascular leakage, including models of uveitis, diabetic retinopathy and choroidal neovascularization (CNV). Furthermore, in the JR5558 mouse model of CNV, we observed with anti-VEGF post-treatment a longitudinal reduction in permeability, in the same animal eyes. We conclude fluorophotometry is a useful method for measuring vascular permeability in the mouse eye, and can be used over multiple time points, without the need to sacrifice the animal. This method has the potential to be used in both basic research for studying the progression and factors underlying disease, but also for drug discovery and development of novel therapeutics.

ANG2 Blockade Diminishes Proangiogenic Cerebrovascular Defects Associated With Models of Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by arteriovenous malformations and blood vessel enlargements. However, there are no effective drug therapies to combat arteriovenous malformation formation in patients with HHT. Here, we aimed to address whether elevated levels of ANG2 (angiopoietin-2) in the endothelium is a conserved feature in mouse models of the 3 major forms of HHT that could be neutralized to treat brain arteriovenous malformations and associated vascular defects. In addition, we sought to identify the angiogenic molecular signature linked to HHT. METHODS: Cerebrovascular defects, including arteriovenous malformations and increased vessel calibers, were characterized in mouse models of the 3 common forms of HHT using transcriptomic and dye injection labeling methods. RESULTS: Comparative RNA sequencing analyses of isolated brain endothelial cells revealed a common, but unique proangiogenic transcriptional program associated with HHT. This included a consistent upregulation in cerebrovascular expression of ANG2 and downregulation of its receptor Tyr kinase with Ig and EGF homology domains (TIE2/TEK) in HHT mice compared with controls. Furthermore, in vitro experiments revealed TEK signaling activity was hampered in an HHT setting. Pharmacological blockade of ANG2 improved brain vascular pathologies in all HHT models, albeit to varying degrees. Transcriptomic profiling further indicated that ANG2 inhibition normalized the brain vasculature by impacting a subset of genes involved in angiogenesis and cell migration processes. CONCLUSIONS: Elevation of ANG2 in the brain vasculature is a shared trait among the mouse models of the common forms of HHT. Inhibition of ANG2 activity can significantly limit or prevent brain arteriovenous malformation formation and blood vessel enlargement in HHT mice. Thus, ANG2-targeted therapies may represent a compelling approach to treat arteriovenous malformations and vascular pathologies related to all forms of HHT.

Enterohepatic Takeda G-Protein Coupled Receptor 5 Agonism in Metabolic Dysfunction-Associated Fatty Liver Disease and Related Glucose Dysmetabolism.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a major health concern with no approved pharmacological therapies. Molecules developed to activate the bile acid-receptor TGR5 regulate pathways involved in MALFD pathogenesis, but the therapeutic value of TGR5 activation on the active form of MAFLD, non-alcoholic steatohepatitis (NASH), still needs to be evaluated. As TGR5 agonism is low in MAFLD, we used strategies to promote the production of endogenous TGR5 ligands or administered pharmacological TGR5 agonists, INT-777 and RO5527239, to study the effect of TGR5 activation on liver and metabolic diseases in high-fat diet-fed foz/foz mice. Although described in the literature, treatment with fexaramine, an intestine-restricted FXR agonist, did not raise the concentrations of TGR5 ligands nor modulate TGR5 signaling and, accordingly, did not improve dysmetabolic status. INT-777 and RO5527239 directly activated TGR5. INT-777 only increased the TGR5 activation capacity of the portal blood; RO5527239 also amplified the TGR5 activation capacity of systemic blood. Both molecules improved glucose tolerance. In spite of the TGR5 activation capacity, INT-777, but not RO5527239, reduced liver disease severity. In conclusion, TGR5 activation in enterohepatic, rather than in peripheral, tissues has beneficial effects on glucose tolerance and MAFLD.

Lysophosphatidic Acid Is a Proinflammatory Stimulus of Renal Tubular Epithelial Cells.

Chronic kidney disease (CKD) refers to a spectrum of diseases defined by renal fibrosis, permanent alterations in kidney structure, and low glomerular-filtration rate. Prolonged epithelial-tubular damage involves a series of changes that eventually lead to CKD, highlighting the importance of tubular epithelial cells in this process. Lysophosphatidic acid (LPA) is a bioactive lipid that signals mainly through its six cognate LPA receptors and is implicated in several chronic inflammatory pathological conditions. In this report, we have stimulated human proximal tubular epithelial cells (HKC-8) with LPA and 175 other possibly pathological stimuli, and simultaneously detected the levels of 27 intracellular phosphoproteins and 32 extracellular secreted molecules with multiplex ELISA. This quantification revealed a large amount of information concerning the signaling and the physiology of HKC-8 cells that can be extrapolated to other proximal tubular epithelial cells. LPA responses clustered with pro-inflammatory stimuli such as TNF and IL-1, promoting the phosphorylation of important inflammatory signaling hubs, including CREB1, ERK1, JUN, IκΒα, and MEK1, as well as the secretion of inflammatory factors of clinical relevance, including CCL2, CCL3, CXCL10, ICAM1, IL-6, and IL-8, most of them shown for the first time in proximal tubular epithelial cells. The identified LPA-induced signal-transduction pathways, which were pharmacologically validated, and the secretion of the inflammatory factors offer novel insights into the possible role of LPA in CKD pathogenesis.

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Elevated Levels of Growth/Differentiation Factor-15 in the Aqueous Humor and Serum of Glaucoma Patients.

Dysregulated levels of growth/differentiation factor-15 (GDF15), a divergent member of the transforming growth factor-beta super family, have been found to be associated with the pathology of various diseases. In this study, we evaluated the levels of GDF15 in aqueous humor (AH) and serum samples derived from primary open-angle glaucoma (POAG) and age- and gender-matched non-glaucoma (cataract) patients to assess the plausible association between GDF15 and POAG. GDF15 levels were determined using an enzyme-linked immunosorbent assay, and data analysis was performed using the Wilcoxon rank sum test, or the Kruskal-Wallis test and linear regression. GDF15 levels in the AH (n = 105) of POAG patients were significantly elevated (by 7.4-fold) compared to cataract patients (n = 117). Serum samples obtained from a subgroup of POAG patients (n = 41) also showed a significant increase in GDF15 levels (by 50%) compared to cataract patients. GDF15 levels were elevated in male, female, African American, and Caucasian POAG patients. This study reveals a significant and marked elevation of GDF15 levels in the AH of POAG patients compared to non-glaucoma cataract control patients. Although serum GDF15 levels were also elevated in POAG patients, the magnitude of difference was much smaller relative to that found in the AH.

TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.

BACKGROUND & AIMS: As the composition of the bile acid (BA) pool has a major impact on liver pathophysiology, we studied its regulation by the BA receptor Takeda G protein coupled receptor (TGR5), which promotes hepatoprotection against BA overload. METHODS: Wild-type, total and hepatocyte-specific TGR5-knockout, and TGR5-overexpressing mice were used in: partial (66%) and 89% extended hepatectomies (EHs) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (CA)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied the impact of TGR5 on: BA composition, liver injury, regeneration and survival. We also performed analyses on the gut microbiota (GM) and gallbladder (GB). Liver BA composition was analysed in patients undergoing major hepatectomy. RESULTS: The TGR5-KO hyperhydrophobic BA composition was not directly related to altered BA synthesis, nor to TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and co-housing experiments, respectively. The TGR5-dependent control of GB dilatation was crucial for BA composition, as determined by experiments including RO treatment and/or cholecystectomy. The poor TGR5-KO post-EH survival rate, related to exacerbated peribiliary necrosis and BA overload, was improved by shifting BAs toward a less toxic composition (CT treatment). After either BDL or a CA-enriched diet with or without cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic liver BA composition was correlated with an unfavourable outcome after hepatectomy. CONCLUSIONS: BA composition is crucial for hepatoprotection in mice and humans. We indicate TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions. LAY SUMMARY: Through multiple in vivo experimental approaches in mice, together with a patient study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function, and liver protection. We showed that hepatic bile acid composition is crucial for optimal liver repair, not only in mice, but also in human patients undergoing major hepatectomy.

Synthesis, Characterization, and in vivo Evaluation of a Novel Potent Autotaxin-Inhibitor.

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect.

New Technologies in Clinical Trials in Corneal Diseases and Limbal Stem Cell Deficiency: Review from the European Vision Institute Special Interest Focus Group Meeting.

To discuss and evaluate new technologies for a better diagnosis of corneal diseases and limbal stem cell deficiency, the outcomes of a consensus process within the European Vision Institute (and of a workshop at the University of Cologne) are outlined. Various technologies are presented and analyzed for their potential clinical use also in defining new end points in clinical trials. The disease areas which are discussed comprise dry eye and ocular surface inflammation, imaging, and corneal neovascularization and corneal grafting/stem cell and cell transplantation. The unmet needs in the abovementioned disease areas are discussed, and realistically achievable new technologies for better diagnosis and use in clinical trials are outlined. To sum up, it can be said that there are several new technologies that can improve current diagnostics in the field of ophthalmology in the near future and will have impact on clinical trial end point design.

Tear Proteomic Predictive Biomarker Model for Ocular Graft Versus Host Disease Classification.

Purpose: Diagnosis of ocular graft-versus-host disease (oGVHD) is hampered by a lack of clinically-validated biomarkers. This study aims to predict disease severity on the basis of tear protein expression in mild oGVHD. Methods: Forty-nine patients with and without chronic oGVHD after AHCT were recruited to a cross-sectional observational study. Patients were stratified using NIH guidelines for oGVHD severity: NIH 0 (none; n = 14), NIH 1 (mild; n = 9), NIH 2 (moderate; n = 16), and NIH 3 (severe; n = 10). The proteomic profile of tears was analyzed using liquid chromatography-tandem mass spectrometry. Random forest and penalized logistic regression were used to generate classification and prediction models to stratify patients according to disease severity. Results: Mass spectrometry detected 785 proteins across all samples. A random forest model used to classify patients by disease grade achieved F1-measure values for correct classification of 0.95 (NIH 0), 0.8 (NIH 1), 0.74 (NIH 2), and 0.83 (NIH 3). A penalized logistic regression model was generated by comparing patients without oGVHD and those with mild oGVHD and applied to identify potential biomarkers present early in disease. A panel of 13 discriminant markers achieved significant diagnostic accuracy in identifying patients with moderate-to-severe disease. Conclusions: Our work demonstrates the utility of tear protein biomarkers in classifying oGVHD severity and adds further evidence indicating ocular surface inflammation as a main driver of oGVHD clinical phenotype. Translational Relevance: Expression levels of a 13-marker tear protein panel in AHCT patients with mild oGVHD may predict development of more severe oGVHD clinical phenotypes.

Identification and Preclinical Development of a 2,5,6-Trisubstituted Fluorinated Pyridine Derivative as a Radioligand for the Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

Despite the broad implications of the cannabinoid type 2 receptor (CB2) in neuroinflammatory processes, a suitable CB2-targeted probe is currently lacking in clinical routine. In this work, we synthesized 15 fluorinated pyridine derivatives and tested their binding affinities toward CB2 and CB1. With a sub-nanomolar affinity (Ki for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18-d6 exhibited outstanding in vitro performance characteristics and was radiofluorinated with an average radiochemical yield of 10.6 ± 3.8% (n = 16) and molar activities ranging from 52 to 65 GBq/µmol (radiochemical purity > 99%). [18F]RoSMA-18-d6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [18F]RoSMA-18-d6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [18F]RoSMA-18-d6 is a promising CB2 PET radioligand for clinical translation.

Pharmacological activation of TGR5 promotes intestinal growth via a GLP-2-dependent pathway in mice.

Glucagon-like peptide (GLP)-1 and -2-secreting L cells have been shown to express the bile acid receptor Takeda G protein-receptor-5 (TGR5) and increase secretion upon receptor activation. Previous studies have explored GLP-1 secretion following acute TGR5 activation, but chronic activation and GLP-2 responses have not been characterized. In this study, we aimed to investigate the consequences of pharmacological TGR5 receptor activation on L cell hormone production in vivo using the specific TGR5 agonist RO5527239 and the GLP-2 receptor knockout mouse. Here, we show that 1) TGR5 receptor activation led to increased GLP-1 and GLP-2 content in the colon, which 2) was associated with an increased small intestinal weight that 3) was GLP-2 dependent. Additionally, we report that TGR5-mediated gallbladder filling occurred independently of GLP-2 signaling. In conclusion, we demonstrate that pharmacological TGR5 receptor activation stimulates L cells, triggering GLP-2-dependent intestinal adaption in mice.NEW & NOTEWORTHY Using the specific Takeda G protein-receptor-5 (TGR5) agonist RO5527239 and GLP-2 receptor knockout mice, we show that activation of TGR5 led to the increase in colonic GLP-1 and GLP-2 concomitant with a GLP-2 dependent growth response in the proximal portion of the small intestine.

Highly Selective, Amine-Derived Cannabinoid Receptor 2 Probes.

The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB2 R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood-likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB2 R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB2 R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB1 R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB2 R to date.

TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function.

OBJECTIVE: We explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability. DESIGN: Experiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied. RESULTS: In vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice. CONCLUSION: The BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.

Role of the autotaxin-lysophosphatidic acid axis in glaucoma, aqueous humor drainage and fibrogenic activity.

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1ß and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.

Structure-Activity Relationship Studies of Pyridine-Based Ligands and Identification of a Fluorinated Derivative for Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [11C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a Ki (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [18F]3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/µmol. Specific binding of [18F]3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405 833. Metabolite analysis revealed only intact [18F]3 in the rat brain. [18F]3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.

Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.