Novel Effects of Combination Therapy Through Inhibition of Caspase-1/Gasdermin D Induced-Pyroptosis in Lupus Nephritis.

Objectives: Combination therapy with mycophenolate mofetil, tacrolimus and steroids are effective in achieving complete remission in lupus nephritis (LN). Combination therapy uniquely downregulated caspase-1 compared with monotherapies, which can cleave gasdermin D (GSDMD) and was recently identified as the pyroptosis executioner. We therefore investigated whether combination therapy enabled the suppression of caspase-1/GSDMD-mediated pyroptosis in LN. Methods: Expression and activation of GSDMD were detected in kidney specimens of the human and mouse with LN using immunohistochemical staining and immunoblotting. Primary podocytes isolated from MRL/lpr mice were incubated with LPS+ATP, and pretreated with monotherapy or combination therapy. Inhibition of caspase-1/GSDMD-induced pyroptosis by combination therapy were assessed in MRL/lpr mice and human specimens. Pyroptosis was examined using a FAM caspase-1 kit and flow cytometry. The correlation between pyroptosis in peripheral blood and the systemic lupus erythematosus disease activity index (SLEDAI) was analyzed. Results: Kidney tissue specimens from LN patients and mice exhibited greatly increased expression levels and cleavage of GSDMD. In cultured podocytes, combination treatment significantly suppressed the activation of NLRP3 and caspase-1 and reduced GSDMD N-terminal levels. Combination therapy repressed disease progression through inhibition of caspase-1/GSDMD-mediated pyroptosis in both humans and MRL/lpr mice. Caspase-1/PI positive cell numbers in peripheral blood were positively correlated with SLE-DAI. LN patients with complete remission and partial remission had remarkably reduced caspase-1/PI positive cell numbers compared to baseline. Ac-FLTD-CMK, a GSDMD-derived inhibitor, prevented the development of LN. Conclusion: Combination therapy suppressed caspase-1/GSDMD-mediated pyroptosis in vitro and in vivo and reduced disease progression.

Emricasan to prevent new decompensation in patients with NASH-related decompensated cirrhosis.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis is a leading cause of end-stage liver disease. Hepatic steatosis and lipotoxicity cause chronic necroinflammation and direct hepatocellular injury resulting in cirrhosis, end-stage liver disease and hepatocellular carcinoma. Emricasan is a pan-caspase inhibitor that inhibits excessive apoptosis and inflammation; it has also been shown to decrease portal pressure and improve synthetic function in mice with carbon tetrachloride-induced cirrhosis. METHODS: This double-blind, placebo-controlled study randomized 217 individuals with decompensated NASH cirrhosis 1:1:1 to emricasan (5 mg or 25 mg) or placebo. Patients were stratified by decompensation status and baseline model for end-stage liver disease-sodium (MELD-Na) score. The primary endpoint comprised all-cause mortality, a new decompensation event (new or recurrent variceal hemorrhage, new ascites requiring diuretics, new unprecipitated hepatic encephalopathy ≥grade 2, hepatorenal syndrome, spontaneous bacterial peritonitis), or an increase in MELD-Na score ≥4 points. RESULTS: There was no difference in event rates between either of the emricasan treatment groups and placebo, with hazard ratios of 1.02 (95% CI 0.59-1.77; p = 0.94) and 1.28 (95% CI 0.75-2.21; p = 0.37) for 5 mg and 25 mg of emricasan, respectively. MELD-Na score progression was the most common outcome. There was no significant effect of emricasan treatment on MELD-Na score, international normalized ratio, total serum bilirubin, albumin level or Child-Pugh score. Emricasan was generally safe and well-tolerated. CONCLUSIONS: Emricasan was safe but ineffective for the treatment of decompensated NASH cirrhosis. However, this study may guide the design and conduct of future clinical trials in decompensated NASH cirrhosis. LAY SUMMARY: Patients with decompensated cirrhosis related to non-alcoholic steatohepatitis are at high risk of additional decompensation events and death. Post hoc analyses in previous pilot studies suggested that emricasan might improve portal hypertension and liver function. In this larger randomized study, emricasan did not decrease the number of decompensation events or improve liver function in patients with a history of decompensated cirrhosis related to non-alcoholic steatohepatitis. CLINICALTRIALS. GOV IDENTIFIER: NCT03205345.

Caspase-1 inhibition prevents neuronal death by targeting the canonical inflammasome pathway of pyroptosis in a murine model of cerebral ischemia.

AIMS: The involvement of pyroptosis in ischemic stroke remains to be established. Therefore, we used the specific pyroptosis inhibitor Vx765 as an experimental intervention target in a murine model of stroke. METHODS: A total of 564 C57BL/6 mice were subjected to photothrombotic procedures and treated via gavage with Vx765 at 1-hour post-ischemia. We subsequently assessed the expression of Gasdermin D (GSDMD), inflammasomes, caspase-1, and interleukin-1ß (IL-1ß) using immunofluorescence (IF) and Western blot (WB) analyses. We also examined ultrastructural changes of cortical neurons with transmission electron microscopy (TEM) and measured infarct volumes dynamically by magnetic resonance imaging (MRI). Moreover, we evaluated the neurologic deficits by modified neurological severity scores, the rotarod test, and Treadscan. RESULTS: Elevated expression of GSDMD and GSDMD p30, the pore-forming subunit, was evident in the peri-ischemic region on days one and three post-ischemia. The neuronal plasma, nuclear, and mitochondrial membranes showed ultrastructural damage at day three post-stroke. Elevated expression of inflammasomes, caspase-1, and IL-1ß was also present on days one and three post-injury. There were significant differences between Vx765-treated and vehicle groups in mean infarct volumes (14.36 vs 21.52 mm3 ; 12.34 vs 18.56 mm3 ; 4.13 vs 10.06 mm3 ; P < .05 at day one, three, and seven post-surgery, respectively). Mice treated with Vx765 showed better motor recovery as assessed by serial behavior tests and had better neuronal survival, which was attributable to pyroptosis inhibition, as illustrated by downregulated expression of the effector protein GSDMD, inflammasomes, caspase-1, and IL-1ß. Besides, treatment with Vx765 preserved neuronal membrane structures after the ischemic injury. CONCLUSIONS: Pyroptosis emerges as an important pathway for neuronal death in an acute ischemic stroke. Vx765, a low molecular weight drug that has proven safe in clinical epilepsy trials, has potential therapeutic value for cerebral ischemia by targeting the canonical inflammasome pathway of pyroptosis.

Neuroprotective Effects of Necrostatin-1 Against Oxidative Stress-Induced Cell Damage: an Involvement of Cathepsin D Inhibition.

Necroptosis, a recently discovered form of non-apoptotic programmed cell death, can be implicated in many pathological conditions including neuronal cell death. Moreover, an inhibition of this process by necrostatin-1 (Nec-1) has been shown to be neuroprotective in in vitro and in vivo models of cerebral ischemia. However, the involvement of this type of cell death in oxidative stress-induced neuronal cell damage is less recognized. Therefore, we tested the effects of Nec-1, an inhibitor of necroptosis, in the model of hydrogen peroxide (H2O2)-induced cell damage in human neuroblastoma SH-SY5Y and murine hippocampal HT-22 cell lines. The data showed that Nec-1 (10-40 µM) attenuated the cell death induced by H2O2 in undifferentiated (UN-) and neuronal differentiated (RA-) SH-SY5Y cells with a higher efficacy in the former cell type. Moreover, Nec-1 partially reduced cell damage induced by 6-hydroxydopamine in UN- and RA-SH-SY5Y cells. The protective effect of Nec-1 was of similar magnitude as the effect of a caspase-3 inhibitor in both cell phenotypes and this effect were not potentiated after combined treatment. Furthermore, the non-specific apoptosis and necroptosis inhibitor curcumin augmented the beneficial effect of Nec-1 against H2O2-evoked cell damage albeit only in RA-SH-SY5Y cells. Next, it was found that the mechanisms of neuroprotective effect of Nec-1 against H2O2-induced cell damage in SH-SY5Y cells involved the inhibition of lysosomal protease, cathepsin D, but not caspase-3 or calpain activities. In HT-22 cells, Nec-1 was protective in two models of oxidative stress (H2O2 and glutamate) and that effect was blocked by a caspase inhibitor. Our data showed neuroprotective effects of the necroptosis inhibitor, Nec-1, against oxidative stress-induced cell damage and pointed to involvement of cathepsin D inhibition in the mechanism of its action. Moreover, a cell type-specific interplay between necroptosis and apoptosis has been demonstrated.

A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by hepatocyte steatosis, ballooning, and lobular inflammation which may lead to fibrosis. Lipotoxicity activates caspases, which cause apoptosis and inflammatory cytokine (IL-1ß and IL-18) production. Emricasan is a pan-caspase inhibitor that decreases serum aminotransferases and caspase activation in patients with NASH. This study postulated that 72 weeks of emricasan treatment would improve liver fibrosis without worsening of NASH. METHODS: In this double-blind, placebo-controlled study 318 patients were randomized 1:1:1 to twice-daily treatment with emricasan (5 mg or 50 mg) or matching placebo for 72 weeks. Patients had definite NASH and NASH CRN fibrosis stage F1-F3, as determined by a central reader, on a liver biopsy obtained within 6 months of randomization. RESULTS: Emricasan treatment did not achieve the primary objective of fibrosis improvement without worsening of NASH (emricasan 5 mg: 11.2%; emricasan 50 mg: 12.3%; placebo: 19.0%; odds ratios vs. placebo 0.530 and 0.588, with p = 0.972 and 0.972, respectively) or the secondary objective of NASH resolution without worsening of fibrosis (emricasan 5 mg: 3.7%; emricasan 50 mg: 6.6%; placebo: 10.5%; odds ratios vs. placebo 0.334 and 0.613, with p = 0.070 and 0.335, respectively). In the small subset of patients with consistent normalization of serum alanine aminotransferase over 72 weeks, emricasan may have improved histologic outcomes. CONCLUSIONS: Emricasan treatment did not improve liver histology in patients with NASH fibrosis despite target engagement and may have worsened fibrosis and ballooning. Caspase inhibition lowered serum alanine aminotransferase in the short-term but may have directed cells to alternative mechanisms of cell death, resulting in more liver fibrosis and hepatocyte ballooning. CLINICAL TRIAL NUMBER: Clinical Trials.gov #NCT02686762. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is characterized by fat accumulation in liver cells, which leads to inflammation and fibrosis. Emricasan was previously shown to inhibit some of the liver enzymes which lead to liver inflammation and fibrosis. In this study, emricasan did not improve liver inflammation or fibrosis in patients with NASH and pre-existing liver fibrosis.

Randomized placebo-controlled trial of emricasan for non-alcoholic steatohepatitis-related cirrhosis with severe portal hypertension.

BACKGROUND & AIMS: Emricasan, an oral pan-caspase inhibitor, decreased portal pressure in experimental cirrhosis and in an open-label study in patients with cirrhosis and severe portal hypertension, defined as a hepatic venous pressure gradient (HVPG) ≥12 mmHg. We aimed to confirm these results in a placebo-controlled study in patients with non-alcoholic steatohepatitis (NASH)-related cirrhosis. METHODS: We performed a multicenter double-blinded study, randomizing 263 patients with NASH-related cirrhosis and baseline HVPG ≥12 mmHg to twice daily oral emricasan 5 mg, 25 mg, 50 mg or placebo in a 1:1:1:1 ratio for up to 48 weeks. The primary endpoint was change in HVPG (ΔHVPG) at week 24. Secondary endpoints were changes in biomarkers (aminotransferases, caspases, cytokeratins) and development of liver-related outcomes. RESULTS: There were no significant differences in ΔHVPG for any emricasan dose vs. placebo (-0.21, -0.45, -0.58 mmHg, respectively) adjusted for baseline HVPG, compensation status, and non-selective beta-blocker use. Compensated patients (n = 201 [76%]) tended to have a greater decrease in HVPG (emricasan all vs. placebo, p = 0.06), the decrease being greater in those with higher baseline HVPG (p = 0.018), with a significant interaction between baseline HVPG (continuous, p = 0.024; dichotomous at 16 mmHg [median], p = 0.013) and treatment. Biomarkers decreased significantly with emricasan at week 24 but returned to baseline levels by week 48. New or worsening decompensating events (∼10% over median exposure of 337 days), progression in model for end-stage liver disease and Child-Pugh scores, and treatment-emergent adverse events were similar among treatment groups. CONCLUSIONS: Despite a reduction in biomarkers indicating target engagement, emricasan was not associated with improvement in HVPG or clinical outcomes in patients with NASH-related cirrhosis and severe portal hypertension. Compensated patients with higher baseline HVPG had evidence of a small treatment effect. Emricasan treatment appeared safe and well-tolerated. LAY SUMMARY: Cirrhosis (scarring of the liver) is the main consequence of non-alcoholic steatohepatitis (NASH). Cirrhosis leads to high pressure in the portal vein which accounts for most of the complications of cirrhosis. Reducing portal pressure is beneficial in patients with cirrhosis. We studied the possibility that emricasan, a drug that improves inflammation and scarring in the liver, would reduce portal pressure in patients with NASH-related cirrhosis and severe portal hypertension. Our results in a large, prospective, double-blind study could not demonstrate a beneficial effect of emricasan in these patients. CLINICAL TRIAL NUMBER: Clinical Trials.gov #NCT02960204.

Pan-caspase inhibitor F573 mitigates liver ischemia reperfusion injury in a murine model.

BACKGROUND: Liver ischemia reperfusion injury (IRI) remains a challenge in liver transplantation. A number of compounds have previously demonstrated efficacy in mitigating IRI. Herein, we applied three specific additive strategies to a mouse IRI screening model to determine their relative potencies in reducing such injury, with a view to future testing in a large animal and clinical ex situ normothermic perfusion setting: 1) F573, a pan-caspase inhibitor, 2) anti-inflammatory anakinra and etanrecept and 3) BMX-001, a mimetic of superoxide dismutase. METHODS: A non-lethal liver ischemia model in mice was used. Additives in the treatment groups were given at fixed time points before induction of injury, compared to a vehicle group that received no therapeutic treatment. Mice were recovered for 6 hours following the ischemic insult, at which point blood and tissue samples were obtained. Plasma was processed for transaminase levels. Whole liver tissue samples were processed for histology, markers of apoptosis, oxidative stress, and cytokine levels. RESULTS: In an in vivo murine IRI model, the F573 treatment group demonstrated statistically lower alanine aminotransferase (ALT) levels (p = 0.01), less evidence of apoptosis (p = 0.03), and lower cytokine levels compared to vehicle. The etanercept with anakinra treatment group demonstrated significantly lower cytokine levels. The BMX-001 group demonstrated significantly decreased apoptosis (p = 0.01) evident on TUNEL staining. CONCLUSIONS: The administration of pan-caspase inhibitor F573 in a murine in vivo model likely mitigates liver IRI based on decreased markers of cellular injury, decreased evidence of apoptosis, and improved cytokine profiles. Anakinra with etanercept, and BMX-001 did not demonstrate convincing efficacy at reducing IRI in this model, and likely need further optimization. The positive findings set rational groundwork for future translational studies of applying F573 during normothermic ex situ liver perfusion, with the aim of improving the quality of marginal grafts.

Bivalent SMAC Mimetics for Treating Cancer by Antagonizing Inhibitor of Apoptosis Proteins.

Inhibitors of apoptosis proteins (IAPs) inhibit caspase activity, allowing various cancers to reduce programmed cell death (apoptosis) and resist drug treatment. The second mitochondrial-derived activator of caspases (SMAC) protein is an endogenous IAP antagonist, which can be considered as a potential anticancer therapy. Small-molecule SMAC mimetics based on the Ala-Val-Pro-Ile motif have been validated as potent IAP antagonists. In particular, most bivalent SMAC mimetics, which target both the baculovirus IAP repeat 2 (BIR2) and BIR3 domains in X-linked IAP (XIAP), antagonize IAPs better than the corresponding monovalent mimetics. Here we focus on strategies for designing bivalent small-molecule SMAC mimetics and progress in using them to antagonize IAPs. We also consider their clinical potential. Our discussion will hopefully help guide further study of these interesting mimetics.

Dietary Compounds for Targeting Prostate Cancer.

Prostate cancer is the third most common cancer worldwide, and the burden of the disease is increased. Although several chemotherapies have been used, concerns about the side effects have been raised, and development of alternative therapy is inevitable. The purpose of this study is to prove the efficacy of dietary substances as a source of anti-tumor drugs by identifying their carcinostatic activities in specific pathological mechanisms. According to numerous studies, dietary substances were effective through following five mechanisms; apoptosis, anti-angiogenesis, anti-metastasis, microRNA (miRNA) regulation, and anti-multi-drug-resistance (MDR). About seventy dietary substances showed the anti-prostate cancer activities. Most of the substances induced the apoptosis, especially acting on the mechanism of caspase and poly adenosine diphosphate ribose polymerase (PARP) cleavage. These findings support that dietary compounds have potential to be used as anticancer agents as both food supplements and direct clinical drugs.

Rhein Augments Antiproliferative Effects of Atezolizumab Based on Breast Cancer (4T1) Regression.

Rhein, an anthraquinone extracted from rhubarb, is used in traditional Chinese medicine for diuresis, diarrhoea, inflammation, and immune regulation. Atezolizumab, a programmed cell death ligand 1 monoclonal antibody, is mainly used to treat bladder cancer and non-small cell lung cancer unresponsive to chemotherapy. We explored the effects of rhein and atezolizumab in combination on breast cancer. Mice with established 4T1 breast cancer xenografts were administered rhein (10 mg/kg) and atezolizumab (10 mg/kg), alone and in combination, and the effects on tumour growth were evaluated. The proportion of CD8+ T cells in the spleen and tumour tissue, the levels of TNF-α, and interleukin-6 in serum as well as the mRNA levels of apoptotic factors (caspase-3, caspase-8, caspase-9, and Bax/Bcl-2) were also evaluated. All of the treatment groups had inhibitory effects on the xenograft tumour growth, with results that were significantly different from those in the control group. In addition, the proportion of CD8+ T cells in the spleen and tumour was significantly increased in the combination therapy group and was significantly different from the other treatment groups. The serum levels of TNF-α and IL-6 were significantly increased in the rhein and combination therapy groups. Finally, the levels of various apoptotic factors in tumour tissues were significantly higher in the combination treatment group than those in the other groups. Administration of rhein, atezolizumab, or their combination all had therapeutic effects on 4T1 breast cancer xenografts in mice, with the combination treatment having stronger effects.

Mesenchymal Stem Cell Capping on ECM-Anchored Caspase Inhibitor-Loaded PLGA Microspheres for Intraperitoneal Injection in DSS-Induced Murine Colitis.

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)-coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single-step method for ECM functionalization on poly(lactic-co-glycolic acid) (PLGA) microspheres using a novel compound, dopamine-conjugated poly(ethylene-alt-maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM-functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad-spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC-microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4+ T cells in colon-draining mesenteric lymph nodes. Therefore, drug-loaded ECM-coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.

Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation.

PURPOSE: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation. METHODS: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 µM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 µM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks. RESULTS: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue. CONCLUSIONS: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.

Caspase 1/11 Deficiency or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in Mice.

Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.

Pyroptosis, a novel mechanism implicated in cataracts.

An understanding of the mechanism of cataract formation may reduce its burden on medical care worldwide. It is established that pyroptosis is associated with oxidative stress, one of the causes of cataracts, and may provide novel therapeutic targets for the treatment of cataracts. The present study therefore investigated the role of pyroptosis in cataract formation. SRA01/04 human lens epithelium cells (HLECs) were treated with H2O2 and cell viability was assessed by an MTT assay. Pyroptosis in HLECs was examined by TUNEL staining, and the expression of caspase­1 and interleukin (IL)­1ß was determined using reverse transcription­quantitative polymerase chain reaction (RT­qPCR), western blot analysis and immunostaining. A caspase­1 inhibitor was used to investigate the effects of caspase­1 downregulation. In addition, the expression of caspase­1 and IL­1ß in lens anterior capsule tissue samples from patients with cataracts and normal controls was also analyzed by immunostaining, RT­qPCR and western blot analysis. The results demonstrated that pyroptosis in H2O2­treated HLECs, and the mRNA and protein expression of caspase­1 and IL­1ß, was significantly increased compared with control cells. Furthermore, caspase­1 and IL­1ß expression was significantly increased in cataract tissue samples compared with normal controls. When HLECs were cotreated with a caspase­1 inhibitor and 100 µM H2O2, caspase­1 and IL­1ß expression were decreased compared with the 100 µM H2O2­only group. In conclusion, the results of the present study demonstrate that pyroptosis may have a role in cataract formation, and the caspase­1 and IL­1ß pathways may be involved in this pathological process. Pyroptosis appears to be a promising target in the prevention of cataract formation.

G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection.

There is currently no efficient method available for the delivery of full length functional proteins into the cytoplasm of retinal cells in vivo. Historically, the most successful approach for the treatment of retinal diseases has been intravitreal injection of antibodies or recombinant proteins, but this approach is not yet utilized for the delivery of proteins that require intracellular access for a therapeutic effect. Here we describe a platform for the delivery of functional proteins into ganglion cells, photoreceptors and retinal pigment epithelium via intravitreal injection. A nucleolin binding aptamer, AS1411, was biotinylated and complexed with traptavidin and utilized as a platform for the delivery of GFP or X-linked inhibitor of apoptosis (XIAP) proteins by intravitreal injection in BALB/c mice. Retinal sections were analyzed for uptake of proteins in the retina. Apoptosis was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Retinas were harvested for analysis of TUNEL and caspase 3/7 activity. Intravitreal injection of AS1411-directed GFP or XIAP complexes enabled delivery of these proteins into ganglion cells, photoreceptors and retinal pigment epithelium in vivo. AS1411-XIAP complexes conferred significant protection to cells in the outer and inner nuclear layers following NMDA induced apoptosis. A concomitant decrease in activity of Caspase 3/7 was observed in eyes injected with the AS1411-XIAP complex. In conclusion, AS1411 can be used as a platform for the delivery of therapeutic proteins into retinal cells. This approach can potentially be utilized to introduce a large variety of therapeutically relevant proteins that are previously well characterized to maintain the structural integrity and function of retina, thus, preventing vision loss due to ocular trauma or inherited retinal degeneration.

Rational Drug Design of Topically Administered Caspase 1 Inhibitors for the Treatment of Inflammatory Acne.

The use of an interleukin ß antibody is currently being investigated in the clinic for the treatment of acne, a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1ß into active IL-1ß, caspase-1, could be an alternative small molecule approach. This report describes the discovery of uracil 20, a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration, topical administration challenges such as phototoxicity, organic and aqueous solubility, chemical stability in solution, and skin metabolic stability are discussed and successfully resolved.

Caspase inhibitors: a review of recently patented compounds (2013-2015).

INTRODUCTION: Although many caspase inhibitors have been patented, caspase inhibitors have not entered the market due to their toxicity and poor pharmacokinetic profile. AREAS COVERED: In this article, we review patents (2013-2015) for peptide and non-peptide caspase inhibitors and their compositions. EXPERT OPINION: Noteworthy patents include a peptidic caspase-2 inhibitor for nasal administration and a peptidomimetic caspase-6 inhibitor that can be administered via several routes for the treatment of neurodegenerative diseases. Furthermore, caspase-1 inhibitors for contact dermatitis and inflammation, cardiovascular diseases, and liver diseases and a caspase-3 inhibitor for cerebral stroke have been patented. Of particular interest is the novel use of tyrosine kinase inhibitors (sunitinib and its derivatives) for the prevention and treatment of age-related ocular diseases via inhibition of the caspase-3, dual-leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) pathways. However, for effective clinical application of caspase inhibitors, novel peptidic and nonpeptidic caspase inhibitors with lower toxicity and improved efficacy should be developed via structural modifications, and further animal studies and preclinical and clinical trials are needed. In addition, the poor pharmacokinetic properties of classic caspase inhibitors may be improved by using advanced drug delivery systems that employ liposomes, polymers, and nanoparticles through effective administration routes.

Der f1 induces pyroptosis in human bronchial epithelia via the NLRP3 inflammasome.

Damage to the bronchial epithelium leads to persistent inflammation and airway remodelling in various respiratory diseases, such as asthma and chronic obstructive pulmonary disease. To date, the mechanisms underlying bronchial epithelial cell damage and death by common allergens remain largely unknown. The aim of the present study was to investigate Der f1, an allergen of Dermatophagoides farinae, which may result in the death of human bronchial epithelial cells (HBECs). Der f1 induces BECs to undergo the inflammatory cell death referred to as pyroptosis, induced by increasing lactate dehydrogenase release and propidium iodide penetration. Stimulation by Der f1 enhances interleukin (IL)­1ß cleavage and release, which is associated with caspase­1 activation. In addition, the NOD­like receptor family pyrin domain­containing 3 (NLRP3), is required for the activation of caspase­1 through increasing the formation of the inflammasome complex. Consistent with these findings, pre­treatment of HBECs with a caspase­1 inhibitor, or silencing of NLRP3 by siRNA transfection, reduced Der f1­mediated IL­1ß and pyroptosis. Therefore, the common allergen Der f1 was not only found to induce allergy, but also led to pyroptosis and IL­1ß secretion via the NLRP3­caspase­1 inflammasome in HBECs. This newly identified connection of the Der f1 allergen with BEC damage and inflammation may play an important role in the pathogenesis of asthma.

Anti-Inflammatory Role of Langerhans Cells and Apoptotic Keratinocytes in Ultraviolet-B-Induced Cutaneous Inflammation.

UV radiation, particularly UVB, is the major risk factor for the induction of skin cancer, and it induces skin inflammation and immunosuppression. Although reports documented that Langerhans cells (LCs) play various roles in photobiology, little is known about whether they contribute to UVB-induced cutaneous inflammation. Recently, the anti-inflammatory effect of apoptotic cells was noted. This study focuses on the roles of LCs and apoptotic cells in UVB-induced cutaneous inflammation. We show that LCs are essential for resolution of UVB-induced cutaneous inflammation. Administration of quinolyl-valyl-O-methylaspartyl-[2,6-difluophenoxy]-methyl ketone, a broad-spectrum caspase inhibitor with potent antiapoptotic properties, inhibited the formation of UVB-induced apoptotic cells and aggravated UVB-induced cutaneous inflammation in wild-type mice. In contrast, exacerbation of UVB-induced cutaneous inflammation following quinolyl-valyl-O-methylaspartyl-[2,6-difluophenoxy]-methyl ketone administration was not observed in LC-depleted mice. These results suggest that the interaction between LCs and apoptotic cells is critical for resolution of UVB-induced cutaneous inflammation. Interestingly, UVB-induced apoptotic keratinocytes were increased in LC-depleted mice. In addition, we revealed that UVB-induced apoptotic keratinocytes were phagocytosed by LCs ex vivo and that prolongation of UVB-induced cutaneous inflammation following treatment with Cytochalasin D, an inhibitor of phagocytosis, was partially attenuated in LC-depleted mice. Collectively, our findings demonstrate that the interaction between LCs and apoptotic cells, possibly via LC-mediated phagocytosis of apoptotic keratinocytes, has an essential anti-inflammatory role in the resolution of UVB-induced cutaneous inflammation.

A Plant-Derived Nucleic Acid Reconciles Type I IFN and a Pyroptotic-like Event in Immunity against Respiratory Viruses.

Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1ß signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1ß and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options.