John C. R. Randle

Inactivation of Caspase-1 in Rodent Brain : A Novel Anticonvulsive Strategy

Summary:  Purpose: Cytokines and related inflammatory mediators are rapidly synthesized in the brain during seizures. We previously found that intracerebral administration of interleukin‐1 (IL‐1)‐β has proconvulsant effects, whereas its endogenous receptor antagonist (IL‐1Ra) mediates potent anticonvulsant actions in various models of limbic seizures. In this study, we investigated whether seizures can be effectively inhibited by blocking the brain production of IL‐1β, by using selective inhibitors of interleukin‐converting enzyme (ICE/caspase‐1) or through caspase‐1 gene deletion.

IL-Converting Enzyme/Caspase-1 Inhibitor VX-765 Blocks the Hypersensitive Response to an Inflammatory Stimulus in Monocytes from Familial Cold Autoinflammatory Syndrome Patients

Familial cold autoinflammatory syndrome (FCAS) and the related autoinflammatory disorders, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, are characterized by mutations in the CIAS1 gene that encodes cryopyrin, an adaptor protein involved in activation of IL-converting enzyme/caspase-1. Mutations in cryopyrin are hypothesized to result in abnormal secretion of caspase-1-dependent proinflammatory cytokines, IL-1β and IL-18. In this study, we examined cytokine secretion in PBMCs from FCAS patients and found a marked hyperresponsiveness of both IL-1β and IL-18 secretion to LPS stimulation, but no evidence of increased basal secretion of these cytokines, or alterations in basal or stimulated pro-IL-1β levels. VX-765, an orally active IL-converting enzyme/caspase-1 inhibitor, blocked IL-1β secretion with equal potency in LPS-stimulated cells from FCAS and control subjects. These results further link mutations in cryopyrin with abnormal caspase-1 activation, and support the clinical testing of caspase-1 inhibitors such as VX-765 in autoinflammatory disorders.

(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an Orally Available Selective Interleukin (IL)-Converting Enzyme/Caspase-1 Inhibitor, Exhibits Potent Anti-Inflammatory Activities by Inhibiting the Release of IL-1β and IL-18

(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1β and IL-18, but it had little effect on the release of several other cytokines, including IL-1α, tumor necrosis factor-α, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

VX-765, an orally available selective interleukin converting enzyme (ICE)/caspase-1 inhibitor exhibits potent anti-inflammatory activities by inhibiting the release of IL-1b and IL-18

(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1β and IL-18, but it had little effect on the release of several other cytokines, including IL-1α, tumor necrosis factor-α, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

ICE/Caspase-1 inhibitors as novel anti-inflammatory drugs.

In recent years, several strategies that selectively inhibit pro-inflammatory cytokines, have yielded effective protein-based therapies for inflammatory disorders, validating the therapeutic hypothesis that intervention in cytokine signalling can provide clinical benefit. However, these protein-based products must be administered by injection, a constraint associated with inconvenience, adverse effects and expense for patients, caregivers and insurers. Besides interfering with the effects of cytokines such as TNF-α or IL-1β that have already been produced, inhibition of pro-inflammatory cytokine production or signalling with low-molecular weight orally-active drugs would combine the convenience of conventional pharmaceuticals with the focused efficacy of the protein therapies. Reducing IL-1β and IL-18 production by inhibition of IL-1β converting enzyme (ICE, caspase-1) is one promising strategy because of the key roles of these cytokines in many inflammatory diseases. Pralnacasan, the first orally available, potent and selective ICE inhibitor to enter clinical trials, is currently under investigation in rheumatoid arthritis.

Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor‐mediated IL‐1β release

We investigated the consequences of transient application of specific stimuli mimicking inflammation to hippocampal tissue on microglia activation and neuronal cell vulnerability to a subsequent excitotoxic insult. Two‐week‐old organotypic hippocampal slice cultures, from 7‐day‐old C57BL/6 donor mice, were exposed for 3 h to lipopolysaccharide (LPS; 10 ng/mL) followed by 3 h co‐incubation with 1 mM ATP, or 100 μM 2′3′‐O‐(4‐benzoyl‐benzoyl) adenosine 5′‐triphosphate triethylammonium, a selective P2X7 receptor agonist. These treatments in combination, but not individually, induced a pronounced activation and apoptotic‐like death of macrophage antigen‐1 (MAC‐1)‐positive microglia associated with a massive release of interleukin (IL)‐1β exceeding that induced by LPS alone. Antagonists of P2X7 receptors prevented these effects. Transient pre‐exposure of slice cultures to a combination of LPS and P2X7 receptor agonists, but not either one or the other alone, significantly exacerbated CA3 pyramidal cell loss induced by subsequent 12 h exposure to 8 μM α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propinate (AMPA). Potentiation of AMPA toxicity was prevented when IL‐1β production or its receptor signaling were blocked by an inhibitor of interleukin‐converting‐enzyme or IL‐1 receptor antagonist during application of LPS + ATP. The same treatments did not prevent microglia apoptosis‐like death. These findings show that transient exposure to specific pro‐inflammatory stimuli in brain tissue can prime neuronal susceptibility to a subsequent excitotoxic insult. P2X7 receptor stimulation, and the consequent IL‐1β release, is mandatory for exacerbation of neuronal loss. These mechanisms may contribute to determine cell death/survival in acute and chronic neurodegenerative conditions associated with inflammatory events.

A multi-variant, viral dynamic model of genotype 1 HCV to assess the in vivo evolution of protease-inhibitor resistant variants.

Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.

Impaired release of IL‐18 from fibroblast‐like synoviocytes activated with protein I/II, a pathogen‐associated molecular pattern from oral streptococci, results from defective translation of IL‐18 mRNA in pro‐IL‐18

Proinflammatory cytokines such as tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and IL‐18 are key mediators of joint inflammation during rheumatoid arthritis (RA). This chronic inflammation may result from a non‐specific innate immune response that could be triggered by a wide variety of microorganisms, because numerous bacterial fragments have been identified in the joints of RA patients. As we have demonstrated previously that protein I/II, a pathogen‐associated molecular pattern (PAMP) from oral streptococci, triggers IL‐6 and IL‐8 gene expression and release from either THP‐1 cells or fibroblast‐like synoviocytes (FLSs), we next explored the capacity of protein I/II to induce the synthesis and release of IL‐18 in THP‐1 cells and in FLSs isolated from either RA or osteoarthritis (OA) patients. We demonstrate that protein I/II induced IL‐18 mRNA in both THP‐1 cells and FLSs but, in contrast to THP‐1 cells, gene expression was not associated with the synthesis of the corresponding protein in FLSs. Furthermore, our studies revealed that FLSs did not express the biologically inactive precursor, pro‐IL‐18, in response to protein I/II. Using actinomycin D, we also showed that IL‐18 mRNA is unstable in FLSs. Taken together, these data indicate that lack of IL‐18 release from activated FLSs results from a defect in translation of IL‐18 mRNA into pro‐IL‐18 because of rapid degradation of IL‐18 mRNA.