Soluble Inhibitor Research Articles

Soluble epoxide hydrolase inhibitors counteract microglia pro-inflammatory response induced by monomeric C-reactive protein

Soluble epoxide hydrolase inhibitors counteract microglia pro-inflammatory response induced by monomeric C-reactive protein

Polymer-prodrug conjugates as candidates for degradable, long-acting implants, releasing the water-soluble nucleoside reverse-transcriptase inhibitor emtricitabine.

Circulating, soluble polymer-drug conjugates have been utilised for many years to aid the delivery of sensitive, poorly-soluble or cytotoxic drugs, prolong circulation times or minimise side effects. Long-acting therapeutics are increasing in their healthcare importance, with intramuscular and subcutaneous administration of liquid formulations being most common. Degradable implants also offer opportunities and the use of polymer-prodrug conjugates as implant materials has not been widely reported in this context. Here, the potential for polymer-prodrug conjugates of the water soluble nucleoside reverse transciption inhibitor emtricitabine (FTC) is studied. A novel diol monomer scaffold, allowing variation of prodrug substitution, has been used to form polyesters and polycarbonates by step-growth polymerisation. Materials have been screened for physical properties that enable implant formation, studied for drug release to provide mechanistic insights, and tunable prolonged release of FTC has been demonstrated over a period of at least two weeks under relevant physiological conditions.

Detection of Free Bioactive IL-18 and IL-18BP in Inflammatory Disorders.

The interleukin (IL)-18 cytokine plays an important driver role in a range of autoimmune and inflammatory diseases, as well as cancer. IL-18 is a potent inducer of interferon gamma (IFN-γ), and the bioactivity of IL-18 is regulated by its natural soluble inhibitor, IL-18-binding protein (IL-18BP), which is present at high concentrations in the circulation. Many cell types have been described to secrete IL-18BP, constitutively or under the influence of IFN-γ, thus generating a negative feedback loop for IL-18. Therefore, solely measuring total IL-18 protein levels does not allow to evaluate its biological activity, especially in the context of systemic inflammatory diseases or other circumstances where IL-18BP is present (e.g., samples containing plasma, cells constitutively expressing IL-18BP). Considering there is a critical need to accurately measure the protein levels of both mature, biologically active IL-18 and IL-18BP as biomarkers of disease activity in patients and also stratification for potential anti-IL-18 therapy, in this chapter we provide the latest techniques to measure mature, free, and bioactive IL-18 and IL-18BP in different samples.

Platelet-Rich Plasma Contains High Concentrations of DKK1, A Potent Inhibitor of Wnt Signaling that Limits Bone Regeneration and Hair Growth

Platelet-rich plasma (PRP) is a popular autologous therapy in the tissue regenerative technology space based on its enriched milieu of platelet-released growth factors (GFs), including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). PRP is frequently used for hair restoration, wound healing, tendon and ligament repairs, and other soft-tissue settings, but PRP did not fulfill its original promise for enhancing bone grafts and bone augmentation in dentistry or orthopedics because it does not consistently promote osteogenesis. The effects of PRP on bone regeneration are often described as controversial, contradictory, and confusing, including evidence that greater platelet enrichment often leads to lesser osteogenic responses. The latter paradox suggests that limited PRP efficacy in bone regeneration may relate to platelet-derived factors that inhibit osteoblasts rather than inadequate GF concentrations. One candidate platelet-derived anti-osteogenic factor is Dickkopf-1 (DKK1), a potent soluble inhibitor of Wnt signaling that limits bone regeneration after bone damage. Recognizing that platelets store DKK1 in their alpha granules and rapidly release it upon platelet activation, we evaluated levels of DKK1, PDGF, and VEGF in human serum, plasma, platelet-poor plasma (PPP), and PRP. Sclerostin, another inhibitor of Wnt signaling that limits bone formation in undamaged bone, was also evaluated because platelets are not known to secrete sclerostin. We show that DKK1, PDGF, and VEGF concentrations are much higher in serum versus plasma, probably due to platelet activation during serum preparation. DKK1, PDGF, and VEGF concentrations were also higher in PRP versus plasma, and in plasma versus PPP. Activation of plasma or PRP with thrombin resulted in marked increases in DKK1, PDGF, and VEGF concentrations, with DKK1 levels in activated PRP being over 40-fold higher than the minimum concentration reported to inhibit osteogenesis in cell culture models. Conversely, sclerostin levels in plasma and PRP were unaffected by platelet activation and were significantly lower in serum versus standard plasma, providing new evidence that platelets do not secrete sclerostin. These results suggest that the beneficial effects of high GF levels in PRP may be limited by the presence of high levels of DKK1 released from the same platelet alpha granules.

Molecular docking and ADMET studies of soluble epoxide hydrolase inhibitors from the leaves of Paederia foetida L.

Cardiovascular diseases are one of the leading cause of mortality and morbidity worldwide. Although many efforts have been made in the drug discovery and development process through decades, the number of approved drugs has been declining. In the recent years, soluble epoxide hydrolase (sEH) has been considered as promising target for drug development since inhibiting sEH function would prevent the formation of arterial thrombosis. Paederia foetida L. is a folk medicine distributed commonly in Vietnam which is well known for its uses in the treatment of various diseases. This study conducted in silico assessment of 21 isolated compounds from leaves of Paederia foetida L. against sEH enzyme for potential inhibition activity. Obtained results demonstrated that compound 4 and 9 could be potent, safe and novel inhibitors based on docking conformation and druglikeness properties.

Interleukin-18 binding protein regulates the apoptosis and necroptosis of fibroblast-like synoviocytes and chondrocytes in rheumatoid arthritis.

Interleukin (IL)-18 plays a pro-inflammatory role in rheumatoid arthritis (RA), and its soluble inhibitor IL-18 binding protein (IL-18BP) has a potential therapeutic role. We investigated the role of IL-18BP on the joint destruction process of RA by accessing the effects of IL-18BP on fibroblast-like synoviocytes (FLSs) and chondrocytes. Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were cultured under T cell proliferative conditions with 10, 50, or 100 ng/mL of IL-18BP. After three days of culture, flow cytometry for CD4+ T cells was performed using various IL-18BP concentrations. The apoptosis and necroptosis of FLSs and chondrocytes were measured by flow cytometry using annexin V and propidium iodide (PI) and western blot under TNF-α stimulation with IL-18BP (10, 50, and 100 ng/mL). Differentiation of CD4+ IL-17A+ and CD4+ IL-4+ cells decreased and that of CD4+ CD25high Foxp3+ and CD4+ interferon (IFN)-γ+ cells increased on addition of IL-18BP to cultured RA patient-driven PBMCs. RA-FLS migration ability was not suppressed by IL-18BP after 12 or 24 h. IL-18BP increased annexin V+ FLS level and reduced annexin V+ chondrocyte level in a dose-dependent manner, whereas PI+ annexin V- FLS and chondrocyte levels were suppressed by 50, 100 ng/mL IL-18BP in culture. The administration of IL-18BP regulated the type 17 helper T cell/ regulatory T cell imbalance and attenuated the production of pro-inflammatory cytokines. IL-18BP further increased FLS apoptosis and decreased the necroptosis of FLS/chondrocytes and apoptosis of chondrocytes suggesting the joint preservative potential of IL-18BP.

Neuroprotective Epigenetic Changes Induced by Maternal Treatment with an Inhibitor of Soluble Epoxide Hydrolase Prevents Early Alzheimer′s Disease Neurodegeneration

Modulation of Alzheimer's disease (AD) risk begins early in life. During embryo development and postnatal maturation, the brain receives maternal physiological influences and establishes epigenetic patterns that build its level of resilience to late-life diseases. The soluble epoxide hydrolase inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl] urea (TPPU), reported as ant-inflammatory and neuroprotective against AD pathology in the adult 5XFAD mouse model of AD, was administered to wild-type (WT) female mice mated to heterozygous 5XFAD males during gestation and lactation. Two-month-old 5XFAD male and female offspring of vehicle-treated dams showed memory loss as expected. Remarkably, maternal treatment with TPPU fully prevented memory loss in 5XFAD. TPPU-induced brain epigenetic changes in both WT and 5XFAD mice, modulating global DNA methylation (5-mC) and hydroxymethylation (5-hmC) and reducing the gene expression of some histone deacetylase enzymes (Hdac1 and Hdac2), might be on the basis of the long-term neuroprotection against cognitive impairment and neurodegeneration. In the neuropathological analysis, both WT and 5XFAD offspring of TPPU-treated dams showed lower levels of AD biomarkers of tau hyperphosphorylation and microglia activation (Trem2) than the offspring of vehicle-treated dams. Regarding sex differences, males and females were similarly protected by maternal TPPU, but females showed higher levels of AD risk markers of gliosis and neurodegeneration. Taken together, our results reveal that maternal treatment with TPPU impacts in preventing or delaying memory loss and AD pathology by inducing long-term modifications in the epigenetic machinery and its marks.

The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility

Wheat is the staple crop for 35% of the world’s population, providing a major source of calories, mainly in the form of starch. The digestibility of wheat starch varies between different flours and products. Wheat products that are rapidly digested elicit large post-prandial glucose peaks associated with metabolic disorders. We investigated the impact of protein on starch digestion in three commercial flours with different grain hardness. A soluble extract of wheat proteins reduced starch digestion, even following gastric proteolysis. This extract was enriched in proteinaceous α-amylase inhibitors which were partially degraded during gastric proteolysis. Starch digestion kinetic analysis was carried out for flour samples pre-treated with different pepsin activities. The rate of starch digestion was altered following pepsin pre-digestion, and the extent of starch digestion increased in response to pepsin pre-digestion. We conclude that soluble proteinaceous alpha-amylase inhibitors present in wheat can escape gastric digestion and significantly contribute to reducing starch digestion in the small intestine.

Screening and Identification of Novel Soluble Epoxide Hydrolase Inhibitors from Corn Gluten Peptides.

The objective of this study was to investigate the soluble epoxide hydrolase (sEH) inhibitory properties of corn gluten peptides. In total, 400 dipeptides and 8000 tripeptides were first virtually screened by molecular docking and 30 potential sEH inhibitory peptides were selected. Among them, WEY, WWY, WYW, YFW, and YFY showed the highest sEH inhibitory activities with IC50 values of 55.41 ± 1.55, 68.80 ± 7.72, 70.66 ± 9.90, 96.00 ± 7.5, and 94.06 ± 12.86 μM, respectively. These five peptides all behaved as mixed-type inhibitors and were predicted to form hydrogen bond interactions mainly with Asp333, a key residue located in the catalytic active site of sEH. Moreover, it was found that the corn gluten hydrolysates of Alcalase, Flavourzyme, pepsin and pancreatin all exhibited high sEH inhibitory activities, with IC50 values of 1.07 ± 0.08, 1.19 ± 0.24, and 1.46 ± 0.31 mg/mL, respectively. In addition, the sEH inhibitory peptides WYW, YFW, and YFY were successfully identified from the corn gluten hydrolysates by Alcalase using nano-LC-MS/MS. This study demonstrated the sEH inhibitory capacity of peptides for the first time and corn gluten might be a promising food protein source for discovering novel natural sEH inhibitory peptides.

High Glycemia and Soluble Epoxide Hydrolase in Females: Differential Multiomics in Murine Brain Microvasculature.

The effect of a high glycemic diet (HGD) on brain microvasculature is a crucial, yet understudied research topic, especially in females. This study aimed to determine the transcriptomic changes in female brain hippocampal microvasculature induced by a HGD and characterize the response to a soluble epoxide hydrolase inhibitor (sEHI) as a mechanism for increased epoxyeicosatrienoic acids (EETs) levels shown to be protective in prior models of brain injury. We fed mice a HGD or a low glycemic diet (LGD), with/without the sEHI (t-AUCB), for 12 weeks. Using microarray, we assessed differentially expressed protein-coding and noncoding genes, functional pathways, and transcription factors from laser-captured hippocampal microvessels. We demonstrated for the first time in females that the HGD had an opposite gene expression profile compared to the LGD and differentially expressed 506 genes, primarily downregulated, with functions related to cell signaling, cell adhesion, cellular metabolism, and neurodegenerative diseases. The sEHI modified the transcriptome of female mice consuming the LGD more than the HGD by modulating genes involved in metabolic pathways that synthesize neuroprotective EETs and associated with a higher EETs/dihydroxyeicosatrienoic acids (DHETs) ratio. Our findings have implications for sEHIs as promising therapeutic targets for the microvascular dysfunction that accompanies vascular dementia.

Effect of D4 Dopamine Receptor on Na+-K+-ATPase Activity in Renal Proximal Tubule Cells.

NKA activity, nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were measured in RPT cells treated with the D4 receptor agonist PD168077 and/or the D4 receptor antagonist L745870, the NO synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) or the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ). Total D4 receptor expression and its expression in the plasma membrane were investigated by immunoblotting in RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Activation of D4 receptors with PD168077, inhibited NKA activity in RPT cells from WKY rats in a concentration- and time-dependent manner. The inhibitory effect of PD168077 on NKA activity was prevented by the addition of the D4 receptor antagonist L745870, which by itself had no effect. The NO synthase inhibitor L-NAME and the soluble guanylyl cyclase inhibitor ODQ, which by themselves had no effect on NKA activity, eliminated the inhibitory effect of PD168077 on NKA activity. Activation of D4 receptors also increased NO levels in the culture medium and cGMP levels in RPT cells. However, the inhibitory effect of D4 receptors on NKA activity was absent in RPT cells from SHRs, which could be related to decreased plasma membrane expression of D4 receptors in SHR RPT cells. Activation of D4 receptors directly inhibits NKA activity via the NO/cGMP signaling pathway in RPT cells from WKY rats but not SHRs. Aberrant regulation of NKA activity in RPT cells may be involved in the pathogenesis of hypertension.

IL-22BP production is heterogeneously distributed in Crohn's disease.

Crohn’s disease (CD), a form of inflammatory bowel disease (IBD), is characterized by impaired epithelial barrier functions and dysregulated mucosal immune responses. IL-22 binding protein (IL-22BP) is a soluble inhibitor regulating IL-22 bioactivity, a cytokine proposed to play protective roles during CD. We and others have shown that IL-22BP is produced in IBD inflamed tissues, hence suggesting a role in CD. In this work, we extended the characterization of IL-22BP production and distribution in CD tissues by applying enzyme-linked immunosorbent assays to supernatants obtained from the culture of endoscopic biopsies of patients, and reverse transcription-quantitative polymerase chain reaction on sorted immune cell subsets. We reveal that IL-22BP levels are higher in inflamed ileums than colons. We observe that in a cell-intrinsic fashion, populations of mononuclear phagocytes and eosinophils express IL-22BP at the highest levels in comparison to other sources of T cells. We suggest the enrichment of intestinal eosinophils could explain higher IL-22BP levels in the ileum. In inflamed colon, we reveal the presence of increased IL-22/IL22BP ratios compared to controls, and a strong correlation between IL-22BP and CCL24. We identify monocyte-derived dendritic cells (moDC) as a cellular subtype co-expressing both cytokines and validate our finding using in vitro culture systems. We also show that retinoic acid induces the secretion of both IL-22BP and CCL24 by moDC. Finally, we report on higher IL-22BP levels in active smokers. In conclusion, our work provides new information relevant to therapeutic strategies modulating IL-22 bioactivity in CD, especially in the context of disease location.

Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors.

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

Synthesis, Docking Study and Biological Evaluation of Amide-Based Soluble Epoxide Hydrolase Inhibitors with Novel Secondary Pharmacophore of Pyrimidin-2-ol.

Soluble epoxide hydrolase enzyme (sEH) is one of the most promising and emerging targets to develop drugs for multiple disease indications, including hypertension, diabetes, stroke, dyslipidemia, pain, etc. Most inhibitor scaffolds have a urea or amide moiety to mimic the active-site transition state. In this regard, we developed a series of amide sEH inhibitors with a pyrimidin-2-ol ring as a new secondary pharmacophore, which was subjected to in vitro evaluation. Compound 4w (4-chloro-N-{4-[6-(4-chlorophenyl)-2-hydroxypyrimidin-4-yl]phenyl}benzamide), which contains 4-chloro substituent in both terminal phenyl rings, exhibited the most inhibitory activity against sEH with an IC50 value of 1.2 nM. Molecular docking analysis of the synthesized compounds revealed that the greater number of hydrogen bonding interactions of the amide group as the primary pharmacophore with Asp-353, Tyr-383, and Tyr-466 as the key catalytic residue triad of the enzyme played a critical role and led to a more favorable binding affinity. Pharmacokinetic properties of the synthesized compounds were calculated in silico, and all ADMET indices fell within acceptable ranges. Altogether, the results of this work could provide useful information on 4,6-diphenylpyrimidin-2-olas sEH inhibitors which can be utilized in further development in this area.

Quinazoline-4(3H)-one-7-carboxamide Derivatives as Human Soluble Epoxide Hydrolase Inhibitors with Developable 5-Lipoxygenase Activating Protein Inhibition.

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs), which are endowed with beneficial biological activities as they reduce inflammation, regulate endothelial tone, improve mitochondrial function, and decrease oxidative stress. Therefore, inhibition of sEH for maintaining high EET levels is implicated as a new therapeutic modality with broad clinical applications for metabolic, renal, and cardiovascular disorders. In our search for new sEH inhibitors, we designed and synthesized novel amide analogues of the quinazolinone-7-carboxylic acid derivative 5, a previously discovered 5-lipoxygenase-activating protein (FLAP) inhibitor, to evaluate their potential for inhibiting sEH. As a result, we identified new quinazolinone-7-carboxamides that demonstrated selective sEH inhibition with decreased FLAP inhibitor properties. The tractable SAR results indicated that the amide and thiobenzyl fragments flanking the quinazolinone nucleus are critical features governing the potent sEH inhibition, and compounds 34, 35, 37, and 43 inhibited the sEH activity with IC50 values of 0.30–0.66 μM. Compound 34 also inhibited the FLAP-mediated leukotriene biosynthesis (IC50 = 2.91 μM). In conclusion, quinazolinone-7-carboxamides can be regarded as novel lead structures, and newer analogues with improved efficiency against sEH along with or without FLAP inhibition can be generated.

TIGIT is the central player in T-cell suppression associated with CAR T-cell relapse in mantle cell lymphoma.

BackgroundChimeric antigen receptor (CAR) T-cell therapy using brexucabtagene autoleucel (BA) induces remission in many patients with mantle cell lymphoma (MCL), and BA is the only CAR T-cell therapy approved by the FDA for MCL. However, development of relapses to BA is recognized with poor patient outcomes. Multiple CAR T-cell therapies have been approved for other lymphomas and the resistance mechanisms have been investigated. However, the mechanisms underlying BA relapse in MCL have not been investigated and whether any previously reported resistance mechanisms apply to BA-relapsed patients with MCL is unknown.MethodsTo interrogate BA resistance mechanisms in MCL, we performed single-cell RNA sequencing on 39 longitudinally collected samples from 15 BA-treated patients, and multiplex cytokine profiling on 80 serial samples from 20 patients.ResultsWe demonstrate that after BA relapse, the proportion of T cells, especially cytotoxic T cells (CTLs), decreased among non-tumor cells, while the proportion of myeloid cells correspondingly increased. TIGIT, LAG3, and CD96 were the predominant checkpoint molecules expressed on exhausted T cells and CTLs; only TIGIT was significantly increased after relapse. CTLs expanded during remission, and then contracted during relapse with upregulated TIGIT expression. Tumor cells also acquired TIGIT expression after relapse, leading to the enhanced interaction of tumor cell TIGIT with monocyte CD155/PVR. In myeloid cells, post-relapse HLA-II expression was reduced relative to pretreatment and during remission. Myeloid-derived suppressor cells (MDSCs) were enriched after relapse with elevated expression of activation markers, including CLU (clusterin) and VCAN (versican). Extracellular chemokines (CCL4, CXCL9, CXCL13), soluble checkpoint inhibitors (sPD-L1, sTIM3, s4-1BB), and soluble receptors (sIL-2R, sTNFRII) were decreased during remission but elevated after relapse.ConclusionsOur data demonstrate that multiple tumor-intrinsic and -extrinsic factors are associated with T-cell suppression and BA relapse. Among these, TIGIT appears to be the central player given its elevated expression after BA relapse in not only CTLs but also MCL cells. The acquisition of TIGIT expression on tumor cells is MCL-specific and has not been reported in other CAR T-treated diseases. Together, our data suggest that co-targeting TIGIT may prevent CAR T relapses and thus promote long-term progression-free survival in MCL patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-022-01655-0.

Anti-nociceptive efficacy of the soluble epoxide hydrolase inhibitor t-TUCB in horses with mechanically induced lameness

IntroductionSoluble epoxide hydrolase (sEH) inhibitors are novel anti-inflammatory and analgesic agents that could improve pain management in horses. The objective of the present study was to evaluate the anti-nociceptive effect of a single-dose intravenous administration of the sEH inhibitor trans-4-{4-[3-(4-trifluro-methoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB) using an adjustable heart bar shoe (a-HBS) model of lameness. We hypothesized that t-TUCB would improve objective and subjective lameness measures compared to the control. Materials and methodsReversible lameness was induced in 8 horses for 24 h using an a-HBS in a randomized, crossover design. A vehicle-control placebo (DMSO) or t-TUCB (1 mg/kg) was intravenously administered at time 0 following a baseline induced lameness evaluation. Blood was collected and lameness was objectively measured with an inertial sensor system at 0-, 1-, 3-, 6-, 12-, and 24-h time points. Front-facing videos were obtained at each time point for subjective evaluation by three blinded evaluators using a visual analog scale (VAS). ResultsTreatment with t-TUCB significantly decreased (i.e. improved) lameness compared to placebo at 1-h and compared to baseline at 1-, 3-, and 6-h following administration. Lameness significantly increased (i.e. worsened) from baseline in placebo-treated horses 12 h after administration. All horses returned to baseline soundness within 24 h of reversing lameness. ConclusionTreatment with single-dose IV administration of t-TUCB improved lameness induced by the a-HSB, suggesting that t-TUCB has anti-nociceptive effects in horses. Clinical relevanceThe soluble epoxide hydrolase inhibitor, t-TUCB, is a promising novel analgesic for horses.

Anti-Inflammatory Activity of Soluble Epoxide Hydrolase Inhibitors Based on Selenoureas Bearing an Adamantane Moiety.

The inhibitory potency of the series of inhibitors of the soluble epoxide hydrolase (sEH) based on the selenourea moiety and containing adamantane and aromatic lipophilic groups ranges from 34.3 nM to 1.2 μM. The most active compound 5d possesses aliphatic spacers between the selenourea group and lipophilic fragments. Synthesized compounds were tested against the LPS-induced activation of primary murine macrophages. The most prominent anti-inflammatory activity, defined as a suppression of nitric oxide synthesis by LPS-stimulated macrophages, was demonstrated for compounds 4a and 5b. The cytotoxicity of the obtained substances was studied using human neuroblastoma and fibroblast cell cultures. Using these cell assays, the cytotoxic concentration for 4a was 4.7–18.4 times higher than the effective anti-inflammatory concentration. The genotoxicity and the ability to induce oxidative stress was studied using bacterial lux-biosensors. Substance 4a does not exhibit genotoxic properties, but it can cause oxidative stress at concentrations above 50 µM. Put together, the data showed the efficacy and safety of compound 4a.

Tumour necrosis factor blockade after asphyxia in foetal sheep ameliorates cystic white matter injury.

Cystic white matter injury is highly associated with severe neurodevelopmental disability and cerebral palsy in preterm infants, yet its pathogenesis remains poorly understood and there is no established treatment. In the present study, we tested the hypothesis that slowly evolving cystic white matter injury after hypoxia-ischaemia is mediated by programmed necrosis initiated by tumour necrosis factor. Tumour necrosis factor blockade was begun 3 days after hypoxia-ischaemia to target the tertiary phase of injury, when most secondary cell death is thought to be complete. Chronically instrumented preterm foetal sheep (0.7 gestation) received 25 min of hypoxia-ischaemia induced by complete umbilical cord occlusion or sham-umbilical cord occlusion (controls, n = 10), followed by intracerebroventricular infusion of the soluble TNF inhibitor, Etanercept, at 3, 8 and 13 days after umbilical cord occlusion (n = 9) or vehicle (n = 9). Foetal brains were processed for histology at 21 days after umbilical cord occlusion. Umbilical cord occlusion with vehicle was associated with a spectrum of macroscopic white matter degeneration, including white matter atrophy, ventriculomegaly and overt temporal lobe cystic white matter injury. Oligodendrocyte maturational arrest and impaired labelling of myelin proteins, characteristic of diffuse white matter injury, was observed in the parietal lobe and surrounding the cystic lesions in the temporal lobe. Etanercept markedly attenuated cystic white matter injury on the side of the intracerebroventricular infusion, with partial contralateral protection. Further, Etanercept improved oligodendrocyte maturation and labelling of myelin proteins in the temporal and parietal lobes. The present study shows that cystic white matter injury reflects late-onset tertiary cell death mediated by delayed neuroinflammation through the tumour necrosis factor pathway. Delayed tumour necrosis factor blockade markedly attenuated cystic white matter injury and restored oligodendrocyte maturation and deficits in myelin protein expression. These data suggest that delayed tumour necrosis factor blockade may represent a viable therapeutic strategy to reduce the risk of cystic and diffuse white matter injury and potentially cerebral palsy after preterm birth, with a surprisingly wide therapeutic window.

Soluble epoxide hydrolase inhibitor, t-AUCB, improves salivary gland function by ameliorating endothelial injury

AimsInhibitor of soluble epoxide hydrolase (t-AUCB) has been used in the experimental therapy of hypertension. This study aimed to investigate whether the secretion of submandibular glands (SMGs) altered in renal hypertensive rats, and to explore whether t-AUCB could improve the salivary secretion. Main methods2-kidney 1-clip Sprague-Dawley rats were used as renal hypertensive animals. t-AUCB treatment was given for 1 week after 8 weeks modeling. Blood pressure, blood perfusion and the secretion of SMGs, and endothelium-dependent relaxation of external maxillary artery were measured to investigate the effects of t-AUCB on the vascular tone and the secretion of SMGs in renal hypertensive rats. SMGs were collected for histological evaluation and the internal arteries were dissected for primary endothelial cells culture. Key findingsThe blood perfusion and flow rate of SMGs in the renal hypertensive rats were significantly lower than those in the controls. Endothelium-dependent relaxation of the external maxillary artery and AMPK/Akt/eNOS signaling was impaired in hypertensive rats. The glandular morphology and the concentration of salivary ions did not change obviously. t-AUCB treatment ameliorated the secretion of SMGs, the blood perfusion, and the dysfunction of endothelium-dependent relaxation of the external maxillary artery by activating the AMPK/Akt/eNOS pathway in hypertensive rats. Significancet-AUCB increases the blood perfusion through ameliorating dysfunction of endothelium-dependent relaxation of SMGs arteries and thus improves the hyposecretion of SMGs in hypertensive rats.