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Abstract Background Chemotherapy-induced neuropathic pain (CIPN) describes a pathological pain state that occurs dose-dependently as a side effect and can limit or even impede an effective cancer therapy. Unfortunately, current treatment possibilities for CIPN are remarkably confined and mostly inadequate as CIPN therapeutics themselves consist of low effectiveness and may induce severe side effects, pointing out CIPN as pathological entity with an emerging need for novel treatment targets. Here, we investigated whether the novel and highly specific FKBP51 inhibitor SAFit2 reduces paclitaxel-induced neuropathic pain. Methods In this study, we used a well-established multiple low-dose paclitaxel model to investigate analgesic and anti-inflammatory properties of SAFit2. For this purpose, the behavior of the mice was recorded over 14 days and the mouse tissue was then analyzed using biochemical methods. Results Here, we show that SAFit2 is capable to reduce paclitaxel-induced mechanical hypersensitivity in mice. In addition, we detected that SAFit2 shifts lipid levels in nervous tissue toward an anti-inflammatory and pro-resolving lipid profile that counteracts peripheral sensitization after paclitaxel treatment. Furthermore, SAFit2 reduced the activation of astrocytes and microglia in the spinal cord as well as the levels of pain-mediating chemokines. Its treatment also increased anti-inflammatory cytokines levels in neuronal tissues, ultimately leading to a resolution of neuroinflammation. Conclusions In summary, SAFit2 shows antihyperalgesic properties as it ameliorates paclitaxel-induced neuropathic pain by reducing peripheral sensitization and resolving neuroinflammation. Therefore, we consider SAFit2 as a potential novel drug candidate for the treatment of paclitaxel-induced neuropathic pain.

Abstract Background Neuropathic pain is experienced worldwide by patients suffering from nerve injuries, infectious or metabolic diseases or chemotherapy. However, the treatment options are still limited because of low efficacy and sometimes severe side effects. Recently, the deficiency of FKBP51 was shown to relieve chronic pain, revealing FKBP51 as a potential therapeutic target. However, a specific and potent FKBP51 inhibitor was not available until recently which hampered targeting of FKBP51. Methods In this study, we used the well-established and robust spared nerve injury model to analyze the effect of SAFit2 on nerve injury-induced neuropathic pain and to elucidate its pharmacodynamics profile. Therefore, the mice were treated with 10 mg/kg SAFit2 after surgery, the mice behavior was assessed over 21 days and biochemical analysis were performed after 14 and 21 days. Furthermore, the impact of SAFit2 on sensory neurons and macrophages was investigated in vitro. Results Here, we show that the FKBP51 inhibitor SAFit2 ameliorates nerve injury-induced neuropathic pain in vivo by reducing neuroinflammation. SAFit2 reduces the infiltration of immune cells into neuronal tissue and counteracts the increased NF-κB pathway activation which leads to reduced cytokine and chemokine levels in the DRGs and spinal cord. In addition, SAFit2 desensitizes the pain-relevant TRPV1 channel and subsequently reduces the release of pro-inflammatory neuropeptides from sensory neurons. Conclusions SAFit2 ameliorates neuroinflammation and counteracts enhanced neuronal activity after nerve injury leading to an amelioration of nerve injury-induced neuropathic pain. Based on these findings, SAFit2 constitutes as a novel and promising drug candidate for the treatment of nerve injury-induced neuropathic pain.

A causal contribution of hyperhomocysteinemia to cognitive decline and Alzheimer’s disease (AD), as well as potential prevention or mitigation of the pathology by dietary intervention, have frequently been subjects of controversy. In the present in vivo study, we attempted to further elucidate the impact of elevated homocysteine (HCys) and homocysteic acid (HCA) levels, induced by dietary B-vitamin deficiency, and micronutrient supplementation on AD-like pathology, which was simulated using the amyloid-based AppNL–G–F knock-in mouse model. For this purpose, cognitive assessment was complemented by analyses of ex vivo parameters in whole blood, serum, CSF, and brain tissues from the mice. Furthermore, neurotoxicity of HCys and HCA was assessed in a separate in vitro assay. In confirmation of our previous study, older AppNL–G–F mice also exhibited subtle phenotypic impairment and extensive cerebral amyloidosis, whereas dietary manipulations did not result in significant effects. As revealed by proximity extension assay-based proteome analysis, the AppNL–G–F genotype led to an upregulation of AD-characteristic neuronal markers. Hyperhomocysteinemia, in contrast, indicated mainly vascular effects. Overall, since there was an absence of a distinct phenotype despite both a significant amyloid-β burden and serum HCys elevation, the results in this study did not corroborate the pathological role of amyloid-β according to the “amyloid hypothesis,” nor of hyperhomocysteinemia on cognitive performance. Nevertheless, this study aided in further characterizing the AppNL–G–F model and in elucidating the role of HCys in diverse biological processes. The idea of AD prevention with the investigated micronutrients, however, was not supported, at least in this mouse model of the disease.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union’s Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

SARS-CoV-2 uses the human cell surface protein angiotensin converting enzyme 2 (ACE2) as the receptor by which it gains access into lung and other tissue. Early in the pandemic, there was speculation that a number of commonly used medications—including ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs)—have the potential to upregulate ACE2, thereby possibly facilitating viral entry and increasing the severity of COVID-19. We investigated the influence of the NSAIDS with a range of cyclooxygenase (COX)1 and COX2 selectivity (ibuprofen, flurbiprofen, etoricoxib) and paracetamol on the level of ACE2 mRNA/protein expression and activity as well as their influence on SARS-CoV-2 infection levels in a Caco-2 cell model. We also analysed the ACE2 mRNA/protein levels and activity in lung, heart and aorta in ibuprofen treated mice. The drugs had no effect on ACE2 mRNA/protein expression and activity in the Caco-2 cell model. There was no up-regulation of ACE2 mRNA/protein expression and activity in lung, heart and aorta tissue in ibuprofen-treated mice in comparison to untreated mice. Viral load was significantly reduced by both flurbiprofen and ibuprofen at high concentrations. Ibuprofen, flurbiprofen, etoricoxib and paracetamol demonstrated no effects on ACE2 expression or activity in vitro or in vivo. Higher concentrations of ibuprofen and flurbiprofen reduced SARS-CoV-2 replication in vitro.

Disturbances in the one-carbon metabolism are often indicated by altered levels of the endogenous amino acid homocysteine (HCys), which is additionally discussed to causally contribute to diverse pathologies. In the first part of the present review, we profoundly and critically discuss the metabolic role and pathomechanisms of HCys, as well as its potential impact on different human disorders. The use of adequate animal models can aid in unravelling the complex pathological processes underlying the role of hyperhomocysteinemia (HHCys). Therefore, in the second part, we systematically searched PubMed/Medline for animal studies regarding HHCys and focused on the potential impact on cognitive performance and decline. The majority of reviewed studies reported a significant effect of HHCys on the investigated behavioral outcomes. Despite of persistent controversial discussions about equivocal findings, especially in clinical studies, the present evaluation of preclinical evidence indicates a causal link between HHCys and cognition-related- especially dementia-like disorders, and points out the further urge for large-scale, well-designed clinical studies in order to elucidate the normalization of HCys levels as a potential preventative or therapeutic approach in human pathologies.

Summary: Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptors remain obscure. IL-38 is highly expressed in the skin and downregulated in psoriasis patients. We report an investigation in cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38 knockout (IL-38 KO) mice show delayed disease resolution with exacerbated IL-17-mediated inflammation, which is reversed by the administration of mature IL-38 or γδ T cell-receptor-blocking antibodies. Mechanistically, X-linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) is upregulated upon γδ T cell activation to feedforward-amplify IL-17 production and is required for IL-38 to suppress γδ T cell IL-17 production. Accordingly, psoriatic IL1RAPL1 KO mice show reduced inflammation and IL-17 production by γδ T cells. Our findings indicate a role for IL-38 in the regulation of γδ T cell activation through IL1RAPL1, with consequences for auto-inflammatory disease. : Han et al. report that genetic depletion of IL-38 in mice delays the resolution of imiquimod-induced psoriasis by increasing the production of the inflammatory cytokine IL-17A by skin-infiltrating T cells. Depleting these T cells or the receptor that is targeted by IL-38 reduces psoriatic skin inflammation. Keywords: IL-38, IL1RAPL1, IL-17, γδ T cells, psoriasis, inflammation

Hyperhomocysteinemia has been suggested potentially to contribute to a variety of pathologies, such as Alzheimer’s disease (AD). While the impact of hyperhomocysteinemia on AD has been investigated extensively, there are scarce data on the effect of AD on hyperhomocysteinemia. The aim of this in vivo study was to investigate the kinetics of homocysteine (HCys) and homocysteic acid (HCA) and effects of AD-like pathology on the endogenous levels. The mice received a B-vitamin deficient diet for eight weeks, followed by the return to a balanced control diet for another eight weeks. Serum, urine, and brain tissues of AppNL-G-F knock-in and C57BL/6J wild type mice were analyzed for HCys and HCA using LC-MS/MS methods. Hyperhomocysteinemic levels were found in wild type and knock-in mice due to the consumption of the deficient diet for eight weeks, followed by a rapid normalization of the levels after the return to control chow. Hyperhomocysteinemic AppNL-G-F mice had significantly higher HCys in all matrices, but not HCA, compared to wild type control. Higher serum concentrations were associated with elevated levels in both the brain and in urine. Our findings confirm a significant impact of AD-like pathology on hyperhomocysteinemia in the AppNL-G-F mouse model. The immediate normalization of HCys and HCA after the supply of B-vitamins strengthens the idea of a B-vitamin intervention as a potentially preventive treatment option for HCys-related disorders such as AD.

Background: Hyperhomocysteinemia is considered a possible contributor to the complex pathology of Alzheimer’s disease (AD). For years, researchers in this field have discussed the apparent detrimental effects of the endogenous amino acid homocysteine in the brain. In this study, the roles of hyperhomocysteinemia driven by vitamin B deficiency, as well as potentially beneficial dietary interventions, were investigated in the novel AppNL-G-F knock-in mouse model for AD, simulating an early stage of the disease. Methods: Urine and serum samples were analyzed using a validated LC-MS/MS method and the impact of different experimental diets on cognitive performance was studied in a comprehensive behavioral test battery. Finally, we analyzed brain samples immunohistochemically in order to assess amyloid-β (Aβ) plaque deposition. Results: Behavioral testing data indicated subtle cognitive deficits in AppNL-G-F compared to C57BL/6J wild type mice. Elevation of homocysteine and homocysteic acid, as well as counteracting dietary interventions, mostly did not result in significant effects on learning and memory performance, nor in a modified Aβ plaque deposition in 35-week-old AppNL-G-F mice. Conclusion: Despite prominent Aβ plaque deposition, the AppNL-G-F model merely displays a very mild AD-like phenotype at the investigated age. Older AppNL-G-F mice should be tested in order to further investigate potential effects of hyperhomocysteinemia and dietary interventions.

Cannabinoid CB1 antagonists have been investigated for possible treatment of e.g. obesity-related disorders. However, clinical application was halted due to their symptoms of anxiety and depression. In addition to these adverse effects, we have shown earlier that chronic treatment with the CB1 antagonist rimonabant may induce EEG-confirmed convulsive seizures. In a regulatory repeat-dose toxicity study violent episodes of "muscle spasms" were observed in Wistar rats, daily dosed with the CB1 receptor antagonist SLV326 during 5 months. The aim of the present follow-up study was to investigate whether these violent movements were of an epileptic origin. In selected SLV326-treated and control animals, EEG and behavior were monitored for 24 hours. 25% of SLV326 treated animals showed 1 to 21 EEG-confirmed generalized convulsive seizures, whereas controls were seizure-free. The behavioral seizures were typical for a limbic origin. Moreover, interictal spikes were found in 38% of treated animals. The frequency spectrum of the interictal EEG of the treated rats showed a lower theta peak frequency, as well as lower gamma power compared to the controls. These frequency changes were state-dependent: they were only found during high locomotor activity. It is concluded that long term blockade of the endogenous cannabinoid system can provoke limbic seizures in otherwise healthy rats. Additionally, SLV326 alters the frequency spectrum of the EEG when rats are highly active, suggesting effects on complex behavior and cognition.

Abstract R‐flurbiprofen is the non‐cyclooxygenase inhibiting R‐enantiomer of the non‐steroidal anti‐inflammatory drug flurbiprofen, which was assessed as a remedy for Alzheimer's disease. Because of its anti‐inflammatory, endocannabinoid‐modulating and antioxidative properties, combined with low toxicity, the present study assessed R‐flurbiprofen in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis in mice. Oral R‐flurbiprofen prevented and attenuated primary progressive EAE in C57BL6/J mice and relapsing‐remitting EAE in SJL mice, even if the treatment was initiated on or after the first flare of the disease. R‐flurbiprofen reduced immune cell infiltration and microglia activation and inflammation in the spinal cord, brain and optic nerve and attenuated myelin destruction and EAE‐evoked hyperalgesia. R‐flurbiprofen treatment increased CD4+CD25+FoxP3+ regulatory T cells, CTLA4+ inhibitory T cells and interleukin‐10, whereas the EAE‐evoked upregulation of pro‐inflammatory genes in the spinal cord was strongly reduced. The effects were associated with an increase of plasma and cortical endocannabinoids but decreased spinal prostaglandins, the latter likely due to R to S inversion. The promising results suggest potential efficacy of R‐flurbiprofen in human MS, and its low toxicity may justify a clinical trial.

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA4 on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B4 and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA4 (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B4 was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA4. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.