Neutrophil activation and neutrophil extracellular traps (NETs) in COVID-19 ARDS and immunothrombosis.

Acute respiratory distress syndrome (ARDS) is an acute inflammatory condition with a dramatic increase in incidence since the beginning of the coronavirus disease 19 (COVID-19) pandemic. Neutrophils play a vital role in the immunopathology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by triggering the formation of neutrophil extracellular traps (NETs), producing cytokines including interleukin-8 (CXCL8), and mediating the recruitment of other immune cells to regulate processes such as acute and chronic inflammation, which can lead to ARDS. CXCL8 is involved in the recruitment, activation, and degranulation of neutrophils, and therefore contributes to inflammation amplification and severity of disease. Furthermore, activation of neutrophils also supports a prothrombotic phenotype, which may explain the development of immunothrombosis observed in COVID-19 ARDS. This review aims to describe hyperinflammatory ARDS due to SARS-CoV-2 infection. In addition, we address the critical role of polymorphonuclear neutrophils, inflammatory cytokines, and the potential targeting of CXCL8 in treating the hyperinflammatory ARDS population.

The History of Nerve Growth Factor: From Molecule to Drug.

Nerve growth factor (NGF), the first neurotrophin to be discovered, has a long and eventful research journey with a series of turning points, setbacks, and achievements. Since the groundbreaking investigations led by Nobel Prize winner Rita Levi-Montalcini, advancements in the comprehension of NGF's functions have revolutionized the field of neuroscience, offering new insights and opportunities for therapeutic innovation. However, the clinical application of NGF has historically been hindered by challenges in determining appropriate dosing, administration strategies, and complications related to the production process. Recent advances in the production and scientific knowledge of recombinant NGF have enabled its clinical development, and in 2018, the United States Food and Drug Administration approved cenegermin-bkbj, a recombinant human NGF, for the treatment of all stages of neurotrophic keratitis. This review traces the evolutionary path that transformed NGF from a biological molecule into a novel therapy with potential research applications beyond the eye. Special emphasis is put on the studies that advanced NGF from discovery to the first medicinal product approved to treat a human disease.

Differential Effects of Nonsteroidal Anti-Inflammatory Drugs in an In Vitro Model of Human Leaky Gut.

The intestinal barrier is the main contributor to gut homeostasis. Perturbations of the intestinal epithelium or supporting factors can lead to the development of intestinal hyperpermeability, termed "leaky gut". A leaky gut is characterized by loss of epithelial integrity and reduced function of the gut barrier, and is associated with prolonged use of Non-Steroidal Anti-Inflammatories. The harmful effect of NSAIDs on intestinal and gastric epithelial integrity is considered an adverse effect that is common to all drugs belonging to this class, and it is strictly dependent on NSAID properties to inhibit cyclo-oxygenase enzymes. However, different factors may affect the specific tolerability profile of different members of the same class. The present study aims to compare the effects of distinct classes of NSAIDs, such as ketoprofen (K), Ibuprofen (IBU), and their corresponding lysine (Lys) and, only for ibuprofen, arginine (Arg) salts, using an in vitro model of leaky gut. The results obtained showed inflammatory-induced oxidative stress responses, and related overloads of the ubiquitin-proteasome system (UPS) accompanied by protein oxidation and morphological changes to the intestinal barrier, many of these effects being counteracted by ketoprofen and ketoprofen lysin salt. In addition, this study reports for the first time a specific effect of R-Ketoprofen on the NFkB pathway that sheds new light on previously reported COX-independent effects, and that may account for the observed unexpected protective effect of K on stress-induced damage on the IEB.

Tamoxifen Twists Again: On and Off-Targets in Macrophages and Infections.

Beyond the wide use of tamoxifen in breast cancer chemotherapy due to its estrogen receptor antagonist activity, this drug is being assayed in repurposing strategies against a number of microbial infections. We conducted a literature search on the evidence related with tamoxifen activity in macrophages, since these immune cells participate as a first line-defense against pathogen invasion. Consistent data indicate the existence of estrogen receptor-independent targets of tamoxifen in macrophages that include lipid mediators and signaling pathways, such as NRF2 and caspase-1, which allow these cells to undergo phenotypic adaptation and potentiate the inflammatory response, without the induction of cell death. Thus, these lines of evidence suggest that the widespread antimicrobial activity of this drug can be ascribed, at least in part, to the potentiation of the host innate immunity. This widens our understanding of the pharmacological activity of tamoxifen with relevant therapeutic implications for infections and other clinical indications that may benefit from the immunomodulatory effects of this drug.

Repurposing the estrogen receptor modulator raloxifene to treat SARS-CoV-2 infection.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates strategies to identify prophylactic and therapeutic drug candidates to enter rapid clinical development. This is particularly true, given the uncertainty about the endurance of the immune memory induced by both previous infections or vaccines, and given the fact that the eradication of SARS-CoV-2 might be challenging to reach, given the attack rate of the virus, which would require unusually high protection by a vaccine. Here, we show how raloxifene, a selective estrogen receptor modulator with anti-inflammatory and antiviral properties, emerges as an attractive candidate entering clinical trials to test its efficacy in early-stage treatment COVID-19 patients.

The role of extracellular vesicles and interleukin-8 in regulating and mediating neutrophil-dependent cancer drug resistance.

Tumor drug resistance is a multifactorial and heterogenous condition that poses a serious burden in clinical oncology. Given the increasing incidence of resistant tumors, further understanding of the mechanisms that make tumor cells able to escape anticancer drug effects is pivotal for developing new effective treatments. Neutrophils constitute a considerable proportion of tumor infiltrated immune cells, and studies have linked elevated neutrophil counts with poor prognosis. Tumor-associated neutrophils (TANs) can acquire in fact immunoregulatory capabilities, thus regulating tumor progression and resistance, or response to therapy. In this review, we will describe TANs' actions in the tumor microenvironment, with emphasis on the analysis of the role of interleukin-8 (IL-8) and extracellular vesicles (EVs) as crucial modulators and mediators of TANs biology and function in tumors. We will then discuss the main mechanisms through which TANs can induce drug resistance, finally reporting emerging therapeutic approaches that target these mechanisms and can thus be potentially used to reduce or overcome neutrophil-mediated tumor drug resistance.

Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

The Role of Interleukin-8 in Lung Inflammation and Injury: Implications for the Management of COVID-19 and Hyperinflammatory Acute Respiratory Distress Syndrome.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) has resulted in the global spread of Coronavirus Disease 2019 (COVID-19) and an increase in complications including Acute Respiratory Distress Syndrome (ARDS). Due to the lack of therapeutic options for Acute Respiratory Distress Syndrome, recent attention has focused on differentiating hyper- and hypo-inflammatory phenotypes of ARDS to help define effective therapeutic strategies. Interleukin 8 (IL-8) is a pro-inflammatory cytokine that has a role in neutrophil activation and has been identified within the pathogenesis and progression of this disease. The aim of this review is to highlight the role of IL-8 as a biomarker and prognostic factor in modulating the hyperinflammatory response in ARDS. The crucial role of IL-8 in lung inflammation and disease pathogenesis might suggest IL-8 as a possible new therapeutic target to efficiently modulate the hyperinflammatory response in ARDS.

Emerging Role of C5 Complement Pathway in Peripheral Neuropathies: Current Treatments and Future Perspectives.

The complement system is a key component of innate immunity since it plays a critical role in inflammation and defense against common pathogens. However, an inappropriate activation of the complement system is involved in numerous disorders, including peripheral neuropathies. Current strategies for neuropathy-related pain fail to achieve adequate pain relief, and although several therapies are used to alleviate symptoms, approved disease-modifying treatments are unavailable. This urgent medical need is driving the development of therapeutic agents for this condition, and special emphasis is given to complement-targeting approaches. Recent evidence has underscored the importance of complement component C5a and its receptor C5aR1 in inflammatory and neuropathic pain, indicating that C5a/C5aR1 axis activation triggers a cascade of events involved in pathophysiology of peripheral neuropathy and painful neuro-inflammatory states. However, the underlying pathophysiological mechanisms of this signaling in peripheral neuropathy are not fully known. Here, we provide an overview of complement pathways and major components associated with dysregulated complement activation in peripheral neuropathy, and of drugs under development targeting the C5 system. C5/C5aR1 axis modulators could represent a new strategy to treat complement-related peripheral neuropathies. Specifically, we describe novel C5aR allosteric modulators, which may potentially become new tools in the therapeutic armory against neuropathic pain.

Structure-Activity Relationship of novel phenylacetic CXCR1 inhibitors.

We reported recently the Structure-Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.

Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2.

Chemokines CXCL8 and CXCL1 play a key role in the recruitment of neutrophils at the site of inflammation. CXCL8 binds two membrane receptors, CXCR1 and CXCR2, whereas CXCL1 is a selective agonist for CXCR2. In the past decade, the physiopathological role of CXCL8 and CXCL1 has been investigated. A novel class of small molecular weight allosteric CXCR1 inhibitors was identified, and reparixin, the first drug candidate, is currently under clinical investigation in the prevention of ischemia/reperfusion injury in organ transplantation. Reparixin binding mode to CXCR1 has been studied and used for a computer-assisted design program of dual allosteric CXCR1 and CXCR2 inhibitors. In this paper, the results of modeling-driven SAR studies for the identification of potent dual inhibitors are discussed, and three new compounds (56, 67, and 79) sharing a common triflate moiety have been selected as potential leads with optimized pharmacokinetic characteristics.

Allosteric Modulation of Chemoattractant Receptors.

Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities.

Predicting human serum albumin affinity of interleukin-8 (CXCL8) inhibitors by 3D-QSPR approach.

A novel class of 2-(R)-phenylpropionamides has been recently reported to inhibit in vitro and in vivo interleukin-8 (CXCL8)-induced biological activities. These CXCL8 inhibitors are derivatives of phenylpropionic nonsteroidal antiinflammatory drugs (NSAIDs), high-affinity ligands for site II of human serum albumin (HSA). Up to date, only a limited number of in silico models for the prediction of albumin protein binding are available. A three-dimensional quantitative structure-property relationship (3D-QSPR) approach was used to model the experimental affinity constant (K(i)) to plasma proteins of 37 structurally related molecules, using physicochemical and 3D-pharmacophoric descriptors. Molecular docking studies highlighted that training set molecules preferentially bind site II of HSA. The obtained model shows satisfactory statistical parameters both in fitting and predicting validation. External validation confirmed the statistical significance of the chemometric model, which is a powerful tool for the prediction of HSA binding in virtual libraries of structurally related compounds.

2-Arylpropionic CXC chemokine receptor 1 (CXCR1) ligands as novel noncompetitive CXCL8 inhibitors.

The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.

Targeting FR-expressing cells in ovarian cancer with Fab-functionalized nanoparticles: a full study to provide the proof of principle from in vitro to in vivo.

Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles.

Current status of chemokine receptor inhibitors in development.

The chemokine network plays pivotal role in a large number of inflammatory, allergic and autoimmune diseases, as well as in the promotion of tumor growth and metastasis. Considerable effort has been put in the pharmaceutical industry to identify therapeutic agents that specifically target chemokine receptors. Despite the fact that several promising programs have proven unsuccessful in Phase II trials the research activity both in academia and industry is still highly intense, whereas for some of the chemokine receptors the progress is still at the preclinical stage. In this review the authors discuss possible reasons beyond successes and failures of early clinical development programs and discuss the most relevant and recent pharmacological approaches with the aim to point out new theories, open issues and expectations in this research field.