Pharmacological Tools Research Articles

The isoproterenol-induced myocardial fibrosis: A biochemical and histological investigation

The isoproterenol (ISO)-induced myocardial fibrosis is considered a reliable and repeatable experimental model characterized by a relatively low mortality rate. Although is well-known that ISO stimulates the β1 adrenergic receptors at the myocardial level, a high degree of heterogeneity emerges around the doses and duration of the treatment generating unclear results. Therefore, we propose to gain insights into the progression of ISO-induced myocardial fibrosis, in order to critically analyze and optimize the experimental model. Male Wistar rats (12–14-week-old) were submitted to subcutaneous injection of ISO, in particular, two doses were selected: the commonly used dose of 5 mg/kg and a lower dose of 1 mg/kg, administered for 3 and 6 days. Biochemical and histological examinations were conducted either immediately after the last administration or after a recovering period of 7 or 14 days from the initial administration. Noteworthy, from our investigation emerged that even the lower dose of ISO was able to induce the maximal biochemical and histological alterations, suggesting that lower doses should be considered to control the progression of the damage more precisely and to identify a prodromic phase in which intervention with pharmacological or nutraceutical tools can be effectively attempted.

Aptamers-based Strategies for the Treatment of Microbial Infections

Background: Nowadays, infectious diseases caused by bacteria are one of the major risks for the human population worldwide. Antimicrobial resistance determined the necessity to develop both, new drugs and therapeutic approaches as alternatives to antibiotics and novel methods to detect bacteria. Aptamers have revealed their potential in combating antimicrobial infections. Aptamers are small singlestranded DNA or RNA oligonucleotides obtained through an in vitro process able to bind several targets with high affinity and specificity. Objective: The aim of this review is to provide an overview of the state of the art of aptamer-based antimicrobial therapeutic strategies, new methods of detection of bacteria, and their potential use as delivery systems. Conclusion: Recent applications on research about aptamers suggest their important potential in discovering novel pharmacological tools for the treatment of microbial infections.

Use of tetraethylammonium (TEA) and Tris loading for blocking TRPM7 channels in intact cells

Tetraethylammonium (TEA), a quaternary ammonium compound, is a well-known blocker of potassium channels belonging to various subfamilies, such as KV1-3, KCa1, 2 and prokaryotic KcsA. In many cases, TEA acts from the extracellular side by open pore blockade. TEA can also block transient receptor potential (TRP) cation channels, such as TRPM7, in a voltage-dependent manner. In human T lymphocytes, intracellular (cytosolic) TEA and its analog TMA (tetramethylammonium) inhibit TRPM7 channel currents in the outward but not inward direction. By contrast, intracellular Mg2+, protons and polyamines inhibit both outward and inward current components equally. Likewise, the majority of available pharmacological tools inhibit TRPM7 channels in a voltage-independent manner. Since TRPM7 is a steeply outwardly rectifying conductance, voltage-dependent blockers can be useful for studying the cellular functions of this channel. TRPM7 protein is endogenously expressed in diverse cell lines, including HEK, HeLa, CHO, RBL and Jurkat. Using patch-clamp electrophysiology, we found that incubating HEK293 and Jurkat T cells overnight in the presence of 20 mM TEA-Cl, resulted in the nearly complete blockade of whole-cell TRPM7 outward current, measured at break-in. By contrast, the inward current was unchanged in TEA-loaded cells. The blockade was fully reversible after washout of intracellular solution in whole-cell but not in perforated-patch recording configurations. Overnight incubation with 20 mM TMA-Cl resulted in a more modest blockade of the outward TRPM7 current. Internal 129 mM TMA and TEA eliminated most of the outward current. TEA uptake in transfected HEK293 cells led to blockade of recombinant murine TRPM7 and the Mg2+ and pH insensitive Ser1107Arg variant. Unexpectedly, Tris-HCl, a widely used pH buffer, could similarly be loaded into Jurkat and HEK cells, and preferentially blocked outward TRPM7 currents. 20 mM and 129 mM Tris in the internal solution blocked TRPM7 current in outward but not inward direction. Voltage-dependent channel blockade by TEA, TMA and Tris loading will be useful for studying the properties and functions of TRPM7-mediated ion transport in intact cells.

Polysacharide of Agaricusblazei gel mitigates bone necrosis in model of the jaws related to bisphosphonate via Wnt signaling

To investigate de effect of PAb gel on the bone tissue of rats submitted to Bisphosphonate-related osteonecrosis of the jaws (BRONJ). Initially, 54 animals were submitted to BRONJ model by Zoledronic Acid (ZA) (0.1 mg/kg 3x/wk for 9 wk, ip), followed by the 1st upper left molar extraction at the 8th wk. After tooth removal, the animals were divided into 3 groups, ZA that received placebo gel or PAb gel that received 1% PAb gel, inside the dental alveolus. The control Group (CONTROL) received 0.1 mg/kg of 0.9% saline and then placebo gel. Three weeks after tooth extraction, the animals were euthanized, and maxillae were colleted for macroscopic, radiographic, histological and Raman spectomery assays. Additionally, GSK3b, beta-catenin, and Runx2 mRNA expressions were determined. Blood samples were collected for the analysis of Bone-specific alkaline phosphatase (BALP) levels. PAb gel improved mucosal healing, increased the number of viable osteocytes, while it reduced the number of empty lacunae, as well as the amount of bone sequestration. Furthermore, PAb gel positively influenced the number and functionality of osteoblasts by stimulating Wnt signaling, thereby inducing bone remodeling. Additionally, PAb gel contributed to improved bone quality, as evidenced by an increase in bone mineral content, a decrease in bone solubility, and an enhancement in the quality of collagen, particularly type I collagen. PAb gel mitigated bone necrosis by stimulating of bone remodeling through Wnt signaling and concurrently improved bone quality. PAb gel emerges as a promising pharmacological tool for aiding in BRONJ therapy or potentially preventing the development of BRONJ.

Abstract LB005: Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics

Abstract Hepatocellular carcinoma (HCC) is an aggressive tumor, and treatment options for patients with unresectable HCC are limited to targeted therapies and immunotherapy. The activation and dysregulation of MYC have been implicated in multiple cancers, including HCC, where approximately 30% of human HCC samples show MYC gene amplification. However, it is challenging to target MYC directly. Therefore, we applied a novel approach to circumvent targeting MYC directly by leveraging Pepper’s proprietary transomic analysis platform, COMPASS, to identify novel targets that are predicted to mimic the effect of turning MYC off. Specifically, COMPASS unlocks functional drivers of disease to identify novel drug targets. To study the role of MYC in tumor biology, we utilize an HCC cell line with tunable MYC expression. We generate genomic, transcriptomic, proteomic, and phosphoproteomic data from samples with MYC-on and MYC-off and compare via COMPASS to identify novel targets that mimic turning MYC off. The MYC-conditional HCC cell line (EC4) allows the regulation of MYC expression via the tetracycline regulatory (Tet-Off) system. Four omic datasets were collected from each sample: genomics (next-generation sequencing, NGS), transcriptomics (NGS), proteomics (mass spectrometry), and phosphoproteomics (mass spectrometry). We employed the COMPASS target prioritization algorithm to identify and rank novel targets that mimic turning MYC off. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in the Huh-7 xenograft model of HCC. All targets were kinases or related to kinase activity and pathways. All PTCs inhibited tumor growth from 18.1-78.3%. One multi-tyrosine kinase inhibitor resulted in maximum tumor growth inhibition, resulting in stasis of tumor growth and increased survival compared with control vehicle-treated mice. We have previously shown how the COMPASS transomics analysis approach can identify novel drug targets for drug resistant EGFRm NSCLC. The data presented here on MYC-driven HCC further validates this approach for novel target identification. Ongoing work will investigate novel targets using gene silencing, as PTCs were unavailable for many of these novel high-ranked targets identified in this study. Further validation studies are ongoing against other difficult-to-treat MYC-driven cancers to identify novel clinical targets and drug candidates to circumvent the MYC pathway. Citation Format: Simon P. Fricker, Christopher J. Nicholson, Samuel J. Roth, Arudhir Singh, Caitlin Brown, Jon Hu, Petronela Buiga, Vishnu P. Kanakaveti, Anja Deutzmann, Dean Felsher, Samantha D. Strasser. Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB005.

A novel potent class I HDAC inhibitor reverses the STAT4/p66Shc apoptotic defect in B cells from chronic lymphocytic leukemia patients

Chronic Lymphocytic Leukemia (CLL) patients have a defective expression of the proapoptotic protein p66Shc and of its transcriptional factor STAT4, which evoke molecular abnormalities, impairing apoptosis and worsening disease prognosis and severity. p66Shc expression is epigenetically controlled and transcriptionally modulated by STAT4; epigenetic modifiers are deregulated in CLL cells and specific histone deacetylases (HDACs) like HDAC1, are overexpressed. Reactivation of STAT4/p66Shc expression may represent an attractive and challenging strategy to reverse CLL apoptosis defects. New selective class I HDAC inhibitors (HDACis, 6a-g) were developed with increased potency over existing agents and preferentially interfering with the CLL-relevant isoform HDAC1, to unveil the role of class I HDACs in the upregulation of STAT4 expression, which upregulates p66Shc expression and hence normalizes CLL cell apoptosis. 6c (chlopynostat) was identified as a potent HDAC1i with a superior profile over entinostat. 6c induces marked apoptosis of CLL cells compared with SAHA, which was associated with an upregulation of STAT4/p66Shc protein expression. The role of HDAC1, but not HDAC3, in the epigenetic upregulation of STAT4/p66Shc was demonstrated for the first time in CLL cells and was validated in siRNA-induced HDAC1/HDAC3 knock-down EBV-B cells. To sum up, HDAC1 inhibition is necessary to reactivate STAT4/p66Shc expression in patients with CLL. 6c is one of the most potent HDAC1is known to date and represents a novel pharmacological tool for reversing the impairment of the STAT4/p66Shc apoptotic machinery.

The 2023 Lewis K. Dahl Memorial Lecture: Angiotensin-Converting Enzyme 2 Posttranslational Modifications-Implications for Hypertension and SARS-CoV-2 Infection.

ACE2 (angiotensin-converting enzyme 2), a multifunctional transmembrane protein, is well recognized as an important member of the (RAS) renin-angiotensin system with important roles in the regulation of cardiovascular function by opposing the harmful effects of Ang-II (angiotensin II) and AT1R (Ang-II type 1 receptor) activation. More recently, ACE2 was found to be the entry point for the SARS-CoV-2 virus into cells, causing COVID-19. This finding has led to an exponential rise in the number of publications focused on ACE2, albeit these studies often have opposite objectives to the preservation of ACE2 in cardiovascular regulation. However, notwithstanding accumulating data of the role of ACE2 in the generation of angiotensin-(1-7) and SARS-CoV-2 internalization, numerous other putative roles of this enzyme remain less investigated and not yet characterized. Currently, no drug modulating ACE2 function or expression is available in the clinic, and the development of new pharmacological tools should attempt targeting each step of the lifespan of the protein from synthesis to degradation. The present review expands on our presentation during the 2023 Lewis K. Dahl Memorial Lecture Sponsored by the American Heart Association Council on Hypertension. We provide a critical summary of the current knowledge of the mechanisms controlling ACE2 internalization and intracellular trafficking, the mutual regulation with GPCRs (G-protein-coupled receptors) and other proteins, and posttranslational modifications. A major focus is on ubiquitination which has become a critical step in the modulation of ACE2 cellular levels.

Novel protocol for multiple-dose oral administration of the L-type Ca2+ channel blocker isradipine in mice: A dose-finding pharmacokinetic study

ABSTRACT Studies in genetically modified animals and human genetics have recently provided new insight into the role of voltage-gated L-type Ca2+ channels in human disease. Therefore, the inhibition of L-type Ca2+ channels in vivo in wildtype and mutant mice by potent dihydropyridine (DHP) Ca2+ channel blockers serves as an important pharmacological tool. These drugs have a short plasma half-life in humans and especially in rodents and show high first-pass metabolism upon oral application. In the vast majority of in vivo studies, they have therefore been delivered through parenteral routes, mostly subcutaneously or intraperitoneally. High peak plasma concentrations of DHPs cause side effects, evident as DHP-induced aversive behaviors confounding the interpretation of behavioral readouts. Nevertheless, pharmacokinetic data measuring the exposure achieved with these applications are sparse. Moreover, parenteral injections require animal handling and can be associated with pain, discomfort and stress which could influence a variety of physiological processes, behavioral and other functional readouts. Here, we describe a noninvasive oral application of the DHP isradipine by training mice to quickly consume small volumes of flavored yogurt that can serve as drug vehicle. This procedure does not require animal handling, allows repeated drug application over several days and reproducibly achieves peak plasma concentrations over a wide range previously shown to be well-tolerated in humans. This protocol should facilitate ongoing nonclinical studies in mice exploring new indications for DHP Ca2+ channel blockers.

Design, synthesis, and biological evaluation of first-in-class FABP1 inhibitors for the treatment of NASH

The fatty acid-binding protein 1 (FABP1) is a fatty acid transporter protein that is considered as an emerging target for metabolic diseases. Despite forceful evidence that the inhibition of FABP1 is essential for ameliorating NASH, pharmacological control and validation of FABP1 are hindered by a lack of relevant inhibitors as pharmacological tool. Therefore, the development of effective FABP1 inhibitors is a current focus of research. Herein, we firstly reported the comprehensive structure-activity relationship (SAR) study of novel FABP1 inhibitors derived from high throughput screening of our in-house library, which resulting in the identification of the optimal compound 44 (IC50 = 4.46 ± 0.54 μM). Molecular docking studies revealed that 44 forms stable hydrogen bonds with amino acids around the active pocket of FABP1. Moreover, 44 alleviated the typical histological features of fatty liver in NASH mice, including steatosis, lobular inflammation, ballooning and fibrosis. Additionally, 44 has been demonstrated to have lipid metabolism regulating, anti-oxidative stress and hepatoprotective properties. This study might be provided a promising insight into the field of NASH and inspiration for the development of FABP1 inhibitors.

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions byhuman platelets,understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

CC Chemokine Family Members' Modulation as a Novel Approach for Treating Central Nervous System and Peripheral Nervous System Injury-A Review of Clinical and Experimental Findings.

Despite significant progress in modern medicine and pharmacology, damage to the nervous system with various etiologies still poses a challenge to doctors and scientists. Injuries lead to neuroimmunological changes in the central nervous system (CNS), which may result in both secondary damage and the development of tactile and thermal hypersensitivity. In our review, based on the analysis of many experimental and clinical studies, we indicate that the mechanisms occurring both at the level of the brain after direct damage and at the level of the spinal cord after peripheral nerve damage have a common immunological basis. This suggests that there are opportunities for similar pharmacological therapeutic interventions in the damage of various etiologies. Experimental data indicate that after CNS/PNS damage, the levels of 16 among the 28 CC-family chemokines, i.e., CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL11, CCL12, CCL17, CCL19, CCL20, CCL21, and CCL22, increase in the brain and/or spinal cord and have strong proinflammatory and/or pronociceptive effects. According to the available literature data, further investigation is still needed for understanding the role of the remaining chemokines, especially six of them which were found in humans but not in mice/rats, i.e., CCL13, CCL14, CCL15, CCL16, CCL18, and CCL23. Over the past several years, the results of studies in which available pharmacological tools were used indicated that blocking individual receptors, e.g., CCR1 (J113863 and BX513), CCR2 (RS504393, CCX872, INCB3344, and AZ889), CCR3 (SB328437), CCR4 (C021 and AZD-2098), and CCR5 (maraviroc, AZD-5672, and TAK-220), has beneficial effects after damage to both the CNS and PNS. Recently, experimental data have proved that blockades exerted by double antagonists CCR1/3 (UCB 35625) and CCR2/5 (cenicriviroc) have very good anti-inflammatory and antinociceptive effects. In addition, both single (J113863, RS504393, SB328437, C021, and maraviroc) and dual (cenicriviroc) chemokine receptor antagonists enhanced the analgesic effect of opioid drugs. This review will display the evidence that a multidirectional strategy based on the modulation of neuronal-glial-immune interactions can significantly improve the health of patients after CNS and PNS damage by changing the activity of chemokines belonging to the CC family. Moreover, in the case of pain, the combined administration of such antagonists with opioid drugs could reduce therapeutic doses and minimize the risk of complications.

Guanidine-to-piperidine switch affords high affinity small molecule NPFF ligands with preference for NPFF1-R and NPFF2-R subtypes

The Neuropeptide FF (NPFF) receptor system is known to modulate opioid actions and has been shown to mediate opioid-induced hyperalgesia and tolerance. The lack of subtype selective small molecule compounds has hampered further exploration of the pharmacology of this receptor system. The vast majority of available NPFF ligands possess a highly basic guanidine group, including our lead small molecule, MES304. Despite providing strong receptor binding, the guanidine group presents a potential pharmacokinetic liability for in vivo pharmacological tool development. Through structure-activity relationship exploration, we were able to modify our lead molecule MES304 to arrive at guanidine-free NPFF ligands. The novel piperidine analogues 8b and 16a are among the few non-guanidine based NPFF ligands known in literature. Both compounds displayed nanomolar NPFF-R binding affinity approaching that of the parent molecule. Moreover, while MES304 was non-subtype selective, these two analogues presented new starting points for subtype selective scaffolds, whereby 8b displayed a 15-fold preference for NPFF1-R, and 16a demonstrated an 8-fold preference for NPFF2-R. Both analogues showed no agonist activity on either receptor subtype in the in vitro functional activity assay, while 8b displayed antagonistic properties at NPFF1-R. The calculated physicochemical properties of 8b and 16a were also shown to be more favorable for in vivo tool design. These results indicate the possibility of developing potent, subtype selective NPFF ligands devoid of a guanidine functionality.

Further proof on the role of accumbal nNOS in cocaine-seeking behavior in rats.

Cocaine use disorder (CUD) remains a severe health problem with no effective pharmacological therapy. One of the potential pharmacological strategies for CUD pharmacotherapy includes manipulations of the brain glutamatergic (Glu) system which is particularly involved in drug withdrawal and relapse. Previous research indicated a pivotal role of ionotropic N-methyl-D-aspartate (NMDA) receptors or metabotropic receptors' type 5 (mGlu5) receptors in controlling the reinstatement of cocaine. Stimulation of the above molecules results in the activation of the downstream signaling targets such as neuronal nitric oxide synthase (nNOS) and the release of nitric oxide. In this paper, we investigated the molecular changes in nNOS in the prefrontal cortex and nucleus accumbens following 3 and 10days of cocaine abstinence as well as the effectiveness of nNOS blockade with the selective enzyme inhibitor N-ω-propyl-L-arginine hydrochloride (L-NPA) on cocaine seeking in male rats. The effect of L-NPA on locomotor activity in drug-naïve animals was investigated. Ten-day (but not 3-day) cocaine abstinence from cocaine self-administration increased nNOS gene and protein expression in the nucleus accumbens, but not in the prefrontal cortex. L-NPA (0.5-5mg/kg) administered peripherally did not change locomotor activity but attenuated the reinstatement induced with cocaine priming or the drug-associated conditioned cue. Our findings support accumbal nNOS as an important molecular player for cocaine seeking while its inhibitors could be considered as anti-cocaine pharmacological tools in male rats.

New approaches to second-degree burn healing: Polyvinyl alcohol membrane loaded to arnica combined to laser therapy.

Second-degree burns require greater care, as the damage is more extensive and worrisome and the use of a biomaterial can help in the cell repair process, with better planning, low cost, and better accessibility. Arnica has anti-inflammatory and analgesic properties in skin lesions treatments and laser therapy is another therapeutic alternative for burns. Evaluate the effects of arnica incorporated into PVA associated or not with low intensity laser on burns in rats. PVA and PVA with arnica (PVA+A) were obtained and characterized physicochemically. Through in vivo studies, the effects of PVA and PVA+A with or without the application of laser on the lesions allowed histological and immunohistochemical analyzes. PVA+A was biocompatible and with sustained release of the active, being a promising pharmacological tool and confirmed that laser therapy was effective in accelerating the healing process, due to its potential biomodulator, improving inflammatory aspects, promoting rapid healing in skin lesions.

Therapeutic Potential of Targeting the PERK Signaling Pathway in Ischemic Stroke.

Many pathologic states can lead to the accumulation of unfolded/misfolded proteins in cells. This causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR), which encompasses three main adaptive branches. One of these UPR branches is mediated by protein kinase RNA-like ER kinase (PERK), an ER stress sensor. The primary consequence of PERK activation is the suppression of global protein synthesis, which reduces ER workload and facilitates the recovery of ER function. Ischemic stroke induces ER stress and activates the UPR. Studies have demonstrated the involvement of the PERK pathway in stroke pathophysiology; however, its role in stroke outcomes requires further clarification. Importantly, considering mounting evidence that supports the therapeutic potential of the PERK pathway in aging-related cognitive decline and neurodegenerative diseases, this pathway may represent a promising therapeutic target in stroke. Therefore, in this review, our aim is to discuss the current understanding of PERK in ischemic stroke, and to summarize pharmacologic tools for translational stroke research that targets PERK and its associated pathways.

Forchlorfenuron-Induced Mitochondrial Respiration Inhibition and Metabolic Shifts in Endometrial Cancer.

Forchlorfenuron (FCF) is a widely used plant cytokinin that enhances fruit quality and size in agriculture. It also serves as a crucial pharmacological tool for the inhibition of septins. However, the precise target of FCF has not yet been fully determined. This study reveals a novel target of FCF and elucidates its downstream signaling events. FCF significantly impairs mitochondrial respiration and mediates metabolic shift toward glycolysis, thus making cells more vulnerable to glycolysis inhibition. Interestingly, FCF's impact on mitochondrial function persists, even in cells lacking septins. Furthermore, the impaired mitochondrial function leads to the degradation of HIF-1α, facilitated by increased cellular oxygen. FCF also induces AMPK activation, suppresses Erk1/2 phosphorylation, and reduces the expression of HER2, β-catenin, and PD-L1. Endometrial cancer is characterized by metabolic disorders such as diabetes and aberrant HER2/Ras-Erk1/2/β-catenin signaling. Thus, FCF may hold promise as a potential therapeutic in endometrial cancer.

Kisspeptin and neurokinin B neuroendocrine pathways in the control of human ovulation.

The roles of initially kisspeptin and subsequently neurokinin B pathways in the regulation of human reproduction through the control of GnRH secretion were first identified 20 years ago, as essential for the onset of puberty in both boys and girls. Within that short time we already now have the first licence for clinical use for a neurokinin antagonist in a related indication, for menopausal vasomotor symptoms. Between these two markers of the start and end of the reproductive lifespan, it is clear that these pathways underlie many of the aspects of the hypothalamic regulation of reproduction which had hitherto been enigmatic. In this review, we describe the data currently available from studies designed to elucidate the roles of kisspeptin and neurokinin B in human ovarian function, specifically the regulation of follicle development leading up to ovulation, and in the control of the mid-cycle GnRH/LH surge that triggers ovulation. These studies, undertaken with only very limited pharmacological tools, provide evidence that the neurokinin B pathway is important in controlling the hypothalamic contribution to the precise gonadotropic drive to the ovary that is necessary for mono-ovulation, whereas the switch from negative to positive estrogenic feedback results in kisspeptin-mediated increased GnRH secretion. Potential therapeutic opportunities in conditions characterised by disordered hypothalamic/pituitary function, polycystic ovary syndrome, and functional hypothalamic amenorrhoea, and in the induced LH surge that is a necessary part of IVF treatment are discussed.

Morita-Baylis-Hillman adduct 2-(3-hydroxy-1-methyl-2-oxoindolin-3-il) acrylonitrile (CISACN) ameliorates the pulmonary allergic inflammation in CARAS model by increasing IFN-γ/IL-4 ratio towards the Th1 immune response

Combined allergic rhinitis and asthma syndrome (CARAS) is an airway-type 2 immune response with a profuse inflammatory process widely affecting the world population. Due to the compromise of quality of life and the lack of specific pharmacotherapy, the search for new molecules becomes relevant. This study aimed to evaluate the effectiveness of the Morita-Bailys-Hillman adduct (CISACN) treatment in the CARAS experimental model. Female BALB/c mice were ovalbumin (OVA) -sensitized and -challenged and treated with CISACN. The treatment decreased the eosinophil migration to the nasal and lung cavities and tissues and the goblet cell hyperplasia/hypertrophy, attenuated airway hyperactivity by reducing the hyperplasia/hypertrophy of the smooth muscle and the extracellular matrix's thickness. Also, the treatment reduced the clinical signs of rhinitis as nasal rubbing and sneezing in a histamine-induced nasal hyperreactivity assay. The immunomodulatory effect of CISACN was by reducing OVA-specific IgE serum level, and IL-33, IL-4, IL-13, and TGF-β production, dependent on IFN-γ increase. Furthermore, the effect of CISACN on lung granulocytes was by decreasing the p-p38MAPK/p65NF-κB signaling pathway. Indeed, CISACN reduced the p38MAPK and p65NF-κB activation. These data demonstrated the anti-inflammatory and immunomodulatory effects of the CISACN with scientific support to become a pharmacological tool to treat airway inflammatory diseases.

Cutaneous T-Cell Lymphoma and Microbiota: Etiopathogenesis and Potential New Therapeutic Targets.

To review the scientific literature related to human microbiota and cutaneous T-cell lymphoma. Methodology. An exploratory and systematic review of the articles retrieved from the bibliographic databases MEDLINE (PubMed), Embase, The Cochrane Library, and Scopus, published in the last 10 years with the following descriptors: "lymphoma, T-cell, cutaneous," "microbiota," "Mycosis Fungoides," "Sézary Syndrome," "lymphoma, primary cutaneous anaplastic large cell," "Lymphomatoid Papulosis" and "Microbiota," "microbiota," "Microbial Community," and "Microbial Communities." Of the 87 references retrieved, after applying the inclusion and exclusion criteria, 21 articles were selected. Most studies linking cutaneous T-cell lymphoma and the microbiota focus on the cutaneous microbiome, with Staphylococcus aureus being the main related agent. Skin colonization by this bacterium could be involved in the hyperactivation of the STAT3 inflammatory pathway and in the overproduction of IL-17, both of which are widely related to the development of more aggressive and advanced forms of cutaneous T-cell lymphoma. We also found evidence of a possible relationship between intestinal dysbiosis and the development of cutaneous T-cell lymphoma, observing a decrease in taxonomic variability and an increase in certain genera such as Prevotella in the intestinal microbiome of patients with cutaneous T-cell lymphoma. The possible etiopathogenic mechanism underlying this relationship could be explained by an increase in systemic cytokine release, promoting the hyperactivation of STAT3 at the skin level. There appears to be a relationship between cutaneous T-cell lymphoma and the cutaneous and intestinal microbiome, as well as a possible pathophysiological pathway involved. The possible modulation of the cutaneous and intestinal microbiome or the action on the signaling inflammatory pathway, using pharmacological tools such as JAK inhibitors or IL-17 inhibitors in the latter case, could open the possibility for future therapeutic studies for cutaneous T-cell lymphoma.

The NMDA receptor modulator zelquistinel durably relieves behavioral deficits in three mouse models of autism spectrum disorder

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders characterized by deficient social communication and interaction together with restricted, stereotyped behaviors. Currently approved treatments relieve comorbidities rather than core symptoms. Since excitation/inhibition balance and synaptic plasticity are disrupted in ASD, molecules targeting excitatory synaptic transmission appear as highly promising candidates to treat this pathology. Among glutamatergic receptors, the NMDA receptor has received particular attention through the last decade to develop novel allosteric modulators. Here, we show that positive NMDA receptor modulation by zelquistinel, a spirocyclic β-lactam platform chemical, relieves core symptoms in two genetic and one environmental mouse models of ASD. A single oral dose of zelquistinel rescued, in a dose-response manner, social deficits and stereotypic behavior in Shank3Δex13-16−/− mice while chronic intraperitoneal administration promoted a long-lasting relief of such autistic-like features in these mice. Subchronic oral mid-dose zelquistinel treatment demonstrated durable effects in Shank3Δex13-16−/−, Fmr1−/− and in utero valproate-exposed mice. Carry-over effects were best maintained in the Fmr1 null mouse model, with social parameters being still fully recovered two weeks after treatment withdrawal. Among recently developed NMDA receptor subunit modulators, zelquistinel displays a promising therapeutic potential to relieve core symptoms in ASD patients, with oral bioavailability and long-lasting effects boding well for clinical applications. Efficacy in three mouse models with different etiologies supports high translational value. Further, this compound represents an innovative pharmacological tool to investigate plasticity mechanisms underlying behavioral deficits in animal models of ASD.