Spatial profiling of METex14-altered NSCLC under tepotinib treatment: Shifting the immunosuppressive landscape.

MET inhibitors have demonstrated efficacy in treating patients with non-small cell lung cancer (NSCLC) harboring METex14 skipping alterations. Advancements in spatial profiling technologies have unveiled the complex dynamics of the tumor microenvironment (TME), a crucial factor in cancer progression and therapeutic response. This study uses spatial profiling to investigate the effects of the MET inhibitor tepotinib on the TME in a case of locally advanced NSCLC with a METex14 skipping alteration. A patient with resectable stage IIIB NSCLC, unresponsive to neoadjuvant platinum-based chemotherapy, received tepotinib following the detection of a METex14 skipping alteration. Paired pre- and post-treatment biopsies were subjected to GeoMx Digital Spatial Profiling using the Cancer Transcriptome Atlas and immune-related protein panels to evaluate shifts in the immune TME. Tepotinib administration allowed for a successful lobectomy and a pathological downstaging to stage IA1. The TME was transformed from an immunosuppressive to a more permissive state, with upregulation of antigen-presenting and pro-inflammatory immune cells. Moreover, a marked decrease in immune checkpoint molecules, including PD-L1, was noted. Spatial profiling identified discrete immune-enriched clusters, indicating the role of tepotinib in modulating immune cell trafficking and function. Tepotinib appears to remodel the immune TME in a patient with METex14 skipping NSCLC, possibly increasing responsiveness to immunotherapy. Our study supports the integration of genetic profiling into the management of early and locally advanced NSCLC to guide personalized, targeted interventions. These findings underscore the need to further evaluate combinations of MET inhibitors and immunotherapies.

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.

Novel pyridazinone derivatives bind to KSRP: Synthesis, anti-tumor biological evaluations and modelling insights.

Pyridazinone derivatives have been extensively used as anticancer agents. IMB5036 is a structure specific pyridazinone compound with potential antitumor activity via targeting KSRP protein which controls gene expression at multiple levels. In this study, fifteen IMB5036 analogues were synthesized and preliminary structure-activity relationships were explored. Among them, compounds 8 and 10 exhibited remarkably anti-proliferation of various cancer cells and a good cancer cell selectivity (against human fetal hepatocyte L02 cells). More detailed investigation was included that both 8 and 10 inhibited colony formation and migration in concentration-dependent mode against MCF-7 cells. Additionally, 8 and 10 induced apoptosis and cell cycle arrest, decreased mitochondrial membrane potential, damaged DNA, and increased reactive oxygen species. Moreover, 8 displayed a potent antitumor efficacy (TGI = 74.2 %, at a dose of 30 mg/kg) in MCF-7 xenograft model by i.p. injection. Further, we synthesized a biotinylated probe 16 for identifying the detail domain of KSRP. Through pull down assay and molecular docking study, we validated that the KH23 domain functioned as the binding pocket for the compounds. Thus, compound 8 was identified as a novel targeting KSRP pyridazinone-based compound and exhibited excellent antitumor activity both in vitro and in vivo.

Imatinib­ and ponatinib­mediated cardiotoxicity in zebrafish embryos and H9c2 cardiomyoblasts.

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose­dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose­dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro, H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo, zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription­quantitative PCR (RT­qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose­dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose­dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose­dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI­induced cardiotoxicities.

Explainable machine learning prediction of edema adverse events in patients treated with tepotinib.

Tepotinib is approved for the treatment of patients with non-small-cell lung cancer harboring MET exon 14 skipping alterations. While edema is the most prevalent adverse event (AE) and a known class effect of MET inhibitors including tepotinib, there is still limited understanding about the factors contributing to its occurrence. Herein, we apply machine learning (ML)-based approaches to predict the likelihood of occurrence of edema in patients undergoing tepotinib treatment, and to identify factors influencing its development over time. Data from 612 patients receiving tepotinib in five Phase I/II studies were modeled with two ML algorithms, Random Forest, and Gradient Boosting Trees, to predict edema AE incidence and severity. Probability calibration was applied to give a realistic estimation of the likelihood of edema AE. Best model was tested on follow-up data and on data from clinical studies unused while training. Results showed high performances across all the tested settings, with F1 scores up to 0.961 when retraining the model with the most relevant covariates. The use of ML explainability methods identified serum albumin as the most informative longitudinal covariate, and higher age as associated with higher probabilities of more severe edema. The developed methodological framework enables the use of ML algorithms for analyzing clinical safety data and exploiting longitudinal information through various covariate engineering approaches. Probability calibration ensures the accurate estimation of the likelihood of the AE occurrence, while explainability tools can identify factors contributing to model predictions, hence supporting population and individual patient-level interpretation.

Levosimendan as an Antidote in Experimental Calcium Channel Blocker Intoxication.

ABSTRACT: The effects of the calcium sensitizer levosimendan on hemodynamics and survival in guinea pigs intoxicated with the calcium blockers verapamil or diltiazem were evaluated in a randomized controlled study. One hundred four animals were randomized to be intoxicated with either verapamil (2.0 mg/kg) or diltiazem (4.5 mg/kg) and thereafter further randomized into 6 groups which received either saline (control), 3 different regimes of levosimendan, calcium chloride, and levosimendan combined with calcium chloride. The hemodynamics and survival of the animals were followed for 60 minutes after intoxication.The negative inotropic effect of calcium blockers was seen as a decrease by over 70% of the positive derivative of the left ventricular pressure. This was reversed by levosimendan. Moreover, both verapamil and diltiazem-induced marked hypotension (-69% and -63% of the baseline value, respectively) which was also reversed by levosimendan. The combined levosimendan and calcium chloride treatment had a synergistic effect in reversing verapamil or diltiazem-induced deterioration in hemodynamics.Both verapamil and diltiazem intoxications decreased the survival rate of guinea pigs to 13%. Levosimendan addition improved survival dose-dependently up to a survival rate of 75% and 88% in the verapamil and diltiazem groups, respectively. Low dose of levosimendan combined with calcium chloride improved survival in verapamil and diltiazem group to 88% and 100%, respectively.In conclusion, the administration of levosimendan improved hemodynamics and survival in calcium channel blocker intoxicated guinea pigs. The synergistic effect of levosimendan and calcium chloride suggests that this combination could be an effective antidote in calcium channel blocker intoxications.

In Silico Molecular Modeling of Four New Afatinib Derived Molecules Targeting the Inhibition of the Mutated Form of BCR-ABL T315I.

Four afatinib derivatives were designed and modeled. These derivatives were compared to the known tyrosine-kinase inhibitors in treating Chronic Myeloid Leukemia, i.e., imatinib and ponatinib. The molecules were evaluated through computational methods, including docking studies, the non-covalent interaction index, Electron Localization and Fukui Functions, in silico ADMET analysis, QTAIM, and Heat Map analysis. The AFA(IV) candidate significantly increases the score value compared to afatinib. Furthermore, AFA(IV) was shown to be relatively similar to the ponatinib profile when evaluating a range of molecular descriptors. The addition of a methylpiperazine ring seems to be well distributed in the structure of afatinib when targeting the BCR-ABL enzyme, providing an important hydrogen bond interaction with the Asp381 residue of the DFG-switch of BCR-ABL active site residue and the AFA(IV) new chemical entities. Finally, in silico toxicity predictions show a favorable index, with some molecules presenting the loss of the irritant properties associated with afatinib in theoretical predictions.

Inhibition Effect of STING Agonist SR717 on PRRSV Replication.

The porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the Arteriviridae family and is a single-stranded, positively stranded RNA virus. The currently available PRRSV vaccines are mainly inactivated and attenuated vaccines, yet none of the commercial vaccines can provide comprehensive, long-lasting, and effective protection against PRRSV. SR717 is a pyridazine-3-carboxamide compound, which is commonly used as a non-nucleoside STING agonist with antitumor and antiviral activities. Nevertheless, there is no evidence that SR717 has any antiviral effects against PRRSV. In this study, a dose-dependent inhibitory effect of SR717 was observed against numerous strains of PRRSV using qRT-PCR, IFA, and WB methods. Furthermore, SR717 was found to stimulate the production of anti-viral molecules and trigger the activation of the signaling cascade known as the stimulator of interferon genes (STING) pathway, which contributed to hindering the reproduction of viruses by a certain margin. Collectively, these results indicate that SR717 is capable of inhibiting PRRSV infection in vitro and may have potential as an antiviral drug against PRRSV.

Oxidative lipid damage by naringenin selectively sensitizes chronic myeloid leukemia cell lines and patient samples to Bcr-Abl tyrosine kinase inhibitors.

Chronic myeloid leukemia (CML) treatment with Bcr-Abl tyrosine kinase inhibitors (TKIs) has significantly improved patient outcomes, yet challenges such as drug resistance and persistence of leukemic stem cells persist. This study explores the potential of naringenin, a natural flavonoid, to enhance the efficacy of Bcr-Abl TKIs in CML therapy. We showed that naringenin reduces viability of a panel of CML cell lines regardless of varying cellular origin and genetic mutations, and acts synergistically with dasatinib and ponatinib. Importantly, naringenin is effective in targeting blast crisis CML CD34+ cells by decreasing their colony formation, self-renewal and viability. Compared to CML, naringenin is significantly less effective against normal bone marrow (NBM) counterparts. In addition, naringenin significantly enhances the inhibitory effects of dasatinib in CML but not NBM CD34+ cells. Mechanism studies showed that naringenin's inhibitory effects were associated with the induction of oxidative stress and lipid damage, as evidenced by increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Notably, naringenin upregulated genes related to mitochondrial biogenesis while downregulating antioxidant defense genes. Pretreatment with α-tocopherol, which inhibits lipid-mediated ROS production, completely abolished the ROS increase and restored cell viability, indicating that lysosomal lipid peroxidation plays a crucial role in naringenin's mechanism of action. In a CML xenograft mouse model, the combination of naringenin and dasatinib resulted in remarkably more tumor growth suppression compared to single drug alone. Importantly, this combination was well-tolerated, with no adverse effects on body weight observed. These findings suggest that naringenin, by inducing oxidative lipid damage, enhances the anti-leukemic effects of Bcr-Abl TKIs, offering a promising therapeutic strategy for CML.

Overcoming clinical BCR-ABL1 compound mutant resistance with combined ponatinib and asciminib therapy.

BCR-ABL1 compound mutations can lead to resistance to ABL1 inhibitors in chronic myeloid leukemia (CML), which could be targeted by combining the ATP-site inhibitor ponatinib and the allosteric inhibitor asciminib. Here, we report the clinical validation of this approach in a CML patient, providing a basis for combination therapy to overcome such resistance.

Empagliflozin mitigates ponatinib-induced cardiotoxicity by restoring the connexin 43-autophagy pathway.

BACKGROUND: Empagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AIMS: To assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. METHODS AND RESULTS: Male C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20 %, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. CONCLUSION: EMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.

Development of Brain Penetrant Pyridazine Pantothenate Kinase Activators.

Conversion of pantothenate to phosphopantothenate in humans is the first dedicated step in the coenzyme A (CoA) biosynthesis pathway and is mediated by four isoforms of pantothenate kinase. These enzymes are allosterically regulated by acyl-CoA levels, which control the rate of CoA biosynthesis. Small molecule activators of the PANK enzymes that overcome feedback suppression increase CoA levels in cultured cells and animals and have shown great potential for the treatment of pantothenate kinase-associated neurodegeneration and propionic acidemias. In this study, we detail the further optimization of PANK pyridazine activators using structure-guided design and focus on the cellular CoA activation potential, metabolic stability, and solubility as the primary drivers of the structure-activity relationship. These studies led to the prioritization of three late-stage preclinical lead PANK modulators with improved pharmacokinetic profiles and the ability to substantially increase brain CoA levels. Compound 22 (BBP-671) eventually advanced into clinical testing for the treatment of PKAN and propionic acidemia.

Association between cytokines, nitric oxide, hemodynamic and microcirculation in a porcine model of sepsis.

Systemic inflammation and hemodynamic or microvascular alterations are a hallmark of sepsis and play a role in organs hypoperfusion and dysfunction. Pimobendan, an inodilator agent, could be an interesting option for inotropic support and microcirculation preservation during shock. The objectives of this study were to evaluate effect of pimobendan on cytokine and nitric oxide (NO) release and investigate whether changes of macro and microcirculation parameters are associated with the release of cytokines and NO in pigs sepsis model. After circulatory failure, induced by intravenous inoculation of live Pseudomonas aeruginosa, eight animals were treated with pimobendan and eight with placebo. Pimobendan did not affect cytokines secretion (TNF-α, IL-6 and IL-10), but decreased time-dependently NO release. Data of macro and microcirculation parameters, NO and TNF- α recorded at the time of circulatory failure (Thypotension) and the time maximum of production cytokines was used for analyses. A positive correlation was observed between TNF-α and cardiac index (r = 0.55, p = 0.03) and a negative with systemic vascular resistance (r = -0.52, p = 0.04). Positive correlations were seen both between IL-10, 30 min after resuscitation (T30min), and systolic arterial pressure (r = 0.57, p = 0.03) and cardiac index (r = 0.67, p = 0.01), and also between IL-6, taken 2 h after resuscitation and systolic arterial pressure (r = 0.53, p = 0.04). Negative correlations were found between IL-10 and lactate, measured resuscitation time (r = -0.58, p = 0.03). Regarding microcirculation parameters, we observed a positive correlation between IL-6 and IL-10 with the microvascular flow index (r = 0.52, p = 0.05; r = 0.84, p = 0.0003) and a negative correlation with the heterogeneity index with TNF-α and IL-10 (r = -0.51, p = 0.05; r = -0.74, p = 0.003) respectively. NO derivatives showed a positive correlation with temperature gradient (r = 0.54, p = 0.04). Pimobendan did not show anti-inflammatory effects in cytokines release. Our results also, suggest changes of macro- and microcirculation are associated mainly with low levels of IL-10 in sepsis.

Pyridaben inhibits cell cycle progression and delays early embryonic development in zebrafish (Danio rerio).

Pyridaben is a broad-spectrum, contact-killing acaricide that can be used to control a variety of harmful food and plant mites. Pyridaben displays cardiotoxicity and liver toxicity toward fish, but the effects on fish embryonic development have not been characterized. We exposed early zebrafish embryos to 20, 30, and 40 µg/L concentrations of pyridaben. The exposure caused developmental abnormalities, including delayed embryonic shield formation, yolk sac resorption, decreases in body length, reduced pigmentation, and delays in hatching. Pyridaben caused a significant increase in the transcription level of the endoderm marker foxa2, but the transcription levels of the ectoderm development marker foxb1a and the mesoderm development marker snaila were not significantly altered. The transcription levels of the genes SOX17 in early embryos were significantly reduced. After exposure to pyridaben, catalase (CAT) activity and glutathione (GSH) content were increased, and cyclin D1, that is involved in early embryonic development, was abnormally expressed. This study shows that pyridaben causes anomalous development in zebrafish embryos by interfering with the cell cycle order of early embryonic development and inducing excessive oxidative stress. Colivelin, an agonist of the STAT3 signaling pathway, acted as a salvage drug to restore the cell cycle order during embryonic development following exposure to pyridaben. Thus, the toxic effects may be caused by pyridaben's regulation of the STAT3 signaling pathway.

Emerging therapeutics: The imidazo[1,2-b]pyridazine scaffold as a novel drug candidate for eumycetoma, a neglected tropical disease.

Mycetoma is a neglected invasive infection endemic in tropical and subtropical regions, presenting as a chronic subcutaneous inflammatory mass that can spread to deeper structures, leading to deformities, disabilities, and potentially mortality. The current treatment of eumycetoma, the fungal form of mycetoma, involves antifungal agents, such as itraconazole, combined with surgical intervention. However, this approach has limited success, with low cure rates and a high risk of recurrence. This study addresses to the urgent need for more effective therapeutics by designing and synthesising 47 diversely pharmacomodulated imidazo [1,2-b]pyridazine derivatives using a simple synthetic pathway with good yields and purity. Of these, 17 showed promising in vitro activity against Madurella mycetomatis, the prime causative agent of eumycetoma, with IC50 ≤ 5 µM and demonstrated significantly lower cytotoxicity compared to standard treatments in NIH-3T3 fibroblasts. Notably, compound 14d exhibited an excellent activity with an IC50 of 0.9 µM, in the same order then itraconazole (IC50 = 1.1 µM), and achieved a favourable selectivity index of 16 compared to 0.8 for itraconazole. These promising results warrant further research to evaluate the clinical potential of these novel compounds as safer, more effective treatments for eumycetoma, thus addressing a profound gap in current therapeutic strategies.

Efficacy and safety of Resmetirom, a selective thyroid hormone receptor-ß agonist, in the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD): a systematic review and meta-analysis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an important public health problem owing to its high prevalence and associated morbidity and mortality secondary to progressive liver disease and cardiovascular events. Resmetirom, a selective thyroid hormone receptor-ß agonist has been developed as a therapeutic modality for MASLD. This systematic review and meta-analysis aimed to evaluate the effectiveness and safety of resmetirom compared to a placebo in the treatment of MASLD. Eligible studies were systematically identified by screening PubMed, Scopus, Web of Science, Cochrane library, Embase, and ClinicalTrials.gov from 2014 to 2024. Only randomized controlled trials comparing the efficacy and safety of resmetirom in the treatment of MASLD against placebo were included in the analysis. Meta-analysis was performed using RevMan 5.4 software. Four studies with low risk of bias and involving a total of 2359 participants were identified. The metanalysis included only three clinical trials with 2234 participants. A significant reduction in MRI-proton density fat fraction (MRI-PDFF) with 80 mg Resmetirom compared to that with placebo [SMD - 27.74 (95% CI - 32.05 to - 32.42), p < 0.00001] at 36-52 weeks as well as at 12-16 weeks [SMD - 30.92 (95% CI - 36.44 to - 25.40), p < 0.00001]. With Resmetirom 100 mg dose at 36-52 weeks [SMD - 36.05 (95% CI - 40.67 to - 31.43), p < 0.00001] and 12-16 weeks [SMD - 36.89 (95% CI - 40.73 to - 33.05), p < 0.00001] were observed. Resmetirom treatment was associated with a significant reduction in LDL-c triglyceride, lipoproteins. and liver enzymes. There was significant reduction FT4 and increase in SHBG and sex steroids with Resmetirom compared to placebo. There was no major difference in the overall treatment emergent adverse events at 80 mg [OR 1.55 (95% CI 0.84 to 2.87), and 100 mg [OR 1.13 (95% CI 0.78 to 1.63), doses of Resmetirom compared to placebo. However, gastrointestinal adverse events diarrhoea and nausea occurred in ≥ 10% in the Resmetirom group compared to placebo at < 12 week. Resmetirom treatment showed modest efficacy in treating MASLD with reduction in MRI-PDFF, LDL-c, triglyceride, lipoproteins, liver enzymes and NASH biomarkers without significant safety concerns. Larger and long-term RCTs may further confirm this promising outcomes of Resmetirom use in MASLD.

Discovery of Potent, Specific, and Orally Available NLRP3 Inflammasome Inhibitors Based on Pyridazine Scaffolds for the Treatment of Septic Shock and Peritonitis.

The NLRP3 inflammasome is a multiprotein complex that is a component of the innate immune system, involved in the production of pro-inflammatory cytokines. Its abnormal activation is associated with many inflammatory diseases. In this study, we designed and synthesized a series of NLRP3 inflammasome inhibitors based on pyridazine scaffolds. Among them, P33 exhibited significant inhibitory effects against nigericin-induced IL-1ß release in THP-1 cells, BMDMs, and PBMCs, with IC50 values of 2.7, 15.3, and 2.9 nM, respectively. Mechanism studies indicated that P33 directly binds to NLRP3 protein (KD = 17.5 nM), inhibiting NLRP3 inflammasome activation and pyroptosis by suppressing ASC oligomerization during NLRP3 assembly. Additionally, P33 displayed excellent pharmacokinetic properties, with an oral bioavailability of 62%. In vivo efficacy studies revealed that P33 significantly ameliorated LPS-induced septic shock and MSU crystal-induced peritonitis in mice. These results indicate that P33 has great potential for further development as a candidate for treating NLRP3 inflammasome-mediated diseases.

Tepotinib plus osimertinib in patients with EGFR-mutated non-small-cell lung cancer with MET amplification following progression on first-line osimertinib (INSIGHT 2): a multicentre, open-label, phase 2 trial.

BACKGROUND: Patients with EGFR-mutated non-small-cell lung cancer (NSCLC) and MET amplification as a mechanism of resistance to first-line osimertinib have few treatment options. Here, we report the primary analysis of the phase 2 INSIGHT 2 study evaluating tepotinib, a highly selective MET inhibitor, combined with osimertinib in this population. METHODS: This open-label, phase 2 study was conducted at 179 academic centres and community clinics in 17 countries. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1 and advanced or metastatic EGFR-mutated NSCLC of any histology, with MET amplification by tissue biopsy fluorescence in-situ hybridisation (FISH; MET gene copy number of ≥5 or MET-to-CEP7 ratio of ≥2) or liquid biopsy next-generation sequencing (MET plasma gene copy number of ≥2·3), following progression on first-line osimertinib. Patients received oral tepotinib 500 mg plus oral osimertinib 80 mg once daily. The primary endpoint was independently assessed objective response in patients with MET amplification by central FISH treated with tepotinib plus osimertinib with at least 9 months of follow-up. Safety was analysed in patients who received at least one study drug dose. This study is registered with ClinicalTrials.gov, NCT03940703 (enrolment complete). FINDINGS: Between Feb 13, 2020, and Nov 4, 2022, 128 patients (74 [58%] female, 54 [42%] male) were enrolled and initiated tepotinib plus osimertinib. The primary activity analysis population included 98 patients with MET amplification confirmed by central FISH, previous first-line osimertinib and at least 9 months of follow-up (median 12·7 months [IQR 9·9-20·3]). The confirmed objective response rate was 50·0% (95% CI 39·7-60·3; 49 of 98 patients). The most common treatment-related grade 3 or worse adverse events were peripheral oedema (six [5%] of 128 patients), decreased appetite (five [4%]), prolonged electrocardiogram QT interval (five [4%]), and pneumonitis (four [3%]). Serious treatment-related adverse events were reported in 16 (13%) patients. Deaths of four (3%) patients were assessed as potentially related to either trial drug by the investigator due to pneumonitis (two [2%] patients), decreased platelet count (one [1%]), respiratory failure (one [1%]), and dyspnoea (one [1%]); one death was attributed to both pneumonitis and dyspnoea. INTERPRETATION: Tepotinib plus osimertinib showed promising activity and acceptable safety in patients with EGFR-mutated NSCLC and MET amplification as a mechanism of resistance to first-line osimertinib, suggesting a potential chemotherapy-sparing oral targeted therapy option that should be further investigated. FUNDING: Merck (CrossRef Funder ID: 10.13039/100009945).

Investigations on 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,2-a]pyridazin-1-amines and related compounds: synthesis, chemical behaviour, structure elucidation and iNOS inhibitory activity.

The present work reports on the preparation of the hitherto unknown title compounds 5, with various synthetic routes described. The initially pursued concept of S-N exchange with varioius 1-substituted 3-methylsulfanyl-5,6,7,8-tetrahydro-1 H -[1,2,4]triazolo[1,2- a ]pyridazines 4 by using nitrogen nucleophiles was only marginally successful. The reactions proceeded slowly and the yields were low, mainly because of the pronounced formation of 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,2- a ]pyridazin-1-imines 7 by oxidation of the heterocyclic amines 5 initially formed. The integration of the synthesis of 3-acylsulfanyl analogues with the more reactive leaving groups also failed. On the other hand, the cyclization of the hydrohalides of hexahydropyridazine-1-carboximidamide with aromatic aldehydes and some low molecular weight ketones gives significantly better results in the synthesis of the title compounds 5. The use of the hydrochloride 6b proved to be advantageous in comparison to the hydroiodide 6a because the yields were significantly better and the imines 7 formed at the same time only to a small extent. In addition, the starting compound 6b can be prepared in a single-step synthesis in very good yield from hexahydropyridazine hydrochloride 1 and cyanamide. The cyclization of N' -phenylhexahydropyridazine-1-carboximidamide hydrochloride 6c with substituted benzaldehydes gives the 3-aryl-substituted 2-phenyl-2,3,5,6,7,8-hexahydro -1H -[1,2,4]triazolo[1,2- a ] pyridazin-1-imines 8. In the context with the study of the reaction of hexahydropyridazine-1-carboximidamide hydroiodide 6a with cyclohexanone, the hexahydropyridazine-1-carboxamide 9 was specifically synthesized. This can be reacted with aromatic aldehydes to give the 5,6,7,8-tetrahydro-1 H -[1,2,4]triazolo[1,2- a ]pyridazin-1-ones 10 in very good yields. The results of the biological testing of representatives of the synthesized 5,6,7,8-tetrahydro-[1,2,4] triazolo[1,2-a]pyridazine-1-amines 5 show, in comparison to the already examined thions 3 and 3-methylsulfanyl derivatives 4, significantly less inducible nitric oxide synthase (iNOS) inhibitory activity.

Beneficial effects of MGL-3196 and BAM15 combination in a mouse model of fatty liver disease.

BACKGROUND AND AIM: Metabolic dysfunction-associated steatohepatitis (MASH) is a metabolic disorder with limited treatment options. The thyroid hormone receptor (THR)-ß agonist resmetirom/MGL-3196 (MGL) increases liver fat oxidation and has been approved for treating adult MASH. However, over 60% of patients receiving MGL treatment do not achieve MASH resolution. Therefore, we investigated the potential for combination therapy of MGL with the mitochondrial uncoupler BAM15 to improve fatty liver disease outcomes in the GAN mouse model of MASH. METHODS: C57BL/6J male mice were fed GAN diet for 38 weeks before stratification and randomization to treatments including MGL, BAM15, MGL + BAM15, or no drug control for 8 weeks. Treatments were admixed in diet and mice were pair-fed to control for drug intake. Treatment effectiveness was assessed by body weight, body composition, energy expenditure, glucose tolerance, tissue lipid content, and histological analyses. RESULTS: MGL + BAM15 treatment resulted in better efficacy versus GAN control mice than either monotherapy in the context of energy expenditure, liver fat loss, glucose control, and fatty liver disease activity score. Improvements in ALT, liver mass, and plasma cholesterol were primarily driven by MGL, while improvements in body fat were primarily driven by BAM15. No treatments altered liver fibrosis. CONCLUSIONS: MGL + BAM15 treatment had overall better efficacy to improve metabolic outcomes in mice fed GAN diet than either monotherapy alone. These data warrant further investigation into combination therapies of THR-ß agonists and mitochondrial uncouplers for the potential treatment of disorders related to fatty liver, obesity, and insulin resistance.