Phase 2A Learnings Incorporated into RewinD-LB, a Phase 2B Clinical Trial of Neflamapimod in Dementia with Lewy Bodies.

BACKGROUND: In an exploratory 91-participant phase 2a clinical trial (AscenD-LB, NCT04001517) in dementia with Lewy bodies (DLB), neflamapimod showed improvement over placebo on multiple clinical endpoints. To confirm those results, a phase 2b clinical study (RewinD-LB, NCT05869669 ) that is similar to AscenD-LB has been initiated. OBJECTIVES: To optimize the choice of patient population, primary endpoint, and biomarker evaluations in RewinD-LB. DESIGN: Evaluation of the efficacy results from AscenD-LB, the main results of which, and a re-analysis after stratification for absence or presence of AD co-pathology (assessed by plasma ptau181), have been published. In addition, the MRI data from a prior phase 2a clinical trial in Early Alzheimer's disease (AD), were reviewed. SETTING: 22 clinical sites in the US and 2 in the Netherlands. PARTICIPANTS: Probable DLB by consensus criteria and abnormal dopamine uptake by DaTscan™ (Ioflupane I123 SPECT). INTERVENTION: Neflamapimod 40mg capsules or matching placebo capsules, twice-a-day (BID) or three-times-a-day (TID), for 16 weeks. MEASUREMENTS: 6-test Neuropsychological Test Battery (NTB) assessing attention and executive function, Clinical Dementia Rating Sum-of-Boxes (CDR-SB), Timed Up and Go (TUG), International Shopping List Test (ISLT). RESULTS: Within AscenD-LB, patients without evidence of AD co-pathology exhibited a neflamapimod treatment effect that was greater than that in the overall population and substantial (cohen's d effect size vs. placebo ≥ for CDR-SB, TUG, Attention and ISLT-recognition). In addition, the CDR-SB and TUG performed better than the cognitive tests to demonstrate neflamapimod treatment effect in comparison to placebo. Further, clinical trial simulations indicate with 160-patients (randomized 1:1), RewinD-LB conducted in patients without AD co-pathology has >95% (approaching 100%) statistical power to detect significant improvement over placebo on the CDR-SB. Preliminary evidence of positive treatment effects on beta functional connectivity by EEG and basal forebrain atrophy by MRI were obtained in AscenD-LB and the Early AD study, respectively. CONCLUSION: In addition to use of a single dose regimen of neflamapimod (40mg TID), key distinctions between phase 2b and phase 2a include RewinD-LB (1) excluding patients with AD co-pathology, (2) having CDR-SB as the primary endpoint, and (3) having MRI studies to evaluate effects on basal forebrain atrophy.

A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Comparison of two autologous hematopoietic stem cell mobilization strategies in patients with multiple myeloma: CE plus G-CSF versus G-CSF only: A single-center retrospective analysis.

BACKGROUND: Despite recent advances in the treatment of multiple myeloma, high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) remains an essential therapeutic keystone. As for the stem cell mobilization procedure, different regimens have been established, usually consisting of a cycle of chemotherapy followed by application of granulocyte-colony stimulating factor (G-CSF), although febrile neutropenia is a common complication. Following national guidelines, our institution decided to primarily use G-CSF only mobilization during the COVID-19 pandemic to minimize the patients' risk of infection and to reduce the burden on the health system. STUDY DESIGN AND METHODS: In this retrospective single-center analysis, the efficacy and safety of G-CSF only mobilization was evaluated and compared to a historic control cohort undergoing chemotherapy-based mobilization by cyclophosphamide and etoposide (CE) plus G-CSF. RESULTS: Although G-CSF only was associated with a higher need for plerixafor administration (p < .0001) and a higher number of apheresis sessions per patient (p = .0002), we were able to collect the target dose of hematopoietic stem cells in the majority of our patients. CE mobilization achieved higher hematopoietic stem cell yields (p = .0015) and shorter apheresis sessions (p < .0001) yet was accompanied by an increased risk of febrile neutropenia (p < .0001). There was no difference in engraftment after ASCT. DISCUSSION: G-CSF only mobilization is a useful option in selected patients with comorbidities and an increased risk of serious infections, especially in the wintertime or in future pandemics.

Alpha 7 Nicotinic Acetylcholine Receptor Agonist PHA 568487 Reduces Acute Inflammation but Does Not Affect Cardiac Function or Myocardial Infarct Size in the Permanent Occlusion Model.

Stimulation of the alpha 7 nicotinic acetylcholine receptor (α7nAChR) has shown beneficial effects in several acute inflammatory disease models. This study aims to examine whether treatment with the selective α7nAChR agonist PHA 568487 can dampen inflammation and thereby improve cardiac function after myocardial infarction in mice. The possible anti-inflammatory properties of α7nAChR agonist PHA 568487 were tested in vivo using the air pouch model and in a permanent occlusion model of acute myocardial infarction in mice. Hematologic parameters and cytokine levels were determined. Infarct size and cardiac function were assessed via echocardiography 24 h and one week after the infarction. Treatment with α7nAChR agonist PHA 568487 decreased 12 (CCL27, CXCL5, IL6, CXCL10, CXCL11, CXCL1, CCL2, MIP1a, MIP2, CXCL16, CXCL12 and CCL25) out of 33 cytokines in the air pouch model of acute inflammation. However, α7nAChR agonist PHA 568487 did not alter infarct size, ejection fraction, cardiac output or stroke volume at 24 h or at 7 days after the myocardial infarction compared with control mice. In conclusion, despite promising immunomodulatory effects in the acute inflammatory air pouch model, α7nAChR agonist PHA 568487 did not affect infarct size or cardiac function after a permanent occlusion model of acute myocardial infarction in mice. Consequently, this study does not strengthen the hypothesis that stimulation of the α7nAChR is a future treatment strategy for acute myocardial infarction when reperfusion is lacking. However, whether other agonists of the α7nAChR can have different effects remains to be investigated.

Concomitant treatment with safinamide and antidepressant drugs: Safety data from real clinical practice.

BACKGROUND AND PURPOSE: The aim of this study was to assess the possible pharmacological interactions between safinamide and antidepressants, and in particular the appearance of serotonin syndrome with data from real life. METHODS: We conducted a retrospective observational study of patients with Parkinson's disease from our Movement Disorders Unit, who were under treatment with any antidepressant drug and safinamide. Specifically, symptoms suggestive of serotonin syndrome were screened for. Also, we collected time of simultaneous use, doses of levodopa and other antiparkinsonian drugs. RESULTS: Clinical records were reviewed for the study period of September 2018 to September 2019. Seventy-eight PD patients who were treated with safinamide of which 25 (32.05%) had a concomitant treatment with an antidepressant drug, being sertraline and escitalopram the most frequent. Mean age was 80 years±8.43 and H&Y stage was 3 [2-4]. Mean dose of levodopa used was 703.75mg±233.15. Median duration of concomitant treatment with safinamide and antidepressant drug was 6 months (IQR 20.5), and over eighteen months in 5 cases. No case of serotonin syndrome was recorded, neither was any of its typical manifestations combined or in isolation. CONCLUSIONS: Our real clinical practice study suggests that concomitant use of safinamide with antidepressant drugs in PD patients seemed to be safe and well tolerated, even in the long term. However, caution is warranted, individualizing treatment regimens and monitoring the potential appearance of adverse effects.

Long-Term Safety and Efficacy of Mavacamten in Symptomatic Obstructive Hypertrophic Cardiomyopathy: Interim Results of the PIONEER-OLE Study.

BACKGROUND: The phase 2 PIONEER-HCM (Phase 2 Open-label Pilot Study Evaluating Mavacamten in Subjects With Symptomatic Hypertrophic Cardiomyopathy and Left Ventricular Outflow Tract Obstruction) study showed that mavacamten improved left ventricular outflow tract gradients, exercise capacity, and symptoms in patients with obstructive hypertrophic cardiomyopathy (HCM), but the results of longer-term treatment are less well described. We report interim results from the PIONEER-OLE (PIONEER Open-Label Extension) study, the longest-term study of mavacamten in patients with symptomatic obstructive HCM. METHODS AND RESULTS: Patients who previously completed PIONEER-HCM (n=20) were eligible to enroll in PIONEER-OLE. Patients received oral mavacamten, 5 mg once daily (starting dose), with individualized dose titration at week 6. Evaluations included serial monitoring of safety, echocardiography, Kansas City Cardiomyopathy Questionnaire-Overall Summary Score, and serum NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels. Thirteen patients enrolled and received mavacamten (median study duration at data cutoff, 201 weeks). Most patients (92.3%) received ß-blockers concomitantly. Treatment-emergent adverse events were predominantly mild/moderate. One patient had an isolated reduction in left ventricular ejection fraction to 47%, which recovered and remained normal with continued treatment at a reduced dose. At week 180, mavacamten was associated with New York Heart Association class improvements from baseline (class II to I, n=9; class III to II, n=1; and unchanged, n=2), sustained reductions in left ventricular outflow tract gradients (mean [SD] change from baseline: resting, -50 [55] mm Hg; Valsalva, -70 [41] mm Hg), and serum NT-proBNP levels (median [interquartile range] change from baseline: -498 [-2184 to -76] ng/L), and improved Kansas City Cardiomyopathy Questionnaire-Overall Summary Score (mean [SD] change from baseline: +17 [16]). CONCLUSIONS: This long-term analysis supports the continued safety and effectiveness of mavacamten for >3 years in obstructive HCM. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03496168.

Evaluating the efficacy and safety of mavacamten in hypertrophic cardiomyopathy: A systematic review and meta-analysis focusing on qualitative assessment, biomarkers, and cardiac imaging.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) is a complex cardiac condition characterized by hypercontractility of cardiac muscle leading to a dynamic obstruction of left ventricular outlet tract (LVOT). Mavacamten, a first-in-class cardiac myosin inhibitor, is increasingly being studied in randomized controlled trials. In this meta-analysis, we aimed to analyse the efficacy and safety profile of Mavacamten compared to placebo in patients of HCM. METHOD: We carried out a comprehensive search in PubMed, Cochrane, and clinicaltrials.gov to analyze the efficacy and safety of mavacamten compared to placebo from 2010 to 2023. To calculate pooled odds ratio (OR) or risk ratio (RR) at 95% confidence interval (CI), the Mantel-Haenszel formula with random effect was used and Generic Inverse Variance method assessed pooled mean difference value at a 95% CI. RevMan was used for analysis. P<0.05 was considered significant. RESULTS: We analyzed five phase 3 RCTs including 609 patients to compare mavacamten with a placebo. New York Heart Association (NYHA) grade improvement and KCCQ score showed the odds ratio as 4.94 and 7.93 with p<0.00001 at random effect, respectively. Cardiac imaging which included LAVI, LVOT at rest, LVOT post valsalva, LVOT post-exercise, and reduction in LVEF showed the pooled mean differences for change as -5.29, -49.72, -57.45, -36.11, and -3.00 respectively. Changes in LVEDV and LVMI were not statistically significant. The pooled mean difference for change in NT-proBNP and Cardiac troponin-I showed 0.20 and 0.57 with p<0.00001. The efficacy was evaluated in 1) A composite score, which was defined as either 1·5 mL/kg per min or greater increase in peak oxygen consumption (pVO2) and at least one NYHA class reduction, or a 3·0 mL/kg per min or greater pVO2 increase without NYHA class worsening and 2) changes in pVO2, which was not statistically significant. Similarly, any treatment-associated emergent adverse effects (TEAE), treatment-associated serious adverse effects (TSAE), and cardiac-related adverse effects were not statistically significant. CONCLUSION: Mavacamten influences diverse facets of HCM comprehensively. Notably, our study delved into the drug's impact on the heart's structural and functional aspects, providing insights that complement prior findings. Further large-scale trials are needed to evaluate the safety profile of Mavacamten.

Exogenously applied gibberellic acid and benzylamine modulate growth and chemical constituents of dwarf schefflera: a stepwise regression analysis.

Ornamental foliage plants that have a dense appearance are highly valued. One way to achieve this is by using plant growth regulators as a tool for plant growth management. In a greenhouse with a mist irrigation system, a study was conducted on dwarf schefflera, an ornamental foliage plant, which was exposed to foliar application of gibberellic acid and benzyladenine hormones. The hormones were sprayed on dwarf schefflera leaves at 0, 100, and 200 mg/l concentrations, at 15-day intervals in three stages. The experiment was conducted as a factorial based on a completely randomized design, with four replicates. The combination of gibberellic acid and benzyladenine at 200 mg/l concentration had a significant effect on leaf number, leaf area, and plant height. The treatment also resulted in the highest content of photosynthetic pigments. Furthermore, the highest soluble carbohydrate to reducing sugars ratio was observed in treatments of 100 and 200 mg/l benzyladenine, and 200 mg/l gibberellic acid + benzyladenine. Stepwise regression analysis showed that root volume was the first variable to enter the model, explaining 44% of variations. The next variable was root fresh weight, and the two-variable model explained 63% of variations in leaf number. The greatest positive effect on leaf number was related to root fresh weight (0.43), which had a positive correlation with leaf number (0.47). The results showed that 200 mg/l concentration of gibberellic acid and benzyladenine significantly improved morphological growth, chlorophyll and carotenoid synthesis, and reducing sugar and soluble carbohydrate contents in dwarf schefflera.

G-CSF + plerixafor versus G-CSF alone mobilized hematopoietic stem cells in patients with multiple myeloma and lymphoma: a systematic review and meta-analysis.

AIM: The combination of granulocyte-colony stimulating factor (G-CSF) and plerixafor is one of the approaches for hematopoietic stem cell mobilization in patients with multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), and Hodgkin's lymphoma (HL). This systematic review and meta-analysis aimed to determine the ability of G-CSF + plerixafor to mobilize peripheral blood (PB) CD34+ cells and examine its safety profile. METHODS: We performed a database search using the terms 'granulocyte colony stimulating factor', 'G-CSF', 'AMD3100', and 'plerixafor', published up to May 1, 2023. The methodology is described in further detail in the PROSPERO database (CRD42023425760). RESULTS: Twenty-three studies were included in this systematic review and meta-analysis. G-CSF + plerixafor resulted in more patients achieving the predetermined apheresis yield of CD34+ cells than G-CSF alone (OR, 5.33; 95%, 4.34-6.55). It was further discovered that G-CSF + plerixafor could mobilize more CD34+ cells into PB, which was beneficial for the next transplantation in both randomized controlled (MD, 18.30; 95%, 8.74-27.85) and single-arm (MD, 20.67; 95%, 14.34-27.00) trials. Furthermore, G-CSF + plerixafor did not cause more treatment emergent adverse events than G-CSF alone (OR, 1.25; 95%, 0.87-1.80). CONCLUSIONS: This study suggests that the combination of G-CSF and plerixafor, resulted in more patients with MM, NHL, and HL, achieving the predetermined apheresis yield of CD34+ cells, which is related to the more effective mobilization of CD34+ cells into PB.

Protective effect of liver X receptor on cigarette smoke and lipopolysaccharide induced airway inflammation and emphysema in mice.

OBJECTIVE: The aim of this study is to assess the impact of Liver X receptors (LXRs) on airway inflammation, airway remodeling, and lipid deposition induced by cigarette smoke and lipopolysaccharide (LPS) exposure in the lung. METHODS: Wild mice and LXR-deficient mice were exposed to cigarette smoke and LPS to induce airway inflammation and remodeling. In addition, some wild mice received intraperitoneal treatment with the LXR agonist GW3965 before exposure to cigarette smoke and LPS. Lung tissue and bronchoalveolar lavage fluid were collected to evaluate airway inflammation, airway remodeling and lipid deposition. RESULTS: Exposure to cigarette smoke and LPS resulted in airway inflammation, emphysema and lipid accumulation in wild mice. These mice also exhibited downregulated LXRα and ABCA1 in the lung. Treatment with GW3965 mitigated inflammation, remodeling and lipid deposition, while the deletion of LXRs exacerbated these effects. Furthermore, GW3965 treatment following exposure to cigarette smoke and LPS increased LXRα and ABCA1 expression and attenuated MyD88 expression in wild mice. CONCLUSION: LXRs demonstrate the potential to mitigate cigarette smoke and LPS- induced airway inflammation, emphysema and lipid disposition in mice.

Isolevuglandins Promote Mitochondrial Dysfunction and Electrophysiologic Abnormalities in Atrial Cardiomyocytes.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet the cellular and molecular mechanisms underlying the AF substrate remain unclear. Isolevuglandins (IsoLGs) are highly reactive lipid dicarbonyl products that mediate oxidative stress-related injury. In murine hypertension, the lipid dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) reduced IsoLGs and AF susceptibility. We hypothesized that IsoLGs mediate detrimental pathophysiologic effects in atrial cardiomyocytes that promote the AF substrate. Using Seahorse XFp extracellular flux analysis and a luminescence assay, IsoLG exposure suppressed intracellular ATP production in atrial HL-1 cardiomyocytes. IsoLGs caused mitochondrial dysfunction, with reduced mitochondrial membrane potential, increased mitochondrial reactive oxygen species (ROS) with protein carbonylation, and mitochondrial DNA damage. Moreover, they generated cytosolic preamyloid oligomers previously shown to cause similar detrimental effects in atrial cells. In mouse atrial and HL-1 cells, patch clamp experiments demonstrated that IsoLGs rapidly altered action potentials (AP), implying a direct effect independent of oligomer formation by reducing the maximum Phase 0 upstroke slope and shortening AP duration due to ionic current modifications. IsoLG-mediated mitochondrial and electrophysiologic abnormalities were blunted or totally prevented by 2-HOBA. These findings identify IsoLGs as novel mediators of oxidative stress-dependent atrial pathophysiology and support the investigation of dicarbonyl scavengers as a novel therapeutic approach to prevent AF.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Selective synthesis of an elusive C-functional bis-cyclam and study of its inhibition of the CXCR4 chemokine receptor.

This article presents the controlled synthesis of a rare example of C,C'-linked bis-cyclam architecture in mild conditions through the "bis-aminal" route previously used for the advantageous synthesis of cyclam, N- and C-functional cyclams and N,N'-bis-cyclams. Two synthetic pathways were explored with the smart design of α,ß-unsaturated ketones or alkyl halides bis-cyclizing agents. The first led to the isolation of a key intermediate for the future design of N-functionalized bis-cyclams, whereas the second allowed the preparation of the targeted C,C'-xylylene-bis-cyclam under mild conditions with decent yield. This compound was then studied as a CXCR4 receptor inhibitor, one of the main applications known for bis-macrocyclic compounds, in particular in the context of HIV (human immunodeficiency virus) infection. Although results demonstrated that its potency is lower (i.e. 137-fold higher IC50) than the gold standard AMD3100 against HIV infection, clear evidence of CXCR4 inhibition is presented, confirming the potential of this novel architecture and related compounds in this research field.

trans-IV restriction: a new configuration for metal bis-cyclam complexes as potent CXCR4 inhibitors.

The chemokine receptor CXCR4 is implicated in multiple diseases including inflammatory disorders, cancer growth and metastasis, and HIV/AIDS. CXCR4 targeting has been evaluated in treating cancer metastasis and therapy resistance. Cyclam derivatives, most notably AMD3100 (Plerixafor™), are a common motif in small molecule CXCR4 antagonists. However, AMD3100 has not been shown to be effective in cancer treatment as an individual agent. Configurational restriction and transition metal complex formation increases receptor binding affinity and residence time. In the present study, we have synthesized novel trans-IV locked cyclam-based CXCR4 inhibitors, a previously unexploited configuration, and demonstrated their higher affinity for CXCR4 binding and CXCL12-mediated signaling inhibition compared to AMD3100. These results pave the way for even more potent CXCR4 inhibitors that may provide significant efficacy in cancer therapy.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Discerning the stability behaviour of mavacamten availing liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy: In silico toxicity and mutagenicity prediction of degradation products.

The present study aimed to separate, identify, and characterise the degradation products formed when mavacamten is exposed to stress degradation as well as the stability of the drug in various environments and also to understand its degradation chemistry. Prediction of in silico toxicity and mutagenicity was aimed at the observed degradation products. Stress degradation along with stability studies and degradation kinetics were performed on mavacamten, and separation of degradation products was carried out by high-performance liquid chromatography. Tandem mass spectrometry studies were executed to characterise the structures of degradation products using product ion fragments. Orthogonally, nuclear magnetic resonance experiments were conducted to elucidate the structures having ambiguity in characterising them. Deductive Estimation of Risk from Existing Knowledge and Structure Activity Relationship Analysis using Hypotheses software were used to establish in silico toxicity and mutagenic profiles of mavacamten and its degradation products. Two degradation products of mavacamten found in acidic hydrolytic stress conditions were separated, identified, characterised, and proposed as 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 1-phenylethanamine. Mavacamten was found to be stable under different pH and gastrointestinal conditions. The degradation kinetics of mavacamten under 1 N acidic condition followed zero-order kinetics, and it was degraded completely within 6 h. In silico toxicity and mutagenicity studies revealed that 1-phenylethanamine can be a skin sensitiser. A high-performance liquid chromatography method was developed for the separation of degradation products of mavacamten and characterised by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. During the manufacturing and storage of drug product, precautions need to be taken when dealing with acidic solutions as the drug is prone to hydrolysis in acidic conditions. The formation of 1-phenylethanamine under these conditions is to be monitored as it is a skin sensitiser.