Distinct Mechanisms Of Action Research Articles

Inhibition of fibril formation by polyphenols: molecular mechanisms, challenges, and prospective solutions.

Fibril formation is a key feature in neurodegenerative diseases like Alzheimer's, Parkinson's, and systemic amyloidosis. Polyphenols, found in plant-based foods, show promise in inhibiting fibril formation and disrupting disease progression. The ability of polyphenols to break the amyloid fibrils of many disease-linked proteins has been tested in numerous studies. Polyphenols have their distinctive mechanism of action. They behave differently on various events in the aggregation pathway. Their action also differs for different proteins. Some polyphenols only inhibit the formation of fibrils whereas others break the preformed fibrils. Some break the fibrils into smaller species, and some change them to other morphologies. This article delves into the intricate molecular mechanisms underlying the inhibitory effects of polyphenols on fibrillogenesis, shedding light on their interactions with amyloidogenic proteins and the disruption of fibril assembly pathways. However, addressing the challenges associated with solubility, stability, and bioavailability of polyphenols is crucial. The current strategies involve nanotechnology to improve the solubility and bioavailability, thus showing the potential to enhance the efficacy of polyphenols as therapeutics. Advancements in structural biology, computational modeling, and biophysics have provided insights into polyphenol-fibril interactions, offering hope for novel therapies for neurodegenerative diseases and amyloidosis.

Comparative Analysis of Metformin and Imeglimin: Exploring Therapeutic Implications

The landscape of type 2 diabetes mellitus (T2DM) management is evolving with the emergence of imeglimin alongside the longstanding use of metformin. While both drugs share structural similarities and target mitochondrial complex I, they exhibit distinct mechanisms of action and therapeutic effects. Imeglimin, with its mitochondrial modulation and competitive inhibition of complex I, holds promise in mitigating beta-cell dysfunction, improving insulin sensitivity, and offering potential cardiovascular benefits. Efficacy studies suggest comparable outcomes with metformin, underscoring the utility of imeglimin as a valuable addition to the T2DM armamentarium. There is mixed evidence on the cardio-renal benefits of metformin while imeglimin’s tissue-protective effects in preclinical studies offer a glimpse into potential cardiovascular advantages. Studies suggest imeglimin’s superior gastrointestinal tolerability. Imeglimin’s potential as an anti-diabetic agent is further underscored by its diverse combination options. In clinical practice, metformin remains a cornerstone in the management of T2DM. Imeglimin, with its lesser gastrointestinal side effects and theoretically lower risk of lactic acidosis, offers a compelling alternative, particularly in patients intolerant to metformin. With preliminary signs pointing toward potential cardiovascular and renal benefits of imeglimin, exploring its application in patients with atherosclerotic cardiovascular disease presents an enticing prospect. As the field advances, adopting a nuanced and personalized approach to T2DM management could lead to imeglimin carving out a significant niche in the dynamic landscape of diabetes therapeutics.

"In Vitro Evaluation of the Antiplatelet Activity of Paravat Shakrita"

Platelet aggregation, a crucial process in thrombotic disorders, underscores the necessity for effective preventive and therapeutic measures. This study compares the antiplatelet effects of Aspirin, a well-established drug, with Paravat Shakrita, a lesser-known formulation from Ayurvedic texts. It underwent GCMS and microbiological analysis to determine its chemical composition and microbial load. Using an in vitro model with rat blood samples, we conducted a Platelet Aggregation Inhibition Assay. As expected, Aspirin inhibited platelet aggregation in a concentration-dependent manner, consistent with its known mechanism. Despite limited documentation, Paravat Shakrita exhibited potent antiplatelet properties, also showing dose-dependent effects. Interestingly, Paravat Shakrita demonstrated comparable or even superior effects at higher concentrations compared to Aspirin. The IC50 values revealed similar inhibitory potency between Paravat Shakrita and Aspirin. This comparison unveiled distinct mechanisms of action, providing various therapeutic options against platelet-mediated thrombosis. While promising, further investigation is crucial to understand Paravat Shakrita's mechanism, ensure its safety, and determine its clinical significance.

Immune Checkpoint Inhibitors Combined with Chemotherapy in The Treatment of Lung Cancer

Immune checkpoint inhibitor (ICI) therapy is the most commonly used immunotherapy regimen. The clinical application of this medicine has been authorized for the treatment of several neoplasms such as melanoma, renal, head and neck, bladder, etc. Lung cancer treatment has witnessed significant advancements, positioning it as a pivotal component within the broader framework of comprehensive lung cancer management. However, due to the heterogeneity of tumors and the intricacy of the microenvironment of tumors, the effective rate of ICIs alone is low in non-selected patients. Surgery, chemotherapy, radiotherapy, targeted therapy, and other treatments can have synergistic effects with immunotherapy, and combination therapy has become a hotspot for exploration. This article centers on the research advancements in the therapeutic approach of combining immune checkpoint inhibitors (ICIs) with chemotherapy. This text offers an introductory overview of the distinct mechanisms of action associated with these two treatment modalities, highlighting the significance of their combined use in the context of lung cancer treatment. The objective is to offer a reference point for future clinical treatments of lung cancer.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

Glucagon-like peptide agonists: A prospective review.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have emerged as promising therapeutic options for addressing Type-2 diabetes, obesity, and related conditions. Among these, semaglutide, tirzepatide, liraglutide etc., all notable GLP-1RA, have gained attention owing to their favourable pharmacological properties and clinical efficacy. This comprehensive review aims to provide a detailed analysis of both the currently available GLP-1RAs in the market and those undergoing clinical trials. The focus is on examining their mechanism of action, pharmacokinetics, efficacy in glycemic control and weight management, safety profile, and potential applications. The review employs a systematic approach to gather information on GLP-1RAs. Relevant literature from the currently literature and ongoing clinical trials is thoroughly examined. Detailed scrutiny is applied to understand the mechanism of action, pharmacokinetic properties, and clinical outcomes of these agents. The review presents a comprehensive overview of the GLP-1RAs, highlighting their distinct mechanisms of action, pharmacokinetic profiles, and clinical effectiveness in glycemic control and weight management. Safety profiles are also discussed, providing a holistic understanding of these therapeutic agents. The findings are discussed in the context of advancements in the field of GLP-1RAs. Potential applications beyond diabetes and obesity are explored, shedding light on the broader implications of these agents in managing related conditions. In conclusion, this review underscores the significance of GLP-1RAs, with a specific focus on semaglutide, in the management of type 2 diabetes, obesity, and beyond. By synthesizing information on their mechanisms, pharmacokinetics, efficacy, and safety, this review provides valuable insights into the potential benefits these agents offer, contributing to the ongoing discourse in the field.

Impact of Symptom Benefit and Transfusion Response on Survival in Myelofibrosis Patients Treated with Pacritinib: PERSIST-2 Landmark Survival Analysis

Background: Pacritinib is a JAK1-sparing inhibitor of JAK2/IRAK1/ACVR1 that demonstrated symptom benefit measured by TSS (v2.0, excluding tiredness) vs best available therapy (BAT) in PERSIST-2, which enrolled cytopenic myelofibrosis patients with platelets ≤100×10 9/L. Recent datashowed that in patients with cytopenic MF, a ≥10% reduction in spleen volume at 12 weeks is associated with improved survival in patients treated with pacritinib ( Ajufo H, et al., J Clin Oncol;2023:14(16):Suppl). Surprisingly, this association was not confirmed in patients treated with BAT (82% of whom were on ruxolitinib). The association between other important measures of clinical benefit such as symptom burden reduction and anemia improvement on survival in patients treated with pacritinib has not previously been described. Methods: Patients randomized to pacritinib 200 mg BID and to BAT on PERSIST-2 were included if they were alive and on study at the start of the week 12 efficacy assessment period. Survival was assessed by Total Symptom Score (TSS; v2.0 excluding tiredness) reduction at various thresholds (≥50%, ≥20%, ≥10%, >0%) and transfusion independence (TI) response. TI was assessed among patients who required red blood cell (RBC) transfusion at baseline (within 90 days) and was defined, for the purposes of a landmark survival analysis, as the absence of RBC transfusions over any 12-week period from first dose through week 14 (allowing for a ±2-week window around week 12). Overall survival (OS) was estimated via the K-M method following the landmark approach; survival in responders vs non-responders was compared using the log-rank test. Results: Of the 59 patients treated with pacritinib who had a reduction of TSS≥10%, the median age was 67 years, 25% had a baseline high-risk DIPSS, median platelet count was 61 x10 9/L, median hemoglobin was 9.7 g/dL, and baseline TSS was 19.1. Reduction of TSS≥10% was associated with improved OS, with 1 death among 59 responders vs 4 deaths among 30 non-responders (hazard ratio [HR]=0.12, 95% confidence interval [CI]: 0.01, 0.79] P=0.021, Fig 1a). Similar to spleen volume reduction, the association between TSS response and OS weakened at more stringent TSS response thresholds (Fig 1c, e), with the BAT arm not showing an association between TSS response and OS at any threshold. (Fig 1b, d, f). In order to assess the relationship of specific TSS domains and survival , a landmark OS analysis was performed based on ≥20% reduction in physical function scores (sum of ‘tiredness’ and 'inactivity'), spleen-related symptom scores (sum of 'abdominal discomfort', ‘early satiety’, and 'left rib pain'), and cytokine-related symptom scores (sum of 'itching', ‘night sweats’, and ‘bone pain‘). For cytokine-related symptoms, the number of deaths in PAC responders was significantly different compared to non-responders, with 0 deaths among 35 responders vs 2 among 15 non-responders ( P=0.0325). Conversely the deaths among responders (2/22) vs non-responders (2/20) were not significantly different in the BAT arm. For the physical function and spleen-related symptom subscales, no significant differences between PAC responders and non-responders was observed (HR=0.60; 95% CI [0.05, 6.74] P=0.679) and (HR=0.26; 95% CI 0.02, 2.93, P=0.2435) respectively. Among 36 patients on PAC who received RBC transfusions at baseline, 7 achieved TI over at least a 12-week period up to the time of the landmark analysis. There were no deaths among TI responders compared to 2 (6.9%) among TI non-responders. There were too few TI responders on BAT (only 2 out of 38) to assess the impact on survival. Conclusion: In cytopenic myelofibrosis patients enrolled in PERSIST-2, having a reduction of TSS≥10% on full-dose PAC was associated with OS benefit - a finding that has not been noted with other JAK inhibitors to date. By contrast, this association was not found with BAT, (82% of these patients received ruxolitinib). Thus, reductions in TSS in patients treated with PAC were associated with an OS benefit. It is possible that pacritinib, with its distinctive mechanism of action, may offer a unique survival advantage for MF patients with moderate or severe thrombocytopenia, and further studies are required to understand the potential mechanisms of these distinct clinical observations. Study funded by CTI BioPharma Corp., a Sobi company.

Real-World Effectiveness of Asciminib in Patients with Chronic Myeloid Leukemia (CML) Harboring the T315I Mutation: A Global Chart Review Study of Patients Treated in the Asciminib Managed Access Program (MAP)

Background: In patients (pts) with CML, the BCR::ABL1 T315I gatekeeper mutation confers treatment resistance to all approved ATP-competitive tyrosine kinase inhibitors (TKIs) except ponatinib and olverembatinib; thus pts harboring this mutation have limited treatment options. Asciminib, a first-in-class TKI that works by specifically targeting the ABL myristoyl pocket (STAMP), has a distinct mechanism of action and no known overlapping resistance profile with ATP-competitive TKIs. In clinical trials, asciminib has demonstrated efficacy and acceptable safety in adult pts with Ph+ CML in chronic phase and the T315I mutation. The asciminib MAP (NCT04360005) enables access to asciminib treatment for pts with CML who are resistant or intolerant to available treatments or for whom available treatments are contraindicated. This global chart review study aimed to better understand the effectiveness and safety of asciminib in pts with CML and the T315I mutation treated as part of the MAP. Methods: This non-interventional chart review analyzed data from existing routine medical charts of pts enrolled in the MAP who received at least one dose of asciminib. Pts with CML aged ≥18 years old were included in the study if they had confirmed presence of the T315I mutation detected at any point prior to commencement of asciminib and had received their first dose of asciminib between 01 November 2018 and 30 April 2022. Data was abstracted from CML diagnosis date to the first dose of asciminib (index date) and up until 12 months after the start of asciminib. Treatment effectiveness was assessed in terms of major molecular response (MMR) in pts in MMR at index date or not, at and by 3, 6, 9, and 12 months. Other variables included demographic and disease characteristics, treatment patterns, response to prior TKIs and safety. All analyses were descriptive. Results: 31 pts were included across 8 countries (Netherlands, UK, Italy, Hong Kong, Pakistan, Spain, Australia, and US). Median age at CML diagnosis was 47 (range 18‒74) years and most pts were in chronic phase ( Table 1). Pts were heavily pretreated, with the majority having received ≥2 prior TKIs. Nearly all pts (28/31; 90.3%) had received prior ponatinib treatment at any time before the switch to asciminib. Median prior TKI treatment duration was longest for ponatinib (43.4 months, range 0.3‒117.3) vs other TKIs. The most common reason for discontinuation of all previous TKIs at any time before asciminib was lack of response to treatment, with the exception of ponatinib, where the most common reason was due to adverse events (AEs). Ponatinib was the last TKI prior to commencement of asciminib in 27/31 (87.1%) pts. Overall, 22/31 pts (71.0%) experienced AEs that led to a switch to another TKI at any time prior to asciminib, whereas nearly half of pts discontinued the last TKI before starting asciminib due to intolerance (14 pts, 45.2%). A total of 12/31 patients (38.7%) were already in MMR at index date ( Table 2), confirming that intolerance was an important reason for switching to asciminib. Of the pts not in MMR at the start of asciminib, 4/10 (40.0%) and 5/12 (41.7%) evaluated pts achieved MMR by 6 and 12 months, respectively. Of the 12 pts in MMR at the start of asciminib, 6/12 (50.0%) maintained MMR by 6 months. Overall, 10/31 pts (32.3%) discontinued asciminib before completion of the 12-month follow-up (death, 7 [4 serious AEs, SAEs; 2 CML; 1 unknown]; physician decision, 3). SAEs were reported in 9 pts (29.0%) that received asciminib; 8 SAEs led to hospitalization, 4 to death (1 cerebrovascular accident, 1 CML, 1 pneumonia, 1 fungal infection), 2 to asciminib discontinuation, and 2 were considered life-threatening. SAEs reported for 1 pt (hematotoxicity and procedural hemorrhage) were assessed as asciminib-related. Conclusions: Asciminib demonstrated effectiveness in pts with CML and the T315I mutation in routine medical practice, including ponatinib pre-treated pts, a population with limited available treatment options. Effectiveness estimates were consistent with those observed in clinical trials. Although the number of pts was small, this global study reports on one of the largest cohorts of pts with CML and the T315I mutation.

EPCORE FL-1: Phase 3 Trial of Subcutaneous Epcoritamab with Rituximab and Lenalidomide (R 2) Vs R 2 Alone in Patients with Relapsed or Refractory Follicular Lymphoma

Background: Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma (NHL). Several treatment options are approved by the US Food and Drug Administration for second- or later-line FL therapy, including rituximab + lenalidomide (R 2). However, advanced-stage FL remains incurable and new treatment options for relapsed or refractory (R/R) disease are needed. Epcoritamab, a subcutaneously administered, bispecific antibody that binds CD3 on T lymphocytes and CD20 on B cells, induces potent and selective T-cell-mediated killing of malignant CD20 + B cells (van der Horst et al, Blood Cancer J 2021). In the first-in-human, phase 1/2 EPCORE NHL-1 trial (NCT03625037) in heavily pretreated patients with B-cell NHL (N=68), epcoritamab showed manageable safety and promising single-agent anti-tumor activity (Hutchings et al, Lancet 2021). Among 10 patients with R/R FL, objective response rate (ORR) was 90% (95% CI: 55-100) and complete response (CR) rate was 50%. In the ongoing phase 1b/2 trial evaluating epcoritamab + R 2 for R/R FL (EPCORE NHL-2 arms 2a and 2b; NCT04663347), ORR was 98% among 104 efficacy-evaluable patients, with a complete metabolic response in 87%. Most cytokine release syndrome (CRS) events were low grade (n=111; grade 1-2, 46%; grade 3, 2%) and occurred in cycle 1; all CRS events resolved with routine management. These encouraging data, and the distinct mechanisms of action of epcoritamab and R 2, support ongoing evaluation of this combination for its potential to improve clinical outcomes in patients with R/R FL. The phase 3 EPCORE FL-1 trial was designed to evaluate epcoritamab in combination with R 2 vs R 2 alone in patients with R/R FL with ≥1 prior line of anti-lymphoma therapy. We present an updated protocol, reflecting recent developments and revisions implemented in this study. Study Design and Methods: EPCORE FL-1 (NCT05409066) is a global, randomized, open-label, multicenter phase 3 trial designed to evaluate efficacy and safety of epcoritamab in combination with R 2 vs R 2 alone in patients with R/R FL. Adult patients must have histologically confirmed classic FL (previously grades 1 to 3a FL) stage II, III, or IV with a CD20 + tumor on a representative biopsy based on local pathology report and no evidence of histologic transformation. Patients must have R/R disease after ≥1 prior anti-lymphoma regimen that contained an anti-CD20 monoclonal antibody in combination with chemotherapy; patients receiving only prior anti-CD20 monoclonal antibody monotherapy and/or radiation are not eligible. Other key eligibility criteria include Eastern Cooperative Oncology Group performance status 0 to 2, fluorodeoxyglucose positron emission tomography-avid disease, and ≥1 measurable disease site per Lugano 2014 criteria. Lenalidomide-refractory FL (best response to lenalidomide of stable or progressive disease or progressive disease within 6 months of completion of lenalidomide) is excluded. Approximately 520 patients will be enrolled and randomized 1:1 to receive a full dose of epcoritamab in combination with R 2 or R 2 alone ( Figure); the trial is currently enrolling in 2 arms, Arms A & C. Based on emerging safety and efficacy data, Arm B has been closed to enrollment and the recommended dose of epcoritamab in combination with R 2 is 48 mg. Epcoritamab will be subcutaneously administered using a step-up dosing regimen to full dose in cycle 1 (28 days/cycle). Full dose of epcoritamab will be administered weekly in cycles 2-3 and monthly from cycle 4 onward for up to 12 cycles of treatment. Patients will be serially assessed for disease progression at prespecified intervals. The primary endpoint is progression-free survival assessed by independent review committee (IRC) per Lugano criteria. Key secondary efficacy endpoints include CR rate (by IRC per Lugano criteria), overall survival, and minimal residual disease negativity. Other efficacy endpoints include best overall response, duration of response, duration of complete response, time to progression, event-free survival, time to next anti-lymphoma treatment, and patient-reported outcomes. Safety endpoints include incidence and severity of treatment-emergent adverse events and adverse events of special interest. Exploratory endpoints include assessments of pharmacodynamic and pharmacokinetic data. The study opened for enrollment in 2022 in North America, South America, Europe, Africa, Asia, and Australia.

Actionable Findings from an Unbiased Drug Screen for Novel Single Agent and Combination Therapies Against AML with Mecom Re-Arrangement

EVI1 (Ecotropic Viral Integration Site 1) is a zinc finger transcriptional regulator encoded from the MECOM (MDS1 and EVI1 Complex) locus at chromosome 3q26.2. Aberrant EVI1 overexpression is observed in approximately 10% of de novo AML, of which approximately half are due to chromosomal rearrangement involving either inv3(q21;q26.2) or t(3;3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is relocated and drives EVI1 expression. In addition to the resulting haploinsufficiency of GATA2, monosomy 7 and activating mutations in the RAS pathway and SF3B1 commonly co-occur in 3q26.2-rearranged (r) AML. AML with EVI1 overexpression generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Among the targets of EVI1 are the ERG, MYC, KIT, Bcl-xL and MPL genes. GATA2 repression, coupled with increased EVI1 activity, promotes AML progression and an aggressive phenotype in 3q26.2-rearranged AML. Therefore, there is a glaring need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML with EVI1 overexpression. To identify druggable vulnerabilities in 3q26.2-r AML, we conducted an unbiased high-throughput drug screen, utilizing a library of 2480 oncology-focused, mechanistically annotated drugs (referred as NCATS Mechanism Interrogation Plates or-MIPE 5.0) (Cancer Cell. 2021; 39:566-579), against seven AML cell lines, i.e., four with 3q26.2-r: UCSD-AML1, HNT-34, AML191 and AML194, and three non-3q26.2-r AML: SET-2, MV4-11 and OCI-AML3. The library exploits mechanistic redundancy by including multiple inhibitors against well-validated oncology targets, while simultaneously encompassing mechanistic diversity, altogether targeting more than 850 distinct mechanisms of action. We exploited this redundancy to identify druggable, target-level dependencies in 3q26.2-r AML, both in terms of absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. This was consistent with previous reports that BET inhibitors (e.g., OTX015, mivebresib or ABBV-075 and JQ1) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and PDX models. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, XIAP/cIAPs inhibitors birinapant (10-1000 nM) or SM-164 (30-1000 nM), chosen based on the MIPE screen outcomes, induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines. This was associated on Western analyses with decline in XIAP, c-IAP1/2, p-ERK1/2, MCL1 and Bcl-xL, but increased protein levels of cleaved caspase-3 and PARP in UCSD-AML1 and AML191 cells. Mivebresib (50-250 nM) or OTX015 (100-500 nM) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, XIAP, c-IAP1/2, CDK4/6 and Bcl-xL while increasing protein levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment with birinapant with mivebresib was synergistically lethal in UCSD-AML1 and AML191 cells, with Delta Synergy scores > 1.0 calculated by the ZIP method. Treatment with the dual mTOR/PIK3CA inhibitor NVP-BGT226 (1-30 nM) or navitoclax or Bcl-xL-specific BH3 mimetic A-1155463 also exerted lethality and synergistically induced apoptosis with mivebresib in AML cells with inv3/t(3;3). We previously reported that the TBL1/β-catenin antagonist tegavivint induced apoptosis in 3q26.2-r PD AML cells and PDX models. Co-treatment with birinapant and tegavivint also synergistically induced apoptosis in 3q26.2-r AML cells. Finally, in the xenograft model of AML194-Luc cells in NSG mice, monotherapy with birinapant (30 mg/kg/t.i.w, Intraperitoneal), vs vehicle control, significantly reduced the AML burden, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with birinapant and the BETi OTX015 (30 mg/kg/day, by oral gavage) was more effective in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of IAP protein inhibition against the cellular models of AML with inv3/t(3;3) with EVI1 overexpression, supporting the rationale to further evaluate in vivo efficacy of birinapant and/or BETi-based combinations against this AML sub-type.

2159. Analysis of Resistance to Oral Standard-of-Care Antibiotics for Urinary Tract Infections Caused By Escherichia coli and Staphylococcus saprophyticus Collected in the United States in 2022

Abstract Background Gepotidacin is a novel, bactericidal, first-in-class triazaacenapthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and binding site and provides well-balanced inhibition of 2 different Type II topoisomerase enzymes. This study reports on the in vitro activity of gepotidacin and other oral antibiotics when tested against contemporary Escherichia coli and Staphylococcus saprophyticus clinical isolates collected from patients with UTIs for a gepotidacin uUTI global surveillance study. Methods A total of 1,001 E. coli and 92 S. saprophyticus isolates were collected during 2022 from 45 medical centers located within the United States. Most isolates (77%) tested were cultured from urine specimens collected from patients seen in ambulatory, emergency, family practice, and outpatient medical services. Bacterial identifications were confirmed by MALDI-TOF MS. Isolates were tested for susceptibility by CLSI methods at a central laboratory (JMI Laboratories). MIC results for oral antibiotics licensed for the treatment of uUTI and drug-resistant subsets were interpreted per CLSI guidelines. Results Gepotidacin (MIC50/90, 2/4 µg/mL) displayed good activity against 1,001 E. coli isolates as 98.8% of all observed gepotidacin MICs were ≤4 µg/mL (Table). Other oral agents tested against these isolates demonstrated the following rates of susceptibility (S): amoxicillin-clavulanate (85.2% S), ampicillin (55.6% S), ciprofloxacin (78.8%S), fosfomycin (99.3% S), mecillinam (93.5%S), nitrofurantoin (97.5% S), and trimethoprim-sulfamethoxazole (73.3% S). Gepotidacin maintained similar MIC50 values (ranging from 1 – 2 µg/mL) and MIC90 values (ranging from 2 – 4 µg/mL) against these drug-resistant subsets. Against S. saprophyticus isolates, gepotidacin (MIC50/90, 0.06/0.12 µg/mL) inhibited all isolates at ≤0.12 µg/mL. Most oral agents showed >90% S against S. saprophyticus isolates, except for penicillin (7.6% S). Conclusion Gepotidacin demonstrated in vitro activity against contemporary E. coli and S. saprophyticus from US urine isolates. This activity remained unaffected by resistance to other oral standard-of-care antibiotics. Disclosures S J Ryan Arends, PhD, Cipla: Grant/Research Support|GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Grant/Research Support Didem Torumkuney, PhD, GSK: Grant/Research Support Nicole E. Scangarella-Oman, MS, GSK: Employee and shareholder Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Synergistic anti‐bacterial activity of imidazole‐based azo dyes with polyvinylpyrrolidone‐coated silver nanoparticles

AbstractThe high demand for novel antimicrobial textiles by the medical, healthcare, hygiene, sportswear, personal protective equipment and filtration sectors has promoted the growth of functional textiles. However, the efficacy of antimicrobial agents against different pathogens is a considerable challenge because of the distinctive mechanisms of action and resistance. The development of novel synergistic antimicrobial dyes may offer numerous opportunities to enhance antimicrobial effectiveness. In this work, a novel imidazole‐based azo dye with a p‐methoxyphenyl group in the N‐1 substituent of the imidazole ring (AzoIz‐a), and corresponding amidrazone precursor (AmIz‐a), were combined with polyvinylpyrrolidone‐coated silver nanoparticles (AgNPs). The molecules, alone and combined with the AgNPs, were characterised by ultraviolet‐visible spectrophotometry and zeta potential. Their synergistic effect was assessed against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The results were compared with a previously reported imidazole‐based azo dye and precursor containing a methyl group in the imidazole ring (AmIz‐b and AzoIz‐b). The results showed interesting antimicrobial properties of the novel AzoIz‐a molecules when combined with a very small concentration of AgNPs. The combination showed an additive effect for S. aureus and a synergistic effect for E. coli and P. aeruginosa. Considering the synergistic results, the effective concentration of the AzoIz‐a decreased from more than 128 to 16‐32 μg.mL−1 by the addition of a small concentration of AgNPs (0.6‐1.3 μg.mL−1), which displayed comparable results with the AzoIz‐b molecule. Thus, the application of these conjugates in textiles may lead to highly coloured materials with remarkable anti‐bacterial properties, which warrant further exploration.

DDDR-18. SINGLE-CELL FUNCTIONAL GENOMIC ANALYSIS OF PHARMACOLOGIC RESPONSES IN NF1 MUTANT GLIOBLASTOMA REVEALS DISTINCT MECHANISMS OF ACTION FOR MEK AND SHP2 INHIBITION

Abstract BACKGROUND Glioblastomas harbor recurrent mutations in NF1 that activate Ras/Raf/MEK signaling, motivating pharmacologic approaches targeting downstream (MEK) or upstream (SHP2) of Ras signaling. However, the mechanisms underlying therapeutic response remain unclear. Here, we integrate genome wide CRISPRi screens and single cell sequencing in cell lines and mouse xenografts to identify functional genomic mechanisms underlying responses to the MEK inhibitor selumetinib or the SHP2 inhibitor RMC-4550. METHODS Human NF1 mutant GBM43 cells expressing CRISPRi machinery were used for CRISPRi screens with selumetinib or RMC-4550. Intracranial xenografts were established with GBM43 cells and treated with selumetinib or RMC-4550. Tumor growth kinetics were measured with bioluminescent imaging. RESULTS Genome-wide CRISPRi screens with selumetinib or RMC-4550 in NF1 mutant human GBM43 cells identified 39 genes (14 sensitivity, 25 resistance) mediating selumetinib response and 327 genes (51 sensitivity, 276 resistance) mediating RMC-4550 response. While both selumetinib and RMC-4550 hits were enriched for components of Ras/Raf/MEK, hits sensitizing RMC-4550 response were uniquely enriched for PI3K signaling and focal adhesion components while hits mediating resistance were enriched for cell cycle genes. In NF1 mutant GBM43, RMC-4550 significantly reduced tumor volume (p=0.02) while selumetinib did not. Single cell RNA-sequencing (scRNA-seq) analysis of 90,782 tumor cells from NF1 mutant GBM43 xenografts treated with selumetinib or RMC-4550 identified 12 clusters across all samples. RMC-4550, but not selumetinib, resulted in significant depletion of cells expressing an epithelial-mesenchymal transition (EMT) signature marked by expression of focal adhesion genes mediating RMC-4550 sensitivity, suggesting this signature comprises a distinct mechanism of action in response to RMC-4550 but not selumetinib. CONCLUSIONS In NF1 mutant glioblastoma in vitro and in vivo models, cellular response to both upstream (SHP2) and downstream (MEK) inhibition are mediated by Ras/Raf/MEK signaling while RMC-4550 alone uniquely regulates a focal adhesion network associated with EMT and demonstrates improved efficacy in vivo.

Abstract 18049: Mathematical Modeling Predicts Treatment Efficacy in Transthyretin Cardiac Amyloidosis

Introduction: Increased recognition of transthyretin cardiac amyloidosis (ATTR-CA) led to the development of treatments with different therapeutic targets. Optimal patient selection, treatment choice, timing and duration remain unclear. We evaluated the efficacy and timing of common and emerging ATTR-CA treatments with a mathematical model based on therapeutic mechanisms. Methods: We developed a system of ordinary differential equations describing transthyretin amyloid (ATTR) deposition and myocardial clearance by clusterin (Figure 1A,B). Published data and equilibrium analyses determined parameters for initial maximal ATTR density. Mechanisms of four therapies were modeled and simulated at varying levels of efficacy. Complete success of the mechanism defines 100% efficacy, i.e. full stabilization of tetramer. Expected clinical efficacy (ECE) is based on studies of clinical doses. Minimum effective clearance (MEC) is the theoretical efficacy required to reduce ATTR fibrils below the threshold for complete clearance. Results: All treatments reduced ATTR fibril density, correlating with efficacy (Fig 1C). At ECE, only NI006 demonstrated complete clearance of ATTR fibrils. With other current treatment strategies, amyloid density will be lowered but never cleared. The secondary mechanism of diflusinil did not change equilibrium behavior compared to tafamidis. MEC values and time to clearance at MEC were lowest for NI006 (14.7%; 65 days) and doxycycline (16.1%; 36 months). Tetramer stabilization required almost complete efficacy (99.3%) and about a decade (119 months) for tetramer clearance. Conclusion: This mathematical model predicts ATTR clearance by four distinct mechanisms of action. The novel anti-ATTR antibody NI006 is a promising treatment with predicted faster onset and higher effectiveness for clearance of ATTR fibrils from the myocardium. This model can evaluate future treatment strategies and therapeutic targets.

Fedratinib Treatment Reduces the Inflammatory Cytokine Profile and Decreases Exhausted T Cells Correlating with Clinical Response in Patients with Myelofibrosis: Biomarker Analysis from the Phase 3 FREEDOM2 Trial

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by a hyperactive JAK-STAT pathway, splenomegaly, and constitutional symptoms. JAK inhibitors (JAKi) have been the mainstay in MF treatment over the past decade. Pro-inflammatory cytokines, the levels of which have prognostic value (Tefferi et al. J Clin Oncol 2011), have been shown to be upregulated in MF. However, there is paucity of data on chronic immune dysregulation in pathogenesis of MF. Here, we report the multiplatform biomarker analysis including immune changes from phase 3 (FREEDOM2) trial in patients with MF previously exposed to ruxolitinib (Rux) and treated with fedratinib vs. best available therapy (BAT) (primary efficacy: 35.8% vs 6% responders at the end of cycle 6 (EOC6) in fedratinib vs BAT, respectively). Of note, 78% of the patients on BAT continued RUX therapy until EOC6, allowing direct comparison between RUX and fedratinib. Methods: In the FREEDOM2 trial, patients (N=201) were randomized into two arms, fedratinib (400 mg/day) or BAT in a 2:1 ratio, respectively. The primary endpoint was the proportion of patients with ≥35% reduction of spleen volume (SVR35) on MRI at the end of cycle 6 (EOC6). In the FREEDOM2 biomarker cohort, mutational analysis was performed in enriched CD66b+ cells using the MLL myeloid panel targeting 74 genes. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels from C1D1 to EOC6. Custom Q2 immune panels to detect myeloid and lymphoid subsets were used for immunophenotyping of peripheral blood samples. Results: Baseline mutations in FREEDOM2 were commonly found MPN mutations that include JAK2 V617F (71%), ASXL1 (43%), CALR (22%), TET2 (22%) and MPL (11%). However, baseline mutation status was not a predictor of response. Patients receiving fedratinib demonstrated significant down-regulation of several pro-inflammatory cytokines including CRP, ENRAGE, IL-18, IL-16, TIMP-1, and increase of anti-inflammatory cytokines including adiponectin, MMP2, and CEA (Wilcoxon signed rank test, BH adjusted p <0.001) at EOC6 vs C1D1. In addition, the change in levels of many cytokines correlated with the clinical endpoint (SVR35) demonstrating that the therapeutically relevant changes were mediated by fedratinib (Pearson rho P<0.05) (Figure 1). Importantly, fedratinib was superior to the BAT in its ability to alter levels of key cytokines (p <0.05) at EOC6. PD-1 expression has been shown to be upregulated in CD4+ and CD8+ T cells in MF patients and is associated with poor overall survival. However, RUX did not have a significant effect on PD1+ T cells (Veletic et al. Haematologica 2021). Interestingly, flow cytometric analysis revealed that fedratinib decreased exhausted CD8+ T cells (PD1+) up to 40% (∆ median change -11.4%,p<0.001)) (figure 2). Importantly, the reduction in exhausted T cells correlated with the reduction in spleen volume with fedratinib treatment ( R= -0.51, P=0.00011; Figure 2). Consistent with this, we also observed median increases in NK (+3%, p<0.001) and NK-T (+13.8%, p<0.001) populations when compared to the BAT arm. Conclusions: To our knowledge, this is the first comprehensive study detailing cytokine and immune changes in patients with MF in a clinical trial. In addition, our data for the first time demonstrates the impact of fedratinib on the immune system and its ability to reduce exhausted T cells, highlighting the immune modulatory mechanism of fedratinib. Notably, the cytokine and immune changes correlated with the primary endpoint and were not observed in the BAT arm demonstrating superior efficacy of fedratinib in previously RUX exposed patients. Furthermore, since majority of the patients in the BAT arm continued to receive RUX, these findings highlight the mechanistic differences between fedratinib and RUX on clinical response in MF (SVR35). Overall, our data highlight multiple novel avenues to achieve therapeutic efficacy in MF and positions fedratinib as an effective therapy with a distinctive mechanism of action. Additional analyses on the effect of fedratinib treatment on myeloid immune subsets and transcriptomic changes are underway to gain comprehensive mechanistic understanding.

Patterns of Change in Multiple Myeloma (MM) Clone Size with Autologous Hematopoietic Stem Cell Transplantation (ASCT) Assessed By Next Generation Sequencing (NGS) in Patients (pts) Receiving Modern Therapy

Background Measurable residual disease (MRD) is a quantitative, dynamic prognostic marker of progression free and overall survival in MM. With modern triplet and quadruplet induction strategies deep responses are the norm and the incremental effect of ASCT impact may vary substantially among pts and be influenced by disease and treatment features. However, the accurate characterization of the impact of ASCT is diluted by measuring a disease surrogate (paraprotein) instead of clone size. We examine the quantitative impact of ASCT on MM clone size by serial NGS testing before and after ASCT and evaluate disease and induction treatment features that modulate this impact. Methods We included pts with newly diagnosed MM from five US centers, who received modern triplet and quadruplet induction followed by single melphalan ASCT between 3/2018 and 5/2023 with MRD testing (ClonoSEQ®) pre and post ASCT. ClonoSEQ® platform utilizes NGS to quantify unique immunoglobulin genes rearrangements associated with MM clone, therefore directly capturing clone size. We excluded pts with clone size <10 -5 prior to ASCT. We explored patient-, disease-, and treatment features influencing clone size reduction with AHCT by 1 log 10. We used binary logistic regression to identify factors associated with ≥1 log 10 clone reduction (60-100 days post ASCT). Results One hundred and forty-nine pts with NDMM had pre and post ASCT results and pre ASCT MRD ≥10 -5 and met the inclusion criteria. The median age was 63 years (35-79), 37 pts (25%) ≥70 years, 62% male, 27% Black, 23% ISS3 and 16% RISS3. Seventy-three pts (49%) had high risk chromosomal abnormalities [HRCA, gain/amp(1q), t(4;14), t(14;16), t(14;20) or del(17p)]); 72 pts (48%) had 0 HRCA, 50 (34%) had 1 HRCA, and 23 (15%) had 2+ HRCA. AntiCD38 monoclonal antibody (mAb) was part of induction in 125 (84%) pts [68 pts quadruplet containing carfilzomib; 53 pts quadruplet containing bortezomib], 28 pts received triplets. Eighty-seven pts (58%) had >1log 10 reduction in MRD burden with ASCT. The median log reduction in MRD burden was 1.15 (IQR 0.65-1.75). For pts with 0, 1 and 2+ HRCA, 47% (34/72), 68% (34/50), 74% (17/23) had a >1 log 10 reduction in clone size and the median reduction was 0.96 log 10 (IQR 0.47-1.66), 1.24 log 10 (IQR 0.89-1.91) and 1.5 log 10 (IQR 0.89-2.20), respectively (Figure). Among pts without HRCA, those with hyperdiploid MM had a trend towards less frequent ≥1 log 10 clone size reduction with ASCT (38% vs. 57%, p=0.18). Overall, we saw reduction ≥1 log 10 in clone size in 61% of pts receiving anti-CD38 mAb in induction (N=125) vs. 46% among those who did not (N=24, p= 0.18). Among pts treated with melphalan 200 mg/m 2 (N=113) compared to those who received 140 mg/m 2 (N=36), ≥1 log 10 reduction in clone size was noted in 61% vs. 50% (p=0.25), respectively. In multivariable analysis that included stage, antiCD38 mAb in induction, use of carfilzomib in induction, HRCA and melphalan dose, the presence of HRCA was the only factor associated with greater than 1 log 10 reduction in MRD burden with ASCT [1 HRCA, OR 2.34 (1.07-5.14); ≥2 HRCA, OR 2.71 (0.91-8.03); p=0.049]. Conclusions The impact of high dose melphalan and AHCT on MM clone size is heterogeneous, of greater magnitude in pts with one or more high-risk chromosome abnormalities, and less pronounced in pts with standard risk disease, particularly hyperdiploid MM. The use of antiCD38 mAb in induction regimens or dose of melphalan does not impact the effect of ASCT on MM clone size, likely owing to the distinct mechanism of action of melphalan. This dataset provides context to explore risk- and response-adapted use of ASCT and use of new cellular and non-cellular immunotherapies as post-induction consolidative strategies.

Some reflections on immunosuppressants and their delivery to the lungs by aerosol in the treatment of COVID-19

Disorders of immune homeostasis represent the key pathogenetic link of COVID-19, which often manifests as a hyperimmune response to the pathogen, leading to severe uncontrolled inflammation in lungs, followed by complications and death. Accordingly, a certain therapeutic potential is provided by different pharmacological drugs with distinct mechanisms of action, This class of drugs should, however, act in common direction by suppressing the immune response, thus being often classified as immunosuppressants (IS). Of them, the most promising are immunobiological preparations, which include monoclonal antibodies, as well as purinergic regulatory agents. There are several attempts to use the “classical” IS by a certain way, e.g., cytostatics and calcineurin inhibitors which found clinical application in transplantology and oncology. However, their usage for treatment of uncontrolled inflammation of respiratory tract was abandoned by the end of XX century. Meanwhile, the aerosol route of drug administration optimizes treatment, both in terms of their effectiveness, and the reduction of side effects thus promoting usage of IS for treatment of uncontrolled airway inflammation. Previously, the analysis of therapeutic opportunities for some IS delivered as aerosols to the lungs in COVID-19 therapy was not carried out, thus bein the purpose of our work. Methodological analysis was carried out using various databases of biomedical scientific information, including Index Medicus, PubMed, Embase, Cohrane Clinical Trials gov registry and patent databases.The efficiency of the impact of various IS subgroups in COVID-19, including their administration by inhalations into the respiratory ways, was assessed. The role of regulatory T cells considered the central regulator of immune response, in pathogenesis of COVID-19 was considered, and their therapeutic potential was characterized, dependent on phase and severity of the disease as well as drug dose dependence. Methods and approaches to the use of IP, advantages and disadvantages are discussed. The expediency and future prospects of their application are considered.One may conclude that the effectiveness of cytostatics and calcineurin inhibitors in the treatment of airway inflammation in COVID-19 remains unconfirmed and seems to be unpromising. Meanwhile, biological preparations, including monoclonal antibodies and purinergic regulatory agents, offer great promise in this respect.

Impact of Safinamide on Patient-Reported Outcomes in Parkinson's Disease.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative condition due to the degeneration of nigral dopaminergic cells. Both motor and non-motor symptoms (NMS) of PD produce a marked impairment in PD patients' quality of life (QoL), but contrary to motor features, NMS do not improve with dopamine replacement. Novel therapeutic interventions for PD have successfully controlled most motor manifestations of PD, but the management of NMS is still challenging. Since NMS have a negative impact on the QoL of PD patients, researchers are currently looking for drugs that can modulate the activity of neurotransmitter systems other than dopamine in the hope that can alleviate NMS in PD. Among the recently approved drugs for patients experiencing fluctuations in motor symptoms, safinamide stands out as an effective add-on therapy to levodopa. Safinamide is a monoamine oxidase type-B inhibitor (MAOB-I), with proven efficacy in reducing motor fluctuations. Its distinctive mechanism of action impacts dopaminergic pathways via MAOB inhibition and glutamatergic pathways by blocking sodium and calcium channels. Findings from Phase III clinical trials, meta-analysis, post-hoc analysis, and real-life experiences indicate that safinamide benefits motor symptoms such as tremor, bradykinesia, rigidity, and gait. Additionally, it shows promise for improving NMS like fatigue, pain, mood, and sleep disturbances in patients with PD. In this article, the authors explore the impact of safinamide on patient-reported outcomes in PD. A thorough search was conducted on PubMed focusing on studies published between 2018 and 2023 in English. The inclusion criteria encompassed clinical trials, randomized controlled trials, systematic reviews, meta-analyses, and reviews. The search strategy revolved around the implementation of MeSH terms related to safinamide and its impact on the quality of life in PD. Our data strongly support the improving effect on QoL, reducing the disabling NMS reported in patients with PD.

Proteasome inhibition as a therapeutic target for the fungal pathogen Cryptococcus neoformans.

The current therapeutic challenges for treating fungal diseases demand new approaches and new drugs. A promising strategy involves combination therapy with agents of distinct mechanisms of action to increase fungicidal activity and limit the impact of mutations leading to resistance. In this study, we evaluated the antifungal potential of bortezomib by examining the inhibition of proteasome activity, cell proliferation, and capsule production by Cryptococcus neoformans, the causative agent of fungal meningoencephalitis. Chemical genetic screens with collections of deletion mutants identified potential druggable targets for combination therapy with bortezomib. In vitro assays of combinations of bortezomib with flucytosine, chlorpromazine, bafilomycin A1, copper sulfate, or hydroxyurea revealed antifungal effects against C. neoformans. Furthermore, combination treatment with bortezomib and flucytosine in a murine inhalation model of cryptococcosis resulted in the improvement of neurological functions and reduced fungal replication and dissemination, leading to a delay in disease progression. This study therefore highlights the utility of chemical genetic screens to identify new therapeutic approaches as well as the antifungal potential of proteasome inhibition. IMPORTANCE Fungal diseases of humans are difficult to treat, and there is a clear need for additional antifungal drugs, better diagnostics, effective vaccines, and new approaches to deal with emerging drug resistance. Fungi are challenging to control because they share many common biochemical functions with their mammalian hosts and it is therefore difficult to identify fungal-specific targets for drug development. One approach is to employ existing antifungal drugs in combination with agents that target common cellular processes at levels that are (ideally) not toxic for the host. We pursued this approach in this study by examining the potential of the clinically approved proteasome inhibitor bortezomib to influence the proliferation and virulence of Cryptococcus neoformans. We found that the combination of bortezomib with the anti-cryptococcal drug flucytosine improved the survival of infected mice, thus demonstrating the potential of this strategy for antifungal therapy.

Functional Repetitive Neuromuscular Magnetic Stimulation (frNMS) Targeting the Tibialis Anterior Muscle in Children with Upper Motor Neuron Syndrome: A Feasibility Study.

Non-invasive neurostimulation as an adjunctive intervention to task-specific motor training is an approach to foster motor performance in patients affected by upper motor neuron syndrome (UMNS). Here, we present first-line data of repetitive neuromuscular magnetic stimulation (rNMS) in combination with personalized task-specific physical exercises targeting the tibialis anterior muscle to improve ankle dorsiflexion (functional rNMS (frNMS)). The main objective of this pilot study was to assess the feasibility in terms of adherence to frNMS, safety and practicability of frNMS, and satisfaction with frNMS. First, during 10 training sessions, only physical exercises were performed (study period (SP) A). After a 1 week break, frNMS was delivered during 10 sessions (SPC). Twelve children affected by UMNS (mean age 8.9 ± 1.6 years) adhered to 93% (SPA) and 94% (SPC) of the sessions, and omittance was not related to the intervention itself in any case. frNMS was safe (no AEs reported in 88% of sessions, no AE-related discontinuation). The practicability of and satisfaction with frNMS were high. Patient/caregiver-reported outcomes revealed meaningful benefits on the individual level. The strength of the ankle dorsiflexors (MRC score) clinically meaningfully increased in four participants as spasticity of ankle plantar flexors (Tardieu scores) decreased in four participants after SPC. frNMS was experienced as a feasible intervention for children affected by UMNS. Together with the beneficial effects achieved on the individual level in some participants, this first study supports further real-world, large-scale, sham-controlled investigations to investigate the specific effects and distinct mechanisms of action of frNMS.