OBJECTIVES/SPECIFIC AIMS: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received considerably less attention. Mac-1 is a heterodimeric beta-2 integrin primarily expressed by myeloid-lineage cells. In its activated state, Mac-1 mediates cell-cell interactions by engaging a variety of surface molecules, including the endothelium-expressed glycoprotein ICAM-1. Here, our goals were (1) to determine the extent to which APS neutrophils adhere to healthy, resting endothelial cells under physiologic flow conditions, and (2) to identify potential therapeutic targets by elucidating the molecules required for that adhesion. METHODS/STUDY POPULATION: Primary APS patients (meeting Sydney criteria) and non-autoimmune controls were matched for age and gender. Freshly isolated human umbilical vein endothelial cells (HUVECs) were utilized within five passages. Samples were introduced into a flow channel via a programmable syringe pump, and perfused across a resting HUVEC monolayer. After 15 minutes of perfusion, the chamber was flushed, and the remaining adherent cells were quantified. Flow cytometry was used to identify differentially-expressed molecules on the surface of APS neutrophils. Neutrophil extracellular trap (NET) release was assessed in static neutrophil-HUVEC cultures. RESULTS/ANTICIPATED RESULTS: Pre-treating control neutrophils with APS plasma resulted in increased adhesion as compared with control plasma (>2.5-fold for n = 12 plasma samples; p < 0.05). This was true under both venous conditions (low shear) and conditions representative of the microvasculature (pulsatile flow and higher shear). Control neutrophils treated with APS plasma demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface, as well as shedding of L-selectin. Upregulation of activated Mac-1 and shedding of L-selectin were also triggered by IgG purified from APS plasma. For these changes to be meaningful clinically, we reasoned that they should be present on neutrophils in the peripheral blood of APS patients. Indeed, perfusion of anticoagulated blood through the flow chamber resulted in increased adhesion of patient neutrophils as compared with controls (>5-fold for n = 18 patients; p < 0.05). Similarly, patient neutrophils demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils to HUVECs in the flow-chamber assay (>2-fold reduction for n = 5 patients; p < 0.05). Importantly, the same monoclonal antibody reduced NET release in neutrophil-HUVEC co-cultures. DISCUSSION/SIGNIFICANCE OF IMPACT: APS neutrophils have an increased adhesive potential, which is dependent upon the activated form of Mac-1. This may lower the threshold for both neutrophil-endothelium engagement and NET release in patients, and thereby have implications for events such as venous thrombosis. Studies are underway to determine the extent to which Mac-1 is a viable therapeutic target in preclinical models of APS.

ABSTRACT IMPACT: We hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases. OBJECTIVES/GOALS: The role of IFNLR1 receptor dynamics and plasticity in regulating the type-III IFN response is largely unknown. As a specific, powerful component of innate immunity, understanding how the type-III IFN system is regulated could lead to the development of novel therapeutic targets and strategies to face a multitude of viral illnesses. METHODS/STUDY POPULATION: To facilitate our investigation, we will generate doxycycline-inducible FLAG-tagged IFNLR1-expression plasmids representing all known transcriptional variants. These plasmids will allow us to: 1) Evaluate the effect of IFNLR1 surface abundance on the type-III IFN transcriptional profile and 2) Assess the extent of IFNLR1-FLAG co-localization with several notable intracellular structures using immunofluorescence, before and after stimulation with IFNL3. RESULTS/ANTICIPATED RESULTS: We have successfully generated three IFNLR1-FLAG transcriptional variants and confirmed inducible-expression and function in vitro. We are currently assessing the role of surface abundance, internalization, differential isoform expression, and trafficking. DISCUSSION/SIGNIFICANCE OF FINDINGS: By completing this study, we hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases.

OBJECTIVES/SPECIFIC AIMS: To investigate the performance of a metric for passive localization of central sulcus. METHODS/STUDY POPULATION: We studied 7 patients with intractable epilepsy undergoing intra-cranial EEG (icEEG) monitoring at Yale, in whom central sulcus (CS) localization was obtained by standard methods. Our method takes advantage of inherent properties of the primary motor cortex (MC), which exhibits enhanced icEEG band-power and coherence across the CS. For each contact x we calculated the z-score of a composite power and synchrony value log10(px)x;cx, where px is sum of the root mean square of the icEEG in the high gamma band (80–115 Hz) for contact x over the 6–10 minutes of NREM sleep studied, and cx is the mean magnitude squared coherence in the same band using a 500-ms Hamming window between contact x and all other contacts. z-score values lower than threshold (th) were set to 0. Finally, we calculated a metric m=z/d, where d is the mean Euclidian distance of each contact from contacts with z scores greater than 0. The last step was implemented to emphasize local network activity. RESULTS/ANTICIPATED RESULTS: We report the results of a pilot study to test the performance of a new operator independent method for passive identification of CS with intractable epilepsy undergoing icEEG monitoring at Yale, in whom CS localization was obtained by standard methods. The sensori-motor (SM) cortex exhibited higher EEG-gamma power compared with non-SM cortex (p<0.0002). There was no significant difference between the motor/premotor and sensory cortex (p<0.47). CS was successfully localized in all patients with thresholds between 0.4 and 0.6. In 2 patients, knowledge of anatomy was needed to distinguish the MC from adjacent epileptic foci. The primary hand and leg motor areas exhibited the highest metric values consistently followed by the tongue motor area. Higher threshold values were very specific (94%) for the anterior bank of the CS but not sensitive. Intermediate threshold values achieved a reasonable trade-off (0.4: 89% specific and 70% sensitive). DISCUSSION/SIGNIFICANCE OF IMPACT: We present and successfully implement a rapid procedure for task-free and stimulation free localization of the central sulcus during sleep based on intrinsic electrophysiological properties of the primary motor strip which exhibits increased power and enhanced local connectivity. We successfully localized the central sulcus in all patients. When implementing appropriate thresholds, our proposed metric M is very specific for the anterior lip of the central sulcus which may make it ideal to identify this important anatomical landmark. Our method is sensitive for epileptogenic regions as well, therefore basic knowledge about central sulcus anatomy may be needed in cases where there is an epileptogenic lesion in the vicinity of the central suclus. Our method makes a few a priori assumptions: The regions around the central sulcus are adequately sampled and the occipital or parieto-occipital regions are not included in the analysis. In order for the method to function properly, nonsensori-MC should be sampled adequately as well. In the future, normative data could be generated for the composite product of connectivity×power which may replace within-patient z-scoring. Our method is rapid and can be implemented on short segments of ECoG data. The proposed method may be potentially used for identification of seeds in the motor cortex for subsequent network analysis and further studies may delineate its potential use in the operating room.

OBJECTIVES/SPECIFIC AIMS: Determine PTL’s mechanism(s) of action in a panel of canine hematopoietic cell lines; this will enable us to 1) verify that PTL is working as expected and 2) rationally select combination therapeutics. Characterize the in vitro sensitivity of canine hematopoietic cell lines to PTL in combination with other chemotherapeutic agents. Determine immunohistochemical NFκB expression in tissue microarrays of spontaneous canine neoplasms and correlate with outcome-linked data. Characterize the in vivo sensitivity of canine hematopoietic cell lines to PTL using a murine xenograft model. METHODS/STUDY POPULATION: Growth inhibition assays were performed using a panel of canine mast cell, histiocytic sarcoma, lymphoma, and leukemia cell lines, with PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence and immunoblotting were used to assess NFκB localization and phosphorylation of NFκB p65 (transcriptional activation), respectively. Intracellular glutathione with and without PTL and combination chemotherapeutics will be assessed spectrophotometrically. Archived spontaneous canine tumors will be evaluated immunohistochemically (IHC) for increased NFκB pathway activation relative to normal control tissues. Nude mice will receive intravenous, intraperitoneal, or subcutaneous injections of canine HS cells and will be treated with PTL or with PTL in combination with standard-of-care chemotherapeutics. RESULTS/ANTICIPATED RESULTS: Results: All immortalized canine cell lines evaluated are sensitive to PTL therapy and undergo dose-dependent apoptosis following exposure to drug. PTL exposure leads to inhibition of NFκB, as evidenced by immunofluorescent nuclear exclusion and decreased p65 phosphorylation. Some chemotherapeutics appear to synergize with PTL in vitro. Anticipated results: We expect to find increased IHC NFκB pathway activation in malignantly transformed tissues relative to controls. We expect standard-of-care therapeutics to synergize with PTL in vivo based on preliminary in vitro data. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will determine whether PTL therapy may be beneficial in dogs with a variety of hematopoietic neoplasms, either alone or in combination with other therapeutics that are currently in clinical use. Dogs with mast cell or histiocytic neoplasia are an excellent model for rare and deadly human diseases, which may also benefit from PTL therapy.

OBJECTIVES/GOALS: More young adults (age <50 years) are diagnosed with metastatic gastric cancer (mGC) every year. We will evaluate the association between environmental risk factors (including historical racial residential segregation) clinical, pathologic, molecular features and H.pylori associated early-onset mGC (mE-GC). METHODS/STUDY POPULATION: This retrospective matched case-control study of patients (1:2 by diagnosis year) with mGC (early-onset [E-GC; <50 years]; vs older-onset [O-GC; >50 years]) from 2000-2022 from the Los Angeles Cancer Surveillance Program (LA-CSP) will be enriched by a chart-abstracted cohort from USC Norris Comprehensive Cancer (NCCC). This annotated database captures sociodemographic, medical, and pathologic features of patients treated for mGC at NCCC. It will link to LA-CSP data exploring neighborhood features (obesity rate, poverty, insurance, access to care, ethnic enclave, historical segregation, etc.) of cases and controls to identify, characterize, and compare geographic “hotspot” neighborhoods of mE-GC. We hypothesize younger Hispanic and Asian patients are at higher risk of H.pylori associated mGC. RESULTS/ANTICIPATED RESULTS: From 2000-2022, 339 patients (mE-GC n = 113; mO-GC n=226) were treated at NCCC. We will have characterized clinical and pathological features of mE-GC vs mO-GC. We determined the proportion of H. pylori associated mE-GC vs mO-GC. We will have established the geographical distribution of patients with mE-GC vs mO-GC to identify high-risk neighborhoods. We will link neighborhood risk factors such as food scarcity, poverty, health care access, ethnic enclaves, to the distinct clinical and pathological features of mE-GC, including H. pylori status. Descriptive statistics, chi-square, t-tests, and multivariable regression will be used to compare mE-GC to mO-GC. After controlling for underlying demographics and tumor features, we anticipate clusters of mE-GC and mO-GC in areas of historical racial segregation. DISCUSSION/SIGNIFICANCE: Linking neighborhood and individual risk factors for mE-GC will inform early detection and prevention efforts for vulnerable individuals in high-risk neighborhoods. Building community partnerships within these neighborhoods is essential for developing interventions targeting H. pylori treatment to reduce health disparities in mE-GC.

OBJECTIVES/GOALS: The Colorado Community Engagement Alliance Against COVID-19 Disparities (CO-CEAL) Team has worked with 5 racial/ethnic communities in Colorado to understand COVID-19 vaccine hesitancy and develop messages, materials and dissemination strategies to impact vaccine hesitancy. Relationships have been key facilitators of this work. METHODS/STUDY POPULATION: CO-CEAL was funded for one year as a part of the national CEAL Teams effort. CO-CEAL engaged urban Hispanic/LatinX, rural Hispanic/LatinX, urban African/American, rural African Immigrant and urban American Indian/Alaska Native communities through Community Connectors in each community. Our Community Connectors led recruitment of community members for our community survey data collection (goal of 200 surveys from each community) and participation in our 5 Community (Boot Camp) Translations. Where possible we worked with Community Connectors and leveraged community relationships with whom we had worked over multiple years and projects. In two communities we worked to quickly build and establish new relationships with Community Connectors and their communities. RESULTS/ANTICIPATED RESULTS: The rapid nature of the CO-CEAL project, its timeline and the unusual nature of the funding mechanism required adaptations to usual ways of working on both the academic and community side.In our CO-CEAL partner communities where there was a history of working together, concerns, possible barriers and other issues tended to be rapidly addressed. The prior existing, long-standing relationships fostered trust that enabled open airing of concerns and an ability to address issues as they arose. In CO-CEAL partner communities where new relationships were being forged, trust needed to be build from the ground up. This made adaptations more challenging and the ability to address concerns and issues as they arose more problematic. Although the work has moved forward in all communities, extra care has been required in some. DISCUSSION/SIGNIFICANCE: When crises necessitate rapid action in communities, the depth of prior investments in building and maintaining relationships makes a difference in how rapidly and successfully partners can work together to address a crisis like the COVID-19 pandemic. Funders and Academic researchers should invest broadly in communities to enable rapid responses.

OBJECTIVES/SPECIFIC AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative procedure for hematological malignancies. Chronic graft Versus host disease (cGVHD) is a lethal complication that often develops after allo-HCT. Fli-1 is an aberrantly expressed protein in cancers including erythroleukemia and melanoma, while being implicated in pathogenesis of systemic lupus in mice and humans, a disease with marked similarity to cGVHD. METHODS/STUDY POPULATION: cGVHD was induced using hematopoietic cells from conditional knock-out mice deficient for the fli-1 gene specifically on T cells and progression of cGVHD in murine allo-HCT recipients was monitored using a clinical scoring system, and changes in activation status of hematopoietic cell populations were quantified using flow cytometry. RESULTS/ANTICIPATED RESULTS: Recipients transplanted with fli-1 deficient T cells exhibited reduced cGVHD clinical scores compared with littermate wild-type controls. Donor-grafts containing fli-1 deficient T cells were associated with restrained T-cell responses including reduced Interferon-y cytokine production, PD-1 expression, and differentiation into follicular helper T cells. fli-1 T-cell deficient donor-grafts also improved donor B-cell reconstitution and reduced plasma cells in allo-HCT recipients relative to littermate wild-type control donor-graft recipients. DISCUSSION/SIGNIFICANCE OF IMPACT: Thus, inhibiting Fli-1 represents a promising therapeutic strategy for the goal of preventing cGVHD after allo-HCT while also directly targeting cancers which aberrantly express Fli-1.

OBJECTIVES/GOALS: The Diabetes Prevention Program (DPP) has been shown to reduce diabetes risk by 58%. Men, particularly men of color, are underrepresented in DPP, while they experience higher diabetes-related morbidity. We examine whether race, ethnicity, and gender disparities in engagement are associated with the risk of referral to DPP in primary care METHODS/STUDY POPULATION: Using electronic health record (EHR) from a large urban health system in the Bronx, NY, with an in-house DPP, we examined patient, visit, referral data for DPP-eligible, adult patients with a primary care visit between July 2015 and December 2017. Eligibility included: hemoglobin A1c between 5.7-6.4%; a body mass index (BMI)≥24 kg/m2 (≥22 if Asian); and having no prior diagnosis of diabetes. A total of 26987 patients were included in this study. We examined patient race, ethnicity, preferred language, visit and prescription history, and health payer, among other characteristics. SPSS was used for univariate and bivariate analyses to examine associations between patient characteristics and referral followed by a logistic regression to examine the multivariate association between predictors and referrals. RESULTS/ANTICIPATED RESULTS: Of all DPP-eligible patients, 49% were Hispanic/Latino, and 39% were non-Hispanic Black. Around one-third (34%) of all eligible patients were men. Among all eligible patients in the sample, only 10% were referred to DPP. There were significant differences in the proportion of eligible patients who were referred versus those who were not referred. Women were referred at more than twice the prevalence of men (8.26% to 2.41%), with Hispanic women being referred most frequently (3.59%), and non-Hispanic white men being referred least frequently (.07%) Ethnicity, race, sex, age, number of provider visits, and number of chronic conditions all impacted healthcare provider referral rates to DPP. The health system’s unique free-of-charge DPP likely influenced the lack of significance of patient health insurance. DISCUSSION/SIGNIFICANCE: Given the implementation of DPP at-scale there is an urgent need to understand the patient and systems-level factors that are associated with referring individuals in the DPP. By detecting characteristics of health systems and patients that warrant special attention, we can improve equitable access to evidence-based diabetes prevention.

A crucial reckoning was initiated when the COVID-19 pandemic began to expose and intensify long-standing racial/ethnic health inequities, all while various sectors of society pursued racial justice reform. As a result, there has been a contextual shift towards broader recognition of systemic racism, and not race, as the shared foundational driver of both societal maladies. This confluence of issues is of particular relevance to Black populations disproportionately affected by the pandemic and racial injustice. In response, institutions have initiated diversity, equity, and inclusion (DEI) efforts as a way forward. This article considers how the dual pandemic climate of COVID-19-related health inequities and the racial justice movement could exacerbate the “time and effort tax” on Black faculty to engage in DEI efforts in academia and biomedicine. We discuss the impact of this “tax” on career advancement and well-being, and introduce an operational framework for considering the interconnected influence of systemic racism, the dual pandemics, and DEI work on the experience of Black faculty. If not meaningfully addressed, the “time and effort tax” could contribute to Black and other underrepresented minority faculty leaving academia and biomedicine – consequently, the very diversity, equity, and inclusion work meant to increase representation could decrease it.

OBJECTIVES/SPECIFIC AIMS: It has been reported that birth mode affects development, with cesarean section born mice gaining more body weight during development. Since mice C-sections involve fostering and nest change, we sought to determine whether changing only the nest and cage would have an effect on development. METHODS/STUDY POPULATION: A total of 53 mice were born to 9 dams, and 21 babies (4 litters) were exchanged in pairs to foreign cages and nests. Litters were followed for body weight and mothers were observed during periods for maternal and nonmaternal behaviors. RESULTS/ANTICIPATED RESULTS: The results show that average body weight was significantly higher in the experimental group in both genders, with 20% higher body weights at weaning. The mothers from the litters that were changed to a new nest showed significantly more non-maternal behavior in the first 2 days if life, than the controls. DISCUSSION/SIGNIFICANCE OF IMPACT: The results suggest that changes in maternal behavior may be linked to the increased weight gain in their babies.

OBJECTIVES/GOALS: The purpose of this work is to investigate natural buffering capacity of liver tissue and tumors, to understand and exploit differences for therapy. Using this work, we will determine the concentrations of reagents (acids or bases) used in ablation treatment to optimize treatment by increasing tumor toxicity and minimizing healthy tissue toxicity. METHODS/STUDY POPULATION: For this preliminary study, two methods will be used: benchtop pH experiments ex vivo and non-invasive imaging using acidoCEST MRI in vivo. For ex vivo, two types of tissues will be tested: non-cancerous liver and tumor tissue from HepG2 inoculated mice (n = 10). After mice are euthanized, pH will be measured in tissue homogenates at baseline and then the homogenates will be placed in either acidic (acetic acid) or basic (sodium hydroxide) solutions with varied concentrations (0.5–10M) and time recorded until pH returns to baseline. For in vivo imaging, Mia PaCA-2 flank model mice (n = 10) will be imaged with acidoCEST MRI to quantify pH at baseline. Mice will then be injected intratumorally with (up to 100 μL of) acid or base at increasing concentrations and imaged to quantify pH changes in the tumor. RESULTS/ANTICIPATED RESULTS: For this study, buffering capacity is defined as the concentration threshold for which tissue can buffer pH back to within normal range. Non-cancerous tissue is likely to buffer a wider range of concentrations compared to tumor tissue. From the benchtop experiment, comparison of time-to-buffer will be made for each concentration of acid/base for the two tissue types. AcidoCEST MRI will provide in vivo buffering capacity and potentially demonstrate tumor heterogeneity of buffering capacity. For both experiments, a pH vs. concentration curve for the two tissue types will allow for comparison of ex vivo to in vivo experiments, which will differentiate contributions of local tissue buffering capacity from the full body’s natural bicarbonate buffer system that depends on respiration and blood flow. DISCUSSION/SIGNIFICANCE OF IMPACT: The pH of the body must be maintained within a narrow range. With cancer, impairment in regulation of tumor metabolism causes acidosis, lowering extracellular pH in tumors. It remains unclear if pH plays a role in local recurrence or tumor toxicity. This work will determine if acidoCEST MRI can measure deliberate alteration of pH and how this change affects biology.

OBJECTIVES/GOALS: High-grade gliomas (HGG) are among the rarest, most aggressive tumors in neurosurgical practice. We aimed to identify the clinical predictors for 30-day readmission and reoperation following HGGs surgery using the NSQIP database and seek to create web-based applications predicting each outcome. METHODS/STUDY POPULATION: We conducted a retrospective, multicenter cohort analysis of patients who underwent resection of supratentorial HGG between January 1, 2016, and December 31, 2020, using the NSQIP database. Demographics and comorbidities were extracted. The primary outcomes were 30-day unplanned readmission and reoperation. A stratified 80:20 split of the available data was carried out. Supervised machine learning algorithms were trained to predict 30-day outcomes. RESULTS/ANTICIPATED RESULTS: A total of 9,418 patients were included in our cohort. The rate of unplanned readmission within 30 days of surgery was 14.9%.Weight, chronic steroid use, pre-operative BUN, and WBC count were associated with a higher risk of readmission. The rate of early unplanned reoperation was 5.47%. Increased weight, higher operative time, and a longer period between hospital admission and the operation were linked to increased risk of early reoperation. Our Random Forest algorithm showed the highest predictive performance for early readmission (AUC = 0.967), while the XG Boost algorithm showed the highest predictive performance for early reoperation (AUC = 0.985).Web-based tools for both outcomes were deployed: https://glioma-readmission.herokuapp.com/, https://glioma-reoperation.herokuapp.com/. DISCUSSION/SIGNIFICANCE: A high fraction of documented early unplanned readmission and reoperation were considered preventable and related to surgery. Machine learning allows better prediction of resected HGG’s prognosis based on findings from baseline methods leading to more personalized patient care.

OBJECTIVES/SPECIFIC AIMS: Listening effort is needed to understand speech that is degraded by hearing loss and/or a noisy environment. Effortful listening reduces cognitive spare capacity (CSC). Predictive contexts aid speech perception accuracy, but it is not known whether the use of context reduces or preserves CSC. Here, we compare the impact of predictive context and cognitive load on behavioral indices of CSC in elderly, hearing-impaired adults. METHODS/STUDY POPULATION: Elderly, hearing-impaired adults listened in a noisy background to spoken sentences in which sentence-final words were either predictable or not predictable based on the sentence context. Cognitive load was manipulated by asking participants to remember either short or long sequences of visually presented digits. Participants were divided into low or high cognitive capacity groups based on a pretest of working memory. Accuracy and response times were examined for report of both sentence-final words and digit sequences. RESULTS/ANTICIPATED RESULTS: Preliminary results indicate that accuracy and response times for both words and digits were facilitated by sentence predictability, suggesting that the use of predictive sentence context preserves CSC. Response times for both words and digits and accuracy for digits were impaired under cognitive load. Trends were similar across high and low cognitive capacity groups. The preliminary results support the idea that habilitation strategies involving context use could potentially support CSC in elderly, hearing-impaired adults. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary results support the concept that habilitation strategies involving context use could potentially support CSC in elderly, hearing-impaired adults.

OBJECTIVES/GOALS: As mortality and morbidity from acute COVID-19 decline, the impact of COVID-19 on short- and long-term quality of life (QoL) becomes critical to address. We assessed the impact of re-purposed COVID-19 therapies on QoL as a secondary outcome measure in ACTIV-6, a decentralized platform trial. METHODS/STUDY POPULATION: Adults aged ≥30 with mild-to-moderate COVID-19 enroll in ACTIV-6 online or through a study site. Patients are randomized to a medication of interest or placebo. Medications are mailed and symptoms are tracked using electronic diaries. QoL is measured#_msocom_1 using the PROMIS-29 questionnaire. Adjusted Bayesian logistic regression models are used to measure effects of treatment on the seven PROMIS-29 QoL domains at days 7, 14, 28#_msocom_2 and 90. Covariates are treatment, age, gender, symptom duration and severity, vaccination status, geographic region, call center#_msocom_3#_msocom_4, and calendar time. Treatment effects are described using ORs, 95% credible intervals, and posterior probabilities of efficacy, P(eff). RESULTS/ANTICIPATED RESULTS: There are 5,362 patients included, representing four of the study arms in ACTIV-6. We report results where P(eff)<0.025 and P(eff)>0.975 in the table below. Table 1. Scale Day: OR* (95% credible interval, P(eff)) Therapy Physical Anxiety Depression Fatigue Sleep Social Pain Ivermectin 400 — Ivermectin 600 D7: 0.77 (0.61-0.96, 0.01) D14: 0.65 (0.49-0.85, <0.01) D28: 0.69 (0.52-0.92, 0.01) — D7: 0.79 (0.64-0.97, 0.01) — D14 0.78 (0.60-1.00, 0.02) D28: 0.66 (0.50-0.87, <0.01) Fluticasone - D14: 0.77 (0.60-0.99, 0.02) — D7: 0.76 (0.62-0.93, <0.01) D90: 0.79 (0.64-0.98, 0.01) — D7: 0.74 (0.59-0.93, 0.01) Fluvoxamine D7: 0.66 (0.51-0.84, 0.01) — D28: 1.38 (1.02, 1.85, 0.98) D7: 0.78 (0.63-0.97, 0.01) D7: 0.77 (0.62-0.95, 0.01) — *OR > 1 favors active intervention DISCUSSION/SIGNIFICANCE: Results suggest fluvoxamine may improve depression scores by day 28, while placebo is favored in several other scales across treatments. Differences between treatment and placebo are not seen at most other timepoints. This trial is ongoing and future work will include results from additional ACTIV-6 study arms.

Objectives/Goals: The overarching objective of this study is to inform clinicians, patients, and other stakeholders of the level of evidence and the real-world risk-to-benefit profiles associated with older antidepressant drugs that are frequently used off-label. Methods/Study Population: A PubMed literature review was performed to identify clinical trials conducted in the USA between 2013 and 2023 for trazodone and 2000 and 2023 for escitalopram and citalopram. These studies were examined for robustness, due to sample size, study design, and generalizability. Findings were compared with information provided on UpToDate® LexiDrug™, a primary database used by clinicians to inform prescribing practice. To explore risks associated with off-label use, the FDA adverse event reporting system was probed to identify adverse events reported for each drug; results were systematically categorized by reason for use. To compare the volume of on-label to off-label prescriptions, data will be extracted from electronic health records from University of Southern California-affiliated hospitals. Results/Anticipated Results: Studies conducted on off-label prescriptions of these drugs show primarily small sample sizes, pointing to a limitation in generalizability. For citalopram (N = 77) and trazodone (N = 42), over half of their off-label studies had samples of 50 participants or less. These two drugs also showed low evidence rating for off-label prescription on LexiDrug due to limited power studies. Multiple health agencies recommend against off-label prescriptions for trazodone due to insufficient evidence. There is limited data in the US regarding the volume of off-label prescriptions; however, trazodone’s FAERS analysis indicated a large proportion of adverse event reports (1099/7239) come from cases where trazodone was used for insomnia, an off-label indication, compared to depression, the on-label indication (464/7239). Discussion/Significance of Impact: With 1 in 6 Americans taking antidepressants and 40%–80% of these psychiatric prescriptions being employed off-label, there is a serious and present risk for patients regarding the safety and efficacy of these medications. Awareness must be brought to clinicians to protect patients and encourage evidence-based practice.