Preclinical Research Articles

Abstract A002: Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy

Abstract Despite promising results with immune checkpoint blockade (ICB) therapy, brain metastases (BrM) remain a deadly complication for cancer patients. Understanding the highly specialized brain metastatic tumor microenvironment (BrTME) is crucial for identifying determinants of response, however, progress has been hindered by limited access to patient samples and scarcity of relevant preclinical models. Here, we developed two mouse melanoma BrM models by intracardiac injection of cells derived from our previously established M4-B2905 melanoma mouse cell line. We characterized their mutational landscape and performed single-cell phenotypic and transcriptomic analysis, demonstrating that these models recapitulate the cellular and molecular features of human melanoma BrMs. Comparative and interactome analysis of our models revealed key factors contributing to ICB response and resistance. We found that the responsive model (BR1) was characterized by tumor cells that polarize microglia toward reactive states via inflammatory programs and response to IFN signaling. These microglia express high levels of antigen presentation and immunostimulatory molecules, triggering T cell recruitment and activation and promoting ICB therapy response. In contrast, in the resistant model (BR3), the tumor cells express neurological molecular signatures and ligands that sustain microglia homeostasis. This results in poor T cell infiltration predominantly composed of naïve cells, evading immune reaction and promoting ICB resistance. While we showed that systemic ICB therapy induced BrTME changes in both models, differences between responder and resistant models were maintained, emphasizing the importance of tumor cell-intrinsic programs in dictating the BrTME. We validated the translational relevance of our findings and demonstrated that BR1 signatures positively correlate with T cell infiltration and are associated with improved patient outcomes, whereas BR3 signatures negatively correlate with T cells and are found in patients with worse prognosis. Here we address an important challenge in the field by providing clinically relevant BrM models that recapitulate both the cellular and molecular features of human disease and the variability of ICB response seen in patients. We further reveal mechanistic insights into BrM ICB response and identify potential therapeutic targets to modulate the BrTME. Citation Format: Amelie Daugherty-Lopes, Eva Perez-Guijarro, Vishaka Gopalan, Jessica Rappaport, Quanyi Chen, April Huang, Khiem C. Lam, Sung Chin, Jessica Ebersole, Emily Wu, Gabriel Needle, Isabella Church, George Kyriakopoulos, Shaojun Xie, Yongmei Zaho, Charli Gruen, Antonella Sassano, Romina E Araya, Andres Thorkelsson, Cari Smith, Maxwell P. Lee, Sridhar Hannenhalli, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid, Shaojun Xie. Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A002.

Abstract PR008: Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia

Abstract Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer with a high mortality rate, partly due to late diagnosis often accompanied by metastasis. Cachexia, a complex syndrome marked by severe weight loss and muscle wasting, frequently accompanies advanced cancers including OSCC. Notably, 50-70% of OSCC patients exhibit cachexia at diagnosis, which persists post-treatment, hinting at a metastatic contribution to this syndrome. One significant challenge in studying cancer cachexia is the lack of well-characterized pre-clinical models that accurately reflect human disease. Traditional mouse models often involve subcutaneously transplanted cells, which fail to mimic the natural tumor-host interactions and metastatic progression seen in patients. These models do not typically exhibit metastasis and differ significantly from the genetic and molecular landscape of human cancers. To address these limitations, we developed a syngeneic mouse model by orthotopically transplanting MOC2 murine OSCC cells into the tongues of B6J mice, thereby closely mimicking the tumor microenvironment and metastatic behavior of human OSCC. By enhancing metastasis with a palm-enriched high-fat diet, we observed progressive tumor growth, metastasis, and body weight loss, independent of food intake reduction, indicating metabolic dysregulation rather than anorexia. Comprehensive analyses, including single-cell RNA sequencing (scRNAseq) of immune cells and transcriptomic and metabolomic profiling of tumor and host tissues, identified significant gene expression changes associated with metastasis and cachexia. Enhanced metastasis correlated with lipid metabolism changes in lung metastases, suggesting that metastatic cells exploit lipids for growth. Further, immune profiling revealed neutrophils' metabolic adaptation in metastasis, with implications for both tumor progression and cachexia. Targeting key metabolic and inflammatory pathways in tumor and immune cells holds potential for mitigating metastasis and cachexia. Early-stage serum metabolomics indicated systemic metabolic shifts, emphasizing the need to understand the interplay between tumor metastasis and host metabolism. These findings underscore the complex mechanisms underlying OSCC-induced cachexia, and the need for new targeted therapeutic strategies to disrupt this lethal synergy between cancer progression and metabolic deterioration of the host. Citation Format: Blanca Majem, Josefina Martin, Liudmila Shevkova, Claudia Bigas, Gloria Pascual, Salvador Aznar-Benitah. Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR008.

Abstract C022: Exercise reduces pancreatic tumor in a gut microbiome dependent manner

Abstract Preclinical studies suggest that aerobic exercise influences the tumor microenvironment by normalizing vasculature and reducing immunosuppression in Pancreatic Ductal Adenocarcinoma (PDAC). Gut microbes can influence PDAC progression by modulating metabolic and immune pathways. However, a knowledge gap remains regarding the role of the gut microbiome in the anti-tumor effects of exercise. We hypothesized that the exercise-induced anti-tumor effect in PDAC is regulated by gut microbiome. We compared the effect of treadmill running on growth of orthotopic PDAC in C57BL/6 mice from Taconic Biosciences (Tac) and Jackson Laboratories (Jax), which have different microbiomes. Tumor-bearing mice performed treadmill exercise (ex, 12 meters/minute (m/min) for 45 minutes (min) daily) or were non-exercised controls (no-ex). Exercise reduced tumor weight compared to no-ex controls in Jax (no-ex, 0.72±0.05 grams (g); ex, 0.54±0.04g; p=0.04) but not in Tac mice (no-ex, 0.72±0.05g; ex, 0.78±0.05g; p=0.47). Interestingly, in Tac mice treated with antibiotics (ATBx) ad libitum, treadmill exercise reduced tumor growth (no-ex, 0.74±0.04 g; ex, 0.53±0.03g; p= 0.006). Taken together, this data indicates that the anti-tumor effect of exercise is affected by gut microbiome. We considered potential mechanisms by which exercise suppresses tumor growth. Tumor infiltrating immune cells were measured by flow cytometry and immunohistochemistry. No significant differences in the tumor infiltrating immune cells were observed between no-ex and ex mice from Tac or Jax, regardless of ATBx, indicating that gut microbiome mediated anti-tumor effect is independent of immune pathways. Previous studies have demonstrated that exercise reduces tumor growth in an intensity dependent manner. Therefore, to determine exercise intensity as percentage of Peak Velocity (VPeak), exercise capacity (ExCap) test was performed. VPeak (m/min), time to exhaustion (min), and distance to exhaustion (m) were used as indicators for treadmill ExCap. Treadmill ExCap was significantly lower in tumor bearing Tac mice (24.86±3.55m/min, 12.56±1.79min, 217.17±31.02m) compared to Jax mice (30.50±3.81m/min; 16.58±2.07min; 320.15±40.02m; p=0.01). However, ATBx treatment did not change the treadmill ExCap in the Tac mice (25.50±3.19m/min; 12.62±1.58min; 209.26±26.16m; p=0.97) despite significantly changing the effect of exercise on tumor growth, indicating that ExCap may not regulate differences observed in the tumor weight. Overall, these results indicate that gut microbiome regulates anti-tumor effects of exercise, that are independent of tumor infiltrating immune cells or exercise intensity. Analysis of serum and tumor metabolites, 16s rRNAseq to evaluate microbial differences, and bulk RNAseq on tumors, is underway with the goal of identifying microbial-derived metabolites which mediate the anti-tumor effect of exercise. Understanding how the gut microbiome mediates the efficacy of exercise to suppress tumor growth will be critical for maximizing the benefits of exercise for patients with PDAC. Citation Format: Sumedha Pareek, Vidhi Chandra, Le Li, Hetal Patel, Truong Lam, Olivereen LeRoux, Haoyue Liu, Jonghae Lee, Florencia McAllister, Keri Schadler. Exercise reduces pancreatic tumor in a gut microbiome dependent manner [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C022.

Machine learning prediction of brain metastasis invasion pattern on brain MRI scans

Abstract Background Brain metastasis invasion pattern (BMIP) is an emerging biomarker associated with recurrence-free and overall survival in patients, and differential response to therapy in preclinical models. Currently, BMIP can only be determined from the histopathological examination of surgical specimens, precluding its use as a biomarker prior to therapy initiation. The aim of this study was to investigate the potential of machine learning (ML) approaches to develop a non-invasive magnetic resonance imaging (MRI)-based biomarker for BMIP determination. Methods From an initial cohort of 329 patients, a subset of 132 patients met inclusion criteria for this retrospective study. We evaluated the ability of an expert neuroradiologist to reliably predict BMIP. Thereafter, the dataset was randomly divided into training/validation (80% of cases) and test subsets (20% of cases). Ground truth for BMIP was histopathologic evaluation of resected specimens. Following MRI sequence co-registration, advanced feature extraction techniques deriving hand-crafted radiomic features with traditional ML classifiers and convolution-based deep learning (CDL) models were trained and evaluated. Different ML approaches were used individually or using ensembling techniques to determine the model with the best performance for BMIP prediction. Results Expert evaluation of brain MRI scans could not reliably predict BMIP, with an accuracy of 44-59% depending on the semantic feature used. Among the different ML and CDL models evaluated, the best performing model achieved an accuracy of 85% and an F1-score of 90%. Conclusion ML approaches can effectively predict BMIP, representing a non-invasive MRI-based approach to guide management of patients with brain metastases.

Sex-specific GABAergic microcircuits that switch vulnerability into resilience to stress and reverse the effects of chronic stress exposure.

Clinical and preclinical studies have identified somatostatin (SST)-positive interneurons as critical elements that regulate the vulnerability to stress-related psychiatric disorders. Conversely, disinhibition of SST neurons in mice results in resilience to the behavioral effects of chronic stress. Here, we established a low-dose chronic chemogenetic protocol to map these changes in positively and negatively motivated behaviors to specific brain regions. AAV-hM3Dq-mediated chronic activation of SST neurons in the prelimbic cortex (PLC) had antidepressant drug-like effects on anxiety- and anhedonia-like motivated behaviors in male but not female mice. Analogous manipulation of the ventral hippocampus (vHPC) had such effects in female but not male mice. Moreover, the activation of SST neurons in the PLC of male mice and the vHPC of female mice resulted in stress resilience. Activation of SST neurons in the PLC reversed prior chronic stress-induced defects in motivated behavior in males but was ineffective in females. Conversely, activation of SST neurons in the vHPC reversed chronic stress-induced behavioral alterations in females but not males. Quantitation of c-Fos+ and FosB+ neurons in chronic stress-exposed mice revealed that chronic activation of SST neurons leads to a paradoxical increase in pyramidal cell activity. Collectively, these data demonstrate that GABAergic microcircuits driven by dendrite targeting interneurons enable sex- and brain-region-specific neural plasticity that promotes stress resilience and reverses stress-induced anxiety- and anhedonia-like motivated behavior. The data provide a rationale for the lack of antidepressant efficacy of benzodiazepines and superior efficacy of dendrite-targeting, low-potency GABAA receptor agonists, independent of sex and despite striking sex differences in the relevant brain substrates.

Preclinical Experimental Study on New Cervical Implant Design to Improve Peri-Implant Tissue Healing

Objectives: In this preclinical study, we used an experimental rabbit model to investigate the effects of a new implant design that involves specific changes to the cervical portion, using a conventional implant design in the control group. Materials and Methods: We used 10 rabbits and 40 dental implants with two different macrogeometries. Two groups were formed (n = 20 per group): the Collo group, wherein implants with the new cervical design were used, which present a concavity (reduction in diameter) in the first 3.5 mm, the portion without surface treatment; the Control group, wherein conical implants with the conventional design were used, with surface treatment throughout the body. All implants were 4 mm in diameter and 10 mm in length. The initial implant stability quotient (ISQ) was measured immediately after the implant insertion (T1) and sample removal (T2 and T3). The animals (n = five animals/time) were euthanized at 3 weeks (T1) and 4 weeks (T2). Histological sections were prepared and the bone–implant contact (BIC%) and tissue area fraction occupancy (TAFO%) percentages were analyzed in the predetermined cervical area; namely, the first 4 mm from the implant platform. Results: The ISQ values showed no statistical differences at T1 and T2 (p = 0.9458 and p = 0.1103, respectively) between the groups. However, at T3, higher values were found for the Collo group (p = 0.0475) than those found for the Control group. The Collo samples presented higher BIC% values than those of the Control group, with statistical differences of p = 0.0009 at 3 weeks and p = 0.0007 at 4 weeks. There were statistical differences in the TAFO% (new bone, medullary spaces, and the collagen matrix) between the groups at each evaluation time (p < 0.001). Conclusions: Considering the limitations of the present preclinical study, the results demonstrate that the new implant design (the Collo group) had higher implant stability (ISQ) values in the samples after 4 weeks of implantation. Furthermore, the histomorphometric BIC% and TAFO% analyses showed that the Collo group had higher values at both measurement times than the Control group did. These findings indicate that changes made to the cervical design of the Collo group implants may benefit the maintenance of peri-implant tissue health.

Antitumorigenic Effect of Combination Treatment with BRAF Inhibitor and Cisplatin in Colorectal Cancer In Vitro and In Vivo

AbstractIn colorectal cancer (CRC), BRAF inhibitor (BRAFi) monotherapy appears ineffective, while cisplatin treatment is associated with adverse effects, drug resistance, and reduced efficacy. Herein, a combinatorial approach is being explored to increase the likelihood of effectively killing colorectal cancer cells. The combined effect of BRAFi (PLX4720, Vemurafenib, Dabrafenib, Encorafenib) and cisplatin treatment is examined in BRAFV600E‐mutated (RKO, HT29, Colo‐205) and BRAFwt (Caco‐2) cell lines, as well as in mouse xenografts of RKO cells. Following cisplatin‐only treatment, all cell lines show accumulation within subG1 (apoptotic cells) and G2/M phases, as well as phosphorylation of ERK1/2 and H2AX. Following BRAFi‐only treatment, BRAFV600E‐mutated cells show accumulation within G0/G1 phase, reduced distribution in the S and G2/M phases, inhibition of ERK1/2 phosphorylation, and increased phosphorylation of H2AX. Combined BRAFi and cisplatin treatment synergistically decrease RKO cells viability, reduce phosphorylation of ERK1/2, and increase phosphorylation of H2AX. Importantly, in mouse xenografts of RKO cells, combined PLX4720 and cisplatin treatment show superior therapeutic potential than each monotherapy (P < 0.001). Taken together, in in vitro and in vivo preclinical models, BRAFi and cisplatin combined treatment has shown an improved antitumor effect, rendering it a potential anticancer treatment strategy for BRAF‐mutant colon cancer patients.

Methods for Modeling Early Life Stress in Rodents.

Animal models of early life stress/adversity (ELS) have provided a foundation from which our understanding of the psychoneuroimmunology of childhood trauma has expanded over recent decades. Rodent models are a cornerstone of the ELS literature with many studies utilizing paradigms based on early life separation/deprivation protocols and manipulating the cage environment. However, no animal model is perfect. In particular, the lack of standardization across ELS models has led to inconsistent results and raised questions regarding the translational value of common preclinical models. In this chapter, we present an overview of the history of ELS rodent models and discuss considerations relevant to the ongoing efforts to both improve existing models and generate novel paradigms to meet the evolving needs of molecular- and mechanism-based ELS research.

Transcriptome signatures of the medial prefrontal cortex underlying GABAergic control of resilience to chronic stress exposure.

Analyses of postmortem human brains and preclinical studies of rodents have identified somatostatin (SST)-positive, dendrite-targeting GABAergic interneurons as key elements that regulate the vulnerability to stress-related psychiatric disorders. Conversely, genetically induced disinhibition of SST neurons (induced by Cre-mediated deletion of the γ2 GABAA receptor subunit gene selectively from SST neurons, SSTCre:γ2f/f mice) results in stress resilience. Similarly, chronic chemogenetic activation of SST neurons in the medial prefrontal cortex (mPFC) results in stress resilience but only in male and not in female mice. Here, we used RNA sequencing of the mPFC of SSTCre:γ2f/f mice to characterize the transcriptome changes underlying GABAergic control of stress resilience. We found that stress resilience of male but not female SSTCre:γ2f/f mice is characterized by resilience to chronic stress-induced transcriptome changes in the mPFC. Interestingly, the transcriptome of non-stressed SSTCre:γ2f/f (stress-resilient) male mice resembled that of chronic stress-exposed SSTCre (stress-vulnerable) mice. However, the behavior and the serum corticosterone levels of non-stressed SSTCre:γ2f/f mice showed no signs of physiological stress. Most strikingly, chronic stress exposure of SSTCre:γ2f/f mice was associated with an almost complete reversal of their chronic stress-like transcriptome signature, along with pathway changes suggesting stress-induced enhancement of mRNA translation. Behaviorally, the SSTCre:γ2f/f mice were not only resilient to chronic stress-induced anhedonia - they also showed an inversed, anxiolytic-like behavioral response to chronic stress exposure that mirrored the chronic stress-induced reversal of the chronic stress-like transcriptome signature. We conclude that GABAergic dendritic inhibition by SST neurons exerts bidirectional control over behavioral vulnerability and resilience to chronic stress exposure that is mirrored in bidirectional changes in the expression of putative stress resilience genes, through a sex-specific brain substrate.

Treating metabolic dysfunction-associated steatohepatitis: The fat-trimming FGF21 approach.

Metabolic dysfunction-associated steatohepatitis (MASH) is a condition characterized by hepatosteatosis, inflammation, and tissue damage, with steatosis as the initial stage, which involves chronic, excess deposition of lipids in hepatic lipid droplets. Despite the growing prevalence and serious risks it poses, including liver decompensation, the need for transplantation, and increased patient mortality, MASH currently faces no approved pharmacotherapy. Several promising treatment candidates have emerged from recent clinical trials, including analogs of FGF21 and agonists of the associated FGFR1-KLB complex. These agents were well-tolerated in trials and have demonstrated significant improvements in both histological and biochemical markers of liver fat content, inflammation, injury, and fibrosis in patients with MASH. Endocrine FGF21 plays a vital role in maintaining homeostasis of lipid, glucose, and energy metabolism. It achieves this through pathways that target lipids or lipid droplets in adipocytes and hepatocytes. Mechanistically, pharmacological FGF21 acts as a potent catabolic factor to promote lipid or lipid droplet lipolysis, fatty acid oxidation, mitochondrial catabolic flux, and heat-dissipating energy expenditure, leading to effective clearance of hepatic and systemic gluco-lipotoxicity and inflammatory stress, thereby preventing obesity, diabetes, and MASH pathologies. In this review, we aim to provide an update on the outcomes of clinical trials for several FGF21 mimetics. We compare these outcomes with preclinical studies and offer a lipid-centric perspective on the mechanisms underlying the clinical benefits of these agents for MASH.

Antibiotics as adjunct to non-surgical periodontal therapy in diabetic patients.

A comprehensive literature search was performed via MEDLINE/PubMed, Scopus, and Web of Science, including all relevant publications accessed on March 2023 and subsequently updated up to July 2024. Additionally, grey literature was searched, and there was no restriction on publication data and languages. All randomized clinical trials comparing topical/systemic antibiotics as an adjunct to scaling and root planning (SRP) with SRP alone for the treatment of diabetic patients with periodontitis were included. Literature reviews, case reports and series, preclinical studies or studies lacking periodontal data were excluded. Clinical attachment level was considered as the primary outcome. Bleeding on probing, pocket depth, and gingival index were the secondary outcomes. The risk of bias was evaluated using the Cochrane Collaboration Tool, and for each domain, bias risk was judged as low, unclear and high risk of bias. Selection, removing duplicates, screening, extracting data, and assessing the quality of studies were done by two investigators and findings were tabulated. Meta-analysis was conducted, generating forest plots to compare different treatment modalities, time periods, and antibiotic types. Key periodontal parameters such as probing pocket depth (PPD), clinical attachment level (CAL), plaque index (PI), and bleeding on probing (BoP) were analysed. Upon screening and further evaluation of 690 articles, 43 were considered for full-text eligibility. Finally, 30 studies were included in the systematic review, and 27 were included in meta-analysis. Out of 30 randomized clinical trials, 9 studies focused on topical and antibiotics, and 21 involved systemic administration of antibiotics. Five of the nine studies focussing on topical administration of antibiotics, were classified as having a low risk of bias and four as having a high risk of bias. Out of 21 studies on systemic antibiotics, eight were classified as having a low risk of bias, two were undefined, and eleven showed a high risk of bias. Authors concluded that adjunctive use of antibiotics could lead to improvement in periodontal clinical parameters CONCLUSION: The findings showed that the adjunctive use of antibiotics led to slight improvements in clinical outcomes. However, keeping in mind antibiotic resistance, and side effects of antibiotics, their use should be approached cautiously and only when necessary.

C1-inhibitor to prevent intracerebral hemorrhage-related secondary brain injury.

Preclinical studies indicate that the systemic application of C1-inhibitor, clinically used to treat hereditary angioedema, reduces secondary brain injury after ischemic stroke. This study assessed the effect of C1-inhibitor on secondary brain injury after hemorrhagic stroke. We used an established striatal whole-blood injection mouse model to mimic intracerebral hemorrhage-related secondary brain injury. Based on the spatiotemporal dynamics in our model, we calculated the necessary sample size (n = 24) and determined the most sensitive time point to detect potential group differences (48h) prior to the experiments. The experimental setup, tissue processing and image analysis adhered to our published protocol. We randomized mice into three groups: C1-inhibitor treatment, placebo, and sham. Histology was standardized by taking eight anatomically predefined slices across the entire lesion. Lesion size, vascular leakage, and inflammatory responses were assessed using automated thresholding and dextran/ICAM1/CD45 intensity mapping. Investigators were blinded to group allocation during the experiment, tissue processing, and image analysis. Whole blood injection resulted in significantly larger lesion size and more pronounced vascular leakage and cellular inflammation compared to the sham group. However, there was no difference in lesion size or inflammatory markers between the C1-inhibitor and placebo groups. In addition, there was no difference in the inflammatory response of the choroid plexus, which has been identified as a central organ orchestrating inflammation after intracerebral hemorrhage. The protective effect of C1-inhibitor might be isolated to pathophysiological processes with a predominant thromboinflammatory component, as in ischemia-reperfusion, but less so in permanent ischemia or intracerebral hemorrhage.

Trials and tribulations of cell therapy for heart failure: an update on ongoing trials.

Heart failure (HF) remains a leading cause of mortality, responsible for 13% of all deaths worldwide. The prognosis for patients with HF is poor, with only a 50% survival rate within 5 years. A major challenge of ischaemia-driven HF is the loss of cardiomyocytes, compounded by the minimal regenerative capacity of the adult heart. To date, replacement of irreversibly damaged heart muscle can only be achieved by complete heart transplantation. In the past 20 years, cell therapy has emerged and evolved as a promising avenue for cardiac repair and regeneration. During this time, cell therapy for HF has encountered substantial barriers in both preclinical studies and clinical trials but the field continues to progress and evolve from lessons learned from such research. In this Review, we provide an overview of ongoing trials of cell-based and cell product-based therapies for the treatment of HF. Findings from these trials will facilitate the clinical translation of cardiac regenerative and reparative therapies not only by evaluating the safety and efficacy of specific cell-based therapeutics but also by establishing the feasibility of novel or underexplored treatment protocols such as repeated intravenous dosing, personalized patient selection based on pharmacogenomics, systemic versus intramural cell delivery, and epicardial engraftment of engineered tissue products.

Prospects of anti-GD2 immunotherapy for retinoblastoma

Retinoblastoma is the most common type of eye tumor in infants and children. Current treatments for retinoblastoma include intravenous chemotherapy, intra-arterial chemotherapy, intravitreal chemotherapy, cryotherapy, radiotherapy, and surgery. However, these treatments come accompanied by adverse effects such as the toxic side effects of chemotherapeutic drugs, post-operative complications including blindness after surgery, or other complications caused by radiotherapy. Immunotherapy is more promising for its low toxicity on normal cells and effectively improves the quality of life of patients. Disialoganglioside (GD2), a sphingolipid expressed on the surface of retinoblastoma, is a potential therapeutic target for retinoblastoma. We summarized immunotherapeutic approaches for both preclinical studies and clinical trials of GD2. An anti-GD2 monoclonal antibody (Dinutuximab), which has been approved for the treatment of high-risk neuroblastomas, has shown promising efficacy in improving patients’ prognosis. Additionally, chimeric antigen receptors (CAR)-T therapy, GD2 vaccines and nanoparticles are also potential therapeutics. Finally, we discuss the prospects and current limitations of these immunotherapeutic approaches for treating retinoblastoma, as well as how to address these problems.

New MiniPromoter Ple389 (ADORA2A) drives selective expression in medium spiny neurons in mice and non-human primates.

Compact cell type-specific promoters are important tools for basic and preclinical research and clinical delivery of gene therapy. In this work, we designed novel MiniPromoters to target D1 and D2 type dopaminoceptive medium spiny neurons in the striatum by manually identifying candidate regulatory regions or employing the OnTarget webserver. We then empirically tested the designs in rAAV-PHP.B for specificity and robustness in three systems: intravenous injection in mice, intracerebroventricular injection in mice, and intracerebroventricular injection in non-human primates. Twelve MiniPromoters were designed from eight genes: seven manually and five using OnTarget. When delivered intravenously in mice, three MiniPromoters demonstrated highly selective expression in the striatum, with Ple389 (ADORA2A) showing high levels of dopamine D2-receptor cell co-localization. The same three MiniPromoters also displayed enriched expression in the striatum when delivered intracerebroventricularly in mice with high levels of DARPP32 co-localization. Finally, Ple389 (ADORA2A) was intracerebroventricularly injected in non-human primates and showed enriched expression in the striatum as in the mouse. Ple389 (ADORA2A) demonstrated expression in the medium spiny neurons in all three systems tested and exhibited the highest level of D2-MSNs and DARPP32 co-labeling in mice, demonstrating its potential as a tool for gene therapy approaches for Parkinson and Huntington disease treatment.

Discovery of New 3-(Benzo[b]Thiophen-2-yl)Pyrrolidine-2,5-Dione Derivatives as Potent Antiseizure and Antinociceptive Agents—In Vitro and In Vivo Evaluation

Background/Objectives: To address the unmet clinical needs in the treatment of epilepsy and pain, the continued development of more effective and safer anticonvulsants and analgesics is still necessary. Therefore, herein we report synthesis and antiseizure/antinociceptive evaluation of a focused series of 3-(benzo[b]thiophen-2-yl)pyrrolidine-2,5-dione derivatives. Methods: The anticonvulsant properties were investigated in acute models of seizures, namely the maximal electroshock (MES), the 6 Hz (32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure models, whereas analgesic activity was tested in the model of a tonic pain/formalin test and oxaliplatin-induced neuropathic pain (in CD-1-mice, i.p.). In addition, a number of in vitro assays were performed, aiming at the evaluation of the drug-like properties of the compounds disclosed herein. Results: We identified 33 as a lead compound with the most promising antiseizure properties, i.e., ED50 (MES) = 27.4 mg/kg and ED50 (6 Hz, 32 mA) = 30.8 mg/kg. Furthermore, 33 at a dose of 100 mg/kg significantly prolonged the latency time to the first seizure episode in the scPTZ model and at high doses did not impaire coordination of mice in the rotarod test (TD50 > 200 mg/kg). Apart from broad antiseizure protection, 33 demonstrated a significant analgesic effect in the formalin test (45 mg/kg, i.p.), and effectively alleviated allodynia in the oxaliplatin-induced neuropathic pain model (30 and 45 mg/kg). The binding assays suggest that the most plausible mechanism of action relies on interaction with the neuronal voltage-sensitive sodium channel (site 2). Furthermore, the drug-like potential of 33 supports favorable in vitro results, i.e., no hepatocytotoxicity and neurocytotoxicity at a high concentration of 100 μM, as well as a lack of mutagenicity at a concentration as high as 500 μM. Conclusions: Compound 33 identified in the current studies is proposed to be an interesting candidate for further preclinical development as therapy for epilepsy and neuropathic pain.

Standardisation of Curcuminoids into More Bioavailable form (CurcuminAura™) and Its Pharmacokinetics

Curcuminoids are naturally occurring phytocompound extracted from the turmeric rhizome Curcuma longa, a member of the ginger (Zingiberaceae) family. Through the development of a trademark product called Bio-Curcumin, this study seeks to increase the bioavailability and absorption of regular marketed curcumin by 95%. This finding has important implications for the academic and scientific community. The pharmacokinetic study of CurcuminAura™ and its innovation of biocurcuminoids is detailed in detail. Innovation of BioCurcuminoids and its Pharmacokinetic Study of CurcuminAura™ with regular curcuminoids is significantly described which is the need of the hour. This enhanced sunflower lecithin is an effective ingredient in the trademark product CurcuminAuraTM, which was created by Bio-Med Ingredients Pvt.Ltd. Its potency in the market can be increased by lecithin's capacity to efficiently encapsulate active substances, improve bioavailability, and increase absorption. Regular curcumin's weak solubility and poor absorption in its free form. Regular Curcumin due to its poor solubility and poor absorption in the free form in the gastrointestinal tract and its rapid biotransformation to inactive metabolites can greatly limit its utility as a health-promoting agent and dietary supplement. Hence to make it more readily available in the body. CurcuminovaTM is developed will enhance the properties of Curcumin making it more potent in the market. The Evaluation of the Comparative Pharmacokinetic Study of CurcuminAuraTM with Marketed Curcumin 95.0% was carried out through pre-clinical investigations in Sprague Dawley Rats via Oral Route which is the aim of this study with two groups in the study design, with four rats of each sex. Oral administration of CurcuminAuraTM and Curcumin 95.0% was administered to rats in the G1 and G3 groups, respectively. A dosage volume of 10 milliliters per kilogram of body weight was maintained for the oral route. After Dosage, under isofluorane,the blood samples were taken from the retroorbital sinuses after the dose was administered under varying periods of anaesthesia. Animals were split up into two sets for each group, and blood samples were taken at 30-, 2-, and 4-hour intervals. Samples were taken for analysis after being kept at -800C. Designed to increase the bioavailability of curcuminoids, CurcuminAuraTM is standardized to 60.9% total curcuminoids by HPLC, as opposed to marked conventional curcumin 95%. This has been confirmed by HPLC analysis. According to comparison studies, CurcuminAuraTM has a bioavailability that is 3.8 times greater than the reference standard. Additionally, in this study it is shown that the maximum absorption happens in the timeline 3 hrs after feeding the drug.

A Pleiotropic Nanomedicine Mitigates Splenic Hyperplasia, Ineffective Erythropoiesis, G6PDH Anomaly through Redox Buffering in Preclinical Mice Model.

Here, we present a pleiotropic nanomedicine-a smart, functionalized redox buffering nanoparticle-that may be used to treat hematological diseases, associated splenic hyperplasia, and issues related to restricted erythropoiesis. With a diameter of 5-7 nm, the spherical nanomaterial is made of manganese oxide and citrate. Here, we have produced the novel nanomaterial and, using cutting-edge electron microscopic and spectroscopic techniques, extensively assessed its redox buffering potential in vitrowith its structural integrity. Using an appropriate animal model (phenyl hydrazine, PHz, intoxicated C57BL/6J mice), we assessed the therapeutic efficacy of the redox buffering nanomedicine in the treatment of anemia and related consequences. We have further investigated the intricate molecular mechanism of the nanomedicine and its therapeutic impact, which includes increased erythropoiesis and G6PDH production, decreased inflammatory responses, mitigation of splenic hyperplasia, and synergistic intracellular redox-buffering. To the best of our knowledge, our studies would find relevance in the innovative management of anemia, decreased erythropoiesis, and splenic hyperplasia.

Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

Certain immune-checkpoint inhibitors have a narrow therapeutic window (TW) as cancer therapeutics, and engineered dual-/multi-targeting agents could potentially widen the TW to bring true clinical benefits. We report a new rationally-designed bispecific-antibody (BsAb), HX009, simultaneously targeting PD1 and CD47 to improve both the efficacy and safety over the respective single-targeting agents by grafting the extracellular domain of SIRPα onto the parental anti-PD1-monoclonal antibody, HX008. This resulted in an IgG4-based "2 × 2" symmetric structure but with an intentionally-reduced CD47-binding affinity, suggesting a novel candidate cancer immunotherapy. Specifically, HX009 has binding affinity constant of 8.951 × 10-9M for human PD1 and 2.557 × 10-8M for human CD47, respectively, where the CD47 binding is significantly weaker as compared to the binding affinity of HX008 to PD1 as well as the binding affinity of SIRPα-Fc to CD47, leading to little binding to RBCs and platelets and is contrasting to many CD47-agents in development. However, HX009 effectively and simultaneously binds to the PD1 and CD47 on PD1+CD47+ T-cells via cis-binding and elicits enhanced T cell activation compared to the parental HX008. HX009 caused little cytokine-release in human peripheral blood mononuclear cells. HX009 cross-species binds to cynomolgus monkey PD1/CD47 but not to rodents, making cynomolgus monkeys the choice of species to investigate the pharmacokinetics (PK) and toxicology of HX009. HX009's anti-tumor activities were confirmed in several humanized preclinical mouse models by determining either its anti-PD1 (humanized hu-CD47-MC38 models) or anti-CD47 (HuT-102 lymphoma CDX and three PDX-AML models) functions, although limited available humanized models have hindered broadly demonstration of enhanced anti-tumor activities contributed from the dual targeting of the BsAb. The expanded DLBCL-PDX trial data suggested that both EBV-status and OX40 expression could potentially be two positive predictors for response to HX009. An intravenous (IV) infusion PK study in cynomolgus monkey revealed its largely vasculature distribution, terminal half-life (T1/2) of ~ 50h, and dose-proportional exposure without accumulation. The anti-drug antibody (ADA) was observed in all monkeys as expected, affecting the PK parameters of repeated administration. The IV single-dose toxicology study with a 14-day observation revealed a maximum tolerated dose of 150mg/kg, while the repeated-dose (once weekly for 4weeks, 5 doses in total) study showed a highest non-severely toxic dose (HNSTD) of 15mg/kg. The desired preclinical PK and safety profiles, along with its antitumor activity, support HX009's candidacy for its clinical development.

Excitatory amino acids as therapeutic agents: Reversing neurodegenerative trajectory by tackling excitotoxicity.

Neurodegenerative diseases pose significant challenges to healthcare systems globally due to their complex etiology and relentless progression, often rendering conventional treatments ineffective. Recent advances have spotlighted excitatory amino acids, particularly D-amino acids, once considered as products of metabolism of the microbiota or deriving from food intake. This review explores the role of D-amino acids in mitigating excitotoxicity-a process characterized by excessive calcium influx through aberrant N-methyl-D-aspartate receptor (NMDAR) activation, which is implicated in the pathogenesis of diseases like Alzheimer's disease. By providing alternative pathways for neuronal signaling and protecting against excitotoxic damage, D-amino acids offer a novel approach to reversing neurodegenerative trajectories. Future research should focus on elucidating the detailed mechanisms of action of these compounds, evaluating their therapeutic potential through rigorous preclinical and clinical trials, and developing effective delivery systems to optimize their neuroprotective effects. This emerging field holds promise for developing innovative treatment strategies that could significantly improve outcomes for patients with neurodegenerative disorders.