Abhishek B. Jha

BACKGROUND: Negative clinical trials foster therapeutic nihilism and public skepticism, especially if they follow optimistic preclinical results. One way to increase the likelihood of successful clinical trials is to enhance the preclinical testing of new treatments. Recently, multicenter preclinical testing networks have gained attention as one tool to enhance rigor and reduce the likelihood of erroneously recommending a new candidate treatment for advancement to clinical trial testing. METHODS: The SPAN (Stroke Preclinical Assessment Network) consists of 6 research laboratories performing the experiments and 1 coordinating center at which treatments are prepared in a blinded manner, subjects are randomized, and statistical analysis is performed. We conducted a rigorous trial that included both sexes, multiple comorbidities, randomization, concealment of treatment status, blinded outcome assessments, prespecified sample sizes, and comprehensive data tracking. Five promising treatments were evaluated: BPN-27332, a lipoxygenase inhibitor; uric acid, a potent natural scavenger of free radicals; tatCN19o, a peptide inhibitor of activated CaMKII (Ca ²⁺ /CaM–dependent protein kinase II); GSK2256294, an inhibitor of sEH (soluble epoxide hydrolase); and NVX-058, an oxygen-carrying compound. A behavioral battery was selected as the end point for the primary analysis. The primary analysis used the modified intention-to-treat population. RESULTS: In total, 1768 rodents (882 females and 886 males) were enrolled: 441 young healthy mice, 444 aging mice, 450 mice with obesity-induced hyperglycemia and metabolic syndrome, and 433 spontaneously hypertensive rats. Data missing due to death before 30 days of follow-up were imputed from the same treatment group, animal model, sex, and stage. Although none of the candidates met the prespecified efficacy boundary, GSK2256294 showed an odds ratio for benefit of 1.28 (95% CI, 0.99–1.65), which was significant at P <0.05. CONCLUSIONS: The second SPAN study reaffirms the importance and feasibility of rigorous, multilaboratory preclinical testing networks to potentially reduce false positives in the development pipeline.

Ehlers-Danlos syndrome (EDS) is a group of connective tissue disorders characterized by joint hypermobility, skin hyperelasticity, perivascular tissue fragility, easy bruising, and increased bleeding risk. Abnormal bleeding in EDS ranges from mild ecchymoses to life-threatening hemorrhage. Platelet function abnormalities have been reported in people with EDS, but the broad nature and extent of these defects remain poorly defined. Herein, we evaluated blood samples from people with the hypermobile, classical, classical-like, and vascular types of EDS, and used a Col5a1+/− mouse model of classical EDS to characterize the extent of platelet dysfunction. Our findings suggest that platelet dysfunction in EDS is an outcome of reduced integrin αIIbβ3 activation resulting from decreased phosphorylation of talin-1, leading to defects in aggregation and spreading. The observed platelet dysfunction was associated with reduced expression of the platelet surface receptors glycoprotein VI (GPVI) and proteinase-activated receptor 1 (PAR1) and impaired downstream signaling. Col5a1+/− mice demonstrated increased tail bleeding time, reproduced the signaling defects observed in platelets from people with EDS, and exhibited decreased susceptibility to FeCl3-induced carotid artery thrombosis. Collectively, our data indicate that platelet dysfunction in EDS is likely contributing to hemorrhagic complications.

Current translational rodent stroke models induce middle cerebral artery occlusion (MCAo) using nylon filaments, injected emboli, intraluminal thrombin, or perivascular endothelin-1 to simulate human stroke. Among these methods, thromboemboli injection followed by thrombolysis best mimics the neuroinflammatory events seen in human patients and may be preferable to the inert nylon filament method most widely used. The standard thromboembolic models, used in leading single laboratories, however, can be time-consuming, produce variable results, and require considerable skill to master. To address these limitations, we developed a thromboembolic MCAo model that targets vessel occlusion and uses intravenous thrombolysis to achieve recanalization, paralleling the systemic thrombolysis administered in clinical scenarios. To reduce the number of animals, we developed a method to store blood from donor animals for later emboli preparation for multiple subjects. Our utilization of prefabricated Doccol microcatheters simplifies thrombus preparation and injection by pre-loading thromboemboli into the microcatheters that are then inserted into the internal carotid artery. To achieve recanalization, we infused intravenous Tenecteplase at a dose of 1.5 mg/kg. To promote reproducibility, we prepared and field-tested standard operating procedures, training videos, and hands-on surgical training workshops. The model uses a standard surgical approach that should be familiar to all investigators who use the widely accepted nylon filament model. Passage of the microcatheter into the distal internal carotid artery, avoiding the pterygopalatine artery, is accomplished in a manner similar to the nylon filament insertion. Recovery and post-stroke assessments may be done with typical behavioral, radiographic, and histologic protocols. This model offers a practical, reproducible, and accessible approach for investigators seeking a thromboembolic MCAo model with controlled recanalization.

Current translational rodent stroke models induce middle cerebral artery occlusion (MCAo) using nylon filaments, injected emboli, intraluminal thrombin, or perivascular endothelin-1 to simulate human stroke. Among these methods, thromboemboli injection followed by thrombolysis best mimics the neuroinflammatory events seen in human patients and may be preferable to the inert nylon filament method most widely used. The standard thromboembolic models, used in leading single laboratories, however, can be time-consuming, produce variable results, and require considerable skill to master. To address these limitations, we developed a thromboembolic MCAo model that targets vessel occlusion and uses intravenous thrombolysis to achieve recanalization, paralleling the systemic thrombolysis administered in clinical scenarios. To reduce the number of animals, we developed a method to store blood from donor animals for later emboli preparation for multiple subjects. Our utilization of prefabricated Doccol microcatheters simplifies thrombus preparation and injection by pre-loading thromboemboli into the microcatheters that are then inserted into the internal carotid artery. To achieve recanalization, we infused intravenous Tenecteplase at a dose of 1.5 mg/kg. To promote reproducibility, we prepared and field-tested standard operating procedures, training videos, and hands-on surgical training workshops. The model uses a standard surgical approach that should be familiar to all investigators who use the widely accepted nylon filament model. Passage of the microcatheter into the distal internal carotid artery, avoiding the pterygopalatine artery, is accomplished in a manner similar to the nylon filament insertion. Recovery and post-stroke assessments may be done with typical behavioral, radiographic, and histologic protocols. This model offers a practical, reproducible, and accessible approach for investigators seeking a thromboembolic MCAo model with controlled recanalization.

BACKGROUND: Past failures in translating stroke cerebroprotection provoked calls for a more rigorous methodological approach, leading to the stroke preclinical assessment network SPAN (Stroke Preclinical Assessment Network), where uric acid (UA) treatment exceeded a prespecified efficacy boundary for the primary functional outcome. Still, successful translation to humans requires confirmation of the effect of UA across key biological variables relevant to patients with stroke. METHODS: We measured the effects of intravenous UA treatment (16 mg/kg) versus intravenous saline in groups of animals enrolled in the SPAN network with diverse comorbidities, sex, and age. The masked study drug or placebo was administered during reperfusion in rodents undergoing a transient middle cerebral artery filament occlusion. The primary outcome was the modified corner test index at day 30 poststroke, and numerous secondary outcomes were collected. A modified intention-to-treat population was used in the analysis. We tested for any interactions with sex, age, and comorbidities (obesity-induced hyperglycemia and hypertension). RESULTS: In total, 710 animals were randomized to receive either intravenous UA or saline. After accounting for procedural dropouts and exclusions from treatment, a total of 687 animals were qualified and analyzed, including 458 assigned to UA and 229 to intravenous saline control. UA-treated animals exhibited a better primary functional outcome at day 30 (probability, 0.56 [95% CI, 0.52–0.60]; P =0.006). UA-treated animals also had a better corner test index at day 7 (probability, 0.55 [95% CI, 0.5–0.59]; P =0.035) and a higher survival rate at day 30 (hazard ratio, 1.41 [95% CI, 1.08–1.83]; P =0.011). Brain morphometry at day 2 and 30 was comparable between the treatment groups. The improved functional outcome and survival in UA-treated animals were preserved across different species, sexes, ages, and comorbidities. CONCLUSIONS: UA provides ischemic stroke cerebroprotection across key relevant biological variables, making it a promising intervention to be further tested in human clinical trials.

Respiratory tract infections (RTIs) caused by bacteria or viruses are associated with stroke severity. Recent studies have revealed an imbalance in the von Willebrand factor (VWF)–ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) axis in patients with RTIs, including coronavirus disease 2019. We examined whether this imbalance contributes to RTI-mediated stroke severity. Wild-type (WT), Vwf−/−, or Adamts13−/− mice with respective littermate controls (Vwf+/+ or Adamts13+/+) were infected intranasally with sublethal doses of Staphylococcus aureus (on days 0, 2, and 5) or mouse-adapted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; on day 0) and subjected to transient (30 or 45 minutes) cerebral ischemia followed by reperfusion. In S aureus–infected mice, infarct volumes were assessed on day 2 and functional outcomes on weeks 1 and 4 after reperfusion. In SARS-CoV-2–infected mice, infarct volumes and functional outcomes (Bederson score) were assessed on day 1 after reperfusion. We demonstrated that S aureus or SARS-CoV-2 RTI was accompanied by an imbalance in the VWF-ADAMTS13 axis and an increase in plasma levels of interleukin-6, C-X-C motif chemokine ligand 1, and monocyte chemoattractant protein-1, which was associated with larger infarcts and worse functional outcomes (P < .05 vs mock infection). S aureus– or SARS-CoV-2–infected Vwf−/− mice exhibited reduced infarcts and improved functional outcomes, whereas infected Adamts13−/− mice displayed greater stroke severity (P < .05 vs control). In the models of RTI preceding stroke, VWF contributes to stroke severity, whereas ADAMTS13 is protective.

Backgrounds: This study explores the design of substituted tetrahydroquinoline (THQ) derivatives and their synthesis as possible inhibitors of mTOR inhibitors for targeted cancer therapy. Methods: Inspired by the structural characteristics of known mTOR inhibitors, eight novel derivatives were synthesized, characterized using mass spectroscopy, ¹H, and ¹³C NMR, and evaluated for anticancer activity. Results: Computational studies, including molecular docking and molecular dynamics (MD) simulations, highlighted the derivative’s strong binding interaction and stability within the mTOR active site. Assays for in vitro cytotoxicity showed strong and specific anticancer action against cell lines of triple-negative breast cancer, lung cancer, and breast cancer while causing negligible impact on healthy cells. Conclusions: Compound 10e emerged as the most promising candidate, displaying exceptional activity against A549 cells (IC₅₀ = 0.033 µM) and inducing apoptosis in a dose-dependent manner, surpassing standard agents, like Everolimus and 5-flurouracil. Structure–activity relationship analysis revealed that incorporating trifluoromethyl and morpholine moieties significantly enhanced selectivity and potency. MD simulations further validated these findings, confirming stable protein-ligand interactions and favorable dynamics over a 100-ns simulation period. Collectively, this study underscores the therapeutic potential of THQ derivatives, particularly compound 10e, as promising mTOR inhibitors with potential applications in lung cancer treatment.