ENDOTHELIAL DYSFUNCTION UNDER EXPERIMENTAL SUBARACHNOID HEMORRHAGE. POSSIBLE WAYS OF PHARMACOCORRECTION

Purpose of the article: to study the effect of acelysine and nimodipine on certain endothelial dysfunction indicators and to evaluate their therapeutic efficacy after subarachnoid hemorrhage in rats. Materials and Methods: an experimental study was carried out using 50 Wistar rats of both sexes. Spontaneous subarachnoid hemorrhage was modeled in animals. Three groups of animals were identified: a control group, a group of animals received a standard therapeutic dose of acelysine, and a group received a standard therapeutic dose of nimotop. Each group included 15 animals. There were also 5 intact animals. The animals were withdrawn from the experiment on days 4 and 7 after the motor and exploratory activity determination. Motor and exploratory activity determination was carried out following SAH with the “Open Field” technique. Determination of biochemical markers of endothelial dysfunction was performed in a rat brain homogenate. Results and discussion. It was found that modeling of subarachnoid hemorrhage (SAH) led to the oxidative stress development development and the product of oxidative modification of proteins (nitrotyrosine (Ntz) on the 4th and especially on the 7th day of the experiment) increase in the brain tissues. Starting from the 4th day, we registered a compensatory increase in the activity of NO-synthase (NOS) - by 56%, followed by a decrease in its activity on the 7th day, by more than 33% against the intact group of animals. It was registered a compensatory increase in VEGF-A in rats with SAH modeling on the 4th day of the experiment and its further decrease on the 7th day. The established pathobiochemical changes in the brain tissue were accompanied by the cognitive deficit development in experimental animals, especially on the 7th day of the SAH. SAH led to a significant decrease in the total activity of animals by 2.63 times, a decrease in the distance traveled by animals by 1.89 times, the number of freezes increased by 1.86 times and the immobility of animals increased when moving from the periphery to the center and immobility in the center of the arena (anxiety, fear, disorientation), as well as a decrease in the distance traveled and the speed of movement in illuminated center of the arena 2 and 2.6 times, respectively. Experimental therapy with acelysine 15 mg/kg led to the normalization of biochemical indicators of endothelial dysfunction: concentration of nitrotyrosine, starting from the 4th day of the experiment, increased eNOS activity and VEGF-A concentration (by 75% and 64% on 7th day). The administration of namidopine led to less pronounced effects, statistically significant changes occurred only in relation to the VEGF-A concentration. Administration of namidopine resulted in only a slight increase in VEGF-A concentration. Acelysine and nimotope significantly increased the total activity of rats on the 7th day after SAH by 76.3% and 48.8%, respectively. In animals treated with acelysine, anxiety and fear decreased. The animals were less aggressive and more empathic - long-term grooming increased 3 times. The administration of nimotop in rats survived SAH had a less pronounced positive effect on behavior. Nimotop did not effect on indicators of general activity and did not increase the total distance traveled. Animals received nimtop were inactive by the 7th day of treatment. Conclusions: Experimental therapy with acelysine led to the normalization of biochemical parameters of endothelial dysfunction, namely nitrotyrosine concentration, starting from the 4th day of the experiment and increased eNOS activity and VEGF-A concentration. It should be noted that, in contrast to the rats of the control group, under prescription of acelysine, there was an increase in the concentration of eNOS and VEGF-A both on the 4th and 7th days of the experiment. The administration of namidopine led to less pronounced effects, statistically significant changes occurred only in relation to the VEGF-A concentration. The administration of acelysin to animals after SAH had a beneficial effect on the emotional status and behavior of animals, and also led to the normalization of their general activity and orientation-exploratory activity. The mechanism of edotheliotropic effect of acelysine, in our opinion, is associated with its antioxidant effects, modulating impact on endothelial NOS, as well as its property, indirectly, to influence on increase VEGF content. Nimotope therapy had no effect on the emotional status and behavior of the animals. The use of calcium channel blockers revealed such side effects as depression, drowsiness, diplopia, and disorientation

Adjuvant Therapy with Budesonide Post-Kasai Reduces the Need for Liver Transplantation in Biliary Atresia

Based on the hypothesis that autoimmunological factors coregulate the pathomechanism in biliary atresia (BA), adjuvant therapy with steroids has become routine, although its efficacy has never been proven. In 2010, a study on the advantages of budesonide compared to prednisolone in autoimmune hepatitis gave rise to experimental therapy using budesonide as an adjuvant BA treatment. Ninety-five BA patients prospectively received a budesonide 2 mg/dose rectal foam daily for three months (SG). A case-matched control group (CG: 81) was retrospectively recruited. The outcome measures were survival with native liver (SNL), determined at six months and two years after the Kasai procedure. The follow-up rate was 100%. At six months, SNL was statistically not different but became so after two years (SG: 54%; CG: 32%; p < 0.001). No steroid-related side effects were observed, except for eight patients with finally caught-up growth retardation. This study demonstrates for the first time a significantly longer survival with native liver in patients with BA after adjuvant therapy. However, indication, dosage, and duration of any budesonide application is not given in neonates with BA. Hence, we suggest extending the postoperative use of budesonide in a multicenter observational study with a clearly defined follow-up protocol, particularly in terms of potentially underestimated side effects.

The features of the course of virus-associated acute lung injury in mice with induced immunosuppression

BACKGROUND: Among all groups of patients with virus-associated acute lung injury with influenza infection, the most severe course is observed in patients with immunosuppression. In this case, despite the studied mechanism of the course of combined pathology, the question of therapy in this group of patients remains unclear. AIM: To study the features of the course of acute lung injury in influenza infection with secondary immunosuppression in an experiment for the possibility of searching for experimental therapy for this combined pathology. MATERIALS AND METHODS: The study was performed on 115 outbred female mice. The mouse-adapted pandemic influenza virus A/California/7/09MA (H1N1)pdm09 was used for modeling viral acute lung injury. Experimental immunosuppression was reproduced by administration of methotrexate (1.25 mg/kg intraperitoneally, once every 3 days during 3 weeks before infection). During the experiment, mortality, blood oxygen saturation, the concentration of pro-inflammatory cytokines in the lungs, and the severity of lung injury were measured. RESULTS: The presence of experimental immunosuppression led to an exacerbation of acute lung injury in infected animals in terms of mortality and lung damage. Changes in the dynamics of proinflammatory cytokines (TNF-, IL-6, IL-1) in the lungs were observed during acute lung injury. Retarded recovery of the lungs functional activity was noted. CONCLUSIONS: The experimental immunosuppression contributed to the exacerbation of acute lung injury and to an increase in the duration of the pathology. These changes could be associated with an altered process of elimination of the pathogen. The reproduced model of combined pathology was used for searching a therapy for these complications.

Evaluation of new or repurposed treatments for COVID-19: protocol for the phase Ib/IIa DEFINE trial platform

IntroductionCOVID-19 is a new viral-induced pneumonia caused by infection with a novel coronavirus, SARS-CoV-2. At present, there are few proven effective treatments. This early-phase experimental medicine protocol describes an overarching and adaptive trial designed to provide safety data in patients with COVID-19, pharmacokinetic (PK)/pharmacodynamic (PD) information and exploratory biological surrogates of efficacy, which may support further development and deployment of candidate therapies in larger scale trials of patients positive for COVID-19.Methods and analysisDefine is an ongoing exploratory multicentre-platform, open-label, randomised study. Patients positive for COVID-19 will be recruited from the following cohorts: (a) community cases; (b) hospitalised patients with evidence of COVID-19 pneumonitis; and (c) hospitalised patients requiring assisted ventilation. The cohort recruited from will be dependent on the experimental therapy, its route of administration and mechanism of action. Randomisation will be computer generated in a 1:1:n ratio. Twenty patients will be recruited per arm for the initial two arms. This is permitted to change as per the experimental therapy. The primary statistical analyses are concerned with the safety of candidate agents as add-on therapy to standard of care in patients with COVID-19. Secondary analysis will assess the following variables during treatment period: (1) the response of key exploratory biomarkers; (2) change in WHO ordinal scale and National Early Warning Score 2 (NEWS2) score; (3) oxygen requirements; (4) viral load; (5) duration of hospital stay; (6) PK/PD; and (7) changes in key coagulation pathways.Ethics and disseminationThe Define trial platform and its initial two treatment and standard of care arms have received a favourable ethical opinion from Scotland A Research Ethics Committee (REC) (20/SS/0066), notice of acceptance from The Medicines and Healthcare Products Regulatory Agency (MHRA) (EudraCT 2020-002230-32) and approval from the relevant National Health Service (NHS) Research and Development (R&D) departments (NHS Lothian and NHS Greater Glasgow and Clyde). Appropriate processes are in place in order to be able to consent adults with and without capacity while following the necessary COVID-19 safe procedures. Patients without capacity could be recruited via a legal representative. Witnessed electronic consent of participants or their legal representatives following consent discussions was established. The results of each study arm will be submitted for publication in a peer-reviewed journal as soon as the treatment arm has finished recruitment, data input is complete and any outstanding patient safety follow-ups have been completed. Depending on the results of these or future arms, data will be shared with larger clinical trial networks, including the Randomised Evaluation of COVID-19 Therapy trial (RECOVERY), and to other partners for rapid roll-out in larger patient cohorts.Trial registration numberISRCTN14212905, NCT04473053.

Methods for modeling tumor growth in mice in experimental studies of human gastric cancer

Gastric cancer (GC) is a group of malignant tumors originating from the gastric mucosa cells. The highest incidence of GC is recorded in Japan, China and Russia, and the lowest one in the USA and New Zealand. Extensive molecular genetic research of GC has revealed its heterogeneity associated with the genomic instability of the tumor and the complexity of its phenotype due to simultaneous changes in several oncogenes and suppressors. This was the basis for the creation of the GC classification by molecular subtypes. The creation of a realistic preclinical model is essential for translational GC studies. Cancer cell lines and xenografts derived from them are among the most common preclinical models. They are easy to generate, but they also have limitations, since these models cannot sufficiently reproduce the unique characteristics of each cancer patient. Patient-derived xenografts (PDX) are currently the best model for testing targets and predictors of response to therapy. PDX models are created by transplanting surgically resected human tumors into immunodeficient mice. These models maintain morphological similarity and replicate the molecular characteristics of parental tumors providing an indispensable tool for assessing anticancer drug response. Statistical data from preclinical studies with PDX models can significantly save the time and resources required for clinical trials. Transgenic and knockout mouse models are also widely used in scientific laboratories in order to study specific genetic pathways of oncogenesis and develop experimental therapy for GC. This review discusses the molecular classifications of GC and experimental murine models that reproduce cancer in situ and are a universal platform for preclinical research in experimental oncology.

CTNI-40. EVALUATING FEASIBILITY AND EFFICIENCY OF PHASE II ADAPTIVE PLATFORM TRIAL DESIGNS BASED ON THE INDIVIDUALIZED SCREENING TRIAL OF INNOVATIVE GLIOBLASTOMA THERAPY (INSIGhT) EXPERIENCE

BACKGROUND The Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) is a phase II platform trial with Bayesian adaptive randomization and deep genomic profiling to more efficiently test experimental agents in newly diagnosed glioblastoma and to prioritize therapies for late-stage testing. METHODS In the ongoing INSIGhT trial, patients with newly diagnosed MGMT-unmethylated glioblastoma are randomized to the control arm or one of three experimental therapy arms (CC-115, abemaciclib, and neratinib). The control arm therapy is radiotherapy with concomitant and adjuvant temozolomide, and primary endpoint is overall survival. Randomization has been adapted based on Bayesian estimation of biomarker-specific probability of treatment impact on progression-free survival (PFS). All tumors undergo detailed molecular sequencing, and this is facilitated with the companion ALLELE protocol. To evaluate feasibility of this approach, we assessed the status of this ongoing trial. RESULTS Since INSIGhT was activated 4.3 years ago, it has expanded to include 12 sites across the United States. A total of 247 patients have been enrolled. Randomization probabilities have been repeatedly adjusted over time based upon early PFS results to alter the randomization ratio from standard 1:1:1:1 randomization. All three arms have completed accrual and efficacy estimates are available based upon comparison to the common control arm in context of relevant biomarkers. There are 87 patients alive and in follow-up, and there are ongoing plans to add additional arms to evaluate further treatments in the future. CONCLUSION The INSIGhT trial demonstrates that a multi-center Bayesian adaptive platform trial is a feasible and effective approach to help prioritize therapies and biomarkers for newly diagnosed GBM. The trial has maintained robust accrual, and the simultaneous testing of multiple agents, sharing a common control arm and adaptive randomization serve as features to increase trial efficiency relative to traditional clinical trial designs.

CTNI-20. FEASIBILITY OF SINGLE-PASS STEREOTACTIC NEEDLE BIOPSY IN RECURRENT GLIOBLASTOMA PATIENTS TO SUPPORT CLIA-CERTIFIED TUMOR PHARMACODYNAMICS FOR A PHASE 0/2 CLINICAL TRIAL

BACKGROUND In neuro-oncology clinical trials, pharmacodynamic (PD) analysis of tumor tissue before and after experimental therapy is challenging. Today, Phase 0 studies in brain tumor patients typically employ archival tissue from a prior resection as the baseline PD comparator. However, the time-interval between tissue acquisitions can be months to years and often includes confounding exposure to other therapies. Here, we demonstrate the feasibility of a pre-treatment, single-pass stereotactic needle biopsy to support CLIA-certified genetic screening and tumor PD analysis in a Phase 0/2 clinical trial. METHODS In support of an ongoing Phase 0/2 ‘trigger’ trial testing CDK4/6 plus ERK1/2 inhibition in recurrent glioblastoma (GBM), a single-pass stereotactic biopsy was completed in select patients prior to enrollment. Each biopsy yields six tissue cores for immunohistochemistry (IHC) and genetic characterization. Using CLIA-certified protocols, genomic DNA (gDNA) was extracted from two cores, followed by library preparation and next-generation sequencing of a customized panel of 37 genes (IvySeq). In the four remaining cores, IHC staining was performed for RB, pRB, pERK, pS6, CDKN2A, MIB-1, ClCas-3 and pH2AX. RESULTS Single-pass stereotactic needle biopsies were collected from five recurrent GBM patients. No intraoperative, perioperative, or radiographic evidence of morbidity was observed. 800-2400ng of gDNA was isolated from each stereotactic biopsy. IvySeq analysis detected CDKN2A deletion, ATRX loss, and mutations in IDH, PTEN and TP53 genes. Based on IHC and genetic analyses of the biopsied samples, three patients were enrolled into the Phase 0/2 clinical trial and baseline value were used for PD analysis. CONCLUSION Single-pass stereotactic needle biopsy is a feasible and safe strategy to collect pre-treatment tissue in support of a Phase 0 clinical trial, enabling CLIA-certified genetic analysis for trial screening and tumor PD analysis.

Liposomal form of dexamethasone in the correction of experimental acute lung injury

Objective. To investigate the possibility of pharmacological correction of acute lung injury of aspiration genesis with a liposomal form of dexamethasone in experiment. Materials and methods. For the experiment, simple liposomes were prepared from phosphatidylcholine and cholesterol with an average size of 320±50 nm and a dexamethasone concentration of 2.98±0.02 mg/ml. The study used outbred white rats, divided into four groups of 16 animals. 1st group Control (without experimental therapy), 2nd group - Experiment 1, where a solution of dexamethasone was injected intravenously at a dose of 6 mg/kg, 3rd group - Experiment 2, where an intravenous combination of dexamethasone solution (6 mg/kg) and hypertonic (7.5%) NaCl solution was administered once, and group 4 - Experiment 3, where liposomes with dexamethasone (6 mg/kg) were injected intravenously once in hypertensive (7.5%) NaCl solution. The main functional parameters of the animals (heart rate, blood pressure, saturation of hemoglobin with oxygen, partial pressure of blood oxygen and respiration rate) were subject to analysis. Functional parameters were analyzed before modeling acute lung injury and after 5 min, 1, 4, 24 hours, and 6 days. At the end of the experiment (day 6) the degree of pulmonary edema and histological signs of acute lung injury were assessed. Morphology was assessed quantitatively in each group. Results. The study found that liposomal dexamethasone in hypertonic NaCl solution, when administered intravenously, was more effective than aqueous dexamethasone solution in correcting functional impairment in acute lung injury. The combination of hypertonic sodium chloride solution with dexamethasone more markedly increases blood pressure and reduces the degree of pulmonary oedema. In acidine pepsin aspiration, liposomal dexamethasone in hypertonic NaCl solution most effectively increased animal survival. Conclusion. Compared with dexamethasone in hypertonic NaCl solution, liposomal dexamethasone is more effective in increasing animal survival and protecting lung tissue from aspiration damage by acidine pepsin.

The DNA co-vaccination using Sm antigen and IL-10 as prophylactic experimental therapy ameliorates nephritis in a model of lupus induced by pristane

Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies such as anti-Sm. Studies in patients with SLE and murine models of lupus reveal that the most critical anti-Sm autoantibodies are predominantly direct against D1(83–119), D2, and B´/B epitopes. Objectives The present study aimed to analyze the induction of antigen-specific tolerance after prophylactic immunization with a DNA vaccine encoding the epitopes: D183-119, D2, B´/B, and B´/BCOOH in co-vaccination with IFN-γ or IL-10 in a murine model of lupus induced by pristane. Material and methods To obtain endotoxin-free DNA vaccines, direct cloning techniques using pcDNA were performed: D183-119, D2, B´/B, B´/BCOOH, IFN-γ, or IL-10. Lupus was induced by 0.5 mL of pristane via intraperitoneal in BALB/c female mice. Immunoprecipitation with K562 cells was metabolically labeled with 35S and ELISA to detect serum antibodies or mice IgG1, IgG2a isotypes. ELISA determined IL-10 and IFN-γ from splenocytes supernatants. Proteinuria was assessed monthly, and lupus nephritis was evaluated by immunofluorescence, and electron microscopy. Results The prophylactic co-vaccination with D2/IL-10 reduced the expression of kidney damage observed by electron microscopy, direct immunofluorescence, and H & E, along with reduced level of anti-nRNP/Sm antibodies (P = 0.048). Conclusion The prophylactic co-vaccination of IL-10 with D2 in pristane-induced lupus ameliorates the renal damage maybe by acting as prophylactic DNA tolerizing therapy.

Phase III Trial Adding Vincristine-Topotecan-Cyclophosphamide to the Initial Treatment of Patients With Nonmetastatic Ewing Sarcoma: A Children's Oncology Group Report

PURPOSE The primary aim of this phase III randomized trial was to test whether the addition of vincristine, topotecan, and cyclophosphamide (VTC) to interval compressed chemotherapy improved survival outcomes for patients with previously untreated nonmetastatic Ewing sarcoma. METHODS Patients were randomly assigned to receive standard five-drug interval compressed chemotherapy (regimen A) for 17 cycles or experimental therapy with five cycles of VTC within the 17 cycles (regimen B). Patients were stratified by age at diagnosis (< 18 years and ≥18 years) and tumor site (pelvic bone, nonpelvic bone, and extraosseous). Tumor volume at diagnosis was categorized as < 200 mL or ≥ 200 mL. Local control occurred following six cycles. Histologic response was categorized as no viable or any viable tumor. Event-free survival (EFS) and overall survival (OS) were compared between randomized groups with stratified log-rank tests. RESULTS Of 642 enrolled patients, 309 eligible patients received standard and 320 received experimental therapy. The 5-year EFS and OS were 78% and 87%, respectively. There was no difference in survival outcomes between randomized groups (5-year EFS regimen A v regimen B, 78% v 79%; P = .192; 5-year OS 86% v 88%; P = .159). Age and primary site did not affect the risk of an EFS event. However, age ≥ 18 years was associated with an increased risk of death at 5 years (hazard ratio 1.84; 95% CI, 1.15 to 2.96; P = .009). The 5-year EFS rates for patients with pelvic, nonpelvic bone, and extraosseous primary tumors were 75%, 78%, and 85%, respectively. Tumor volume ≥ 200 mL was significantly associated with lower EFS. CONCLUSION While VTC added to five-drug interval compressed chemotherapy did not improve survival, these outcomes represent the best survival estimates to date for patients with previously untreated nonmetastatic Ewing sarcoma.