Preclinical Program Research Articles

No difference in cerebral perfusion between the wild-type and the 5XFAD mouse model of Alzheimer’s disease

Neuroimaging with [2,2-dimethyl-3-[(2R,3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(2R,3E)-3-hydroxyiminobutan-2-yl]azanide;oxo(99Tc)technetium-99(3+) ([99mTc]HMPAO) single photon emission computed tomography (SPECT) is used in Alzheimer’s disease (AD) to evaluate regional cerebral blood flow (rCBF). Hypoperfusion in select temporoparietal regions has been observed in human AD. However, it is unknown whether AD hypoperfusion signatures are also present in the 5XFAD mouse model. The current study was undertaken to compare baseline brain perfusion between 5XFAD and wild-type (WT) mice using [99mTc]HMPAO SPECT and determine whether hypoperfusion is recapitulated in 5XFAD mice. 5XFAD and WT mice underwent a 45 min SPECT scan, 20 min after [99mTc]HMPAO administration. Whole brain and regional standardized uptake values (SUV) and regional relative standardized uptake values (SUVR) with whole brain reference were compared between groups. Brain perfusion was similar between WT and 5XFAD brains. Whole brain [99mTc]HMPAO retention revealed no significant difference in SUV (5XFAD, 0.372 ± 0.762; WT, 0.640 ± 0.955; p = 0.536). Similarly, regional analysis revealed no significant differences in [99mTc]HMPAO metrics between groups (SUV: 0.357 ≤ p ≤ 0.640; SUVR: 0.595 ≤ p ≤ 0.936). These results suggest apparent discrepancies in rCBF between human AD and the 5XFAD model. Establishing baseline perfusion patterns in 5XFAD mice is essential to inform pre-clinical diagnostic and therapeutic drug discovery programs.

Lactobacillus gasseri LA806 Supplementation in Patients with Irritable Bowel Syndrome: A Multicenter Study.

The potential benefits of Lactobacillus gasseri LA806 in IBS were previously identified in a comprehensive preclinical research program. The purpose of this multicenter study was to explore in real-life conditions changes in IBS symptoms and quality of life in patients receiving a 4-week supplementation with L. gasseri LA806. Altogether 119 patients meeting Rome IV criteria for IBS were included, of whom 118 received the supplement. The majority of patients (71.8% (95% CI 63.6−79.9%)) manifested a ≥30% decrease in abdominal pain at 4 weeks, the mean abdominal pain score diminishing by 54.2% (from 5.3 ± 2.2 to 2.2 ± 2.4, p < 0.0001). A statistically significant decrease in abdominal pain was seen as early as the first week. A decrease of ≥30% in both abdominal pain score and global IBS symptom score was attained in 61.5% of patients (95% CI 51.7−71.2%). The mean IBS-SSS score fell by 152 ± 112 points (p = 0.001), with symptoms being attenuated in 85% of patients (CGI-I). Supplementation led to a 10-fold decrease in the number of patients reporting severe IBS symptoms. The concomitant intake of antidiarrheals, antispasmodics and analgesics decreased and quality of life scores significantly improved. These preliminary results warrant confirmation by a randomized, placebo-controlled study that this study will allow a better design.

Students' opinions on tutor-supported comprehensive care training in clinical dental education.

Tutor-supported comprehensive care training at the University of Bern School of Dental Medicine (SDM) has been used for many years. Therefore, the aim of this study was to evaluate dental students' opinions on tutor-supported training to identify key aspects of future course organisation that are important for students to achieve the minimum requirements for their graduation. A digital survey was developed and distributed among all fourth- and fifth-year dental students enrolled in the SDM in 2014 and 2016. A total of 28 (41.2%) and 21 (36.2%) students participated in the survey in 2014 and 2016, respectively. The average age of all respondents was 25.8 (±4.0) years. The proportion of females was 75% with no differences between groups, neither among classes nor years of the survey. The students felt well prepared following the bachelor's degree pre-clinical programme and a two-week introduction immediately preceding the clinical course. During clinical training, the students' experiences with their assigned tutors were positive even though waiting times for tutors during patient care as well as organisational efforts to manage attestations and logbooks were mentioned. For each discipline, patient assignment (ρ=0.54, p<0.0001) and frequently meeting with their tutors (ρ=0.56, p<0.0001) revealed the highest correlation with 1) achieving minimum requirements and 2) improving treatment planning skills in both fourth- and fifth-year dental students. In conclusion, tutor-supported comprehensive care training is well accepted by dental students while focusing on both patient assignment and frequent discussions with tutors may help students to better achieve minimum requirements in clinical dental education.

Students’ opinions on tutor-supported comprehensive care training in clinical dental education

Tutor-supported comprehensive care training at the University of Bern School of Dental Medicine (SDM) has been used for many years. Therefore, the aim of this study was to evaluate dental students’ opinions on tutor-supported training to identify key aspects of future course organisation that are important for students to achieve the minimum requirements for their graduation. A digital survey was developed and distributed among all fourth- and fifth-year dental students enrolled in the SDM in 2014 and 2016. A total of 28 (41.2%) and 21 (36.2%) students participated in the survey in 2014 and 2016, respectively. The average age of all respondents was 25.8 (±4.0) years. The proportion of females was 75% with no differences between groups, neither among classes nor years of the survey. The students felt well prepared following the bachelor’s degree pre-clinical programme and a two-week introduction immediately preceding the clinical course. During clinical training, the students’ experiences with their assigned tutors were positive even though waiting times for tutors during patient care as well as organisational efforts to manage attestations and logbooks were mentioned. For each discipline, patient assignment (rho=0.54, p&lt;0.0001) and frequently meeting with their tutors (rho=0.56, p&lt;0.0001) revealed the highest correlation with 1) achieving minimum requirements and 2) improving treatment planning skills in both fourth- and fifth-year dental students. In conclusion, tutor-supported comprehensive care training is well accepted by dental students while focusing on both patient assignment and frequent discussions with tutors may help students to better achieve minimum requirements in clinical dental education.&#x0D; Accepted for publication: September 5, 2022&#x0D; Published online: September 8, 2022

Abstract IA007: PREVENT agent development pipeline

Abstract The NCI’s PREVENT Cancer Preclinical Drug Development Program is a peer-reviewed program designed to support the preclinical development of promising agents and biomarkers for cancer interception/prevention towards clinical applications. PREVENT is not a grant program but allocates NCI contract resources to advance approved projects in a milestone-driven manner. Results obtained through NCI contract resources are returned to the applicant PIs and used to support further development by the applicants or in partnership with NCI. Resources available to PREVENT Program applicants include preclinical efficacy testing, CGMP manufacturing, GLP pharmacokinetic and IND-enabling toxicology studies, and IND filings. The PREVENT Program is focused on preventive agent development in the areas of Immunoprevention (cancer vaccines and immunomodulatory agents), Chemoprevention (novel mechanisms, anti-inflammatory agents, drug repurposing, toxicity reduction via alternative dosing regimens and agent combinations) and clinically translatable mechanistic biomarkers (pharmacodynamics, immune correlates, and tumor preventive efficacy). Submission deadlines for PREVENT Concept Applications occur twice per year on the second Monday in January and July. Further information can be obtained at the PREVENT Program website: https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical Citation Format: Mark Stevn Miller, Brian Cholewa, John Clifford, Vignesh Gunasekharan, Altaf Mohammed, Shanker Gupta, Robert Shoemaker, Shizuko Sei. PREVENT agent development pipeline [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA007.

Development and Effects of Pre-clinical Research Nurse Programs of Nursing Students

Objectives: Clinical research nurses play an important role in clinical trials. The purpose of this study was to identify the effect of pre-clinical research nurse program on biomedical ethics awareness, critical thinking disposition and teamwork in nursing students. Methods: This study was a non-equivalent control group pretest-posttest quasi-experimental design: the participants included 55 nursing students (28 in the experimental and 27 in the control group). Biomedical ethics awareness, critical thinking disposition and teamwork between the experimental and control groups were measured before and after the program participation. Data were collected from November 1 to 19, 2021 by taking a self reported survey and analyzed by using Chi-square test, Fisher’s exact test, Independent t-test, and Paired t-test with SPSS WIN 25.0 program. Results: The experimental group showed higher biomedical ethics awareness (t=2.15, &lt;i&gt;p&lt;/i&gt;=0.036) relative to those of the conrol group after the program participation. However, there were no statistical differences in critical thinking disposition (t=1.29, &lt;i&gt;p&lt;/i&gt;=0.204) and teamwork (t=1.97, &lt;i&gt;p&lt;/i&gt;=0.054) between the twp groups. Conclusions: According to these results, pre-clinacal research nurse program developed in this study could be used to increase biomedical ethics awareness of nursing students. In addition, there is a need to develop modified programs with various teaching methods to increase the level of critical thinking disposition and teamwork as well as biomedical ethics awareness in nursing students who may become clinical research nurses in the future.

MODL-28. DEVELOPING A STRATEGY FOR MODELING HIGH-GRADE GLIOMA IN GӦTTINGEN MINIPIGS

Abstract BACKGROUND The current literature does not describe a reproducible large animal model of intracranial high-grade glioma (HGG). Prior work has demonstrated the feasibility of inducing HGG de-novo in rodents by targeting specific oncogenic pathways. Here we report our approach to the production of supratentorial HGG in a series of minipigs through lentiviral gene transfer and subsequent initial characterization of a porcine glioma cell line. METHODS Four minipigs received injections into the subcortical white matter using a combination of lentiviral vectors expressing platelet-derived growth factor beta (PDGF-B), HRAS, and shRNA-p53. Animals underwent behavioral monitoring through porcine neurobehavioral scoring (PNS) and veterinary monitoring. Magnetic resonance imaging (MRI) was conducted at endpoint prior to necropsy. Post-mortem tissue biopsies underwent tissue culture and neuropathologic evaluation with hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and immunofluorescent staining. Data are presented using appropriate statistical tests where relevant and descriptive statistics. RESULTS Two pigs received 50ul injections and reached endpoint by the end of post-operative week 1 and 2. Two pigs received 25 ul injections and were asymptomatic until a pre-determined endpoint of 4 weeks. MRI scans at endpoint demonstrated contrast enhancing, mass forming lesions at the site of injection with evidence of hemorrhage and perilesional edema, consistent with high-grade glioma. On H&amp;E staining high-grade glioma growth was identified in 100% of animals. We observed immunopositivity for tumor markers GFAP, OLIG2, NG2, SOX2, and PDGFRA, as well as redox markers, and microenvironmental features consistent with high-grade glioma. Porcine glioma cell cultures were found to have significantly greater proliferative rate compared to control, and demonstrated GFAP, OLIG2, PDGFRA, and CD68 immunopositivity. CONCLUSIONS Lentiviral gene transfer represents a feasible strategy for glioma modeling in the Gӧttingen minipig. With our described methodology, we present a realistic strategy for reproducible modeling of intracranial glioma as a platform for preclinical neurosurgical development programs.

Advancing cell therapy manufacturing towards industry 4.0

David McCall, Commissioning Editor, BioInsights, speaks to Thomas Bollenbach, Chief Technology Officer, Advanced Regenerative Manufacturing Institute (ARMI)Thomas Bollenbach, PhD is Chief Technology Officer at the Advanced Regenerative Manufacturing Institute (ARMI) BioFabUSA. Tom is responsible for providing leadership to the institute’s technical programs, which include the development of enabling platform technologies for human cell and tissue manufacturing, Quality by Design-based process development methodologies for scalability and GMP-readiness, and development and demonstration of modular, automated and closed manufacturing systems. Prior to joining ARMI, Tom served as Vice President of Research and Development at Harvard Apparatus Regenerative Technology, where he led the development of cell- and biomaterial-based tracheal, bronchial and esophageal implants. Tom joined HART from Organogenesis, where he implemented IND-enabling preclinical programs for bioengineered living skin, and maintained strong cross-functional interactions with Clinical Operations, Manufacturing and Business Units to provide scientific support to corporate strategies. Tom received his BSc in Biochemistry from the University of Waterloo, a PhD in Biochemistry from the University of Notre Dame.

Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation Covid-19 Vaccine.

Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Applying Patient and Public Involvement in preclinical research: A co-created scoping review.

Patient and Public Involvement (PPI) in research aims to improve the quality, relevanceand appropriateness of research. PPI has an established role in clinical research where there is evidence of benefit, and where policymakers and funders place continued emphasis on its inclusion. However, for preclinical research, PPI has not yet achieved the same level of integration. As more researchers, including our team, aim to include PPI in preclinical research, the development of an evidence-based approach is important. Therefore, this scoping review aimed to identify and map studies where PPI has been used in preclinical research and develop principles that can be applied in other projects. A scoping review was conducted to search the literature in Medline (PubMed), EMBASE, CINAHL, PsycInfoand Web of Science Core Collection to identify applied examples of preclinical PPI. Two independent reviewers conducted study selection and data extraction separately. Data were extracted relating to PPI in terms of (i) rationale and aims, (ii) approach used, (iii) benefits and challenges, (iv) impact and evaluationand (v) learning opportunities for preclinical PPI. Findings were reviewed collaboratively by PPI contributors and the research team to identify principles that could be applied to other projects. Nine studies were included in the final review with the majority of included studies reporting PPI to improve the relevance of their research, using approaches such as PPI advisory panels and workshops. Researchers report several benefits and challenges, although evidence of formal evaluation is limited. Although currently there are few examples of preclinical research studies reporting empirical PPI activity, their findings may support those aiming to use PPI in preclinical research. Through collaborative analysis of the scoping review findings, several principles were developed that may be useful for other preclinical researchers. This study was conducted as part of a broader project aiming to develop an evidence base for preclinical PPI that draws on a 5-year preclinical research programme focused on the development of advanced biomaterials for spinal cord repair as a case study. A PPI Advisory Panel comprising seriously injured rugby players, clinicians, preclinical researchersand PPI facilitators collaborated as co-authors on the conceptualization, executionand writing of this review, including refining the findings into the set of principles reported here.

Are We Facing the End of Gross Anatomy Teaching as We Have Known It for Centuries?

The status of anatomy education in undergraduate medical education has dramatically changed over the course of the past century. From the most important and time-consuming component of the preclinical program, anatomy education has reduced in size and status, and yielded in curricular space to accommodate other disciplines and topics. Meanwhile, radiology has become more prominent, as a means to visualize anatomy, not only in clinical care but also in education. For this perspective paper, the authors, all with backgrounds in anatomy, radiology and/or medical education, conducted structured conversations with several academic colleagues with similar backgrounds, reviewed pertinent literature and analyzed the causes of the historical decline of a knowledge domain of medical education, that nevertheless is widely considered essential for medical students and graduates. After this analysis, the authors propose four ways forward. These directions include systematic peer teaching and development of anatomy education as a scholarly domain, further vertical integration with postgraduate medical education, full integration with radiology education, and capitalizing on educational technology. Schools in several industrialized countries have made steps in these directions, which can be further strengthened. In less affluent countries, and in countries with curricula strongly determined by tradition, these steps are less easy to make. To respond to changes in global health and health care, combined with the inevitable technological progress, and international mobility, we believe all schools will move in these directions, slower or faster.

A hybrid educational approach to service learning: impact on student attitudes and readiness in working with medically underserved communities

ABSTRACT Medical students engage with medically underserved communities (MUC) and vulnerable populations but often lack preparation to advocate appropriately for these communities. While preclinical programs with an experiential community component effectively increase knowledge about serving MUC, the pandemic limited clinical opportunities in community settings for learners. We examined the impact of a streamlined, hybrid service learning curriculum on first-year medical student attitudes towards MUC and their readiness and interest in addressing health barriers faced by this population. The redesigned curriculum for the student-led program required participants to attend nine virtual seminars led by faculty and community members with expertise serving MUC. Students partnered with one of three community agencies to organize service projects and gain exposure to the life experiences of MUC using virtual and in-person approaches. Of the fifteen first year medical students who participated in the program, positive attitudes were sustained across all scales using the Medical Student Attitudes Toward the Underserved (MSATU) questionnaire after one year. A majority (≥50%) of students reported a large increase in their knowledge of the health challenges faced by underserved populations after each didactic session. Despite the mostly virtual nature of community partnerships, students reported increased confidence in their ability to direct MUC patients to local resources (p < 0.01). The program also had a positive impact on student interest in working with medically underserved patients in the future, with 71% of participants indicating a significant impact on their interest in working in a medically underserved area. Our redesigned elective curriculum provided participants with foundational knowledge to advocate appropriately for underserved populations and demonstrated the efficacy of virtual approaches for community service and service learning. Our findings suggest hybrid and virtual experiential learning opportunities are a viable and non-inferior curricular approach to teaching health equity and community health.

Clinical observership as an aid to learning and understanding of human embryology

Hands-on learning approach such as ‘Clinical observership’ is globally applicable in learning. This study evaluated clinical observership as an aid to pre-clinical medical students’ understanding of human embryology. Students were grouped into A and B; group B took calls as studentobservers in the labour ward of Ekiti State University Teaching Hospital in addition to taking regular classes in embryology while group A was the control group. Previously validated selfadministered questionnaires were used to test students’ intelligence quotient and knowledge of embryology before and after lectures and clinical observership. The short-term effect of clinical observership was tested by comparing the pre-field and post-field responses of the studentvolunteers while the long-term effect was tested by evaluating their performance in the first professional exams after the completion of pre-clinical program. The perception of group B participants on the observership program was assessed with a pre-tested questionnaire. There was no significant difference (p=0.4162) in the IQ between the control (77±1.2) and the experimental group (79±1.4). There was no significant difference (p= 0.0795) in the mean preobservation test score between the control (33±1.7) and the experimental group (38±1.7). However, a significant increase (p=0.0282) was observed in the mean post-observation test score in the experimental (29±2.4) when compared with the control (22±1.9). Although there was an increase in the mean score in the MBBS exam in the experimental group (59±2.7) when compared with the control (57±2.0), it was not statistically significant. On the perception of the method in improving learning, majority of the students were affirmative. Clinical observership enhances the learning of embryology hence we recommend its incorporation into the teaching methods of embryology in medical schools.

Angiotensinogen Suppression: A New Tool to Treat Cardiovascular and Renal Disease.

Multiple types of renin-angiotensin system (RAS) blockers exist, allowing interference with the system at the level of renin, angiotensin-converting enzyme, or the angiotensin II receptor. Yet, in particular, for the treatment of hypertension, the number of patients with uncontrolled hypertension continues to rise, either due to patient noncompliance or because of the significant renin rises that may, at least partially, overcome the effect of RAS blockade (RAS escape). New approaches to target the RAS are either direct antisense oligonucleotides that inhibit angiotensinogen RNA translation, or small interfering RNA (siRNA) that function via the RNA interference pathway. Since all angiotensins stem from angiotensinogen, lowering angiotensinogen has the potential to circumvent the RAS escape phenomenon. Moreover, antisense oligonucleotides and small interfering RNA require injections only every few weeks to months, which might reduce noncompliance. Of course, angiotensinogen suppression also poses a threat in situations where the RAS is acutely needed, for instance in women becoming pregnant during treatment, or in cases of emergency, when severe hypotension occurs. This review discusses all preclinical data on angiotensinogen suppression, as well as the limited clinical data that are currently available. It concludes that it is an exciting new tool to target the RAS with high specificity and a low side effect profile. Its long-term action might revolutionize pharmacotherapy, as it could overcome compliance problems. Preclinical and clinical programs are now carefully investigating its efficacy and safety profile, allowing an optimal introduction as a novel drug to treat cardiovascular and renal diseases in due time.

LB1027 An adipose-derived stem cell-engineered patch represents a promising treatment for chronic wounds

Mesenchymal stem cell (MSC)-based therapies are emerging as promising approach to treat chronic wounds, although uncontrolled/sub-optimal delivery represents a major threat to their wide-spread clinical adoption. Here we report a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates MSC extracted from the human fat (ASCs) in a clinical-grade sponge made of porcine crosslinked-gelatin. ASC were derived from the abdominal/skin fat of four ischemic patients. Microarray data revealed that ASC grown within the patch upregulate the transcription of genes important for wound healing, including VEGF, CXCL12 and FGF7. The patch acted both as a concentrator and a reservoir of ASC-derived factors and sponge-derived fragments having healing capacity. The ASC-patch promoted dermal fibroblast survival, epidermal epithelialization and neovascularization, as shown in a chick chorioallantoic membrane model. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a pe-clinical rat animal model of ischemic wounds. Together, we provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.

Optimization of the production process for the anticancer lead compound illudin M: process development in stirred tank bioreactors

BackgroundThe fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown improved in vitro selectivity towards cancer cells encouraging further investigation. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus. We have recently reported a robust shake flask process for the production of gram quantities of illudin M from Omphalotus nidiformis aiming to transfer that process into stirred tank bioreactors, which can be used in a commercial production set-up. However, process transfer across different systems is not straightforward and particularly challenging when the producer is morphologically complex. There are only a few reports that address the development of bioprocesses for the production of compounds from Basidiomycota as these organisms have not been extensively studied because of their complex life cycles and often are difficult to cultivate under laboratory conditions.ResultsThe recently developed shake flask process delivering stable titers of ~ 940 mg L−1 of illudin M was investigated using off-gas analysis to identify critical parameters which facilitated the transfer from shaken into stirred tank bioreactors. Comparable titers to the shake flask process were achieved in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in 523 mg L−1 and is the starting point for optimization of the identified parameters at that scale.ConclusionBy identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into 2 L stirred tank bioreactors reaching a comparable titer (> 900 mg L−1), which is significantly higher than any previously reported. The insights obtained from 10 L scale pave the way towards further scale-up studies that will enable a sustainable supply of illudin M to support preclinical and clinical development programs.

A Review of NeuroAiDTM II (MLC901) Development in Alzheimer’s Disease Treatment: Promises of A Multimodal Pathway

Background: Alzheimer’s disease (AD) is a clinical and economic burden on society. Without new treatment, the impact of AD on society could triple by 2050. Aim: After a brief overview of treatments and challenges of new drug developments for AD, we reviewed the preclinical and clinical development program of NeuroAiD (MLC901, MLC601). Method: A literature search was conducted by using different web sources. The initial screening was based on keywords contained in the subtitles of each corresponding paragraphs of this article. We sorted the reviewed publications by relevance and publication date selecting 74 references out of the 319 initially shortlisted for review. Review: Since 1998, only symptomatic drugs were marketed. Intensive research has continued, aimed at delaying the onset of the disease and/or slowing its progression. However, the predictive value of delaying the onset of AD remains debated. Since 2003, aducanumab is the first new treatment approved and registered by US-FDA as an amyloid beta-directed antibody indicated for the treatment of Alzheimer’s disease under post-marketing conditions. Traditional medicines (TM) have shown interesting results, but many of TM clinical studies leave much to be desired from a methodological point of view. Among TM, NeuroAiD (MLC901/601), a botanical-derived combination, acts in a multimodal pathway combining neuroprotective and neuroregenerative properties. It has demonstrated sustained symptomatic benefits, slowing the disease progression in AD with a good safety profile. Discussion/Conclusions: The discovery of treatments preventing or slowing down the disease progression, are necessary to get reliable diagnostic tools to confirm AD diagnosis, and follow its evolution and long-term therapy. A growing consensus is emerging on the need for a multi-factorial approach to the treatment and the development of suitable AD drug combinations. Such an approach has been that of TM for a long time. This is the case for NeuroAiD, that it may be integrated safely either after symptomatic treatments have failed or on top of symptomatic treatments.

Abstract LB165: Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer

Abstract Background: There is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Currently, anti-hormonal therapies are the only option in individuals previously diagnosed with hormone receptor positive breast cancer. However, these agents are associated with many side effects. Furthermore, there are currently no preventive options for hormone receptor negative breast cancers. To study DCIS progression, we developed a model referred to as MIND (Mouse-INtraDuctal) in which patient-derived DCIS cells and cell lines are injected intraductally and studied as they progress to invasive ductal carcinoma (IDC). Previously we provided strong evidence for the role of B Cell Lymphoma-9 (BCL9) as a potential driver of DCIS malignancy. We chose to target BCL9 using RE40CA and its active ingredient CA previously identified in a screen for inhibitors of β-catenin binding to BCL9. Experimental Strategy: CA and RE40CA were administered to DCIS.COM MIND animal models for four weeks by oral gavage or mixed in a powder diet formulation. Drug plasma levels were evaluated by LC-MS. Four weeks following drug administration, mammary glands were excised and evaluated for efficacy and expression of biomarkers of aggressive DCIS, nuclear β-catenin and BCL9. For efficacy studies, mammary glands containing DCIS like lesions were processed and subjected to immunofluorescence staining using anti-human CK-5, anti-SMA, Dapi, β-catenin and BCL9. Extent of DCIS in vivo growth, number of invasive lesions, tumor number and tumor volume were evaluated for each mammary gland. Results: Efficacy studies showed a significant reduction in the number of invasive lesions by administration of CA by oral gavage (CA OG:10mg/kg), CA at 200mg/kg diet (CA200), CA400 and RE40CA 500. A significant reduction in extent of DCIS in vivo growth was achieved by CA OG and RE40CA 500. A significant reduction in the number of tumors was achieved by CA OG, RE40CA OG (25mg/kg equivalent to CA 10mg/kg), RE40CA 100, RE40CA 500 and RE40CA 1000. A significant reduction in tumor volume was achieved by CA OG, RE40CA OG, RE40CA 100, RE40CA 500 and RE40CA 1000. Linear regression modelling was used to compare statistical differences among the groups and differences that reached p-value &amp;lt;0.05 were considered significant. Administration of CA or RE40CA by oral gavage was also associated with a significant loss of nuclear BCL9/β-catenin expression in DCIS epithelial cells. Dietary administration of CA and RE40CA as OG or mixed in powder diet showed an increasing trend in plasma concentrations as the dose was increased. Evaluation of HE stained sections of animal organs by histopathology showed no evidence in any tissue of necrosis, degeneration, inflammation or neoplasia. Additionally, no consistent lesions attributable to treatment were observed in any of the animals or groups. Conclusion: Administration of CA and RE40CA are safe and effective in prevention of DCIS malignancy. Future studies are aimed at testing the efficacy of CA/RE40CA using DCIS cell lines representing other DCIS subtypes (ER+/PR+ and HER2 overexpressing) as well as patient derived DCIS MIND animal models. Supported by NCI PREVENT Cancer Preclinical Drug Development Program, 75N91019D00016/75N91020F00001. Citation Format: Yan Hong, Aditi Rastogi, Aryamitra Banerjee, Brandon L. Plattner, Matthew Lindeblad, Alexander V. Lyubimov, Timothy Fields, Seema A. Khan, Altaf Mohammed, Jeremy J. Johnson, Fariba Behbod. Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB165.

Abstract 715: Targeting the mTOR pathway for the prevention of er-negative breast cancer

Abstract Background: Triple-Negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. These cancers have a poor prognosis and are treated predominantly with chemotherapy. Therefore, we are working to find preventive therapies for these potentially lethal cancers. Dysregulation of PI3K-mTOR pathway is commonly associated with TNBC and other ER-negative breast cancers. We hypothesized that targeting mTOR may prevent development of ER-negative breast cancers and in this study tested whether the mTOR inhibitor, everolimus, prevents cancer in several mouse models of ER-negative breast cancer. Methods: MMTV-erbB2 mice were purchased from the Jackson Laboratory. C3(1)/SV40TAg and BRCA1co/co;MMTV-Cre;p53+/- were generated through breeding. P53-null and p53-mutant mammary gland mice were generated by transplanting p53-null or p53-mutant mammary glands into cleared fat pads of p53-wild type mice. The mice were treated with either control or everolimus (5mg/kg), administered by oral gavage daily (5X/week). All mice develop palpable mammary tumors within 8-12 months. Mice were observed daily for tumor formation and drug toxicity. The percentage of tumor free mice was recorded, and time to tumor development was visualized using Kaplan-Meier curves and analyzed using the generalized Wilcoxon test. Results: Treatment with everolimus significantly increased median survival of all mouse models. Everolimus treatment was also able to completely prevent mammary tumor formation in 27% of C3(1)/SV40Tag mice and 44% p53-mutant mammary gland mice. In p53-null mammary gland mice, 46% of control mice had developed tumors after 420 days, compared to 7% of everolimus treated mice. Long term treatment of everolimus was associated with mild toxicity that includes slight weight loss (&amp;lt;10%) and skin changes (matted hair, skin erythema). Conclusions: Everolimus significantly delayed mammary tumorigenesis in all five breast cancer mouse models. Our results suggest that everolimus is a promising cancer preventive drug for ER-negative tumors, and that further studies of everolimus in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB). Citation Format: Abhijit Mazumdar, Jamal Hill, William Tahaney, Yanxia Ma, Alejandro Contreras, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR pathway for the prevention of er-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 715.

DIPG-49. International preclinical drug discovery and biomarker program informing an adoptive combinatorial trial for DMG

INTRODUCTION: DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) will implement an innovative clinical trial design of combinatorial arms for patients with DMG at all disease stages, that is adaptive to pre-clinical and correlate data generated in eight collaborating institutions. The goal of the team is to rapidly identify and validate i) promising drugs and drug combinations for clinical use, and ii) predictive biomarkers of promising drugs. METHODS: In vitro (n=30) and in vivo (n=8) models of DMG across fourteen institutions were used to assess single and combination treatment of over 80 drugs and drug combinations. Predictive biomarkers of response for top candidate drugs were identified using extensive molecular assays including proteomics, CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS: Inhibitory concentration (IC50) of all drugs were established and validated across all participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), ONC201+panobinostat (p=0.01). Marizomib was highly toxic in murine PDX and zebrafish larvae assays. Murine pharmacokinetic analysis showed peak brain levels of ONC201, and ONC206 above pre-clinical IC50 concentrations. Molecular testing and analyses of existing drug screen across 578 cancer cells validated mitochondrial stress and additional proteins, as the main targets induces by ONC201/6. CONCLUSION: Thorough preclinical testing in a multi-site laboratory setting identified promising therapeutics for DMGs, resulting in launch of two clinical trials (PNOC022, ONOC023). Validation of identified biomarkers are ongoing using clinical specimen as well as in vivo PDX models.