Preclinical Proof Of Concept Research Articles

Enhancement of Glutamate Uptake as Novel Antiseizure Approach: Preclinical Proof of Concept.

Excitotoxicity is a common hallmark of epilepsy and other neurological diseases associated with elevated extracellular glutamate levels. Thus, here, we studied the protective effects of (R)-AS-1, a positive allosteric modulator (PAM) of glutamate uptake in epilepsy models. (R)-AS-1 was evaluated in a range of acute and chronic seizure models, while its adverse effect profile was assessed in a panel of standard tests in rodents. The effect of (R)-AS-1 on glutamate uptake was assessed in COS-7 cells expressing the transporter. WAY 213613, a selective competitive EAAT2 inhibitor, was used to probe the reversal of the enhanced glutamate uptake in the same transporter expression system. Confocal microscopy and Western blotting analyses were used to study a potential influence of (R)-AS-1 on GLT-1 expression in mice. (R)-AS-1 showed robust protection in a panel of animal models of seizures and epilepsy, including the maximal electroshock- and 6 Hz-induced seizures, corneal kindling, mesial temporal lobe epilepsy, lamotrigine-resistant amygdala kindling, as well as seizures induced by pilocarpine or Theiler's murine encephalomyelitis virus. Importantly, (R)-AS-1 displayed a favorable adverse effect profile in the rotarod, the minimal motor impairment, and the Irwin tests. (R)-AS-1 enhanced glutamate uptake in vitro and this effect was abolished by WAY 213613, while no influence on GLT-1 expression in vivo was observed after repeated treatment. Collectively, our results show that (R)-AS-1 has favorable tolerability and provides robust preclinical efficacy against seizures. Thus, allosteric enhancement of EAAT2 function could offer a novel therapeutic strategy for treatment of epilepsy and potentially other neurological disorders associated with glutamate excitotoxicity. ANN NEUROL 2024.

Rapid differentiation of cystic fibrosis-related bacteria via reagentless atmospheric pressure photoionisation mass spectrometry

Breath analysis is an area of significant interest in medical research as it allows for non-invasive sampling with exceptional potential for disease monitoring and diagnosis. Volatile organic compounds (VOCs) found in breath can offer critical insight into a person’s lifestyle and/or disease/health state. To this end, the development of a rapid, sensitive, cost-effective and potentially portable method for the detection of key compounds in breath would mark a significant advancement. Herein, we have designed, built and tested a novel reagent-less atmospheric pressure photoionisation (APPI) source, coupled with mass spectrometry (MS), utilising a bespoke bias electrode within a custom 3D printed sampling chamber for direct analysis of VOCs. Optimal APPI-MS conditions were identified, including bias voltage, cone voltage and vaporisation temperature. Calibration curves were produced for ethanol, acetone, 2-butanone, ethyl acetate and eucalyptol, yielding R2 > 0.99 and limits of detection < 10 pg. As a pre-clinical proof of concept, this method was applied to bacterial headspace samples of Escherichia coli (EC), Pseudomonas aeruginosa (PSA) and Staphylococcus aureus (SA) collected in 1 L Tedlar bags. In particular, PSA and SA are commonly associated with lung infection in cystic fibrosis patients. The headspace samples were classified using principal component analysis with 86.9% of the total variance across the first three components and yielding 100% classification in a blind-sample study. All experiments conducted with the novel APPI arrangement were carried out directly in real-time with low-resolution MS, which opens up exciting possibilities in the future for on-site (e.g., in the clinic) analysis with a portable system.

Abstract C040: Discovery and development of novel ATG13 degrading compounds that inhibit autophagy and treat Non-Small Cell Lung Cancer

Abstract Autophagy is the process by which a cell maintains homeostasis by removing and recycling cytoplasmic debris. Autophagy provides lung cancer cells with essential nutrients for their increased proliferation and metabolism and aids in survival during anti-cancer treatments. CRISPR knockout or siRNA knockdown of the autophagy protein ATG13 inhibits autophagy and reduces viability of non-small cell lung cancer (NSCLC) cell lines. These data show that ATG13 is essential for autophagy and essential for lung cancer cell survival, making it a viable therapeutic target for cancer treatment. To identify compounds that degrade ATG13 and inhibit autophagy, we established a high-throughput screening (HTS) compatible Nano-Glo HiBiT assay where endogenous ATG13 was labeled on the C-terminus with HiBiT peptide via CRISPR. Optimization of this assay allowed for a signal to background Z’ factor of 0.71 and confirmed a positive control compound that induced up to 50% ATG13 degradation, thus indicating that this assay was suitable for compound screening. Currently, over 50,000 compounds from a variety of libraries have been screened in 1536-well format and 15 positive hits for ATG13 degradation have been identified. A series of counter-screens and secondary assays including In Cell Western Blot and Protein Thermal Shift assays will be used to confirm these hits. At the end of this testing funnel, we will have characterized novel compounds that degrade ATG13 and inhibit autophagy. These hits will provide a starting point for further chemical optimization to provide lead ATG13 degraders to evaluate in advanced preclinical proof-of concept studies of lung cancer. Citation Format: Patrick M Hagan, Atoosa Emami, Hilarie Austin, Ian Pass, Allison Limpert, Douglas Sheffler, Nicholas Cosford. Discovery and development of novel ATG13 degrading compounds that inhibit autophagy and treat Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C040.

Guanine nucleotide exchange factors for Rho GTPases (RhoGEFs) as oncogenic effectors and strategic therapeutic targets in metastatic cancer

Metastatic cancer cells dynamically adjust their shape to adhere, invade, migrate, and expand to generate secondary tumors. Inherent to these processes is the constant assembly and disassembly of cytoskeletal supramolecular structures. The subcellular places where cytoskeletal polymers are built and reorganized are defined by the activation of Rho GTPases. These molecular switches directly respond to signaling cascades integrated by Rho guanine nucleotide exchange factors (RhoGEFs), which are sophisticated multidomain proteins that control morphological behavior of cancer and stromal cells in response to cell-cell interactions, tumor-secreted factors and actions of oncogenic proteins within the tumor microenvironment. Stromal cells, including fibroblasts, immune and endothelial cells, and even projections of neuronal cells, adjust their shapes and move into growing tumoral masses, building tumor-induced structures that eventually serve as metastatic routes. Here we review the role of RhoGEFs in metastatic cancer. They are highly diverse proteins with common catalytic modules that select among a variety of homologous Rho GTPases enabling them to load GTP, acquiring an active conformation that stimulates effectors controlling actin cytoskeleton remodeling. Therefore, due to their strategic position in oncogenic signaling cascades, and their structural diversity flanking common catalytic modules, RhoGEFs possess unique characteristics that make them conceptual targets of antimetastatic precision therapies. Preclinical proof of concept, demonstrating the antimetastatic effect of inhibiting either expression or activity of βPix (ARHGEF7), P-Rex1, Vav1, ARHGEF17, and Dock1, among others, is emerging.

18F]F-DED PET imaging of reactive astrogliosis in neurodegenerative diseases: preclinical proof of concept and first-in-human data

ObjectivesReactive gliosis is a common pathological hallmark of CNS pathology resulting from neurodegeneration and neuroinflammation. In this study we investigate the capability of a novel monoamine oxidase B (MAO-B) PET ligand to monitor reactive astrogliosis in a transgenic mouse model of Alzheimer`s disease (AD). Furthermore, we performed a pilot study in patients with a range of neurodegenerative and neuroinflammatory conditions.MethodsA cross-sectional cohort of 24 transgenic (PS2APP) and 25 wild-type mice (age range: 4.3–21.0 months) underwent 60 min dynamic [18F]fluorodeprenyl-D2 ([18F]F-DED), static 18 kDa translocator protein (TSPO, [18F]GE-180) and β-amyloid ([18F]florbetaben) PET imaging. Quantification was performed via image derived input function (IDIF, cardiac input), simplified non-invasive reference tissue modelling (SRTM2, DVR) and late-phase standardized uptake value ratios (SUVr). Immunohistochemical (IHC) analyses of glial fibrillary acidic protein (GFAP) and MAO-B were performed to validate PET imaging by gold standard assessments. Patients belonging to the Alzheimer’s disease continuum (AD, n = 2), Parkinson’s disease (PD, n = 2), multiple system atrophy (MSA, n = 2), autoimmune encephalitis (n = 1), oligodendroglioma (n = 1) and one healthy control underwent 60 min dynamic [18F]F-DED PET and the data were analyzed using equivalent quantification strategies.ResultsWe selected the cerebellum as a pseudo-reference region based on the immunohistochemical comparison of age-matched PS2APP and WT mice. Subsequent PET imaging revealed that PS2APP mice showed elevated hippocampal and thalamic [18F]F-DED DVR when compared to age-matched WT mice at 5 months (thalamus: + 4.3%; p = 0.048), 13 months (hippocampus: + 7.6%, p = 0.022) and 19 months (hippocampus: + 12.3%, p < 0.0001; thalamus: + 15.2%, p < 0.0001). Specific [18F]F-DED DVR increases of PS2APP mice occurred earlier when compared to signal alterations in TSPO and β-amyloid PET and [18F]F-DED DVR correlated with quantitative immunohistochemistry (hippocampus: R = 0.720, p < 0.001; thalamus: R = 0.727, p = 0.002). Preliminary experience in patients showed [18F]F-DED VT and SUVr patterns, matching the expected topology of reactive astrogliosis in neurodegenerative (MSA) and neuroinflammatory conditions, whereas the patient with oligodendroglioma and the healthy control indicated [18F]F-DED binding following the known physiological MAO-B expression in brain.Conclusions[18F]F-DED PET imaging is a promising approach to assess reactive astrogliosis in AD mouse models and patients with neurological diseases.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively totumorprogression and therapeutic resistance. We first presentthe interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 rolesin different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progressionand provides preclinical proof of concept that a combinationof ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

Abstract 330: Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate

Abstract Introduction: Prostate cancer is the second most common cancer among men worldwide. In 2021, it is estimated that 248,530 men in the United States will be diagnosed with prostate cancer, and 34,130 will die from the disease. Although current treatments have success initially, development of resistance commonly leads to recurrence of an incurable castrate-resistant form of the disease. Thus, significant need for novel therapies to improve the outcome of castrate-resistant prostate cancer remains. B7-H3 (CD276), a member of the B7 family of immunomodulatory molecules, is overexpressed in primary and metastatic prostate cancer, and correlates with disease severity and poor clinical outcome. MGC018, a duocarmycin-based B7-H3 antibody-drug conjugate, is currently being evaluated in clinical studies. Here, MGC018 was explored preclinically to assess the potential for targeting B7-H3 in prostate cancer. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in prostate cancer tissue microarrays (TMA). In vivo efficacy studies were conducted with human prostate cancer cell line-derived xenograft (CDX) models to explore the antitumor activity of MGC018 as a single agent and in combination with Poly (ADP-ribose) polymerase (PARP) and androgen receptor (AR) inhibitors. Based on the results in the CDX studies, in vivo efficacy studies were extended to a panel of metastatic prostate cancer patient-derived xenograft (PDX) models, which exhibit heterogenous expression of B7-H3 and more closely mimic the biological characteristics of patient tumors. Results: Staining of prostate tumor TMAs revealed high expression of B7-H3 in primary and metastatic prostate cancer. Of the prostate samples evaluated, 95% (38/40) of the tumor samples were positive for B7-H3 (H-score ≥ 20): 65% had H-scores greater than 200, while 20% and 10% had H-scores between 101-200 and 1-100, respectively. MGC018 demonstrated in vitro cytotoxicity toward B7-H3-positive human prostate cancer cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo toward prostate cancer CDX models, and the antitumor activity of MGC018 was enhanced when combined with inhibitors of PARP or AR. In PDX models of metastatic prostate cancer, MGC018 was active as a single agent toward heterogeneous B7-H3-expressing tumors, and combining MGC018 with inhibitors of PARP or AR led to a greater response in some models. Conclusion: B7-H3 is frequently overexpressed in prostate cancer. MGC018 demonstrated potent antitumor activity in vivo toward CDX and PDX models of prostate cancer, and enhanced antitumor activity when combined with inhibitors of PARP or AR. These results support prostate cancer as an indication that may be responsive to ADC-based treatments directed toward B7-H3. MGC018 is being investigated in metastatic prostate cancer in a Phase 1/2 clinical study. Citation Format: Juniper A. Scribner, Francine Z. Chen, Anushka De Costa, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Jonathan Li, Scott Koenig, Chet Bohac, Ezio Bonvini, Paul A. Moore, Deryk Loo. Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate [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 330.

Abstract 3592: Targeting SWI/SNF ATPases in enhancer-addicted human cancers

Abstract In mammalian cells, DNA is wrapped around histone octamers (collectively referred to as nucleosomes) which form a physical barrier to all DNA-based processes. The switch/sucrose non-fermentable (SWI/SNF) is a multi-subunit chromatin remodeling complex that uses energy from ATP hydrolysis to reposition or eject nucleosomes at non-coding regulatory elements, thereby enabling access to the underlying DNA for transcriptional activation. Notably, the SWI/SNF complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers, with the revised complex in cancer cells enabling central oncogenic gene programs. Yet, no studies have assessed the therapeutic efficacy of complete SWI/SNF inactivation across human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. In a panel with over 90 normal and cancer cell lines from 18 different lineages, we found MYC-driven multiple myeloma and androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate and breast cancers to be preferentially sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including cancer cell lines with inactivating SMARCA4 mutations. We found complete SWI/SNF ATPase degradation to instantaneously compact the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely MYC, IRF4, TCF3, AR, FOXA1, and ERG. This ensued in parallel untethering of these oncogenic drivers from the chromatin, with subsequent chemical decommissioning of their core enhancer circuitry and attenuation of downstream gene programs. Furthermore, using chromatin conformation assays we found SWI/SNF inactivation to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the MYC, AR, ERG, IRF4, and TCF3 oncogenes themselves, thereby tempering their expression in cancer cells. Treatment with the SMARCA2/4 degrader alone induced potent inhibition of tumor growth in cell line-derived xenograft models of multiple myeloma, as well as prostate cancer, and synergized with AR antagonists, inducing disease remission in several drug-resistant disease models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration—all being major toxicities associated with the BRD4-targeting therapeutics. To our knowledge, this study is the first preclinical proof of concept that targeted obstruction of chromatin accessibility at non-coding regulatory elements can be a potent therapeutic strategy in enhancer-addicted tumors, warranting the safety and efficacy assessments of SWI/SNF inhibitors and degraders in human clinical trials. Citation Format: Abhijit Parolia, Lanbo Xiao, Yuanyuan Qiao, Pushpinder Bawa, Sanjana Eyunni, Eleanor Young, Rahul Mannan, Sandra E. Carson, Yu Chang, Yuping Zhang, James George, Mustapha Jaber, Fengyun Su, Rui Wang, Sanjita Sasmal, Leena Khare, Subhendu Mukerjee, Chandrasekhar AbbinenI, Kiran Aithal, Xuhong Cao, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted human cancers [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 3592.

Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.

131P Translating a novel autotaxin inhibitor from preclinical proof of concept in pancreatic cancer to a biomarker response in human subjects

Autotaxin (ATX) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). LPA signaling modulates tumor cell function and contributes to the development of the fibrotic tumor microenvironment, resulting in reduced host immunity and impaired response to therapy. The expression of ATX and LPA is elevated in plasma of pancreatic cancer patients and correlates with soluble CA19-9. IOA-289 is a potent and orally available autotaxin inhibitor being developed as a novel treatment for pancreatic cancer and other highly fibrotic tumors.

Transient Transfection of the Respiratory Epithelium with Gamma Interferon for Host-Directed Therapy in Pulmonary Tuberculosis

Nebulized gamma interferon (IFN-γ) protein has been studied for clinical safety and efficacy against pulmonary tuberculosis (TB). The protein is expensive, requires a cold chain, and is difficult to deploy in limited-resource, high-incidence settings. We generated a preclinical proof of concept (PoC) for a dry powder inhalation (DPI) containing DNA constructs to transiently transfect the lung and airway epithelium of mice with murine IFN-γ. Bacterial colony-forming units (CFU) in the lungs of mice infected with Mycobacterium tuberculosis (Mtb) reduced from about 106/g of tissue to ~104 after four doses given once a week. Nodular inflammatory lesions in the lungs reduced significantly in number. Immunohistochemistry of infected lung sections for LC3-1 and LAMP-1 indicated autophagy induction between 18 and 48 h after inhalation. ELISA on bronchoalveolar lavage (BAL) fluid showed differences in kinetics of IFN-γ concentrations in the epithelial lining fluid of healthy versus infected mice. Uninfected mice receiving DNA constructs expressing a fluorescent protein were live-imaged. The fluorescence signals from the intracellular protein peaked at about 36 h after inhalation and declined by 48 h. These results establish preclinical PoC of the efficacy of a DPI and dosing regimen as a host-directed and transient gene therapy of experimental pulmonary TB in mice, justifying preclinical development.

Systematic target actionability reviews of preclinical proof-of-concept papers to match targeted drugs to paediatric cancers

BackgroundChildren with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. MethodsA sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for paediatric tumour type and categorised into nine separate PoC data modules. Each experimental finding was scored for experimental outcome and quality independently by two reviewers; discrepancies were assessed by a third reviewer and resolved by adjudication. Scores corresponding to one PoC module were merged for each tumour type and visualised in a heat map matrix in the publicly available R2 data portal [r2.amc.nl]. Results and conclusionsTo test our TAR methodology, we conducted a pilot study on MDM2 and TP53. The heat map generated from analysis of 161 publications provides a rationale to support drug development in specific paediatric solid and brain tumour types. Furthermore, our review highlights tumour types where preclinical data are incomplete or lacking and for which additional preclinical testing is advisable.

P.149The MYODA operational seamless clinical trial design phase I to III: a new approach for rare diseases to evaluate the safety, efficacy, pharmacokinetics, and pharmacodynamics of BIO101 (MAS activator) in paediatric patients with a genetically confirmed diagnosis of Duchenne muscular dystrophy

Clinical development of new medications is traditionally broken down into 3 phases. This prevents patients from participating in more than one of the development phases and comes with a burden on site staff (with repeated site initiation and training) leading to prolonged development timelines (up to 10 years) and difficulties in patient recruitment, especially in rare diseases such as DMD. There is a significant need, therefore, for utilization of novel designs, which will make clinical development more amenable for patients and more efficient for study site staff. Biophytis is developing BIO101 (20-hydroxyecdysone, MAS activator) for DMD. The pre-clinical proof-of-concept (PoC) for BIO101 demonstrates combined beneficial effects on skeletal muscle and respiratory functions in DMD animal models, suggesting a range of benefit in a broad DMD population. To ensure an optimal and patient-centered clinical development of BIO101, a seamless methodology is intended to be utilized. The trial design should take into consideration the needs and struggle of the patients who participate in the study. An operational seamless design is one that can cover the requirements, in terms of needed evidence, to support a new medicine (i.e. from phase I to phase III), while offering participants an access to the trial medication and reduce the need for repeated screening procedures. Biophytis proposes a 3-part study that combines Phase I to III of the clinical development in one operational seamless study with adaptive elements. Patients with DMD with various degrees of loss of ambulation will be recruited in one of the four-ascending multiple-dose cohorts of 1 week-duration (Part1), they will continue at the same dose for 24 weeks (Part2=phase II) in order to support dose(s) that will be tested for the pivotal Part 3. Continuing patients from Part 1 and 2 with additional patients will enter a 48 week-period (Part 3) to establish longer term safety and efficacy of BIO101.

EP.87MYODA clinical program: composite score for assessing the efficacy of BIO101 (MAS activator) in ambulatory and non-ambulatory Duchenne boys

The purpose of the clinical program MYODA is to evaluate muscle strength and function in boys suffering from Duchenne Muscular Dystrophy (DMD) under long-term oral administration of BIO101 (20-hydroxyecdysone, MAS activator). Ambulant and non-ambulant DMD boys will be recruited. The heterogeneity of the clinical course of DMD has long been recognised and more recently has been captured in registry studies (Ricotti et al. 2013, 2016, Mercuri et al. 2016). Moreover, the mode of action of BIO101 and the pre-clinical proof-of-concept (PoC) suggest that DMD patients who will benefit from BIO101 is quite broad and that this benefit can be shown in a variety of functions: mobility, strength and respiratory. Due to study size limitation, using multiple outcomes and studies to assess totality of evidence for efficacy and safety is desirable for rare disease drug development. In comparing a new treatment with a control for DMD under a randomized clinical trial setting, each study patient has multiple efficacy outcomes collected over time (NSAA, PUL, 6MWT, MyoPinch, MyoGrip, PEF, FVC). Instead of defining a primary endpoint using a single outcome and identifying several secondary endpoints like most conventional studies, Biophytis presents several approaches to utilize multiple outcomes simultaneously to evaluate BIO101 effect. The resulting procedures should be more powerful to detect the treatment difference than the conventional design and also provide clinically meaningful interpretation on the totality of treatment efficacy evidence (Ricotti et al.2019). Biophytis hypothesizes that a composite score, which covers both muscle strength, skeletal muscle and respiratory functions and captures changes across the loss of ambulation might be best adapted to detect changes across the wide disease spectrum. Biophytis will study how the change in the endpoint may be clinically meaningful. Biophytis will extend the analysis using Matching population from DMD databases.

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

BackgroundSmoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction.ResultsThis paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2- to 19-fold enhanced catalytic activity compared to wt at 10 μM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen’s access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity.ConclusionsSystematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme’s immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

Quantitative temporal diffusion spectroscopy as an early imaging biomarker of radiation therapeutic response in gliomas: A preclinical proof of concept

PurposeThis study aims to test the ability of quantitative temporal diffusion spectroscopy (qTDS) to assess cellular changes associated with radiation-induced cell death in a rat glioma model. Methods and MaterialsTumor response to a single fraction of 20 Gy of x-ray radiation was investigated in a rat glioma (9L) model. Tumor response was monitored longitudinally at postinoculation days 21, 23, and 25, using a specific implementation of qTDS with acronym IMPULSED (Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion), as well as conventional diffusion and high-resolution anatomic imaging. IMPULSED method combines diffusion-weighted signals acquired over a range of diffusion times that are then analyzed and interpreted using a theoretical model of water diffusion in tissues, which generates parametric maps depicting cellular and subcellular structural information on a voxel-wise basis. Results from different metrics were compared statistically. ResultsA single dose of 20 Gy x-ray radiation significantly prolonged survival of 9L-bearing rats. The mean cell sizes of irradiated tumors decreased (P < .005) after radiation treatment, which we associate with cell shrinkage and the formation of small cellular bodies during apoptosis and necrosis. A combination of IMPULSED-derived parameters (mean cell size d and extracellular structural parameter βex) separated 90% of irradiated tumors from the nonirradiated cases at post inoculation day 23, whereas a combination of tumor growth and conventional apparent diffusion coefficient did not differentiate irradiated tumors from nonirradiated tumors. ConclusionsThis proof-of-concept study demonstrates the IMPULSED method to be a new method for deriving quantitative microstructural parameters in a preclinical tumor model. The method provides unique information based on the diffusion time dependency of diffusion magnetic resonance imaging, which cannot be obtained by conventional diffusion weighted imaging methods, and the results have a close correlation with primary biologic markers of treatment efficacy, such as cell death and survival.

A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.

Abstract 3841: Biologic evaluation of heterocyclic pyrimidines as novel tubulin inhibitors targeting the colchicine binding site

Abstract Interfering with tubulin dynamics is an attractive strategy for cancer therapy, and tubulin-targeting agents are some of the most widely used chemotherapeutic drugs. Current FDA-approved tubulin inhibitors target either the taxane site (e.g., paclitaxel) or the vinca alkaloid site (e.g., vinblastine). However, their clinical efficacy is often limited by the development of multidrug resistance and a narrow therapeutic index. Tubulin inhibitors interacting with the colchicine site have shown potent anticancer activities, are generally less susceptible to multidrug resistance, and can disrupt tumor vasculature, leading to rapid tumor cell apoptosis. Here, we report on the biologic evaluation of heterocyclic pyrimidines as a new class of tubulin inhibitors targeting the colchicine binding site with a significantly improved therapeutic index. One of the most promising compounds of the series, SB157, was able to strongly inhibit tubulin polymerization and induce cytotoxicity in cancer cells in vitro. We have also obtained the high-resolution X-ray crystal structure for SB157, confirming its direct binding to the colchicine binding site in tubulin, and elucidated the detailed molecular interactions. SB157 significantly inhibited tumor growth in an A375 melanoma xenograft model, and the tumors revealed elevated apoptotic activity and extensive disruption of tumor vasculature. Finally, we demonstrated that SB157 significantly overcame clinically relevant multidrug resistance in a paclitaxel-resistant PC-3/TxR prostate cancer xenograft mouse model. Collectively, these studies provide preclinical proof of concept and structural basis to support the continued development of SB157 and optimization of this scaffold as an improved class of tubulin inhibitors for cancer therapy. Citation Format: Kinsie Arnst. Biologic evaluation of heterocyclic pyrimidines as novel tubulin inhibitors targeting the colchicine binding site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3841.

Dedicated bifurcated balloon for coronary bifurcation lesions: a preclinical proof of concept.

Dedicated bifurcated balloon for coronary bifurcation lesions: a preclinical proof of concept.

Kv1.3 inhibition as a potential microglia-targeted therapy for Alzheimer's disease: preclinical proof of concept.

Microglia significantly contribute to the pathophysiology of Alzheimer's disease but an effective microglia-targeted therapeutic approach is not yet available clinically. The potassium channels Kv1.3 and Kir2.1 play important roles in regulating immune cell functions and have been implicated by in vitro studies in the 'M1-like pro-inflammatory' or 'M2-like anti-inflammatory' state of microglia, respectively. We here found that amyloid-β oligomer-induced expression of Kv1.3 and Kir2.1 in cultured primary microglia. Likewise, ex vivo microglia acutely isolated from the Alzheimer's model 5xFAD mice co-expressed Kv1.3 and Kir2.1 as well as markers traditionally associated with M1 and M2 activation suggesting that amyloid-β oligomer induces a microglial activation state that is more complex than previously thought. Using the orally available, brain penetrant small molecule Kv1.3 blocker PAP-1 as a tool, we showed that pro-inflammatory and neurotoxic microglial responses induced by amyloid-β oligomer required Kv1.3 activity in vitro and in hippocampal slices. Since we further observed that Kv1.3 was highly expressed in microglia of transgenic Alzheimer's mouse models and human Alzheimer's disease brains, we hypothesized that pharmacological Kv1.3 inhibition could mitigate the pathology induced by amyloid-β aggregates. Indeed, treating APP/PS1 transgenic mice with a 5-month oral regimen of PAP-1, starting at 9 months of age, when the animals already manifest cognitive deficits and amyloid pathology, reduced neuroinflammation, decreased cerebral amyloid load, enhanced hippocampal neuronal plasticity, and improved behavioural deficits. The observed decrease in cerebral amyloid deposition was consistent with the in vitro finding that PAP-1 enhanced amyloid-β uptake by microglia. Collectively, these results provide proof-of-concept data to advance Kv1.3 blockers to Alzheimer's disease clinical trials.