SBIR-STTR Award

Immunometabolic NLRX1 Therapeutics for IBD Biotherapeutics Inc (BTI) is an emerging biotech company that synergistically combines the power of advanced computational modeling with translational experimentation to accelerate the development of novel products for precision medicine and health. This SBIR application stems from data showing a vital role for nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) as a new therapeutic target for IBD. Our Product: BTI has identified the first family of small-molecule compounds that bind and activate the novel regulatory molecule, NLRX1. The goal of this project is to validate NLRX1 as a target and develop NX-13 as the lead NLRX1-based, oral first-in-class therapeutic for IBD. Background: IBD is a chronic widespread and debilitating illness that afflicts over 5 million people worldwide. Current treatments are only modestly successful with significant adverse side effects, including cancer, infection and death. Thus, there is an unmet clinical need for safer, more efficacious IBD therapeutics. NLRX1 can suppress intestinal inflammation during infections and autoimmune disorders. BTI has validated that loss of NLRX1 causes reduced mucosal healing, increased fibrosis, >5-fold up-regulation in inflammatory cytokine biomarkers, and complete restructuring of gut microbiome ecology during IBD. Additionally, our top NLRX1 ligand exerts therapeutic actions in models of IBD. This SBIR Phase I application will characterize ligand- protein interactions of NX-13 and NLRX1, define the NX-13 cellular mechanism of action and validate its safety and specificity to NLRX1. The Specific Aims are to: AIM 1. Evaluate NX-13 interactions with purified and in situ NLRX1 by CD-spectroscopy and isothermal calorimetry on purified WT and mutant NLRX1 and by cellular thermal shift assays. AIM 2. Determine the cellular mechanism of action of NX-13 in vivo in the DSS model of colitis using cre- lox cell specific (CD4+, myeloid, epithelial) knockouts of NLRX1. AIM 3. Conduct preliminary ADME-Tox and specificity assays with NX-13 in off-target binding assays, 7- d dose-range finding toxicity studies in rats and ADME-Tox assays (hERG, Ames, CYP450, P-gp). Expected Outcomes: Validation of NX-13 as a lead molecule for targeting NLRX1 through: i) engagement of NLRX1 in situ at concentrations ? 0.1 µM); ii) identification of cellular mechanism of action by loss of efficacy in cell specific KO mice; iii) a benign safety profile with NOAEL ? 1,000 mg/kg oral in rats. SBIR Phase II will validate the implication of NLRX1 in human IBD, determine PK in rats, define anti-fibrotic effects in mouse models of intestinal fibrosis and advance NX-13 to IND-enabling GLP toxicology studies. Commercial Application: Success in this project will launch a new drug development pipeline at BTI centered on NLRX1-activating therapeutics with anti-inflammatory and anti-fibrotic effects. BTI's new NLRX1-targeting oral therapeutics could disrupt a market of over $10B annually growing at 25% annual rates.

Public Health Relevance Statement:
Public Health Relevance The spread of Inflammatory bowel disease (IBD) is growing globally and it afflicts over 2 million people in the United States and over 5 million people worldwide. This SBIR Phase I has the potential to develop a novel class of potent oral drugs for IBD that exert their therapeutic actions by acting on the novel target, NLRX1. In addition to the mechanism of action and efficacy work, this project will perform preliminary safety and ADME studies to advance the compound along the FDA regulatory pipeline into IND. The proposed studies will lay the groundwork for developing NX-13 a small molecule therapeutic with the potential to selectively engage and activate NLRX1 in the gut and discovering how NX-13 modulates immune and inflammatory responses in the gastrointestinal (GI) tract in IBD.

Project Terms:
ADME Study; Aerobic; Anti-inflammatory; Autoimmune Diseases; base; Benign; Binding; Binding Proteins; Biological Assay; Biological Markers; Biological Response Modifier Therapy; Biotechnology; Calorimetry; Cells; Cessation of life; Chronic; Clinical; Clinical Trials; Colitis; Colorectal Cancer; commensal bacteria; commercial application; Computer Simulation; cytokine; Data; Development; Disease; Disease Management; Disease Pathway; Dose; drug development; Ecology; Epithelial; Epithelial Cells; Family; Fibrosis; Gastrointestinal tract structure; Gene Expression; Goals; gut microbiome; gut microbiota; healing; Human; Immune; immune checkpoint; Immune response; In Situ; In Vitro; in vivo; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Inflammatory Response; innovation; Intestinal Fibrosis; Knock-out; Knockout Mice; Knowledge; Lead; Legal patent; Leucine-Rich Repeat; Ligands; Long-Term Effects; Lung diseases; Malignant Neoplasms; Mediating; Medical; Methods; Mitochondria; Modeling; mouse model; Mucous Membrane; Mus; mutant; Myelogenous; Myeloid Cells; new therapeutic target; No-Observed-Adverse-Effect Level; novel; novel therapeutics; Nucleotides; off-patent; Oral; Outcome; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Plasma Proteins; Positioning Attribute; Precision Health; precision medicine; preference; Program Development; Proteins; public health relevance; Rattus; receptor; Recombinants; research and development; Role; Safety; safety study; Severity of illness; side effect; single cell sequencing; Site; Small Business Innovation Research Grant; small molecule; small molecule therapeutics; Specificity; Spectrum Analysis; stem; success; T-Lymphocyte; Testing; Therapeutic; Toxic effect; Toxicology; Treatment Efficacy; United States; Up-Regulation; Validation; Viral; Work

Validation of Immunometabolic NLRX1 Therapeutics for IBD Biotherapeutics Inc (BTI) is an emerging biotech company that synergistically combines the power of advanced computational modeling with translational experimentation to accelerate the development of novel products for precision medicine and health. This SBIR application stems from data showing a vital role for nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) as a new therapeutic target for IBD. Our Product: BTI has identified the first family of small-molecule compounds that bind and activate the novel regulatory molecule, NLRX1. In a Phase I SBIR, we successfully established NX-13 as a NLRX1-specific agonist that reduces inflammatory lesions by 90% in CD4+ T cell-specific immunometabolic mechanisms. Background: IBD is a chronic widespread and debilitating illness that afflicts over 5 million people worldwide with total expenses exceeding $15 billion annually in the U.S alone. Current treatments are only modestly successful with significant adverse side effects. Thus, there is an unmet clinical need for safer, more efficacious IBD therapeutics. NLRX1 can suppress intestinal inflammation during infections and autoimmune disorders. BTI has validated that loss of NLRX1 causes reduced mucosal healing, increased fibrosis, >5-fold up-regulation in inflammatory cytokine biomarkers, and complete restructuring of gut microbiome ecology during IBD. This SBIR Phase II application will characterize microbial and anti-fibrotic mechanisms of NX-13, demonstrate translational efficacy in human UC primary cells and conduct IND-enabling safety studies. The Specific Aims are to: AIM 1. Determine the effect of NX-13 on the intestinal microbiome during colitis through 16S gut microbiome sequencing and signatures of gut-microbiome interactions. AIM 2. Characterize the anti-fibrotic mechanisms of NX-13 in chronic colitis models and in vitro effects on fibroblast gene expression and extracellular matrix deposition. AIM 3. Validate the translational efficacy of NX-13 in PBMCs and LPMCs from UC patients through evaluation of overall cellular response and CD4-specific immunometabolic mechanisms. AIM 4. Conduct an IND-enabling GLP toxicity study in a non-rodent species with a 28-day repeat dose toxicity study at three dose levels (250, 500, 1000 mg/kg) compared to vehicle. Expected Outcomes: Validation of NX-13 as a lead agonistic molecule for targeting NLRX1 through: i) identification of gut microbial and anti-fibrotic mechanisms of NX-13; ii) 50% reduction in TNF? and IFN? production in human PBMCs and LPMCs; and iii) a benign safety profile with oral NOAEL ? 1,000 mg/kg. Commercial Application: Success in this project will launch a new drug development pipeline centered on NLRX1-activating therapeutics with anti-inflammatory and anti-fibrotic effects. BTI’s new NLRX1-targeting oral therapeutics could disrupt a market of over $10B annually growing at 25% annual rates.

Public Health Relevance Statement:
Public Health Relevance The spread of Inflammatory bowel disease (IBD) is growing globally and it afflicts over 2 million people in the United States and over 5 million people worldwide. This SBIR Phase I has the potential to develop a novel class of potent oral drugs for IBD that exert their therapeutic actions by acting on the novel target, NLRX1, and advance them along the FDA regulatory pipeline into IND and clinical testing. The proposed studies will lay the groundwork for developing NX-13 a small molecule therapeutic with the potential to selectively engage and activate NLRX1 in the gastrointestinal (GI) tract and discovering how NX-13 modulates immune and inflammatory responses in the GI tract in IBD.

Project Terms:
Aerobic; Agonist; Anti-Inflammatory Agents; Autoimmune Diseases; Bacteroidetes; Benign; Binding; Biological Assay; Biological Availability; Biological Markers; Biological Response Modifier Therapy; Biotechnology; Blood; Calorimetry; Canis familiaris; CD4 Positive T Lymphocytes; Cells; Cessation of life; Chronic; Clinical; Clinical Trials; Colitis; Colorectal Cancer; commensal bacteria; commercial application; Computer Models; cytokine; Data; Deposition; Development; Disease; Disease Management; Dose; drug development; Drug Kinetics; Ecology; Epithelial Cells; Evaluation; Extracellular Matrix; Family; Fibroblasts; Fibrosis; Firmicutes; Gastrointestinal tract structure; Gene Expression; Glutamine; Goals; gut microbiome; gut microbiota; healing; Human; Hydrogen Peroxide; immune checkpoint; Immune response; Immunophenotyping; In Situ; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; inflammatory disease of the intestine; Inflammatory Response; innovation; Interferon Type II; Knock-out; Knowledge; Lead; Legal patent; Lesion; Leucine-Rich Repeat; Ligands; Long-Term Effects; Lung diseases; Malignant Neoplasms; Measures; Mediating; Medical; metabolic phenotype; Metabolism; Methods; microbial; microbiome sequencing; Mitochondria; mitochondrial metabolism; Modeling; mouse model; Mucous Membrane; Mus; Myeloid Cells; new therapeutic target; No-Observed-Adverse-Effect Level; novel; novel therapeutics; Nucleotides; Operative Surgical Procedures; Oral; Outcome; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phase; Phenotype; Positioning Attribute; Precision Health; precision medicine; preference; Production; Program Development; prophylactic; Proteins; public health relevance; Rattus; receptor; Recombinants; research and development; research clinical testing; response; Role; Safety; safety study; Severity of illness; side effect; Signal Pathway; Small Business Innovation Research Grant; small molecule; small molecule therapeutics; Specificity; Spectrum Analysis; stem; success; T-Lymphocyte; Testing; Therapeutic; TNF gene; Toxic effect; Treatment Efficacy; Trichrome stain method; United States; Up-Regulation; Validation; Viral