Exploratory studies with NX-13: oral toxicity and pharmacokinetics in rodents of an orally active, gut-restricted first-in-class therapeutic for IBD that targets NLRX1.

Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is an emerging therapeutic target for a spectrum of human diseases. NX-13 is a small molecule therapeutic designed to target and activate NLRX1 to induce immunometabolic changes resulting in lower inflammation and therapeutic responses in inflammatory bowel disease (IBD). This study investigates the safety of NX-13 in a seven-day, repeat-dose general toxicity study in male and female Sprague Dawley rats at oral doses of 500 and 1000 mg/kg. Weights, clinical signs, functional observational battery, clinical pathology and histopathology were used for evaluation. Daily oral dosing of NX-13 up to 1000 mg/kg did not result in any changes in weight, abnormal clinical signs or behavior. No significant differences were observed between treated and control rats in hematology or blood biochemistry. Histopathological evaluation of 12 tissues demonstrated no differences between controls and treated rats. There were no changes in weights of brain, heart, kidney, liver or spleen. Pharmacokinetic analysis of a single oral dose of NX-13 at 10 mg/kg in Sprague Dawley rats provided a maximum plasma concentration of 57 ng/mL at 0.5 h post-dose. Analysis of colon tissue after oral dosing with 1 and 10 mg/kg indicated high peak concentrations (10 and 100 µg/g, respectively) that scale in a dose-proportional manner. These experiments suggest that NX-13 is safe and well-tolerated in rats given oral doses as high as 1000 mg/kg with a favorable gastrointestinal localized pharmacokinetic profile, confirming NX-13 as a promising therapeutic for Crohn's disease and ulcerative colitis.

Oral Treatment with BT-11 Ameliorates Inflammatory Bowel Disease by Enhancing Regulatory T Cell Responses in the Gut.

Inflammatory bowel disease (IBD) is an expanding autoimmune disease afflicting millions that remains difficult to treat due to the accumulation of multiple immunological changes. BT-11 is an investigational new drug for IBD that is orally active, gut restricted, and targets the lanthionine synthetase C-like 2 immunometabolic pathway. CD25+ FOXP3+ CD4+ T cells are increased locally within the colon of BT-11-treated mice in Citrobacter rodentium and IL-10-/- mouse models of colitis. The maintained efficacy of BT-11 in the absence of IL-10 combined with the loss of efficacy when direct cell-cell interactions are prevented suggest that the regulatory T cell (Treg)-related elements of suppression are cell contact-mediated. When PD-1 is inhibited, both in vitro and in vivo, the efficacy of BT-11 is reduced, validating this assertion. The depletion of CD25+ cells in vivo abrogated the retention of therapeutic efficacy postdiscontinuation of treatment, indicating that Tregs are implicated in the maintenance of tolerance mediated by BT-11. Furthermore, the involvement of CD25 suggested a role of BT-11 in IL-2 signaling. Cotreatment with BT-11 and IL-2 greatly enhances the differentiation of CD25+ FOXP3+ cells from naive CD4+ T cells relative to either alone. BT-11 enhances phosphorylation of STAT5, providing a direct linkage to the regulation of FOXP3 transcription. Notably, when STAT5 is inhibited, the effects of BT-11 on the differentiation of Tregs are blocked. BT-11 effectively enhances the IL-2/STAT5 signaling axis to induce the differentiation and stability of CD25+ FOXP3+ cells in the gastrointestinal mucosa to support immunoregulation and immunological tolerance in IBD.

Activation of NLRX1 by NX-13 Alleviates Inflammatory Bowel Disease through Immunometabolic Mechanisms in CD4+ T Cells.

Inflammatory bowel disease (IBD) is a complex autoimmune disease with dysfunction in pattern-recognition responses, including within the NLR family. Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a unique NLR with regulatory and anti-inflammatory functions resulting in protection from IBD in mouse models. NX-13 is an orally active, gut-restricted novel drug candidate that selectively targets and activates the NLRX1 pathway locally in the gut. In vitro and in vivo efficacy of NLRX1 activation by NX-13 was examined. Oral treatment with NX-13 alleviates disease severity, colonic leukocytic infiltration, and cytokine markers of inflammation in three mouse models of IBD (dextran sulfate sodium, Mdr1a-/-, and CD45RBhi adoptive transfer). Treatment of naive CD4+ T cells with NX-13 in vitro decreases differentiation into Th1 and Th17 subsets with increased oxidative phosphorylation and decreased NF-κB activation and reactive oxygen species. With stimulation by PMA/ionomycin, TNF-α, or H2O2, PBMCs from ulcerative colitis patients treated with NX-13 had decreased NF-κB activity, TNF-α+ and IFN-γ+ CD4+ T cells and overall production of IL-6, MCP1, and IL-8. NX-13 activates NLRX1 to mediate a resistance to both inflammatory signaling and oxidative stress in mouse models and human primary cells from ulcerative colitis patients with effects on NF-κB activity and oxidative phosphorylation. NX-13 is a promising oral, gut-restricted NLRX1 agonist for treating IBD.

Cooperation of Gastric Mononuclear Phagocytes with Helicobacter pylori during Colonization.

Helicobacter pylori, the dominant member of the human gastric microbiota, elicits immunoregulatory responses implicated in protective versus pathological outcomes. To evaluate the role of macrophages during infection, we employed a system with a shifted proinflammatory macrophage phenotype by deleting PPARγ in myeloid cells and found a 5- to 10-fold decrease in gastric bacterial loads. Higher levels of colonization in wild-type mice were associated with increased presence of mononuclear phagocytes and in particular with the accumulation of CD11b+F4/80hiCD64+CX3CR1+ macrophages in the gastric lamina propria. Depletion of phagocytic cells by clodronate liposomes in wild-type mice resulted in a reduction of gastric H. pylori colonization compared with nontreated mice. PPARγ-deficient and macrophage-depleted mice presented decreased IL-10-mediated myeloid and T cell regulatory responses soon after infection. IL-10 neutralization during H. pylori infection led to increased IL-17-mediated responses and increased neutrophil accumulation at the gastric mucosa. In conclusion, we report the induction of IL-10-driven regulatory responses mediated by CD11b+F4/80hiCD64+CX3CR1+ mononuclear phagocytes that contribute to maintaining high levels of H. pylori loads in the stomach by modulating effector T cell responses at the gastric mucosa.

NLRX1 Regulates Effector and Metabolic Functions of CD4+ T Cells.

Nucleotide oligomerization domain-like receptor X1 (NLRX1) has been implicated in viral response, cancer progression, and inflammatory disorders; however, its role as a dual modulator of CD4+ T cell function and metabolism has not been defined. The loss of NLRX1 results in increased disease severity, populations of Th1 and Th17 cells, and inflammatory markers (IFN-γ, TNF-α, and IL-17) in mice with dextran sodium sulfate-induced colitis. To further characterize this phenotype, we used in vitro CD4+ T cell-differentiation assays and show that NLRX1-deficient T cells have a greater ability to differentiate into an inflammatory phenotype and possess greater proliferation rates. Further, NLRX1-/- cells have a decreased responsiveness to immune checkpoint pathways and greater rates of lactate dehydrogenase activity. When metabolic effects of the knockout are impaired, NLRX1-deficient cells do not display significant differences in differentiation or proliferation. To confirm the role of NLRX1 specifically in T cells, we used an adoptive-transfer model of colitis. Rag2-/- mice receiving NLRX1-/- naive or effector T cells experienced increased disease activity and effector T cell populations, whereas no differences were observed between groups receiving wild-type or NLRX1-/- regulatory T cells. Metabolic effects of NLRX1 deficiency are observed in a CD4-specific knockout of NLRX1 within a Citrobacter rodentium model of colitis. The aerobic glycolytic preference in NLRX1-/- effector T cells is combined with a decreased sensitivity to immunosuppressive checkpoint pathways to provide greater proliferative capabilities and an inflammatory phenotype bias leading to increased disease severity.

Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11.

BT-11 is an orally active, gut-restricted investigational therapeutic targeting the lanthionine synthetase C-like 2 pathway with lead indications in ulcerative colitis (UC) and Crohn disease (CD), 2 manifestations of inflammatory bowel disease (IBD). In 5 mouse models of IBD, BT-11 is effective at oral doses of 8 mg/kg. BT-11 was also efficacious at nanomolar concentrations in primary human samples from patients with UC and CD. BT-11 was tested under Good Laboratory Practice conditions in 90-day repeat-dose general toxicity studies in rats and dogs, toxicokinetics, respiratory, cardiovascular and central nervous system safety pharmacology, and genotoxicity studies. Oral BT-11 did not cause any clinical signs of toxicity, biochemical or hematological changes, or macroscopic or microscopic changes to organs in 90-day repeat-dose toxicity studies in rats and dogs at doses up to 1,000 mg/kg/d. Oral BT-11 resulted in low systemic exposure in both rats (area under the curve exposure from t = 0 to t = 8 hours [AUC0-8] of 216 h × ng/mL) and dogs (650 h × ng/mL) and rapid clearance with an average half-life of 3 hours. BT-11 did not induce changes in respiratory function, electrocardiogram parameters, or behavior with single oral doses of 1,000 mg/kg/d. There was no evidence of mutagenic or genotoxic potential for BT-11 up to tested limit doses using an Ames test, chromosomal aberration assay in human peripheral blood lymphocytes, or micronucleus assay in rats. Therefore, nonclinical studies show BT-11 to be a safe and well-tolerated oral therapeutic with potential as a potent immunometabolic therapy for UC and CD with no-observed adverse effect level >1,000 mg/kg in in vivo studies.

Gut commensals require Peyer's patches to induce protective systemic IgA responses.

Gut educated IgA secreting plasma cells that disseminate beyond the mucosa and into systemic tissues have been described as providing beneficial effects from disease in several contexts. Several bacteria have been implicated in the induction of systemic IgA, however the mechanisms that result in differential levels of induction by each bacterial species are still unknown. Here we show, the commensal bacteria, Bacteroides fragilis (Bf), is an efficient inducer of systemic IgA responses. The ability of Bf to induce the production of bone marrow IgA plasma cells and high levels of serum IgA relied on high levels of gut colonization in a dose-dependent manner. Colonization induced Bf-specific IgA responses were severely diminished in the absence of Peyer's patches, but not the murine cecal patch. Colonization of mice with Bf, a natural human commensal, resulted in few changes within the microbiome and the host transcriptional profile in the gut, suggesting a commensal relationship with the host. Bf colonization did benefit the mice by inducing systemic IgA that led to increased protection in a bowel perforation model resulting in lower peritoneal abscess formation. These findings demonstrate a critical role for bacterial colonization and Peyer's patches in the induction of robust systemic IgA responses that confer protection from bacterial dissemination outside of the gut.

Switched and unswitched memory B cells detected during SARS-CoV-2 convalescence correlate with limited symptom duration.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the pandemic human respiratory illness COVID-19, is a global health emergency. While severe acute disease has been linked to an expansion of antibody-secreting plasmablasts, we sought to identify B cell responses that correlated with positive clinical outcomes in convalescent patients. We characterized the peripheral blood B cell immunophenotype and plasma antibody responses in 40 recovered non-hospitalized COVID-19 subjects that were enrolled as donors in a convalescent plasma treatment study. We observed a significant negative correlation between the frequency of peripheral blood memory B cells and the duration of symptoms for convalescent subjects. Memory B cell subsets in convalescent subjects were composed of classical CD24+ class-switched memory B cells, but also activated CD24-negative and natural unswitched CD27+ IgD+ IgM+ subsets. Memory B cell frequency was significantly correlated with both IgG1 and IgM responses to the SARS-CoV-2 spike protein receptor binding domain (RBD) in most seropositive subjects. IgM+ memory, but not switched memory, directly correlated with virus-specific antibody responses, and remained stable over 3 months. Our findings suggest that the frequency of memory B cells is a critical indicator of disease resolution, and that IgM+ memory B cells may play an important role in SARS-CoV-2 immunity.

The Safety, Tolerability, and Pharmacokinetics Profile of BT-11, an Oral, Gut-Restricted Lanthionine Synthetase C-Like 2 Agonist Investigational New Drug for Inflammatory Bowel Disease: A Randomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial.

BT-11 is a new oral, gut-restricted, first-in-class investigational drug for Crohn disease (CD) and ulcerative colitis (UC) that targets the lanthionine synthetase C-like 2 (LANCL2) pathway and immunometabolic mechanisms. Oral BT-11 was assessed for safety, tolerability, and pharmacokinetics (PK) in normal healthy volunteers (n = 70) in a randomized, double-blind, placebo-controlled trial. Subjects (n = 70) were randomly assigned to one of five single ascending dose cohorts (up to 100 mg/kg, p.o.) and three multiple ascending dose cohorts [up to 100 mg/kg daily (QD) for seven days, orally]. Safety and tolerability were assessed by adverse event (AE) reporting, vital signs, electrocardiogram, hematology, and clinical chemistry. BT-11 did not increase total or gastrointestinal AE rates, as compared with placebo, and no serious adverse events were observed. Oral BT-11 dosing does not result in any clinically significant findings by biochemistry, coagulation, electrocardiogram, hematology, or urinalysis as compared with placebo. Mean fecal concentrations of BT-11 increased linearly with increasing oral doses, with 2.39 mg/g at 7.7 mg/kg on day 7 of the multiple ascending dose (MAD). Analysis of plasma pharmacokinetics indicates that maximum systemic concentrations are approximately 1/6000th of observed concentrations in feces and the distal gastrointestinal tract. Fecal calprotectin levels were lower in BT-11 treated groups as compared to placebo. BT-11 significantly decreases interferon gamma positive (IFNγ+) and tumor necrosis factor alpha positive (TNFα+) cluster of differentiation 4 positive (CD4+) T cells and increases forkhead box P3 positive (FOXP3+) CD4+ T cells in colonic lamina propria mononuclear cells from patients with CD and patients with UC at concentrations of 0.01 µM when treated ex vivo. BT-11 treatment is well-tolerated with no dose-limiting toxicities up to daily oral doses of 100 mg/kg (16 tablets); whereas the efficacious dose is a single tablet (8 mg/kg). Phase II studies in CD and UC patients are ongoing.

Abscisic acid enriched fig extract promotes insulin sensitivity by decreasing systemic inflammation and activating LANCL2 in skeletal muscle.

Abscisic acid is a phytohormone found in fruits and vegetables and is endogenously produced in mammals. In humans and mice, lanthionine synthetase C-like 2 (LANCL2) has been characterized as the natural receptor for ABA. Herein, we characterize the efficacy of a fig fruit extract of ABA in promoting glycemic control. This ABA-enriched extract, at 0.125 µg ABA/kg body weight, improves glucose tolerance, insulin sensitivity and fasting blood glucose in diet-induced obesity (DIO) and db/db mouse models. In addition to decreasing systemic inflammation and providing glycemic control without increasing insulin, ABA extract modulates the metabolic activity of muscle. ABA increases expression of important glycogen synthase, glucose, fatty acid and mitochondrial metabolism genes and increases direct measures of fatty acid oxidation, glucose oxidation and metabolic flexibility in soleus muscle cells from ABA-treated mice with DIO. Glycolytic and mitochondrial ATP production were increased in ABA-treated human myotubes. Further, ABA synergized with insulin to dramatically increase the rate of glycogen synthesis. The loss of LANCL2 in skeletal muscle abrogated the effect of ABA extract in the DIO model and increased fasting blood glucose levels. This data further supports the clinical development of ABA in the treatment of pre-diabetes, type 2 diabetes and metabolic syndrome.