LANCL2 (lanthionine synthetase C-like 2). No IC50, Ki, EC50, or KD values are provided in the manuscripts. Surface plasmon resonance (SPR) was used to measure direct binding, and the compound showed typical small molecule-protein interaction with fast on/off rates [1].

BT-11 is a first-in-class, potent, orally available and selective modulator of LANCL2 (Lanthionine synthetase C-like 2) which is a novel therapeutic target for inflammatory and autoimmune diseases and diabetes, exerts anti-inflammatory and insulin-sensitizing effects. BT-11 at high doses has an excellent safety profile up to 1000 mg/kg/day. It has demonstrated therapeutic efficacy in 3 validated mouse models of colitis at doses as low as 8 mg/kg/d; It protects from IBD by targeting LANCL2. BT-11 has potential to be a safe and effective orally active therapeutic for IBD. The oral treatment with BT-11 (8 mg/kg/d) in a mouse model of IBD resulted in lowering the disease activity index, decreasing colonic inflammatory lesions by 4-fold, and suppressing inflammatory markers (e.g., TNF-α, and interferon-γ) in the gut. Furthermore, studies in LANCL2-/- mice demonstrated that loss of LANCL2 abrogated beneficial actions of BT-11, suggesting high selectivity for the target. In conclusion, BT-11 merits continued development as a LANCL2-based, first-in-class orally active therapeutic for IBD.

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Kinase Assay: LANCL2 engagement produces an increase of PKA, followed by an accumulation of cAMP in the cytoplasm. BT-11 treatment splenocytes shows a dose−response increase of cAMP production. BT-11 stimulates cAMP production by activating the LANCL2 pathway.

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In vitro, BT-11 was assayed for direct binding to LANCL2 by surface plasmon resonance (SPR). The compound showed a typical small molecule-protein interaction with very fast on rates and very fast off rates. The equilibrium dissociation constant (KD) was measured by plotting equilibrium binding levels against compound concentration, but no numerical KD value is reported in the manuscript [1].
In mouse splenocytes (WT vs LANCL2-/-), treatment with BT-11 at concentrations of 0, 1.25, 2.5, 5, and 10 μM resulted in a dose-dependent increase in cAMP production in WT-derived splenocytes, whereas cAMP production did not correlate with dose in LANCL2-/- splenocytes. This indicates that BT-11 stimulates cAMP production by activating the LANCL2 pathway [1].

Oral treatment with BT-11 at 8 mg/kg/d in a mouse model of inflammatory bowel disease results in lowering the disease activity index, decreasing colonic inflammatory lesions by 4-fold, and suppressing inflammatory markers (e.g., TNF-α, and interferon-γ) in the gut. Furthermore, studies in LANCL2−/− mice demonstrates that loss of LANCL2 abrogates beneficial actions of BT-11, suggesting high selectivity for the target. Oral treatment with BT-11 (8 mg/kg/day) ameliorates colitis in mice. Initial safety assessment in rats indicates that oral treatment with BT-11 at high doses has an excellent safety profile up to 1000 mg/kg/day[1]. BT-11 is well tolerated in rats, and may hold promise as an orally active therapeutic for Crohn’s disease. One hour after oral administration of a single dose of 80 mg/kg, BT-11 has a maximal concentration of 21 ng/mL; the half-life is 3 hours
In a dextran sodium sulfate (DSS) mouse model of IBD (colitis) using WT C57BL/6J mice, oral treatment with BT-11 (8 mg/kg/day, daily by oral gavage starting on day 0 when DSS challenge was initiated) for 7 days significantly improved disease activity index and decreased colonic inflammatory lesions. Histopathological analysis showed that BT-11 significantly decreased leukocytic infiltration (by 5-fold), epithelial erosion, and mucosal thickening compared to vehicle control. Representative photomicrographs demonstrated amelioration of colonic lesion formation [1].
Oral BT-11 treatment significantly upregulated colonic LANCL2 expression on day 3, and downregulated colonic TNF-α mRNA expression while upregulating anti-inflammatory IL-10 mRNA expression on day 7. LANCL2 mRNA was decreased during peak inflammation in DSS colitis, and BT-11 restored it to homeostatic levels [1].
In selectivity studies using WT and LANCL2-/- mice with DSS colitis, the absence of LANCL2 completely abrogated the therapeutic efficacy of BT-11 (8 mg/kg/day, 7 days). Disease activity index scores, colonic histopathology (leukocytic infiltration), TNF-α levels, MHC-II+ CD11c+ granulocytes, and MCP-1 were all improved by BT-11 in WT mice but not in LANCL2-/- mice. Similarly, upregulation of IL-10 by BT-11 was completely abrogated in LANCL2-/- mice [1].

Surface plasmon resonance (SPR) was used to assess direct binding of BT-11 to LANCL2. BT-11 at five different concentrations in triplicate was injected over LANCL2-coated surfaces. Binding sensorsgrams showed a typical small molecule-protein interaction with very fast on rates and very fast off rates. The equilibrium binding level was plotted against compound concentration to determine the equilibrium dissociation constant (KD) [1].

cAMP expression measurement: Splenocytes isolated from WT and LANCL2-/- male mice (over 8 weeks) were incubated in plates precoated with anti-mouse CD28 (1 μg/mL) and anti-mouse CD3 (5 μg/mL) for cell stimulation. Media consisted of cRPMI supplemented with BT-11 at 0, 1.25, 2.5, 5, and 10 μM (four replicates per treatment plus four negative controls without stimulation). After 20 h of incubation, a second 10 min treatment with the same concentrations was done. Cells were washed with PBS and harvested into 0.1 M HCl/H2O. Cell lysates were collected and cAMP intracellular concentration was measured using a Direct cAMP EIA kit [1].

8 mg/kg/d for mouse model; 500 mg/kg and 80 mg/kg/d for rats; oral Male Harlan Sprague Dawley rats and Wild type and LANCL2 / male mice with IBD models

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DSS-induced colitis in mice: Colitis was induced in C57BL/6J or LANCL2-/- mice by administration of DSS (dextran sodium sulfate) in drinking water for 7 days. BT-11 (8 mg/kg/day) was administered daily through orogastric gavage (orally) starting on day 0 when DSS challenge was initiated. Colonic inflammation was assessed 7 days after DSS treatment. Histopathology: Colonic sections were fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned (5 μm), stained with H&E. Colons were graded with a compounded histological score including leukocyte infiltration, mucosal thickening, and epithelial cell erosion (scores 0-4 each) [1].
Rat safety studies (exploratory): Adult male Sprague Dawley rats (200-250 g) were used. For the single dose study, rats received a single oral dose of BT-11 at 500 mg/kg (suspended in Ora Plus vehicle) or vehicle control. For the repeated dose study, rats received 80 mg/kg/day orally for 14 days, followed by a 7-day drug-free observation period. Animals were fasted 12 hours prior to dosing. Behavioral endpoints (Functional Observational Battery, FOB) were assessed at multiple time points (single dose: baseline, 4 h, 1, 7, 14 days; repeated dose: baseline, days 3, 7, 14, 21). FOB included home cage observation, handling, open field, reflexes, and physiological measurements (weight, body temperature, rotarod, grip strength, foot splay). Blood and tissues (brain, kidney, liver, adrenal gland, testes, stomach, small and large intestines, duodenum/pancreas, heart, lungs, spleen, thymus, rib) were collected for clinical pathology and histopathological examination at various time points (single dose: days 2,4,8,14; repeated dose: days 4,8,14,21). Tissues were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned at 5 μm, stained with H&E, and examined by light microscopy [1,2].

Pharmacokinetic study: Adult male Sprague-Dawley rats with indwelling jugular catheters were given a single oral dose of BT-11 at 80 mg/kg (suspended in Ora Plus) after 12 h fasting. Blood samples (0.5 mL) were collected at 0.5, 1, 2, 4, 8, 12, 16, 20, 25, and 48 h after dosing (n=3 per time point). Plasma was separated and analyzed by LC-MS/MS (Luna C18 column, mobile phase with formic acid, ESI source, MRM detection). Pharmacokinetic parameters: Cmax (maximal concentration) = 21 ng/mL at Tmax = 1 h; elimination rate constant (kel) = -0.225 /hr; apparent volume of distribution (Vd) = 3.375 L/kg; clearance (Cl) = 0.760 ng/L/kg (as reported); half-life (T1/2) = 3.08 h; absorption rate constant (ka) = 1.69 /hr. Plasma concentrations at 0.5 h: median 12.18 ng/mL; 1 h: 20.95 ng/mL; 2 h: 16.88 ng/mL; 4 h: 11.07 ng/mL; 8 h: 4.33 ng/mL; 12 h: 1.83 ng/mL; 16 h: 1.40 ng/mL; 20 h: 1.39 ng/mL; 24 h: 1.01 ng/mL; 48 h: 1.90 ng/mL. Following a 500 mg/kg single dose, plasma concentrations were 3.1-32 ng/mL at 2 h, 6.7-17 ng/mL at 4 h, and 3.0-9.6 ng/mL at 22 h. BT-11 is stable in simulated gastric and intestinal fluids (unpublished data) [1,2].

Exploratory safety studies in rats: Single oral dose of 500 mg/kg BT-11 caused no behavioral evidence of detrimental effects in a Functional Observational Battery (FOB) over 14 days. Statistically significant differences were observed for weight on day 14 (LS Mean 254.71 vs 264.46 g, p=0.0006) and hind grip strength on day 1 (LS Mean 0.40 vs 0.46 kg, p=0.0272), but these were not considered biologically significant. Clinical pathology: Transient increases in liver enzymes (ALT, AST, ALP) and total bilirubin on days 2 and 4, but values returned to normal by day 8 and were not statistically significant. No histopathologic differences between vehicle and treated groups at any time point (2,4,8,14 days) [2].
Repeated oral dose of 80 mg/kg/day for 14 days in rats: No behavioral evidence of detrimental effects in FOB. Few categorical differences (posture, cage removal ease, response to touch) considered normal variation. Continuous endpoints showed no differences except rears on day 14 (LS Mean 30.27 vs 23.93, p=0.0273). No test article-associated lesions in histopathology. Clinical pathology showed no clinically or statistically significant differences between control and treated rats. The compound was well-tolerated at 80 mg/kg/day for 14 days (cumulative dose 1,120 mg/kg) [2].

J Med Chem.2016 Nov 23;59(22):10113-10126;Int J Toxicol.2016 Sep;35(5):521-9.

BT-11 is a bis(benzimidazolylaryl) heterocycle, an analogue of the LANCL2 ligand CID247228 (NSC61610). It was identified through molecular docking, synthesis, and SPR binding screening. The compound activates the LANCL2 pathway leading to cAMP elevation, PKA activation, CREB phosphorylation, downregulation of TNF-α, and upregulation of IL-10. Oral administration results in limited systemic absorption and high concentrations in the colon and colonic contents, suggesting local action in the GI tract. The compound is intended for the treatment of Crohn's disease and ulcerative colitis. It has been tested in three validated mouse models of colitis with efficacy at doses as low as 8 mg/kg/day, and shows a favorable safety profile in rats up to 1000 mg/kg/day (no toxicologically relevant differences) [1,2].

C30H24N8O2

528.5640

528.202

1912399-75-7

1912399-91-7 (HCl);1912399-75-7;

121299620

Typically exists as solid at room temperature

3.4

2

6

4

40

840

0

MVHWZNBAQIGPOQ-UHFFFAOYSA-N

InChI=1S/C30H24N8O2/c39-29(25-13-5-11-23(31-25)27-33-19-7-1-2-8-20(19)34-27)37-15-17-38(18-16-37)30(40)26-14-6-12-24(32-26)28-35-21-9-3-4-10-22(21)36-28/h1-14H,15-18H2,(H,33,34)(H,35,36)

[4-[6-(1H-benzimidazol-2-yl)pyridine-2-carbonyl]piperazin-1-yl]-[6-(1H-benzimidazol-2-yl)pyridin-2-yl]methanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)