阅读说明：本技术 基于羊毛硫氨酸合成酶c样2的治疗剂 (Therapeutic agents based on lanthionine synthase C-like 2 ) 是由 J·巴桑甘亚·里尔拉 A·卡尔博巴里奥斯 R·甘朵尔 J·D·库珀 R·霍特西利亚斯 于 2015-03-19 设计创作，主要内容包括：本发明提供靶向羊毛硫氨酸合成酶C样蛋白质2路径的化合物。所述化合物可以用于治疗多种病状,包括传染性疾病、自身免疫性疾病、糖尿病以及慢性发炎性疾病。(The present invention provides compounds that target the lanthionine synthase C-like protein 2 pathway. The compounds may be used to treat a variety of conditions, including infectious diseases, autoimmune diseases, diabetes, and chronic inflammatory diseases.)

1. A compound having the formula a-B-C or a pharmaceutically acceptable salt or ester thereof, wherein:

a is:

b is:

c is:

A₇、A₈、A₉、A₁₀、A₁₁、A₁₂、A₁₃and A₁₄Each independently selected from CH and CR¹⁸And N;

A₁₅、A₁₆、A₁₇、A₁₈、A₁₉and A₂₀Each independently selected from CH and CR¹⁹、N、NR²⁰O and S with the proviso that A₁₅、A₁₆And A₁₇Is NR²⁰O or S, and A₁₈、A₁₉And A₂₀Is NR²⁰O or S;

R¹⁸and R¹⁹Each independently selected from C₁-C₆An alkyl group; c₁-C₆Dialkylamino group of which eachA C₁-C₆Alkyl groups are independently selected; -NH₂(ii) a An alkylamino group; a heterocycloalkyl group; and substituted heterocycloalkyl, wherein said substituted heterocycloalkyl is substituted with one to two substituents independently selected from the group consisting of₁-C₆Alkyl) and C₁-C₆Alkyl groups; and is

R²⁰Is C₁-C₆An alkyl group.

2. The compound of claim 1, wherein:

A₇、A₈、A₉、A₁₀、A₁₁、A₁₂、A₁₃and A₁₄Each independently selected from CH and N;

A₁₅、A₁₆、A₁₇、A₁₈、A₁₉and A₂₀Each independently selected from CH, N, O and S with the proviso that A₁₅、A₁₆And A₁₇Is O or S, and A₁₈、A₁₉And A₂₀Only one of which is O or S.

3. The compound of claim 2, wherein B is:

4. the compound of claim 3, wherein C is:

5. the compound of claim 4, wherein A₁₂And A₁₃Each is CH.

6. The compound of claim 5, wherein A₁₁And A₁₄Is N, and A₁₁And A₁₄Is CH.

7. The compound of claim 3, wherein C is:

8. the compound of claim 7, wherein A₂₀Is O.

9. The compound of claim 8, wherein a₁₈And A₁₉Each is CH.

10. The compound of claim 1, wherein the compound has the structure:

or a salt thereof.

11. Use of a compound according to any one of claims 1-10 for the preparation of a pharmaceutical composition for the treatment of a condition, wherein the condition is selected from the group consisting of: infectious diseases, autoimmune diseases, diabetes, and chronic inflammatory diseases.

12. The use of claim 11, wherein the condition is an infectious disease comprising influenza infection.

13. The use of claim 11, wherein the condition is an autoimmune disease, which is an autoimmune inflammatory disease.

14. The use of claim 13, wherein the autoimmune inflammatory disease comprises inflammatory bowel disease.

15. The use of claim 14, wherein the inflammatory bowel disease comprises ulcerative colitis.

16. The use of claim 14, wherein the inflammatory bowel disease comprises crohn's disease.

17. The use of claim 11, wherein the condition is diabetes.

18. The use of claim 11, wherein the condition is diabetes selected from the group consisting of type 1 diabetes and type 2 diabetes.

19. The use of claim 11, wherein the condition is a chronic inflammatory disease comprising metabolic syndrome.

Technical Field

The present invention relates to the field of medical treatment of diseases and disorders. More specifically, the present invention relates to the class of biologically active compounds for the treatment and prevention of inter alia the following diseases: inflammatory and immune-mediated diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis and type 1 diabetes, as well as chronic inflammatory diseases and conditions such as insulin resistance, impaired glucose tolerance, prediabetes, type 2diabetes and obesity-related inflammation.

Background

Lanthionine C-like protein 2(LANCL2) (also known as "lanthionine synthase C-like protein 2" or "lanthionine synthase component C-like protein 2" is a signal-conducting pathway protein of the expressed immune cell, gastrointestinal tract, neuron, testis and pancreas [1]. Activation of the LANCL2 pathway increases insulin sensitivity and reduces inflammation associated with various autoimmune, inflammatory, and metabolic conditions. The results of in vivo and in vitro tests in mice demonstrate that the use of compounds targeting this pathway reduces glucose levels in the glucose tolerance test by a factor of 2 and provides for prescription versus control(GlaxoSmithKline plc, Brenford, England) of Bronstedford, England) is an effective treatment but has significant side effects. Targeting the LANCL2 pathway also reduced intestinal inflammation by 90% and the number of lesions correspondingly reduced by 4-fold. The results of this testing and other validation of the path are published in 12 peer review articles on duty [2-13 ]]。

Within the category of autoimmune-related inflammation, there is currently a global pandemic of autoimmune disorders, such as Inflammatory Bowel Disease (IBD), systemic lupus, rheumatoid arthritis, type 1 diabetes, psoriasis, multiple sclerosis. There is also a pandemic of chronic metabolic inflammatory diseases including metabolic syndrome, obesity, prediabetes, cardiovascular disease and type 2 diabetes. Current treatments are moderately effective, but are expensive and have serious side effects. The route of administration of the most effective treatment for autoimmune diseases (e.g., anti-TNF antibodies) is via IV or subcutaneous injection, thus requiring visits to the clinic/surgery and frequent monitoring. The unique mode of action of LANCL2 provides an orally administered therapeutic agent that is as effective as an anti-TNF antibody, but without side effects and high cost. Given the overall prevalence of inflammatory and autoimmune diseases, the LANCL2 pathway has the potential to significantly affect millions of patients.

Abscisic acid ("ABA") is a natural compound found in the original screening process that binds to LANCL 2.

A large number of compounds are described in the field of synthetic organic chemistry. Various compounds are provided by the following references: WO1997/036866 to Diana et al, WO 2006/053109 to Sun et al, WO 2006/080821 to Kim et al, WO 2007/019417 to Nunes et al, WO 2009/067600 and WO 2009/067621 to Singh et al, WO2008/079277 to Adams et al, JP 2008/056615 to Urasoe et al, WO 2011/066898 to Stoessel et al, US 2013/0142825 to Basaganya-Riera et al, and U.S. Pat. No. 7,741,367 to Basaganya-Riera et al. Some of the compounds described in these references are known to activate the LANCL2 pathway, while others do not.

There is a need to develop novel ligands for the LANCL2 pathway to allow treatment to be specifically tailored to individual diseases and potentially maximize its efficacy.

The present application thus describes a range of compound classes that have been developed by novel medicinal chemical methods and screened using in silico, in vitro, and in vivo techniques to maximize their ability to bind to the LANCL2 protein and thus achieve beneficial responses in a variety of disease conditions, including (but not limited to) autoimmune, chronic inflammatory, metabolic, and infectious diseases.

Disclosure of Invention

The present invention provides a compound comprising the formula Z-Y-Q-Y '-Z' or a pharmaceutically acceptable salt or ester thereof,

wherein:

z is:

y is:

q is piperazin-1, 4-diyl; 2, 5-diazabicyclo [2.2.1]Heptane-2, 5-diyl; 2, 5-diazabicyclo [2.2.2]Octane-2, 5-diyl; 1, 4-diazepan-1, 4-diyl; benzene-1, 4-diamine-N¹,N⁴-a diradical; ethane-1, 2-diamine-N¹,N²-a diradical; n is a radical of¹,N²Dialkyl ethane-1, 2-diamine-N¹,N²-a diradical; propane-1, 3-diamine-N¹,N³-a diradical; n is a radical of¹,N³1, 3-diamine-N-dialkylpropane¹,N³-a diradical; 1, 4-diaminoanthracene-9, 10-dione-1, 4-diyl; c₆Aromatic-1, 4-diamine-N¹,N⁴-diyl, wherein said arene is substituted at the 2, 3, 5 or 6 position with one to four substituents, and wherein said substituents are independently selected from the group consisting of: -C (O) O (C)₁To C₆) Alkyl, OH, O (C)₁To C₆) Alkyl, (C)₁To C₆) Alkyl, CF₃F, Cl and Br; or substituted piperazin-1, 4-diyl, wherein said piperazine is substituted at the 2, 3, 5, or 6 positions with one to eight substituents, and wherein said substituents are independently selected from the group consisting of: (C)₁To C₆) Alkyl, aryl (C)₁To C₆) Alkyl, C (O) OH and C (O) O (C)₁To C₆) An alkyl group;

y' is:

or a single bond; and is

Z' is:

or R⁵；

Wherein:

only at Z' is R⁵When Y' is a single bond;

A₁and A₁' independently of each other is N, N (C)₁To C₆) Alkyl, O, S or CR⁶；

A₂And A₂' each independently is N or CR⁷；

A₃And A₃' independently of one another are NR⁸O or S;

A₄and A₄' each independently is N or CR⁹；

A₅And A₅' each independently is N or CR¹⁰；

A₆And A₆' each independently is N or CR¹¹；

R¹、R¹'、R²、R²'、R³、R³'、R⁴、R⁴'、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹Each independently selected from the group consisting of: hydrogen; an alkyl group; a halo group; a trifluoromethyl group; dialkylamino, wherein each alkyl is independently selected; -NH₂(ii) a An alkylamino group; an arylalkyl group; a heteroarylalkyl group; a heterocycloalkyl group; a substituted heterocycloalkyl substituted with 1 to 2 substituents independently selected from the group consisting of: -C (O) OH, -C (O) O (C)₁To C₆) Alkyl, (C)₁To C₆) Alkyl, -CF₃F, Cl and Br; and substituted heteroarylalkyl;

wherein the substituted heteroarylalkyl is substituted with 1 to 3 substituents independently selected from the group consisting of: -NH₂；-NH(C₁To C₆) An alkyl group; -N ((C)₁To C₆) Alkyl radical)₂Wherein each alkyl group is independently selected; an alkyl group; a halo group; an aryl group; a substituted aryl group substituted with 1 to 3 substituents independently selected from the group consisting of: -SO₂R¹²、-OR¹³-halo, -CN, -CF₃Aminoalkyl-, -S (O) R¹⁴And an alkyl group; a heterocycloalkyl group; a heteroaryl group; a substituted aryl group substituted with 1 to 3 substituents independently selected from the group consisting of: alkyl, -CF₃F, Cl and Br; alkylamino-; heterocyclic ringsAlkyl-amino-; alkylamino-amino-; -NHC (O) OR¹⁵；-NHC(O)NR¹⁶R¹⁷；-C(O)NR¹⁶R¹⁷(ii) a And substituted heteroaryl substituted with 1 to 3 substituents selected from the group consisting of: alkyl, halo, CN, NH₂、-NH(C₁-C₆Alkyl), -N (C)₁-C₆Alkyl radical)₂(wherein each alkyl group is independently selected), -CF₃And substituted aryl (substituted 1 to 3 independently selected from the group consisting of-S (O)₂R¹⁵and-CN);

wherein R is¹²、R¹³、R¹⁴、R¹⁵、R¹⁶And R¹⁷Each independently selected from the group consisting of: c₁-C₆Alkyl, containing independently selected hair C₁-C₆Dialkylamino radical of alkyl, -NH₂Alkylamino, heterocycloalkyl and substituted heterocycloalkyl (one to two of which are independently selected from the group consisting of-C (O) O (C)₁-C₆Alkyl) and C₁-C₆Substituted with substituents from the group consisting of alkyl).

In some compounds, A₃And A₃At least one of' is O or S. In some compounds, A₁And A₁One or both of' is N. In some compounds, A₂And A₂One or two of' is CH, A₃Is NH, A₄Is N, A₅Is CH, and A₆Is CH. In some compounds, A₂And A₂One or two of' is CH, A₃And A₃One or two of' is NH, A₄And A₄One or two of' is N, A₅And A₅One or two of is CH, and A₆And A₆One or both of' are CH. In some compounds, Q is piperazin-1, 4-diyl; 2, 5-diazabicyclo [2.2.1]Heptane-2, 5-diyl; 2, 5-diazabicyclo [2.2.2]Octane-2, 5-diyl; 1, 4-diazepan-1, 4-diyl; n is a radical of¹,N²Dialkyl ethane-1, 2-diamine-N¹,N²-a diradical;N¹,N³1, 3-diamine-N-dialkylpropane¹,N³-a diradical; 1, 4-diaminoanthracene-9, 10-dione-1, 4-diyl; c₆Aromatic-1, 4-diamine-N¹,N⁴-diyl, wherein the arene is substituted in the 2, 3, 5 or 6 position with one to four substituents, and each substituent is independently selected from the group consisting of: -C (O) O (C)₁To C₆) Alkyl, OH, O (C)₁To C₆) Alkyl, (C)₁To C₆) Alkyl, CF₃F, Cl and Br; or substituted piperazin-1, 4-diyl, wherein said piperazine is substituted at the 2, 3, 5 or 6 positions with one to eight substituents, and each substituent is independently selected from the group consisting of: (C)₁To C₆) Alkyl, aryl (C)₁To C₆) Alkyl, C (O) OH and C (O) O (C)₁To C₆) An alkyl group.

In some compounds, the formula Z-Y-Q-Y '-Z' is:

or

A salt thereof. In some compounds, the members of one or more pairs selected from the group consisting of: a. the₁And A₁'、A₂And A₂'、A₃And A₃'、A₄And A₄'、A₅And A₅'、A₆And A₆'、R₁And R₁'、R₂And R₂'、R₃And R₃' and R₄And R₄'. In some compounds, the members of one or more pairs selected from the group consisting of: a. the₁And A₁'、A₂And A₂'、A₃And A₃'、A₄And A₄'、A₅And A₅'、A₆And A₆'、R₁And R₁'、R₂And R₂'、R₃And R₃' and R₄And R₄'. In some compounds, the members of each pair selected from the group consisting of: a. the₁And A₁'、A₂And A₂'、A₃And A₃'、A₄And A₄'、A₅And A₅'、A₆And A₆'、R₁And R₁'、R₂And R₂'、R₃And R₃' and R₄And R₄'. In some compounds, the members of each pair selected from the group consisting of: a. the₁And A₁'、A₂And A₂'、A₃And A₃'、A₄And A₄'、A₅And A₅'、A₆And A₆'、R₁And R₁'、R₂And R₂'、R₃And R₃' and R₄And R₄'。

In some compounds, the formula Z-Y-Q-Y '-Z' is:

or

A salt thereof.

Some compounds of the invention have the following structure:

or

A salt thereof.

The present invention also provides a pharmaceutical composition comprising a compound of formula A-B-C or a pharmaceutically acceptable salt or ester thereof,

wherein:

a is:

b is:

and is

C is:

wherein:

A₇、A₈、A₉、A₁₀、A₁₁、A₁₂、A₁₃and A₁₄Each independently selected from CH and CR¹⁸And N;

A₁₅、A₁₆、A₁₇、A₁₈、A₁₉and A₂₀Each independently selected from CH and CR¹⁹、N、NR²⁰O and S with the proviso that A₁₅、A₁₆And A₁₇May be N, NR²⁰O or S, and A₁₈、A₁₉And A₂₀May be N, NR²⁰O or S;

R¹⁸and R¹⁹Each independently selected from C₁-C₆An alkyl group; c₁-C₆Dialkylamino group of which each C is₁-C₆Alkyl groups are independently selected; -NH₂(ii) a An alkylamino group; a heterocycloalkyl group; and substituted heterocycloalkyl, wherein said substituted heterocycloalkyl is substituted with one to two substituents independently selected from the group consisting of₁-C₆Alkyl) and C₁-C₆Alkyl groups; wherein there is more than one CR¹⁸In the compound of (1), each R¹⁸Independently selected and having more than one CR¹⁹In the compound of (1), each R¹⁹Independently selected; and is

R²⁰Is C₁-C₆An alkyl group.

In some compounds, B is:

some compounds have the following structure:

or a salt thereof.

The invention also provides methods of treating a condition in an animal with any one or more of the compounds described herein. The methods comprise administering to the animal an effective amount of one or more of the compounds described herein. The condition may be selected from the group consisting of: infectious diseases, autoimmune diseases, diabetes, and chronic inflammatory diseases. In some methods, the infectious disease comprises a viral disease, such as influenza infection. In some methods, the autoimmune disease comprises an autoimmune inflammatory disease, such as inflammatory bowel disease, including ulcerative colitis and/or Crohn's disease. In some methods, the diabetes is selected from the group consisting of type 1 diabetes and type 2 diabetes. In some methods, the chronic inflammatory disease comprises metabolic syndrome. In some methods, the methods comprise administering an amount of a compound effective to increase LANCL2 activity, reduce inflammation, and/or increase anti-inflammatory effects.

The invention also provides compounds for treating a condition in an animal with any one or more of the compounds described herein. Compounds for such use include any of the compounds described herein. Use may comprise administering to the animal an effective amount of one or more of the compounds described herein, wherein the condition is selected from the group consisting of: infectious diseases, autoimmune diseases, diabetes, and chronic inflammatory diseases. In some versions, the infectious disease comprises a viral disease, such as an influenza infection. In some versions, the autoimmune disease comprises an autoimmune inflammatory disease, such as inflammatory bowel disease, including ulcerative colitis and/or crohn's disease. In some versions, diabetes is selected from the group consisting of type 1 diabetes and type 2 diabetes. In some versions, the chronic inflammatory disease comprises metabolic syndrome. In some versions, the compounds are effective to increase LANCL2 activity, reduce inflammation, and/or increase anti-inflammatory effects.

The objects and advantages of the present invention will become more fully apparent from the following detailed description of the preferred embodiments of the invention, taken in conjunction with the accompanying drawings.

Drawings

Fig. 1A and 1b. computational prediction of compound binding to LANCL2 and biochemical experimental validation using SPR.

FIG. 2 Cluster histogram (Cluster histogram) of the first five clusters of NSC 6160. One hundred docking operations were performed with NSC6160 docked with LANCL2 using AutoDockTools. RMSD Cluster tolerance isBinding energies are listed in kJ/mol.

FIG. 3 Cluster histogram of the first five clusters of ABA. One hundred docking operations were performed with ABA docked with LANCL2 using AutoDock Tools. RMSD Cluster tolerance isBinding energies are listed in kJ/mol.

Fig. 4 cluster histogram processing of the first five BT-11 clusters. One hundred docking operations were performed with BT-11 docked with LANCL2 using AutoDock Tools. RMSD Cluster tolerance isBinding energy is listed in kJ/molAnd (6) discharging.

Fig. 5 cluster histograms for the first five BT-6 clusters. One hundred docking operations were performed with BT-6 docked with LANCL2 using AutoDock Tools. RMSD Cluster tolerance isBinding energies are listed in kJ/mol.

Fig. 6 cluster histograms for the first five BT-15 clusters. One hundred docking operations were performed with BT-15 docked with LANCL2 using AutoDock Tools. RMSD Cluster tolerance isBinding energies are listed in kJ/mol.

FIG. 7 Cluster histograms for the first five clusters of BT-ABA-5 a. One hundred docking operations were performed with BT-ABA-5a docked with LANCL2 using AutoDock Tools. RMSD Cluster tolerance isBinding energies are listed in kJ/mol.

FIG. 8 shows the binding kinetics of lanthionine synthase C-like protein 2(LANCL2) to BT-11 and BT-15. Panels A and C show Surface Plasmon Resonance (SPR) sensorgrams for varying concentrations of BT-11(A) and BT-15(C) binding to immobilized LANCL 2. Graphs B and D show plots of maximum Resonance Units (RU) versus BT-11(B) and BT-15(D) concentrations. Indicating steady state dissociation constant (K) using the 1:1 binding model_D)。

FIGS. 9A and 9B kinetics of the binding of lanthionine synthase C-like protein 2(LANCL2) to BT-6 (FIG. 9A) and BT-ABA-5a (FIG. 9B). Surface Plasmon Resonance (SPR) sensorgrams for varying concentrations of BT-6 and BT-ABA-5a binding to immobilized LANCL2 are shown.

Figure 10 effect of oral administration on disease activity and overall pathology in mice with Dextran Sodium Sulfate (DSS) colitis. Panel a shows disease activity index scores in mice treated with BT-11 alone or vehicle. Panels B-C show total pathology scores from (B) spleen, (C) Mesenteric Lymph Node (MLN), and (D) colon in mice treated with vehicle or BT-11. Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

Figure 11. effect of oral BT-11 administration on colonic inflammatory lesions in mice with DSS colitis. Representative micrographs of (A, D) control (B, E) DSS and (C, F) BT-11 treated DSS mice are shown. Histopathological lesions were assessed based on (G) leukocyte infiltration, (H) epithelial cell erosion, and (I) mucosal thickening. Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

Figure 12. dose response effect of oral BT-11 administration on colonic inflammatory lesions in mice with DSS colitis. Histopathological lesions were assessed based on (a) leukocyte infiltration, (B) mucosal thickening, and (C) epithelial cell erosion. Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

FIG. 13 colonic gene expression analysis of TNF α, interleukin 10(IL-10) and LANCL 2. Colonic gene expression was shown to assess the levels of (A) pro-inflammatory TNF α, (B) IL-10, and (C) LANCL 2. Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

Figure 14 dose response effect of orally administered BT-11 on a subset of colonic pro-inflammatory and anti-inflammatory immune cells in mice with DSS colitis. Flow cytometry analysis was used to measure (A) TNFa + cells, (B) IL-10+ CD4+ T cells and (C) FOXP3+ CD4+ T cells in colonic mucosa.

Figure 15. effect of oral BT-11 administration on total pathological lesions in tissues of wild type and LANCL 2-/-mice with DSS colitis. Panel A shows disease activity index scores in wild-type versus LANCL 2-/-mice treated with BT-11 alone or vehicle. Panels B-D show total pathology scores from (B) colon, (C) Mesenteric Lymph Node (MLN), and (D) spleen in wild-type and LANCL 2-/-mice treated with vehicle or BT-11. Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

Figure 16. effect of oral BT-11 administration on colonic inflammatory lesions in wild type and LANCL 2-/-mice with DSS colitis. Histopathological lesions were assessed based on (a) leukocyte infiltration, (B) mucosal thickening, and (C) epithelial cell erosion. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 17. effect of oral administration of BT-11 on a subset of immune cells infiltrating the colonic lamina propria, spleen and Mesenteric Lymph Nodes (MLNs) in wild-type and LANCL 2-/-mice with chronic colitis. Flow cytometry was used to analyze the levels of (A) colonic MCP1+ CD45+ cells, (B) MCP1+ CD45+ cells in MLN, (C) colonic TNFa + CD45+ cells, (D) colonic MHC-II + CD11C + granulocytes, (E) colonic IL-10+ CD45+ cells, and (F) IL-10+ CD45+ splenocytes after treatment with BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 18 Effect of oral BT-11 administration on Disease Activity Index (DAI) score in IL-10-/-mice with chronic colitis. DAI scores (n-10) for IL-10 knockout mice that developed idiopathic colitis and were treated daily with vehicle alone or with 20, 40 and 80mg bt-11 per kg body weight. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 19. effect of oral BT-11 administration on macroscopic tissue score in chronic model of colitis after treatment with BT-11. Macroscopic scoring in (A) spleen, (B) Mesenteric Lymph Node (MLN) and (C) colon of mice treated with vehicle or with BT-11 at three different concentrations (20, 40 and 80 mg/Kg). Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 20. Effect of oral BT-11 administration on colon histopathological changes in the chronic IL-10-/-model of IBD. Histopathological lesions were assessed based on (a) leukocyte infiltration, (B) epithelial cell erosion, and (C) mucosal thickening. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 21. effect of oral administration of BT-11 on a subset of immune cells infiltrating the solid layer of IL-10-/-colon in chronic colitis. Flow cytometry was used to analyze the levels of (A) F4/80+ macrophages, (B) MHC-II + CD11C + Dendritic Cells (DCs), (C) CD4+ FOXP3+ regulatory T cells, and (D) T helper 1(Th1) cells in colon LPs after treatment with BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 22. Effect of oral BT-11 administration on a subset of immune cells infiltrating the spleen and mesenteric lymph nodes in IL-10-/-with chronic colitis. Flow cytometry was used to analyze the levels of (a) CD4+ RORgt + T cells, (B) CD4+ FOXP3+ T cells, (C) CD4+ CD45+ FOXP3+ regulatory T cells, and (D) T helper 1(Th1) cells after treatment with BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 23. Effect of oral treatment with BT-11 on colonic expression of LANCL2 and TNF α. Colon gene expression was used to assess the levels of (A) LANCL2 and (B) TNF α. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 24 effect of oral BT-11 administration on vehicle versus disease activity index score in treated mice in adoptive transfer model of chronic colitis. After intraperitoneal transfer of 400,000 untreated CD4+ T cells, RAG 2-/-mice were treated with vehicle or BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 25 effect of oral BT-11 administration on disease activity index score in vehicle versus treated wild-type versus LANCL 2-/-metastatic mice in the adoptive transfer model of chronic colitis. After intraperitoneal transfer of 400,000 untreated CD4+ T cells from wild-type or LANCL 2-/-donors, RAG 2-/-mice were treated with vehicle or BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 26 effect of oral BT-11 administration on weight loss in a chronic IBD model of CD4+ induced colitis. Mice were weighed and percent weight loss was calculated. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 27 effect of oral BT-11 administration on CD4+ T cells induced macroscopic tissue score in chronic model of colitis after BT-11 treatment. Macroscopic scores in (A) spleen, (B) MLN, (C) colon and (D) ileum of mice treated with vehicle or 80mg/Kg BT-11 are shown. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 28 effect of oral BT-11 administration on CD4+ T cells using wild type and LANCL 2-/-mice after BT-11 treatment induces macroscopic tissue scores in a chronic model of colitis. Macroscopic scores in (A) spleen, (B) MLN and (C) colon of wild type and LANCL 2-/-mice treated with vehicle or 80mg/Kg BT-11 are shown. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 29 effect of oral BT-11 administration on vehicle versus colon and ileum histopathology in the model of adoptive transfer of chronic colitis in treated mice. Histopathological lesions in the colon (a, C, E) and ileum (B, D, F) were assessed based on (a, B) leukocyte infiltration, (C, D) epithelial cell erosion, and (E, F) mucosal thickening. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 30. Effect of oral BT-11 administration on vehicle comparison of Colon histopathology in treated mice transferred with wild-type or LANCL2-/-CD4+ T cells in an adoptive transfer model of chronic colitis. Histopathological lesions were assessed based on (a) leukocyte infiltration, (B) mucosal thickening, and (C) epithelial cell erosion. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 31 effect of oral BT-11 administration on vehicle versus disease activity index score in treated mice in adoptive transfer model of chronic colitis. Flow cytometry was used to analyze the levels of (A) F4/80+ CD11B + macrophages, (B) CD45+ IFNg + cells, (C) CD4+ FOXP3+ regulatory T cells, and (D) CD4+ IL-10+ anti-inflammatory cells after treatment with BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 32. effect of oral BT-11 administration on vehicle versus disease activity index score in treated mice in adoptive transfer model of chronic colitis. Flow cytometry was used to analyze the levels of (A) CD4+ FOXP3+ T cells, (B) CD4+ IL-10+ T cells, (C) CD45+ IFNg + cells and (D) CD4+ FOXP3+ T cells, (E) CD4+ IL-10+ T cells, (F) CD45+ IFNg + cells in the spleen after treatment with BT-11. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 33 effect of oral BT-11 administration on disease activity index score in vehicle versus treated wild-type versus PPAR γ -/-metastatic mice in the model of adoptive transfer of chronic colitis. After intraperitoneal transfer of 400,000 untreated CD4+ T cells from wild-type or PPAR γ -/-donors, RAG 2-/-mice were treated with vehicle or BT-11. (A) Disease activity index scores versus time post-metastasis are shown. Histopathological lesions in the colon were assessed based on (B) leukocyte infiltration, (C) mucosal thickening, and (D) epithelial cell erosion. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 34. Effect of oral BT-11 administration on fasting blood glucose and insulin levels in NOD mice with diabetes. (A) Fasting glucose levels were assessed at weeks 0, 1,3, 4,5, 10 and 11 of treatment with vehicle or BT-11(80 mg/kg/d). (B) Fasting serum insulin levels were assessed at week 5 of treatment with vehicle or BT-11(80 mg/kg/d). Statistically significant differences (P <0.05) (n ═ 10) are indicated by asterisks.

FIG. 35. Effect of oral administration of BT-11 on lesion formation in pancreas of type 1 diabetic mice. Histopathological lesions were assessed based on leukocyte infiltration, lesion formation and tissue erosion. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Figure 36. effect of oral BT-11 administration on (a) fasting blood glucose levels and (B) glucose tolerance test. (A) At weeks 2 and 12 after the experimental setup, mice were fasted for 12h and blood glucose levels were assessed. (B) Mice were also challenged with IP glucose injection (2g/Kg) and glucose was measured. Statistically significant differences (P <0.05) are indicated by asterisks.

FIG. 37. Effect of oral BT-11 administration on infiltration of a proinflammatory population into White Adipose Tissue (WAT). WAT was excised and digested, and immunophenotypic results were assessed by flow cytometry. Exhibits (A) infiltrating macrophages and (B) Ly6c^{Height of}Level of GR1+ infiltrating cells. Statistically significant differences (P) are indicated by asterisks<0.05)。

FIG. 38. Effect of oral BT-11 administration on glucose homeostasis in the db/db model of diabetes. (A) Fasting Blood Glucose (FBG) concentrations from leptin receptor deficient (db/db) mice treated with BT-11 or vehicle at weeks 1 and 3 after experimental setup are shown. (B) Plasma glucose levels after intraperitoneal glucose challenge (1 g per kg body weight) are shown. Blood was collected before (0) glucose loading, followed by 15, 30, 60, 90, 120, 180, 220, and 265 minutes after glucose loading. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

FIG. 39. Effect of oral BT-11 administration on the expression of LANCL2, TNF α, and MCP-1 in White Adipose Tissue (WAT) from mice with diet-induced obesity. Gene expression analysis of LANCL2, TNF α, and MCP-1 was evaluated compared to untreated mice. The zero line represents the baseline of mice receiving vehicle only.

Figure 40. effect of oral BT-11 administration on clinical score and incidence of influenza virus infected mice. Mice were infected with influenza virus and clinical scores were made throughout the experiment. Clinical scores were labeled for (a) activity and (B) body appearance. (C) The percentage of mice that lost more than 15% of body weight was plotted to show the change in morbidity. Statistically significant differences between groups are indicated with asterisks (P < 0.05).

Detailed Description

General definitions

Unless stated otherwise, the following definitions are used throughout this application:

analysis of variance (ANOVA): an arithmetic method for partitioning the overall variation of a data set into specific components based on the source of the variation. Which has been used to determine if the numerical differences between treatment groups are statistically significant.

And (3) fat generation: the process of generating new adipocytes or adipose storage cells.

Allele: one of many possible DNA codes in the same gene.

Conjugated diene: a molecule containing two double bonds separated by one single bond.

Db/Db mice: the term used to define the type of mouse that lacks both alleles of a leptin receptor long isoform. This deficiency causes a high propensity to develop type 2 diabetes. For further discussion of Db/Db mice see examples below.

Enantiomers: optical isomers; molecules are classified chemically based on their ability to rotate the plane of polarization clockwise (+) or counterclockwise (-).

Blood sugar: the concentration of glucose in the blood.

Hyperglycemia is: the concentration of glucose in the blood increases beyond the normal range.

Hyperinsulinemia: the concentration of insulin in the blood increases beyond the normal range.

Blood insulin: the concentration of insulin in the blood.

Insulin resistance: the tissue is unable to respond to insulin and absorb glucose from the blood.

Substantially pure: the purity is at least 90 wt.%, preferably at least 95 wt.%, such as at least 98 wt.%, 99 wt.%, or about 100 wt.%.

Type 2diabetes or non-insulin dependent diabetes mellitus: a term referring to the common type of diabetes that results from the unresponsiveness of cells to insulin action. If the cell is unresponsive to insulin, it cannot absorb glucose from the blood, which causes glucotoxicity (glucotoxicity). In addition, cells lack the energy derived from glucose oxidation.

IBD: inflammatory Bowel Disease (IBD) involves chronic inflammation in all or part of your digestive tract. IBD mainly includes ulcerative colitis and crohn's disease. Both are usually associated with severe diarrhea, pain, fatigue and weight loss. IBD can be debilitating and sometimes life-threatening complications.

Ulcerative Colitis (UC): UC is IBD that causes persistent inflammation and sores (ulcers) in the innermost lining layer in your large intestine (colon) and rectum.

Crohn's disease: crohn's disease is IBD that causes inflammation of the lining of your digestive tract. In crohn's disease, inflammation often spreads deep within the affected tissue. Inflammation may involve different regions of the digestive tract, the large intestine, the small intestine, or both.

IL-10: interleukin-10 (IL-10), also known as human Cytokine Synthesis Inhibitory Factor (CSIF), is an anti-inflammatory cytokine. In humans, IL-10 is encoded by the IL10 gene.

FOXP 3: FOXP3 (forkhead box P3) (also known as scurfin) is a protein involved in immune system responses. The member of the FOX protein family, FOXP3, appears to act as a master regulator (transcription factor) in the development and function of regulatory T cells.

TNF- α: tumor necrosis factor (TNF, cachexin, or cachectin, and previously referred to as tumor necrosis factor alpha or TNF α) is a cytokine involved in systemic inflammation and is a member of the cytokine group that stimulates the acute phase response.

MCP 1: monocyte chemotactic protein-1. The early term for CC cytokines, which are critical for atherosclerotic lesion development, is found in endothelial cells, macrophages and vascular smooth muscle cells of patients undergoing coronary artery bypass procedures. The term preferred by the authorities is now chemokine (C-C motif) ligand 2.

Interferon γ: interferon gamma is a pro-inflammatory dimeric soluble cytokine that is the only member of the type II class of interferons.

Type 1 diabetes mellitus: type 1 diabetes, once referred to as juvenile-onset diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin, a hormone required to allow sugar (glucose) to enter cells to produce energy.

Leukocyte infiltration: leukocyte infiltration refers to the process of moving or infiltrating leukocytes into injured tissue to initiate the repair process.

Chemical definition

Unless otherwise stated, the term "alkyl" alone or as part of another substituent means a fully saturated straight, branched, or cyclic hydrocarbon group or combination thereof (e.g., C) having the indicated number of carbon atoms₁-C₁₀Meaning one to ten carbon atoms, inclusive), and may include divalent and multivalent groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexylAlkyl, (cyclohexyl) ethyl, cyclopropylmethyl, and homologs and isomers thereof, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unless otherwise indicated, the term "alkyl" also includes those alkyl derivatives defined in more detail below as "heteroalkyl" and "cycloalkyl".

The term "alkenyl" means an alkyl group as defined above except that it contains one or more double bonds. Examples of alkenyl groups include ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), and the like, as well as higher carbon number homologs and isomers.

The term "alkynyl" means an alkyl or alkenyl group as defined above except that it contains one or more triple bonds. Examples of alkynyl groups include ethynyl, 1-and 3-propynyl, 3-butynyl, and the like, including higher carbon number homologs and isomers.

The terms "alkylene", "alkenylene" and "alkynylene", alone or as part of another substituent, mean a divalent radical derived from an alkyl, alkenyl or alkynyl group, respectively, as represented by-CH₂CH₂CH₂CH₂-as exemplified.

Typically, alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene groups will have from 1 to 24 carbon atoms. Those having 10 or less carbon atoms are preferred in the present invention. The term "lower carbon number" when applied to any of these groups, as in "lower alkyl" or "lower alkylene," denotes a group having 10 or fewer carbon atoms.

By "substituted" is meant that a chemical group as described herein further includes one or more substituents, such as lower alkyl, aryl, acyl, halogen (e.g., alkyl halide, such as CF)₃) Hydroxyl, amino, alkoxy, alkylamino, amido, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any carbon or substituent of the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene moieties. In addition, theThese groups may be pendant from or integral to the carbon chain itself.

The term "aryl" is used herein to refer to an aromatic substituent, which may be a single aromatic ring or multiple aromatic rings fused together, covalently linked, or linked to a common group (e.g., a diazo, methylene, or ethylene moiety). The common linking group may also be a carbonyl group, as in benzophenone. Aromatic rings may include, for example, phenyl, naphthyl, biphenyl, benzhydryl, and benzophenone, among others. The term "aryl" encompasses "arylalkyl" and "substituted aryl". For phenyl, the aryl ring may be mono-, di-, tri-, tetra-or pentasubstituted. The larger ring may be unsubstituted or carry one or more substituents.

"substituted aryl" refers to an aryl group as just described that includes one or more functional groups such as lower alkyl, acyl, halogen, alkyl halide (e.g., CF)₃) Hydroxyl, amino, alkoxy, alkylamino, amido, acyloxy, phenoxy, mercapto, and saturated and unsaturated cyclic hydrocarbons fused to aromatic rings, covalently linked or linked to a common group (e.g., a diazo, methylene or ethylene moiety). The linking group may also be a carbonyl group, as in cyclohexyl phenyl ketone. The term "substituted aryl" encompasses "substituted arylalkyl".

The term "halogen" or "halo" is used herein to refer to fluorine, bromine, chlorine, and iodine atoms.

The term "hydroxy" is used herein to refer to the group-OH.

The term "amino" is used to refer to NRR ', where R and R' are independently H, alkyl, alkenyl, alkynyl, aryl, or substituted analogs thereof. "amino" encompasses "alkylamino" groups that designate secondary and tertiary amines, and "amido" groups that describe the group RC (O) NR'.

Administration of drugs

In the context of the methods of the present invention, a therapeutically effective amount of a compound of the present invention can be administered to an animal, including mammals and humans, in a variety of ways. While in preferred embodiments, the compounds of the present invention are administered orally or parenterally, other forms of administration are also contemplated, such as via a medicinal compound or an aerosol.

For oral administration, an effective amount of the compound can be administered, for example, in a solid, semi-solid, liquid, or gaseous state. Specific examples include tablets, capsules, powders, granules, solutions, suspensions, syrups, and elixirs. However, the compounds are not limited to these forms.

For formulating the compounds of the invention into tablets, capsules, powders, granules, solutions or suspensions, the compounds are preferably mixed with binders, disintegrants and/or lubricants. If desired, the resulting composition may be mixed with diluents, buffers, wetting agents, preservatives and/or flavoring agents using known methods. Examples of binders include crystalline cellulose, cellulose derivatives, corn starch, cyclodextrins and gelatin. Examples of disintegrants include corn starch, potato starch and sodium carboxymethyl cellulose. Examples of lubricants include talc and magnesium stearate. In addition, additives that have been conventionally used, such as lactose and mannitol, may also be used.

For parenteral administration, the compounds of the invention may be administered rectally or by injection. For rectal administration, suppositories may be used. Suppositories can be prepared by mixing the compounds of the invention with pharmaceutically suitable excipients which melt at body temperature but remain solid at room temperature. Examples include, but are not limited to, cocoa butter, carbowax, and polyethylene glycol. The resulting composition can be molded into any desired form using methods known in the art.

For administration by injection, the compounds of the invention may be injected subcutaneously, intradermally, intravenously, or intramuscularly. The medicinal drugs for such injections can be prepared by dissolving, suspending or emulsifying the compound of the present invention in an aqueous or non-aqueous solvent (such as vegetable oil, synthetic resin acid glyceride, higher fatty acid ester or propylene glycol) by a known method. If necessary, additives which have been conventionally used, such as solubilizers, osmoregulators, emulsifiers, stabilizers or preservatives, may also be added. Although not required, it is preferred that the composition be sterile or sterilized.

For formulating the compounds of the present invention as suspensions, syrups or elixirs, pharmaceutically suitable solvents may be employed. Among these solvents are included the non-limiting example water.

The compounds of the invention can also be used together with other compounds having other pharmaceutically suitable activities for the preparation of medicaments for medical use. Medicaments containing a compound of the invention as a sole compound or as part of a composition may be used to treat a subject in need thereof.

The compounds of the invention may also be administered in the form of an aerosol or an inhalant which is prepared by charging the compound in liquid or finely powdered form together with a gaseous or liquid spray and, if desired, known adjuvants such as bulking agents, into a non-pressurized container such as an aerosol container or a nebulizer. Pressurized gases such as dichlorofluoromethane, propane or nitrogen may be used as a spray.

The compounds of the invention can be administered to animals, including mammals and humans, in need thereof in the form of pharmaceutical compositions, such as tablets, capsules, solutions or emulsions. The invention also encompasses other forms of the compounds described in the present invention, including but not limited to esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, structurally related compounds thereof, analogs thereof, and combinations thereof, administered in single or multiple doses.

The compounds of the present invention can also be administered to an animal in need thereof in the form of a nutritional supplement (food or nutraceutical supplement).

The terms "prevention", "treatment" or "amelioration" and similar terms as used herein include both prevention and complete or partial treatment. The term may also include any other change in the condition of a patient that reduces symptoms, improves symptoms, reduces symptom severity, reduces incidence of disease, or improves treatment outcome.

The compounds described in the present invention are preferably used and/or administered in the form of compositions. Suitable compositions are preferably pharmaceutical compositions, foods or food supplements. These compositions provide a convenient form of delivery of the compounds. The compositions of the present invention may comprise an antioxidant in an amount effective to increase the stability or solubility of the compound with respect to oxidation.

The amount of compound administered in the methods of the invention or for administration in the uses of the invention is any suitable amount. It is preferably from about 0.0001g to about 20g (more preferably from 0.01g to 1g, such as from 0.05g to 0.5g) of compound per day. Suitable compositions may be formulated accordingly. Those skilled in the art of administration of biologically active agents will be able to develop specific dosing regimens for various individuals based on known and well understood parameters.

Preferred compositions according to the invention are pharmaceutical compositions, such as in the form of tablets, pills, capsules, caplets, multiparticulates (including granules, beads, pellets and microencapsulated particles), powders, elixirs, syrups, suspensions and solutions. The pharmaceutical composition will typically comprise a pharmaceutically acceptable diluent or carrier. The pharmaceutical composition is preferably suitable for parenteral or oral administration. Orally administrable compositions may be in solid or liquid form and may take the form of tablets, powders, suspensions and syrups, among others. Optionally, the composition comprises one or more flavouring and/or colouring agents. In general, the therapeutic and nutritional compositions may comprise any substance that does not significantly interfere with the action of the compound on the individual.

Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the pharmaceutical art. The compositions of the invention may contain from 0.01 to 99% by weight of a compound of the invention. The compositions of the present invention are generally prepared in unit dosage form. Preferably, the unit dose of a compound described in the present invention is 1mg to 1000mg (more preferably 50mg to 500 mg). Excipients used in the preparation of these compositions are excipients known in the art.

Other examples of product forms for use in the composition are food supplements, such as in the form of soft or hard capsules comprising an encapsulating material selected from the group consisting of: gelatin, starch, modified starch, starch derivatives such as glucose, sucrose, lactose and fructose. The encapsulating material may optionally contain crosslinking or polymerization agents, stabilizers, antioxidants, light absorbers for protecting photosensitive fillers, preservatives, and the like. Preferably, the unit dose of the compound in the food supplement is from 1mg to 1000mg (more preferably from 50mg to 500 mg).

In general, the term carrier may be used throughout this application to refer to a composition that may be mixed with the described compounds, whether it is a pharmaceutical carrier, food, nutritional supplement, or dietary adjuvant. For the purposes of the present invention, the materials described above may be considered carriers. In certain embodiments of the invention, the carrier has little to no biological activity on the compounds of the invention.

Dosage: the methods of the invention can comprise administering to an animal in need thereof a therapeutically effective amount of the compound. An effective amount of a compound will depend on the form of the compound administered, the length of administration, the route of administration (e.g., oral or parenteral), the age of the animal, including mammals and humans, and the condition of the animal.

For example, the amount of compound effective to treat or prevent type 2diabetes, pre-diabetes, type 1 diabetes, impaired glucose tolerance, insulin resistance, ulcerative colitis or crohn's disease, or any other condition described herein, in an animal may vary from 0.1-10,000mg/kg per day. A preferred effective amount of the compound is 1 to 5,000mg/kg per day, and a more preferred dose is 2 to 100mg/kg per day. The upper limit of the effective amount to be administered is not critical, since the compounds are relatively non-toxic, as demonstrated by our toxicological data. When administered to an animal for a period of time in the range of between about 7 to 100 days (and preferably for a period of time of 15 to 50 days, and most preferably for a period of time of 30 to 42 days), an effective amount of the compound is most effective in treating or preventing ulcerative colitis, crohn's disease, type 2diabetes, type 1 diabetes, prediabetes, metabolic syndrome, impaired glucose tolerance, and insulin resistance in an animal.

The amount of compound most effective in preventing overactivation of the immune system may vary from 0.1 to 500mg/kg per day, and the preferred dose is from 1 to 150mg/kg per day.

When an effective amount of a compound of the present invention is administered in the form of a nutritional, therapeutic, medical or veterinary composition, the preferred dosage ranges from about 0.01% to 2.0% wt/wt relative to the food or nutraceutical product.

In certain other embodiments, the present invention provides the use of a LANCL 2-binding compound as well as a structurally related compound, such as a compound selected from the group consisting of an ester thereof, a pharmaceutically suitable salt thereof, a metabolite thereof, a structurally related compound thereof, or a combination thereof, for the treatment and prevention of IBD and gastrointestinal inflammation.

In addition, the present invention relates generally to inhibiting inflammation in the gastrointestinal tract, wherein the relevant components include the stomach, small intestine, large intestine and rectum. The effect is produced by exposing the compound to various cell types in the body that induce biological effects. Cells may include those from gastrointestinal tissue, immune cells (i.e., macrophages, monocytes, lymphocytes), or epithelial cells. In certain embodiments, the invention provides for treating an individual with a compound of the invention (e.g., in the form of a dietary supplement) to reduce or prevent inflammation associated with inflammatory bowel disease (crohn's disease or ulcerative colitis). The invention also encompasses the administration of a compound of the invention to the gastrointestinal tract in order to inhibit the expression of cell adhesion molecules in the intestinal tract.

When practiced, the methods of the invention can administer the compound to the subject via any acceptable route of administration using any acceptable form, as described above, and allow the subject's body to distribute the compound to the target cells via natural processes. As described above, administration can likewise be by direct injection into a site (e.g., organ, tissue) containing the target cells (i.e., cells to be treated).

Further, administration may follow any number of schemes. Thus, it may comprise a single or single administration of the test compound, or multiple administrations over a period of time. Thus, treatment may comprise repeating the administration step one or more times until the result is achieved. In certain embodiments, treatment may last for a long period of time, such as weeks, months, or years. The person skilled in the art is fully readily able to develop a suitable dosing regimen for an individual based on parameters known in the art. Dosages of the compounds of the invention may be used in the methods of these embodiments of the invention. For treating IBD, gastrointestinal inflammation, or suppressing expression of cell adhesion molecules in the intestinal tract, it is preferred that the compound be administered in an amount of about 1 mg/day to 9,000 mg/day.

The amount to be administered will vary depending on the subject, the stage of the disease or disorder, the age of the subject, the general health of the subject, and a variety of other parameters known to and routinely considered by those of skill in the medical arts. As a general matter, a sufficient amount of the compound will be administered so as to produce a detectable change in the amount of gastrointestinal inflammation, which in the case of IBD is often correlated with the amount of pain experienced by the individual. In cases where the patient is not currently experiencing symptoms of IBD, the changes that can be sought may relate to immune cell parameters such as TNF α expression on immune cells or the percentage of regulatory T cells in the blood. Suitable amounts are disclosed herein, and other suitable amounts can be identified by one of skill in the art without undue or excessive experimentation based on the amounts disclosed herein.

In one aspect, the invention provides a method of treating or preventing the development of IBD in a subject suffering from IBD or otherwise healthy subject that may have a genetic predisposition to crohn's disease or ulcerative colitis. The methods may also involve treating those individuals with a form of remission of IBD. According to the present invention, the term "subject suffering from IBD" is used to mean a subject (e.g. animal, human) suffering from a disease or disorder exhibiting one or more clinical signs typical of IBD. In general, methods of treatment or prevention according to this aspect of the invention comprise administering to a subject an amount of a compound therapy effective to treat or prevent one or more symptoms or clinical manifestations of IBD or to prevent the development of such symptoms or manifestations.

Thus, according to the methods of the present invention, the present invention may provide methods of treating IBD, inflammation associated with intestinal infections, and inflammation associated with autoimmune diseases. The treatment method can be a prophylactic method. In certain embodiments, the method is a method of treating IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune disease. In other embodiments, the method is a method of preventing IBD. In embodiments, the method is a method of preventing a form of remission of IBD from becoming activated. In still other embodiments, the method is a method of improving the health of an individual suffering from IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune disease. Organisms responsible for gastrointestinal infections include (but are not limited to): coli (Escherichia coli), Shigella (Shigella), Salmonella (Salmonella), pathogenic Vibrio (Vibrios), Campylobacter jejuni (Campylobacter jejuni), Yersinia enterocolitica (enterocolitica Yersina), Toxoplasma gondii (Toxoplasma gondii), Entamoeba histolytica (Entamoeba histolytica), and Giardia lamblia (Giardia lamblia). Thus, in certain embodiments, the present invention provides a method of protecting the health, organs and/or tissues of an individual suffering from or having developed IBD, inflammation associated with intestinal infection and inflammation associated with autoimmune disease.

In one embodiment of the invention, the method of treating IBD comprises treating IBD without causing discernible side effects such as significant weight gain, systemic immunosuppression, cushing's disease (cushing oid) appearance, osteopenia/osteoporosis or pancreatitis as is common with currently available IBD treatments (i.e., corticosteroids, tumor necrosis factor α inhibitors). That is, it has been found that the method of treatment according to the present invention, which provides a therapeutic effect at least in part by affecting the expression and/or activation of LANCL2 in some cells, provides a beneficial effect without causing a significant weight increase (e.g., by fluid retention) in the treated individual as compared to an otherwise similar individual not receiving the treatment.

Thus, the methods of the invention can provide a method of reducing inflammation. The methods can reduce inflammation systemically (i.e., throughout the body of the individual) or locally (e.g., at the site of administration or at the site of inflammatory cells, including but not limited to T cells and macrophages). In treating or preventing inflammation according to the methods of the present invention, one effect that may be found is a reduction in the number of blood mononuclear cells or macrophages and lymphocytes that infiltrate the intestine. Another effect may be a regulatory immune cell population (e.g., CD 4)⁺CD25⁺FoxP3⁺Regulatory T cells), or regulatory properties of lymphocytes or macrophages (e.g., increased interleukin 4(IL-4) or IL-10 or decreased TNF- α and IL-6).

The invention also provides methods of treating infectious diseases with the compounds described herein. Non-limiting examples of such infectious diseases include viral infections, bacterial infections, and fungal infections.

Non-limiting examples of viral infections include infections by, inter alia, the following viruses: adenoviridae (adenoviridae) viruses, such as adenoviruses; viruses of the herpes virus family (herpesviridae), such as herpes simplex type 1, herpes simplex type 2, varicella-zoster virus, ebeltan-bal virus (epstein-barr virus), human cytomegalovirus, human herpesvirus and type 8; papillomavirus family (papillomaviridae) viruses, such as human papilloma virus; polyomaviridae (polyomaeridae) viruses such as BK virus and JC virus; viruses of the poxviridae (poxviridae) family, such as smallpox; viruses of the hepadnaviridae (hepadnaviridae) family, such as hepatitis B virus; parvoviridae (parvoviridae) viruses, such as human bocavirus and parvovirus B19; astroviridae (astroviridae) viruses, such as human astrovirus; caliciviridae (caliciviridae) viruses, such as Norwalk virus (norwalk virus); picornaviridae (picornaviridae) viruses, such as coxsackievirus (coxsackievirus), hepatitis a virus, poliovirus, and rhinovirus; coronaviridae (coronaviridae) viruses such as acute respiratory syndrome virus; flaviviridae (flaviviridae) viruses such as hepatitis C virus, yellow fever virus, dengue virus and west nile virus; togaviridae (togaviridae) viruses, such as rubella virus; viruses of the hepaciviridae (hepeviridae) family, such as hepatitis E virus; retroviridae (retroviridae) viruses, such as Human Immunodeficiency Virus (HIV); viruses of the orthomyxoviridae family (orthomyxoviridae), such as influenza viruses; arenaviridae (arenaviridae) viruses, such as the citrullinotus (guanarito virus), junin virus (junin virus), lasar virus (lassa virus), maculo virus (machuto virus) and sabia virus (sabi a virus); bunyaviridae (bunyaviridae) viruses, such as Crimean-Congo hemorrhagic fever virus (Crimean-Congo hemorrhhagi cfevirus); filoviridae (filoviridae) viruses, such as ebola virus (ebola virus) and marburg virus (marburg virus); paramyxoviridae (paramyxoviridae) viruses, such as measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and Nipah virus; rhabdoviridae (rhabdoviridae) viruses, such as rabies virus; unassigned viruses, such as hepatitis D virus; and reoviridae (reoviridae) viruses, such as rotavirus, circovirus, colorado tick fever virus (collevivus) and banna virus (banna virus).

Non-limiting examples of bacterial infections include infections by bacteria described above in addition to the following: bacillus anthracis (Bacillus anthracyclis), Bacillus cereus (Bacillus cereus), Bordetella pertussis (Bordetella pertussis), Bordetella burgdorferi (Bordetella burgdorferi), Brucella bovis (Brucella abortus), Brucella canis (Brucella canis), Brucella caprina (Brucella melitensis), Brucella suis (Brucella suis), Campylobacter jejuni (Campylobacter jejuni), Chlamydia pneumoniae (Chlamydia pneumoniae), Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia psittaci (Chlamydia thermophila), Clostridium botulinum (Clostridium Borrelia), Clostridium difficile (Clostridium difficile), Clostridium difficile (Clostridium perfringens), Clostridium perfringens (Clostridium perfringens), Clostridium (Clostridium perfringens), Clostridium (Clostridium difficile), Clostridium (Clostridium), Clostridium difficile), Clostridium (Clostridium difficile), Clostridium (Clostridium difficile), Clostridium (Clostridium), leptospira (Leptospira interrogans), Listeria monocytogenes (Listeria monocytogenes), Mycobacterium leprae (Mycobacterium leprae), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium ulcerosa (Mycobacterium ulcerosa), Mycoplasma pneumoniae (Mycoplasma pneumoniae), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria menninggidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Rickettsia immediately (Rickettsia ricksii), Salmonella typhi (monella typhi), Salmonella typhi (Salmonella typhi), Salmonella typhi (Streptococcus pneumoniae), Streptococcus pneumoniae (Staphylococcus epidermidis), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pneumoniae (Staphylococcus), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae), Streptococcus (Streptococcus) and Streptococcus (Streptococcus), Yersinia enterocolitica (Yersinia enterocolitica), Yersinia pseudotuberculosis (Yersinia pseudouberculosis), and other species from the genera of the above-mentioned organisms.

Non-limiting examples of fungal infections include infections with the following fungi: aspergillus (Aspergillus) fungi, such as Aspergillus fumigatus (Aspergillus fumigatus), which cause aspergillosis; blastomycosis (blastomycosis) fungi, such as dermatitis blastomycosis (blastomycosis), which causes blastomycosis; candida (Candida) fungi, such as Candida albicans (Candida albicans), which cause candidiasis; coccidioidomycosis (coccidiides) fungi, which cause coccidioidomycosis (river valley fever); cryptococcus (Cryptococcus) fungi, such as Cryptococcus neoformans (Cryptococcus neoformans) and Cryptococcus gattii (Cryptococcus gattii), which cause cryptococcosis; dermatophytes (dermophytophytes) fungi, which cause tinea; fungi causing fungal keratitis, such as Fusarium (Fusarium species), Aspergillus (Aspergillus species), and Candida (Candida species); histoplasma (Histoplasma) fungi, such as Histoplasma capsulatum (Histoplasma capsulatum), which cause Histoplasma disease; mucorales (Mucorales) fungi, which cause mucormycosis; saccharomyces (Saccharomyces) fungi, such as Saccharomyces cerevisiae; pneumocystis (Pneumocystis) fungi, such as yersinia (Pneumocystis jiirovacii), which cause Pneumocystis pneumonia; and Sporothrix fungi, such as scheimpflug Sporothrix schenckii, which cause sporotrichosis.

The invention also provides methods of treating autoimmune inflammatory diseases with the compounds described herein. Non-limiting examples of autoimmune inflammatory diseases include, inter alia, Inflammatory Bowel Disease (IBD), systemic lupus, rheumatoid arthritis, type 1 diabetes, psoriasis and multiple sclerosis.

The invention also provides methods of treating chronic inflammatory diseases with the compounds described herein. Non-limiting examples of chronic inflammatory diseases include metabolic syndrome, obesity, prediabetes, cardiovascular disease, and type 2diabetes, among others.

The invention also provides methods of treating diabetes, including type 1 diabetes, type 2diabetes, and other types of diabetes, with the compounds described herein. The term "diabetes (diabetes/diabetes mellitis)" is used to encompass metabolic disorders in which an individual has hyperglycemia (i.e. hyperglycemia). Hyperglycemic conditions have various etiologies, such as the pancreas not producing enough insulin, or the cells not responding to the insulin produced. There are several recognized subtypes of diabetes. Type 1 diabetes is characterized by the body's complete inability to produce insulin or the body's inability to produce sufficient insulin. Type 2diabetes generally results from insulin resistance, a condition in which cells are unable to use insulin correctly. Type 2diabetes is sometimes co-manifested with insulin deficiency. Gestational diabetes occurs when a pregnant woman, not previously diagnosed with diabetes, develops hyperglycemia. The less common forms of diabetes include congenital diabetes (due to genetic defects associated with insulin secretion), cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several monogenic forms of diabetes (including maturity onset diabetes of the young). Monogenic diabetes encompasses several forms of hereditary diabetes (in contrast to the more complex polygenic pathogens that lead to hyperglycemia) caused by mutations in a single autosomal dominant gene.

In view of the above methods, it should be apparent that the present invention provides LANCL 2-binding compound therapies for contacting cells, such as treating cells of an individual. The discussion above has focused on the use of the compounds of the present invention as part of a composition for situations that may be generally considered a pharmaceutical or medical environment.

As described in more detail above, the compounds described in the present invention for the treatment of IBD, gastrointestinal inflammation and other conditions described may be formulated as pharmaceutical, nutritional compositions, functional food compositions or dietary adjuvants.

The elements and method steps described herein may be used in any combination, whether explicitly described or not.

All method step combinations as used herein may be performed in any order, unless otherwise specified or explicitly implied to the contrary by the context in which the combination is made.

As used herein, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include each number and subset of numbers subsumed within the range, whether or not specifically disclosed. Furthermore, these numerical ranges should be construed as providing support for any number or subset of claims within the range. For example, disclosure of 1 to 10 should be construed to support a range of 2 to 8,3 to 7, 5 to 6, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, and so forth.

All patents, patent publications, and co-pending publications (i.e., "references") cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference. In the event that the present invention conflicts with the incorporated reference, the present invention controls.

It is to be understood that the invention is not limited to the specific constructions and arrangements of parts herein shown and described, but covers such modifications as fall within the scope of the appended claims.

Examples of molecular modeling

Example 1: molecular modeling of LANCL2 ligand binding

Introduction to

Established LANCL2 agonists, such as abscisic acid (ABA) and NSC61610, exert anti-inflammatory activity in a wide range of disease models ranging from IBD to diabetes and influenza. The value of LANCL2 as a novel therapeutic target deserves efforts to discover and develop new classes of orally active drugs for the treatment of chronic metabolic, immune-mediated and infectious diseases. As discussed in this example, other LANCL2 agonists were developed via iterative combination of computational modeling and experimentally validated rational drug design. This example demonstrates a method of increasing rational drug design and medicinal chemistry efforts to increase solubility, increase binding to LANCL2, reduce cost, and understand the LANCL2 protein itself.

Method of producing a composite material

The structure of LANCL 2. The crystal structure of LANCL2 is absent. Thus, to understand the structure and function of LANCL2, homology modeling was performed on human LANCL2 using the LANCL1 crystal structure as a template. The model quality is assessed and improved via an energy minimization procedure. Homology modeling predicts the 3D structure of proteins by identifying their homologous proteins relative to other members of the protein family whose structure has been solved experimentally [52 ]. Proteins are likely to be homologous when they have greater than 35% sequence identity. Lancl1 shares 54% sequence identity with Lancl2 [15 ].

Compound generation and ligand structure. The LANCL2 agonist structures were generated (fig. 1A and 1B). SMILES from these agonists were generated using NIH's online SMILES transducers and converters [53 ]. At the same time, a separately structured pdb file is generated and downloaded. The pdb file is converted to the pdbqt necessary for virtual screening using AutoDock Tools.

And (6) virtual screening. Executing the generated derivative files with AutoDock ToolsAnd (4) butting. A search space is defined, including the grid square center and the x, y, and z dimensions. Docking was applied to the whole protein target and the grid covered the entire protein surface. The grid being a common cube (xx) In which grid points are spaced apartThis grid is centered in the middle of the protein. These dimensions and spacing allow the grid to cover the entire LANCL2 surface. Genetic algorithms are used for random global optimization. For each compound, one hundred binding conformations were generated by AutoDock Tools. By usingThe RMSD clustering tolerances of (a) cluster 100 resulting poses for each derivative.

And analyzing the virtual screening result. Finding the best way to assemble each compound into LANCL2 using AutoDock Vina resulted in a docking record file that contained docking records including the binding energy of each predicted binding pattern for all compounds. Binding energy means the sum of total intermolecular energy, total internal energy and torsional free energy minus the energy of the unbound system. Compounds were rated by the maximum negative energy. The lowest combined energy pose in the first cluster is considered the most favorable docking pose. Lower binding free energies indicate a more stable protein-ligand system and a higher affinity between the protein and the ligand. Exemplary compounds were further validated by in vitro testing and preclinical studies using mouse models of human disease.

Results

NSC61610 docking summary. The histogram of the first five clusters with the lowest energy position energy in NSC61610 is given in fig. 2.NSC 61610 has very high affinity for the' central cleft. The first two clusters representing 7% of the total manipulations were each directed to this site. Due to the two angstrom (angstrom) tolerance, other clusters are likely to be directed to this site. The next two clusters were directed to 'allosteric sites' that approached blue random coil.

ABA docking summary. The histograms of the first five clusters with the lowest energy position energy in ABA are given in figure 3.ABA has moderate affinity but very high specificity for the 'allosteric' site between the light green helix and the light green random coil. 29% of the operations are directed to this top cluster. The second cluster is also directed to this site. Due to the two angstrom tolerance, other clusters are likely to be for this site. The fourth cluster appears to be in the 'central fissure'. This leaves the question of the true treatment site for ABA to remain to be solved.

BT-11 docking overview. The histogram of the first five clusters with the lowest energy position energy in BT-11 is given in fig. 4. The first two clusters of BT-11 were for the 'Central fracture', but represent only 2% of the operations. However, due to the two angstrom tolerance, other clusters are likely to be for this site. BT-11 has slightly less affinity for this site than NSC61610 but greater than ABA. BT-11 has demonstrated therapeutic effects (see examples below).

BT-6 docking overview. The histogram of the first five clusters with the lowest energy position energy in BT-6 is given in fig. 5.BT-6 has the highest affinity among any of the compounds docked. The first two, and possibly the first three clusters are for the 'central fracture'. Due to the two angstrom tolerance, other clusters are likely to be for this site. Cluster 4 is directed to 'allosteric' sites along the blue random coil.

BT-15 docking overview. The histogram of the first five clusters with the lowest energy position energy in BT-15 is given in fig. 6.BT-15 does not have the binding affinity of NSC61610 or BT-11. While it does appear to target the 'central cleft', this effect does not appear to be as pronounced as NSC61610 or BT-11.

BT-ABA-5a docking summary. The histogram of the first five clusters with the lowest energy position energy in BT-ABA-5a is given in FIG. 7. The highest affinity of BT-ABA-5a is at a point not seen in any previously examined docks. However, clusters 2 and 3 represent the vast majority of the operations, 32%. Cluster 2 is for the right posterior allosteric site. Cluster 3 is directed against an ` allosteric ` site of ABA. Cluster 4 is also directed to this site. Due to the two angstrom tolerance, other clusters are likely to be for this site.

Discussion of the related Art

ABA and NSC61610 exert LANCL 2-dependent immunomodulatory, anti-inflammatory and anti-diabetic effects, however, computational predictions indicate that they bind at different sites of LANCL 2. As expected, the rationally designed ligands are directed primarily to the primary binding sites of ABA and NSC 61610. BT-ABA compounds are small in size and have a-COOH functional group; this visually indicates that it will be directed to a hydrophilic surface pocket. BT compounds are much more hydrophobic; thus, this indicates visually that it will be directed to the more hydrophobic central cleft surrounded by the a-helix.

Binding affinity was of moderate correlation with SPR data (FIGS. 1A and 1B; see examples below). SPR data (and K)_DValue) indicates an order of bond strengths of NSC61610 (2.3)&6.3)、BT-11(6.3&7.7)、BT-15(11.4&21.4) and BT-6 (18.2). The modeled data (with lowest BE) showed the order of the binding strengths BT-6(-10.47), NSC61610(-10.27), BT-11(-9.39), BT-15 (-8.87). SPR data and modeled data indicate the same order of binding strengths, except BT-6 flips from worst to first. Molecular modeling data combined with rational drug design is likely to yield a better understanding of the LANCL2 protein, which would allow further development of analogs that target the LANCL2 pathway and activate it to take advantage of its potent anti-diabetic and anti-inflammatory properties.

Examples of medicinal chemistry

Example 2: BT-11 and salts

As shown in scheme 2-1, a solution of 6- (1H-benzimidazol-2-yl) pyridine-2-carboxylic acid (12g) in DMF (100mL) was cooled to 0 ℃, and then EDC · HCl (1.5 equiv), HOBt (1.5 equiv) and DIPEA (1.2eq, taken in volume with the presumed density) were added sequentially. The mixture was stirred at 0 ℃ for 10 minutes. Piperazine (0.5 eq) was added and the reaction mixture was gradually warmed to room temperature and stirred for 16 hours. After the reaction is completed (byTLC monitoring, eluent: DCM containing 10% MeOH), the reaction mixture was poured into ice-cold water (about 300mL), the precipitated solid was filtered, washed with ice-cold water, and dried to give BT-11(10g, 75%) as a light brown solid.¹H NMR(400MHz,DMSO-d₆),13.0(s,1H),12.8(s,1H),8.38(dd,2H),8.13(dt,2H),7.73(dd,2H),7.67(d,2H),7.57(dd,2H),7.25(m,4H),3.90(bs,2H),3.80(bdd,2H),3.65(bdd,2H),3.56(bs,2H)。LCMS-ES 529.44[M+H]⁺,265.46[(M+2H)/2]⁺⁺。

Scheme 2-1

As shown in scheme 2-2, a suspension of BT-11(1.0 equiv) in a minimum amount of MeOH (5mL) was cooled to 0 deg.C and 4M methanolic HCl (methanolic HCl) (excess, 15mL/1g) was added dropwise over a period of 15-20 minutes. The mixture was allowed to gradually warm to room temperature for 3 hours. At the completion of the reaction (monitored by TLC, eluent: CH with 10% MeOH)₂Cl₂) After this time, the volatiles were evaporated under reduced pressure. With CH containing 10% MeOH₂Cl₂The crude material was washed and lyophilized to give an off-white solid (850mg, 75%).¹H NMR(400MHz,DMSO-d₆),8.58(dd,2H),8.29(dt,2H),7.83(m,6H),7.44(bd,4H),3.91(bs,2H),3.81(bm,2H),3.64(bm,2H),3.55(bs,2H)。LCMS-ES 529.56[M+H]⁺。

Scheme 2-2

Example 3: BT-12

As shown in scheme 3-1, 6- (benzoxazol-2-yl) pyridine-2-carboxylic acid (4.05g) was treated with EDC & HCl (1.5 equiv.), HOBt (1.5 equiv.), and DIPEA (1.2 equiv., taken in volume with inferred density) and 0.5 equiv piperazine at 0 deg.C in CH with 10% DMF₂Cl₂The solution of (1). The mixture was allowed to warm to room temperature for 16 hours. A light brown solid formed and was filtered on a sintered glass funnel usingWater washed and lyophilized to give a light brown solid (3.2 g).¹H NMR(300MHz,CDCl₃),8.45(dd,2H),8.05(m,2H),7.9(d,2H),7.8(dd,2H),7.6(dd,2H),7.4(m,2H),7.35(m,2H),4.0(bm,8H)。

Scheme 3-1

Example 4: BT-14 and salts

A solution of 6- (benzoxazol-2-yl) pyridine-2-carboxylic acid (500mg) in DMF (10mL) was treated with EDC & HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (3 equiv.), and piperazine-1-carboxylic acid tert-butyl ester (1.1 equiv.) at 0 deg.C as shown in scheme 4-1. The mixture was allowed to warm to room temperature for 16 hours. After evaporation of the solvent, the residue was extracted into EtOAc and washed with water. The organic layer was evaporated under vacuum and the crude residue washed with pentane to give a light brown solid (120mg, 48%).¹H NMR(400MHz,DMSO-d₆),8.4(d,1H),8.2(t,1H),7.9(t,2H),7.8(d,1H),7.5(dt,2H),3.7(bm,2H),3.5(bm,4H),3.4(bm,2H),1.4(s,9H)。LCMS-ES 409.49[M+H]⁺,431.37[M+Na]⁺,447.36[M+K]⁺。

Scheme 4-1

The resulting compound from scheme 4-1 (200mg) was treated with methanolic hydrochloric acid (6mL) at 0 ℃ as shown in scheme 4-2. The mixture was allowed to warm to room temperature for 3 hours. The solvent was evaporated and washed with pentane and diethyl ether to give a light brown solid (160mg, quantitative).¹H NMR(300MHz,DMSO-d₆) 9.30(bs,2H),8.45(d,1H),8.25(t,1H),7.9(m,3H),7.5 (quintuple, 2H),3.7(bm,2H),3.5(bm,2H),3.3(bm,4H),1.4(s, 9H). LCMS-ES309.26[ M + H ]]⁺。

Scheme 4-2

As shown in scheme 4-3, saturated NaHCO was used₃The resulting salt from scheme 4-2 (25mg) was neutralized in aqueous solution and dried in a lyophilizer to yield 20 mg/96% BT-14 on hand. The yield was 90%.¹H NMR(300MHz,DMSO-d₆) 8.4(d,1H),8.2(t,1H),7.90(t,2H),7.75(d,1H),7.5 (quintuple, 2H),3.95(bm,2H),3.8(bm,2H),3.3(bm,2H),3.2(bm, 2H); 309.37LCMS-ES [ M + H ]]⁺。

Scheme 4-3

Example 5: BT-15

As shown in scheme 5-1, 6- (1H-benzimidazol-2-yl) pyridine-2-carboxylic acid (50mg) in DMF (5mL) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (3 equiv.) and 0.9 equiv BT-14 hydrochloride at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. Filtered through a sintered funnel, washed with water, and lyophilized to remove water, yielding 20mg BT-15.¹H-NMR(400MHz,DMSO-d⁶),12.93(d,1H),8.44(dd,1H),8.36(t,1H)8.25(t,1H),8.17(m,2H),7.87(m,3H),7.72(m,2H),7.54(m,2H),7.31(m,3H),3.90(s,2H),3.82(bm,2H),3.67(bm,2H),3.58(bm,2H)。LCMS-ES 530.48[M+H]⁺,265.94[(M+2H)/2]⁺⁺。

Scheme 5-1

BT-15 has shown LANCL2 binding (FIG. 1A). Its predicted value for binding affinity to LANCL2 was-9.9, and the Kd value for affinity confirmed by SPR was 21.4.

Example 6: BT-13 salt

As shown in scheme 6-1, 6- (1H-benzimidazol-2-yl) pyridine-2-carboxylic acid (500mg) in DMF (10mL) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (3 equiv.), and piperazine-1-carboxylic acid tert-butyl ester (1.1 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. After the reaction mixture was poured into ice-cold water, the precipitate was filtered,and dried to give a light brown solid (600mg, 70%). TLC (100% ethyl acetate). HNMR&LCMS complies.(Yield:70％).¹H NMR(300MHz,DMSO-d₆) 12.90(s,1H),8.4(d,1H),8.15(t,1H),7.65(td,3H),7.25 (quintuple, 2H),3.7(bm,2H),3.5(bm,2H),3.3(bm,4H),1.4(s, 9H). LCMS-ES 408.35[ M + H ]]⁺。

Scheme 6-1

The resulting compound from scheme 6-1 (600mg) was treated with methanolic hydrochloric acid (6mL) at 0 ℃ for 3 hours as shown in scheme 6-2. The mixture was allowed to gradually warm to room temperature for 3 hours. Evaporation of excess methanolic hydrochloric acid gave BT-13 hydrochloride (500mg) as a pale brown solid.

Scheme 6-2

Example 7: BT-4 and salts

As shown in scheme 7-1, DMF (6mL) containing 3- (1H-benzimidazol-2-yl) benzoic acid (100mg) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (1 equiv.) and 0.5 equiv piperazine at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. TLC (10% methanol: DCM) showed formation of a non-polar spot and absence of starting material. After work-up and washing with diethyl ether, 30 mg/95% of BT-4 were isolated.¹H NMR(400MHz,DMSO-d₆),13.0(s,2H),8.3(bm,4H),7.75(bm,4H),7.60(bm,4H),7.2(bm,4H),3.65(bm,8H)。LCMS-ES 527.36[M+H]⁺,264.50[(M+2H)/2]⁺⁺。

Scheme 7-1

As shown in scheme 7-2, 30 mg/95% BT-4 was treated with 4M HCl in dioxane for 3 hours. Evaporation of the solvent and washing with ether gave 10 mg/97% BT-4 hydrochloride.¹H NMR(400MHz,DMSO-d₆),8.45(bm,4H),7.80(bm,8H),7.50(bm,4H),3.65(bm,8H)。LCMS-ES527.44[M+H]⁺,264.50[(M+2H)/2]⁺⁺。

Scheme 7-2

Example 8: BT-6 and salts

As shown in scheme 8-1, 3- (1H-benzimidazol-2-yl) benzoic acid (100mg) in DMF (6mL) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (1 equiv.), and benzene-1, 4-diamine (0.5 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. TLC (10% methanol: DCM) showed formation of a non-polar spot and absence of starting material. After work-up and washing with diethyl ether, a light brown solid (60mg) was isolated.¹H NMR(300MHz,DMSO-d₆) 13.1(s,2H),10.45(s,2H),8.75(s,2H),8.40(d,2H),8.05(d,2H),7.85(s,4H),7.70(t,4H),7.55(d,2H)7.25 (quintuple, 4H). LCMS-ES549.0[ M + H ]]⁺275.1[(M+2H)/2]⁺⁺。

Scheme 8-1

As shown in scheme 8-2, 60 mg/98% BT-6 was treated with 4M HCl in dioxane for 3 hours. After evaporation of the solvent and washing with ether, 50 mg/96% BT-6 hydrochloride was obtained.¹H NMR(300MHz,DMSO-d₆),10.60(s,2H),9.00(s,2H),8.55(d,2H),8.30(d,2H),7.90(s,4H),7.85m,6H),7.50(m,4H)。LCMS-ES549.3[M+H]⁺275.3[(M+2H)/2]⁺⁺。

Scheme 8-2

Example 9: BT-16 and salts

As shown in scheme 9-1, 6- (1H-benzimidazol-2-yl) pyridine-2-carboxylic acid (100mg) in DMF (10mL) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (3 equiv.), and benzene-1, 4-diamine (0.5 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. After the reaction mixture was poured into ice-cold water, the precipitate was filtered and dried to give a light brown solid (60 mg).

Scheme 9-1

Compound BT-16(50mg) was treated with HCl in dioxane (3mL) at 0 ℃ as shown in scheme 9-2. The mixture was allowed to warm to room temperature for 4 hours. Evaporation of excess dioxane hydrochloric acid gave 30mg of a brown solid (30 mg).¹H NMR(300MHz,DMSO-d₆),11.00(s,2H),8.6(bm,2H),8.35(bm,4H),8.05(s,4H),7.85(bm,4H),7.40(bm,4H)。LCMS-ES 551.84[M+H]⁺。

Scheme 9-2

Example 10: BT-3 and salts

As shown in scheme 10-1, DMF (10mL) containing 3- (2-benzoxazolyl) benzoic acid (50mg) was treated with EDC & HCl (1.25 equiv.), HOBt (1.25 equiv.), DIPEA (1 equiv.) and piperazine (1 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. After diluting the reaction mixture with ice-cold water, the resulting solid was discarded, filtered, and then dried to obtain 30mg of BT-3.¹H NMR(300MHz,DMSO-d₆),8.2(bm,4H),7.8(bm,4H),7.7(bm,4H),7.45(bm,4H),3.6(bm,8H)。LCMS-ES 529.32[M+H]⁺。

Scheme 9-1

As shown in scheme 10-2, BT-3(30mg) was treated in methanolic hydrochloric acid (5mL) at 0 ℃. The mixture was allowed to warm to room temperature for 4 hours. After evaporation of excess methanolic hydrochloric acid under vacuum, a brown solid (15mg) formed.

Scheme 10-2

Example 11: BT-5 and salts

As shown in scheme 11-1, 3- (2-benzoxazolyl) benzoic acid (50mg) in DMF (10mL) was treated with EDC & HCl (1.25 equiv.), HOBt (1.25 equiv.), DIPEA (1 equiv.) and benzene-1, 4-diamine (0.5 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. The reaction mixture was diluted with ice-cold water, and the solid was discarded, filtered, and then dried to give a light brown solid (30 mg).¹H NMR(300MHz,TFA),9.2(bs,2H),8.8(bm,2H),8.6(bm,2H),7.9(bm,14H)。

Scheme 11-1

As shown in scheme 11-2, 35mg of BT-5 was treated in dioxane hydrochloride (HCl dioxane) (5mL) at 0 deg.C. The mixture was allowed to warm to room temperature for 4 hours. After evaporation of excess dioxane under vacuum, a light brown solid (15mg) was formed.¹H NMR(300MHz,TFA),9.3(bs,2H),8.8(bm,2H),8.6(bm,2H),7.9(bm,14H)。

Scheme 11-2

Example 12: BT-17 and salts

As shown in scheme 12-1, 6- (benzoxazol-2-yl) pyridine-2-carboxylic acid (100mg) in DMF (10mL) was treated with EDC. HCl (1.5 equiv.), HOBt (1.5 equiv.), DIPEA (1.2 equiv.) and benzene-1, 4-diamine (0.5 equiv.) at 0 ℃. The mixture was allowed to warm to room temperature for 16 hours. The reaction mixture was diluted with ice-cold water, the solid discarded, filtered and dried to give a light brown solid (70 mg).¹H NMR(400MHz,TFA),8.85(dd,4H),8.55(t,2H),8.1(bm,4H),7.95(m,4H),7.85(s,4H).LCMS-ES 553.28[M+H]⁺。

Scheme 12-1

As shown in scheme 12-2, BT-17(60mg) was treated in dioxane hydrochloric acid (10mL) at 0 ℃ to room temperature for 4 hours. After evaporation of the solvent by using a lyophilizer, a light brown solid (45mg) was formed.¹H NMR(400MHz,TFA),8.90(bm,4H),8.6(bm,2H),8.0(bm,10H)。

Scheme 12-2

Example 13: BT-ABA-25

The structure of BT-ABA-25 is shown in scheme 13-1. BT-ABA-25 is a ligand of LANCL2 (FIG. 1B). Its predicted value for binding affinity to LANCL2 was-7.5, and the Kd value for affinity confirmed by SPR was 1.77 e-04.

Scheme 13-1

Example 14: BT-ABA-5a

As shown in scheme 14-1, 8-vinyl-1, 4-dioxaspiro [4.5 ] using argon]A solution of decan-8-ol (200mg, 1 eq) and methyl 5-bromofuran-2-carboxylate (1.5 eq) in Et3N (2mL) was degassed for 10 min. Next, pd (oac)2(0.025 eq), DPPF (0.05 eq) were added and degassed again for 10 minutes. The resulting reaction mixture was heated at 100 ℃ for 16 hours. A light brown solid (130mg) was isolated by column chromatography (EtOAx/hexane 3: 7).¹H NMR(400MHz,DMSO-d₆),7.30(d 1H),6.60(d 1H),6.45(dd,2H),4.75(s,1H),3.85(s,4H),3.80(s,3H),1.85(m,2H),1.65(m,2H),1.50(m,4H),LCMS-ES 291.34[M+H]⁺。

Scheme 14-1

As shown in scheme 14-2, to 100mg of the compound (compound 4) obtained in scheme 14-1 in THF: H₂To a solution of O in MeOH (2:1:0.5mL) was added LiOH (3 equiv.), and the mixture was stirred at room temperature for 16 h. The mixture was then concentrated under reduced pressure and the crude material was dissolved in a minimum amount of water and acidified with 2N HCl until pH 4. The compound was extracted with EtOAc and concentrated to give a light brown solid (54mg) which was used in the next reaction without further purification (scheme 14-3).¹H NMR(400MHz,DMSO-d₆),7.50(d 1H),6.60(d 1H),6.45(dd,2H),4.75(s,1H),3.85(s,4H),3.80(s,3H),1.85(m,2H),1.65(m,2H),1.50(m,4H)。LCMS-ES 277.26[M+H]⁺。

Scheme 14-2

As shown in scheme 14-3, 3N HCl (0.1mL) was added to THF containing Compound 5(50mg) with stirring at 0 deg.C. The mixture was allowed to warm to room temperature for 6 hours. TLC showed the absence of SM and non-polar spots. The mixture was concentrated under reduced pressure, diluted with water, extracted with EtOAc, and re-concentrated to give a brown solid (20 mg).¹H NMR(400MHz,DMSO-d₆),13.00(bs 1H),7.20(d 1H),6.95(d 1H),6.60(d 1H),6.45(d,1H),6.10(t,1H),3.05(m,2H),2.65(t,2H),2.5,(2H)。LCMS-ES 233.21[M+H]⁺LCMS-ES 231.27[M-H]^-463.15[2M-H]^-。

Scheme 14-3

Example 15: BT-ABA-6

As shown in scheme 15-1, 8-vinyl-1, 4-dioxaspiro [4.5 ] using argon]Decan-8-ol (500mg, 1 eq), ethyl 3-iodobenzoate (0.8 eq) and PPh₃(0.02 eq) in Et₃The solution in N (8mL) was degassed for 10 min. Then, Pd (OAc) was added₂(0.02 eq) and degassed again for 10 minutes.The resulting reaction mixture was heated at 95 ℃ for 16 hours. After work-up, a light brown solid (500mg) was isolated by column chromatography (EtOAc/hexane 3: 7).¹H NMR(400MHz,DMSO-d₆),7.95(s 1H),7.80(d 1H),7.71(d1H),7.47(t 1H),6.65(d 1H),6.49(d,1H),4.65(bs 1H),4.32(q,2H),3.68(s,4H),1.99-1.68(m,4H),1.55-1.50(m,4H),1.33(t 3H)。LCMS-ES 315.38[M-17]⁺。

Scheme 15-1

As shown in scheme 15-2, Compound 4(500mg) was dissolved in THF/H₂The solution in O/EtOH (4:2:1, 17.5mL) was cooled to 0 ℃; LiOH (2.5 equivalents) was added and the mixture was stirred while rising to room temperature over 16 hours. The mixture was concentrated under reduced pressure and the crude material was dissolved in a minimal amount of water and acidified with 1N HCl until pH 3-4. Purification by column chromatography (EtOAc/hexanes 1:1) afforded a pale yellow solid (220 mg).¹H NMR(400MHz,DMSO-d₆),13.00(bs 1H),7.95(s 1H),7.78(d 1H),7.67(d 1H),7.44(t 1H),6.64(d 1H),6.48(d,1H),4.65(s 1H),3.86(s,4H),1.87-1.61(m,4H),1.55-1.50(m,4H)。LCMS-ES 287.34[M-17]⁺。

Scheme 15-2

As shown in scheme 15-3, to a mixture of 100mg of Compound 5(100mg) and THF at 0 deg.C under stirring was added 2N HCl (1.5 mL). The mixture was allowed to warm to room temperature for 6 hours. The solution was then concentrated under reduced pressure, diluted with water, extracted with EtOAc, and re-concentrated to give a pale yellow solid (20 mg).¹H NMR(400MHz,DMSO-d₆),13.00(bs 1H),8.00(s,1H),7.80(d 1H),7.65(d 1H),7.45(t 1H),6.75(d 1H),6.45(d,1H),6.10(t,1H),5.15(s 1H),2.65(m,2H),2.15(m,2H),1.90(m,4H),LCMS-ES 259.37[M-H]^-519.48[2M-H]^-。

Scheme 15-3

Example 16: BT-ABA-13

As shown in scheme 16-1, to compound 2(2.5g, 1 eq.) in CH at 0 deg.C with stirring₂Cl₂To a solution in (50mL) was added dihydropyrane (1.3 equiv.) and TsOH (0.1 equiv.). The resulting solution was allowed to gradually warm to room temperature for 14 hours. A pale yellow liquid was isolated by column chromatography (EtOAc/hexane 1: 9). The compound was used in the next step without further purification.

Scheme 16-1

To Et as shown in scheme 16-2₃To N was added compound 3(2.5g, 1.0 equivalent), 4,5, 5-tetramethyl- [1,3, 2]]Dioxaboropentane (1.2 equivalents) and bis (cyclopentadienyl) zirconium hydrochloride (0.15 equivalents) were heated at 60 ℃ to 70 ℃ for 16 hours. The reaction mixture was diluted with hexane. The precipitate was removed by filtration through a short pad of silica gel and washing with hexane. After the hexane solution was concentrated, a colorless oily liquid (1.3g) was obtained.¹H NMR(400MHz,CDCl3),6.60(d1H),5.60(d 1H),6.35(d,1H),4.75(s,1H),3.85(s,3H),2.80(m,2H),2.35(m,2H),2.05(m,4H)。

Scheme 16-2

As shown in scheme 16-3, Compound 4(550mg, 1.1 equiv.), methyl 6-bromopicolinate (1.0 equiv.), K were reacted with argon₂CO₃(2.0 equiv.) in DME/H₂The solution in the O9: 1 mixture (8mL) was degassed for 10 minutes. Next, Pd [ (P (Ph))₃]₄(0.04 eq). The resulting reaction mixture was heated at 100 ℃ for 16 hours. The reaction solution was concentrated, followed by column chromatography (EtOAc/hexane 1:3) to give a pale yellow solid (230 mg). LCMS-ES404.39[ M + H ]]⁺,302.26[M-101]⁺。

Scheme 16-4

As shown in scheme 16-5, to Compound 5(230mg, 1.0 equiv) in acetone/H₂TsOH (0.1 equiv.) was added to a solution of O1: 1(6 mL). The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated and then subjected to column chromatography (EtOAc/hexane 7:3) to give a pale yellow liquid (110 mg).¹H NMR(400MHz,CDCl3),8.00(d 1H),7.80(t,1H),7.50(d,1H),6.90(m 2H),4.00(s,3H),2.80(m,2H),2.35(m,2H),2.10(m,4H),LCMS-ES 276.38[M+H]⁺。

Scheme 16-5

As shown in scheme 16-6, to Compound 6(75mg) at 0 deg.C THF/H with stirring₂LiOH (2.5 equiv.) was added to a solution of O3: 1(3 mL). The mixture was allowed to warm to room temperature for 6 hours. The reaction mixture was acidified with citric acid and extracted with a mixture of THF and EtOAc. The organic solution was concentrated to give an off-white solid (10 mg).¹H NMR(300MHz,DMSO-d₆),13.05(bs,1H),7.90(m,2H),7.65(d,1H),7.05(d,1H),6.80(d,1H),5.20(s,1H),2.65(m,2H),2.20(bd 2H),2.10-1.90(m,4H),LCMS-ES 262.27[M+H]⁺。

Scheme 16-6

Example 17: BT-ABA-16

As shown in scheme 17-1, Compound 4(437mg, 1.2 equiv.), methyl 2-bromoisonicotinate (1.0 equiv.), K were reacted with argon₂CO₃(2.0 equiv.) in DME/H₂The solution in the O9: 1 mixture (8mL) was degassed for 10 minutes. Next, Pd [ (P (Ph))₃]₄(0.04 eq). At 90 deg.CThe resulting reaction mixture was heated for 12 hours. The reaction solution was concentrated, followed by column chromatography (EtOAc/hexane 1:3) to give a pale yellow liquid (300 mg).¹H NMR(300MHz,CDCl₃),8.70(d,1H),7.85(s,1H),7.65(d,1H),6.85(d,1H),6.65(d,1H),4.70(m,1H),3.95(m,4H),2.20-1.40(m,16H),LCMS-ES 404.54[M+H]⁺,302.53[M-101]⁺。

Scheme 17-1

As shown in scheme 17-2, 5(300mg, 1.0 equiv.) is added to acetone/H₂TsOH (0.1 equiv.) was added to a solution of O1: 1(6 mL). The resulting reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was concentrated and then subjected to column chromatography (EtOAc/hexane 7:3) to give an off-white solid (160 mg).¹H NMR(300MHz,DMSO-d6),8.70(d,1H),7.85(s,1H),7.65(d,1H),7.05(d,1H),6.85(d,1H),5.20(s,1H),3.90(s,3H),2.65(td,2H),2.15(bd,2H),2.00(m,2H),1.85(m,2H),LCMS-ES276.22[M+H]⁺。

Scheme 17-2

As shown in scheme 17-3, Compound 6(100mg) was added to THF/H at 0 deg.C with stirring₂LiOH (2.5 equiv.) was added to a solution of O3: 1(3 mL). The mixture was allowed to warm to room temperature for 16 hours. The reaction mixture was acidified with citric acid and extracted with a mixture of THF and EtOAc. Concentration under reduced pressure gave an off-white solid (20 mg).¹H NMR(300MHz,DMSO-d6),13.60(bs,1H),8.70(d,1H),7.85(s,1H),7.60(d,1H),7.00(d,1H),6.85(d,1H),5.20(s,1H),2.65(m,2H),2.20-1.80(m,6H),LCMS-ES 262.28[M+H]⁺。

Scheme 17-3

Example 18: BT-ABA-14

As shown in scheme 18-1, Compound 4(300mg, 1.2 equiv.), methyl 4-bromopicolinate (1.0 equiv.), K were reacted with argon₂CO₃(2.0 equiv.) in DME/H₂The solution in the O9: 1 mixture (8mL) was degassed for 10 minutes. Next, Pd [ (P (Ph))₃]₄(0.04 eq). The resulting reaction mixture was heated at 90 ℃ for 12 hours. The reaction solution was concentrated, followed by column chromatography (EtOAc/hexane 1:3) to give a pale yellow liquid (200 mg).¹H NMR(300MHz,CDCl₃),8.50(d,1H),8.20(bs,1H),7.45(d,1H),6.70(d,1H),6.50(d,1H),4.60(m,1H),3.95(m,4H),2.20-1.40(m,16H),LCMS-ES 390.35[M+H]⁺。

Scheme 18-1

As shown in scheme 18-2, 5(200mg, 1.0 equiv.) is added to acetone/H₂TsOH (0.1 equiv.) was added to a solution of O1: 1(6 mL). The resulting reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was acidified with citric acid and extracted with a mixture of THF and EtOAc. The solution was concentrated to give an off-white solid (18 mg).¹H NMR(300MHz,DMSO-d₆),8.60(d,1H),8.05(s,1H),7.60(d,1H),6.90(d,1H),6.70(d,1H),5.20(bs,1H),2.65(m,2H),2.15(bd,2H),2.05-1.80(m,4H),LCMS-ES262.27[M+H]⁺。

Examples of receptor binding

Example 19: lancl2 binding examples

Computational modeling studies and biochemical validation were combined to guide the selection of compounds that bind to LANCL 2. Recent iterations of Surface Plasmon Resonance (SPR) technology provide real-time assays for unlabeled proteins and small molecules>25Da) between the two molecules. BIACORE^TMT200 (GE Healthcare, Piscataway, NJ) technology further developed by the general electro-medical group of Picscataway, N.J.)Additional benefits of GMP/GLP compliance and spontaneous large scale data acquisition for screening or detailed titration in a period of less than 24 hours are provided. The molecule of interest interacts through BIACORE^TMThe T200 SPR technique was routinely validated.

Method of producing a composite material

High throughput screening via LANCL 2-compound interaction molecule modeling. Auto-Doc Vina [14] is a currently advanced technology software suite that enables high throughput parallel calculations to determine LANCL 2-plant compound binding. The software suite first calculates (i) the force of the free energy associated with the binding complex and then (ii) the conformational space available for complex formation between the target and the ligand. These methods are random in nature and therefore require repeated independent screening to search all parameter spaces exhaustively and provide predictive confidence. Currently, the LANCL2 model is available via homology modeling of LANCL1 [15 ]. The AutoDock and AutoGrid graphics front-end AutoDockTools are used to define the search space, including the grid square center and the x, y, z dimensions [16 ]. AutoDock Vina produced five binding conformations for each compound. Docking was applied to the whole protein target, where the grid covered the entire protein surface. A docking log file is generated consisting of binding energies for each predicted binding pattern of all compounds to all surfaces.

Lancl 2-kinetic determination of small molecule interactions. BIACORE^TMT200 was used to determine the kinetic parameters of binding of small molecules BT-11, BT-ABA-5a, BT-6 and BT-15 (analytes) to LANCL2 (ligands). Data were generated in triplicate in a dose-dependent (5-8 titration points) fashion and analyzed to determine binding models (Langmuir, conformational shifts, etc.), real-time association and dissociation constants, and equilibrium dissociation constants. SPR technology allows to validate specific LANCL 2-phytochemical component interactions and to increase the gold standard for the binding mechanism and rate for profound understanding. Experiments were performed on carboxymethyl polyglucose (CM5) sensor chips by covalent attachment of LANCL2 by amine coupling. Flow of the sensor wafer with a 1:1 mixture of 0.1M N-hydroxysuccinimide (NHS) and 0.5M 1-ethyl-3- (-3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) at 10. mu.l/minThe mobile pools 1 and 2 were activated for 720 seconds. Stock solution LANCL2(0.41mg/mL) was diluted to 8.2. mu.g/mL (1:50 dilution) in 10mM sodium acetate (pH 5.0) and injected at a flow rate of 10. mu.l/min onto the surface of activation flow cell 2 for 1000 seconds. After capture of LANCL2(11000RU) on flow cell 2, the surfaces of flow cells 1 and 2 were deactivated by injection of 1M ethanolamine at 10. mu.l/min for 720 seconds. The working buffer was 25mM MOPS, pH 6.5, containing 0.05% T-20 and 0.15M NaCl. Kinetic studies were performed by injecting different concentrations of BT-11 (25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.13. mu.M, 1.56. mu.M and 0.76. mu.M), BT-ABA-5a (40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M and 1.25. mu.M) and BT-15/BT-6 (20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M, 0.625. mu.M and 0.313. mu.M) in triplicate. Each sample was injected for 60 seconds (contact time) followed by a 60 second dissociation time with a flow rate of 100. mu.L/min. A 180 second stabilization time was used before the next injection. By BIACORE^TMThe T200 evaluation software (version 1) analyzed the data to determine the affinity binding constant (KD) using the 1:1 binding model.

Results

Both BT-11 and BT-15 bind strongly to LANCL 2. To confirm the binding of BT-11 and BT-15 to the LANCL2 protein, we performed BIACORE^TMSPR analysis was performed in a T-200 instrument. Optical technique SPR for detecting molecular interactions was used to measure the binding affinity between LANCL2 and its ligands (i.e., BT-11 and BT-15). We immobilized the purified recombinant LANCL2 protein on BIACORE^TMSensor chip and use instrument microfluidic system to inject small molecules onto the protein surface. The change in total mass on the chip surface was measured, which corresponds to small binding to proteins. By injecting a series of small molecule concentrations, we were able to calculate the steady state binding affinities of BT-11 binding to LANCL2 and BT-15 binding to LANCL 2. The binding sensorgram shows typical small molecule protein interactions with extremely fast association and off rates (fig. 8, panels a and C). These rapid interactions are beyond the technical capabilities of the instrument. Therefore, the reliable association rate constant (k) was not determined_a) And dissociation rate constant (k)_d). Equilibrium dissociation constant (K)_D) Commonly used to describe the affinity between ligands and proteinsForce, such as how tightly the ligand binds to a particular protein. Ligand-protein affinity is affected by non-covalent intermolecular interactions between two molecules, such as hydrogen bonding, electrostatic interactions, hydrophobic forces, and van der waals forces. By plotting the equilibrium binding levels against the compound concentration, we were able to measure the steady-state affinity (K) of each interaction_D) (FIG. 8, FIGS. B and D). Both small molecules showed similar binding affinity to LANCL2 (BT-11: 7.7uM, BT-1511.4 uM).

BT-ABA-5a and BT-6 strongly bind to LANCL 2. Similar to the results described above and to confirm the binding of BT-6 and BT-ABA-5a to LANCL2, we are at BIACORE^TMSPR analysis was performed in a T-200 instrument. In this case, we also immobilized the purified recombinant LANCL2 protein on BIACORE^TMSensor chip and use instrument microfluidic system to inject small molecules onto the protein surface. The change in total mass on the chip surface was measured, which corresponds to small binding to proteins. Looking more closely at the binding sensorgrams (FIGS. 9A and 9B), our results show how BT-6 and BT-ABA-5a bind very quickly but do not dissociate at the same rate, compared to BT-11/BT-15, which associates very quickly and dissociates very quickly. Notably, the occupancy time of BT-ABA-5a exhibited the slowest off-rate, implying that BT-ABA-5a was retained in the LANCL2 binding pocket for the longest time. This longer binding could potentially affect activation of the LANCL2 pathway by triggering more potent anti-inflammatory and anti-diabetic and other therapeutic responses.

Other compounds have been tested by SPR and the results are understandably shown in fig. 1A and 1B.

Examples of Experimental research

Example 20: application of BT-11 to acute model of IBD

Introduction to

Inflammatory Bowel Disease (IBD) is a chronic, recurring disease of the gastrointestinal tract that afflicts over one million and forty thousand people in the united states. IBD comprises two distinct manifestations: ulcerative colitis and Crohn's disease. Current therapies for IBD are moderately successful and have significant adverse side effects on long-term control of the disease [17 ]. Crohn's disease represents the chronic phase of the disease, while acute Ulcerative Colitis (UC) presents an early pathology affecting colon tissue. UC is a chronic idiopathic inflammatory condition of the gastrointestinal tract characterized by mucosal inflammation in the rectum that extends proximally through the colon in a continuous manner to varying degrees. The condition is characterized by a course of recurrence and remission of varying severity. Most patients present with mild to moderate severity of left or distal disease. Most maintain remission for long periods of time with maintenance medication. However, natural history studies indicate that between 10% and 40% of patients will undergo colectomy at some point during their disease course.

Drug treatment of steroid refractory severe UC has expanded somewhat in recent years, with cyclosporine (ciclosporin) and infliximab (infliximab) being available as rescue agents; however, surgery remains the only "curative" option. The present invention provides a novel drug product for the treatment of UC by targeting a novel receptor designated LANCL 2. Our optimized compound BT-11 was administered orally and distributed systemically, and in UC exerted an immunomodulatory effect by targeting LANCL2 in intestinal immune cells. Our preclinical efficacy studies in acute UC in mice demonstrate how administration with BT-11 reduces disease activity index and improves intestinal inflammation by significantly reducing leukocyte infiltration in the intestinal mucosa as well as reducing mucosal thickening and epithelial cell erosion. Gene expression analysis confirmed that oral administration of BT-11 up-regulated IL-10 and LANCL2 expression, and down-regulated TNF α mRNA expression in the acute DSS-induced ulcerative colitis model in mice.

Method of producing a composite material

A mouse. C57BL/6 was purchased from Jackson Laboratory and was housed in a ventilated rack under specific pathogen-free conditions. LANCL 2-/-mice were purchased from the KOMP repository at the University of California Davis school (University of California Davis). All mice were maintained in an animal facility. All protocols were approved by the institutional Animal care and use committee and met or exceeded the guidelines of the National Institutes of Health (National Institutes of Health) Laboratory Animal Welfare and Public Health Service Office (Office of Laboratory Animal and Public Health Service policy) policy.

DSS induces colitis. Colitis was induced in C57BL/6J mice by administration of 5% (w/v) dextran sodium sulfate (DSS; molecular weight 42 kDa; ICN Biochemicals, Aurora, OH) added to drinking water. Colonic inflammation was assessed 7 days after DSS treatment. The group in the DSS project consists of: i. non-DSS and vehicle treated mice, ii.non-DSS and BT-11(80mg/kg) treated mice, iii.dss treated and vehicle treated mice, and iv.dss treated and BT-11(80mg/kg) treated mice. Twelve mice were included in each group.

Histopathology. Colon sections from IBD studies in mice were fixed in 10% buffered neutral formalin, later embedded in paraffin, and then sectioned (5 μm) and stained with H & E stain for histological examination. The colon was graded with mixed histological scores, including (1) leukocyte infiltration, (2) mucosal thickening, and (3) extent of epithelial cell erosion. For each of the previous categories, the slices were ranked with a score of 0-4, and the data was analyzed as a normalized mixed score.

Quantitative real-time PCR. Total RNA was isolated from the mouse colon using the RNEASY PLUS Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA (1. mu.g) for use with ISCRIPT^TMcDNA Synthesis kit (Bio-Rad from Hercules, Eschelles, Calif.) to generate cDNA templates. The total reaction volume was 20. mu.L, where in MJ MINI^TMThe reactions were incubated in a thermal cycler (Bio-Rad) as follows: 5 minutes at 25 ℃, 30 minutes at 52 ℃,5 minutes at 85 ℃ and maintained at 4 ℃. PCR was performed on the cDNA using Taq DNA polymerase (Life Technologies, Calsbad, Calif.). Each gene amplicon was purified using the MINELUTE PCR purification kit (Qiagen) and on an agarose gel by using a DNA quality ladder (Promega, Madison, Wis.) and using a NanometersDrops (nanodrop) were quantified. These purified amplicons are used to optimize real-time PCR conditions and generate standard curves in real-time PCR assays. Primers were designed using Oligo 6 software. For each primer set, primer concentration and binding temperature were optimized for ICYCLER IQ using a systematic gradient scheme^TMThe system (Bio-Rad) maintained PCR efficiencies between 92% and 105% and correlation coefficients for each primer set during optimization and also during sample DNA real-time PCR>0.98. ICYCLER IQ use by real-time qPCR^TMSystem and IQ^TM Green super mix (Green Supermix) (Bio-Rad) to examine the cDNA concentration of the genes of interest. A standard curve for each gene was generated using a 10-fold dilution of the purified amplicon starting with 5pg cDNA and later used to calculate the initial amount of target cDNA in an unknown sample.Green I is a generic double-stranded DNA insertion dye and thus non-specific products and primers/dimers can be detected in addition to the amplicon of interest. To determine the number of products synthesized during real-time PCR, a melting curve analysis was performed on each product. Real-time PCR was used to measure the initial amount of nucleic acid in each unknown cDNA sample on the same 96-well plate.

And (5) carrying out statistical analysis. ANOVA followed by snow fee multiple comparison (Scheffe's multiple comparison method) was used to analyze the parametric data. Nonparametric data were analyzed by using a Mann-Whitney's U test followed by a Dunn's multiple comparisons test (Dunn's multiple comparisons test). ANOVA was performed by using the general linear model program of SAS version 6.0.3(SAS Institute). Statistical significance was assessed as P.ltoreq.0.05.

Results

BT-11 ameliorates disease and histopathology in a colitis DSS model. The objective of this study was to investigate whether administration of BT-11 activates LANCL2 and exerts anti-inflammatory properties in the context of IBD. To assess the efficacy of our exemplary compound BT-11 in an acute model of IBD, we treated C57BL/6J mice with 5% DSS in a 7 day challenge. Treatment with BT-11 significantly improved disease activity scores throughout the challenge period (fig. 10, panel a). Furthermore, macroscopic lesions in spleen (fig. 10, panel B), MLN (fig. 10, panel C) and colon (fig. 10, panel D) were also significantly reduced after activation of the LANCL2 pathway by BT-11 at day 7 post challenge.

BT-11 ameliorates colon histopathology in a dose-responsive manner in mice with acute inflammatory colitis. We next examined the effect of BT-11 on histopathological inflammatory lesions of the colon. Consistent with our observations of disease activity and overall lesion, histopathological analysis confirmed that treatment with BT-11 significantly reduced inflammation in the intestinal mucosa by 5-fold, based on assessment of leukocyte infiltration (fig. 11, panel G), epithelial cell erosion (fig. 11, panel H), mucosal thickening (fig. 11, panel I). Representative colon micrographs show how treatment with BT-11 significantly improved gut mucosal status and infiltration of several immune subgroups during DSS-induced colitis in mice by improving epithelial cell integrity and reducing disruption of gut architecture (figure 11, panels a-F). We performed a dose response study with BT-11 and we interestingly observed how three markers of colonic inflammation (leukocyte infiltration, mucosal thickening and epithelial erosion) decreased in mice with colitis as the BT-11 dose increased from 10 to 80mg/Kg (figure 12, panels a-C).

Oral treatment with BT-11 reduced TNF α expression and upregulated LANCL2 and IL-10. To investigate more closely the effect of BT-11 on the modulation of the immune system, we assessed the genetic expression of IL-10, LANCL2 and TNF α. The results show how treatment with BT-11 down-regulates the expression of tumor necrosis factor alpha (TNF α) (fig. 13, panel a) and up-regulates the levels of interleukin 10(IL-10) (fig. 13, panel B) and LANCL2 receptors (fig. 13, panel C), thus generating a positive feedback loop (positive feedback loop) that promotes anti-inflammatory effects and down-regulates the inflammatory response driven by TNF α by performing a dose response study, we can hypothesize that subsequent activation of our ligand BT-11 and LANCL2 pathways directly increases the production of colonic IL-10 when colonic IL-10 expression assessed by flow cytometry is performed after the kinetics of dose response with BT-11 (fig. 14, panel B). We observed that the reduction of colon TNF α expressing cells was significantly different in both cases of 40 and 80mg/Kg BT-11, but not in the case of lower doses (e.g. 10 or 20mg/Kg) (fig. 14, panel a) we also observed how FOXP3 expression in MLN was dose dependent (fig. 14, panel C).

The effect of BT-11 during acute colitis was dependent on LANCL 2. To demonstrate how the beneficial effects of administration with BT-11 are exerted during acute colitis in mice, we performed a study comparing such effects in wild-type and LANCL2 knockout (LANCL2-/-) mice. Our results demonstrate that LANCL2 is necessary for BT-11 to exert its anti-inflammatory benefits, as loss of LANCL2 prevents mice from recovering from acute DSS-induced colitis (figure 15, panel a). Likewise, when comparing wild-type and LANCL 2-/-littermates, loss of LANCL2 abolished the reduction in macroscopic scores in the colon (fig. 15, panel B), MLN (fig. 15, panel C) and spleen (fig. 15, panel D). Furthermore, the role of BT-11 in lesion formation in colonic mucosa also was LANCL 2-dependent, as we assessed histopathological analysis in LANCL 2-/-mice treated with vehicle or BT-11, and we observed how loss of LANCL2 completely abolished BT-11 effects (fig. 16).

To further characterize the cellular response after treatment with BT-11, we performed a further LANCL2 knockout study to determine whether the reduction of pro-inflammatory proteins and the increase of anti-inflammatory factors were impaired. Our flow cytometry results demonstrated that the reduction of the pro-inflammatory factor MCP1 had a LANCL2 dependence in colon (fig. 17, panel a) and MLN (fig. 17, panel B) because the loss of the LANCL2 gene abrogated the BT-11 effect. We also found that TNF α secretion in the colon was LANCL2 dependent (fig. 17, fig. C), and that upregulation of the MHC-II + CD11C + granulocyte population was LANCL2 dependent (fig. 17, fig. D). Consistent with these results, we found that upregulation of IL-10 secretion following BT-11 treatment was completely abolished in colon (fig. 17, panel E) and spleen (fig. 17, panel F) in LANCL2 knockout mice, again demonstrating the dependence of our optimized compounds on our targets of interest.

Discussion of the related Art

LANCL2 has emerged as a novel therapeutic target for inflammation and immune-mediated diseases [18 ]. Our in vivo results demonstrate for the first time that oral treatment with LANCL2 ligand BT-11 improves intestinal immunopathology in a mouse model of IBD by suppressing inflammation. LANCL2 has recently received some attention as a potential therapeutic target due to its function associated with ABA binding and signaling [19] and the recently discovered alternative membrane-based mechanism of PPAR γ activation [8 ]. Furthermore, we determined the expression of LANCL2 in a series of mouse tissues, which showed that, in addition to brain and testes, LANCL2 is also expressed in other tissues, such as thymus, spleen, colon and Peyer's patch (Peyer's patch), indicating a possible association between LANCL2 and immune responses and suggesting a broader potential of LANCL2 as a therapeutic target.

Previously, we have reported that ABA activates PPAR γ transactivation (transactivate) in vitro and suppresses systemic inflammation similar to other PPAR γ agonists. Since both ABA and NSC61610 target LANCL2, NSC61610 may also act via PPAR γ activation. The experimental results show that NSC61610 treatment activates PPAR γ in the original macrophage, thereby providing evidence of a potential signaling association between LANCL2 and PPAR γ and indicating that NSC61610 may target the LANCL2-PPAR γ axis in vitro. To investigate the importance of LANCL2 in NSC 61610-mediated activation of PPAR γ, we determined whether knocking out the effect of LANCL2 in the original macrophages by using siRNA attenuated or abolished the effect of NSC61610 on PPAR γ reporter activity. Our findings indicate that knockout of LANCL2 significantly attenuated the effect of NSC61610 on PPAR γ activity [12 ]. In this example, we show how administration of BT-11 exerts anti-inflammatory properties by not only reducing disease activity index scores and macroscopic scores in spleen, MLN and colon (fig. 10), but also significantly reducing histopathological lesions (fig. 11). We show how these two specific effects depend on LANCL2 (fig. 15 and 16). We also demonstrated that BT-11 reduced TNFa levels and up-regulated LANCL2 and IL-10 (FIG. 13). We also demonstrated that the effect here is LANCL2 dependent, since we did not observe these trends in LANCL 2-/-mice (fig. 17). These results confirm that LANCL2 is a novel therapeutic target for inflammatory diseases and BT-11 is a compound targeted to it.

Example 21: use of BT-11 in chronic model of Crohn's disease

Introduction to

As stated above, Inflammatory Bowel Disease (IBD) and its two clinical manifestations ulcerative colitis and crohn's disease are immune-mediated diseases characterized by extensive inflammation and immune cell infiltration of the gastrointestinal tract. The etiology of IBD is multifactorial and causes interactions among genetic predisposition, environmental factors and intestinal microbiota.

The present example will focus on chronic IBD performance: crohn's disease. Inflammation in ulcerative colitis is characterized by a continuous pattern involving the superficial mucosa and submucosa but limited to the colon, whereas in crohn's disease, the inflammation is transmural and discontinuous, and beyond the most affected ileum, any region of the intestinal tract may be affected. The pathogenesis of crohn's disease is complex and is influenced by genetic and environmental factors as well as by immune-mediated damage to the intestinal mucosa caused by long-term activation of the mucosal immune system.

Therapies aimed at down-regulating immune and inflammatory responses, such as the corticosteroid prednisone or the anti-TNF- α antibody(Janssen Biotech, Inc., of Horsham, Pa.) has shown promise for reducing disease severity and reducing its recurrence. However, these treatments are also associated with various adverse side effects, such as cushing's-like appearance, weight gain, and systemic immunosuppression, and there is an urgent need to develop safe alternatives for long-term management of IBD [20]。

The present invention provides a novel drug product for the treatment of crohn's disease by targeting a novel receptor known as LANCL 2. The exemplary compound BT-11 was administered orally and distributed systemically, and exerted immunomodulatory effects by targeting LANCL2 in intestinal immune cells, not only in UC, but also in crohn's disease. Our preclinical efficacy studies in a chronic model of crohn's disease in mice demonstrate how administration with BT-11 reduces the disease activity index and improves intestinal inflammation by significantly reducing leukocyte infiltration in the intestinal mucosa as well as reducing mucosal thickening and epithelial cell erosion. Gene expression analysis confirmed that oral administration of BT-11 up-regulated LANCL2 expression and down-regulated TNF α mRNA expression in a chronic model of IBD in mice. In addition, administration of BT-11 reduced proinflammatory macrophage and dendritic cell infiltration into the colonic lamina propria, as well as up-regulated FOXP 3-expressing CD4+ T cells and down-regulated numbers of effector Th1 cells in the colon. We also performed knockout studies to confirm that these effects are LANCL2 dependent. Finally, in the induction site, BT-11 was able to down-regulate the production of Th17 cells and up-regulate the regulatory CD4+ T cell compartment via up-regulation of FOXP3 expression.

Method of producing a composite material

A mouse. C57BL/6 and IL-10 knockout mice were purchased from Jackson Laboratory and housed in ventilated shelves under specific pathogen-free conditions. LanCL 2-/-mice were purchased from the KOMP repository at Davis division, university of California. All mice were maintained in an animal facility. All experimental protocols were approved by the institutional animal care and use committee and met or exceeded guidelines for national institutes of health laboratory animal welfare and public health services office policies.

CD4+ T cells were enriched and sorted. Splenocytes obtained from C57BL/6J (wild-type) mice were enriched with CD4+ T cells by magnetic negative sorting using an I-Mag cell isolation system (BD Pharmingen). Cells were incubated with biotin-labeled Ab mixture followed by a second incubation with streptavidin particles and exposed to a magnet to remove undesirable cells. The purity of the CD4+ enriched cell suspension was between 93% and 96%. CD4+ enriched cells were used for adoptive transfer or further purified by FACS. For FACS sorting, cells were labeled with CD45RB, CD4 and CD25 and sorted in FACSARIA^TMCell sorter (BD Biosciences of San Jose, Calif.) separated into CD4+ CD45RBhigh CD 25-cells (i.e., effector T cells). The purity of FACS sorted CD4+ subset was 98% or more.

Intraperitoneal (i.p.) administration of 4 × 10 to six week old SCID and RAG 2-/-mice⁵CD4+ CD45RBhigh CD 25-from C57BL/6J (wild type) or LANCL 2-/-mice. Mice were weighed weekly and clinical signs of disease were recorded daily for 14 weeks. Mice developing signs of severe wasting disease were killed. Otherwise, mice were sacrificed at 90 days after transfer. The cohorts used for adoptive transfer studies were as follows: i. non-metastatic and vehicle treatment, ii.non-metastatic and BT-11(80mg/kg) treatment, iii.metastatic and vehicle treatment, iv.metastatic and BT-11(80mg/kg) treatment. 12 mice were used in each group.

Histopathology. Colon sections from IBD studies in mice were fixed in 10% buffered neutral formalin, later embedded in paraffin, and then sectioned (5 μm) and stained with H & E stain for histological examination. The colon was graded with mixed histological scores, including (1) leukocyte infiltration, (2) mucosal thickening, and (3) extent of epithelial cell erosion. For each of the previous categories, the slices were ranked with a score of 0-4, and the data was analyzed as a normalized mixed score.

And (5) separating the cells. Spleen and Mesenteric Lymph Nodes (MLN) were excised and crushed in 1 x PBS/5% FBS using the frosted ends of two sterile microscope slides. The single cell suspension was centrifuged at 300 × g for 10 min and washed once with 1 × PBS. The red blood cells are removed by osmotic lysis prior to the washing step. All cell pellets were resuspended in FACS buffer (1 × PBS supplemented with 5% FBS and 0.09% sodium azide) and subjected to flow cytometric analysis. In parallel, the colon is resected and lamina propria white blood cells (LPLs) are isolated. Tissue pieces were washed in CMF (1 × HBSS/10% FBS/25mM Hepes) and the tissues were incubated with CMF/5mM EDTA twice for 15 minutes each, while stirring at 37 ℃. After washing with 1 × PBS, the tissue was further digested in CMF supplemented with 300U/ml collagenase type VIII and 50U/ml DNase I (both Sigma-Aldrich) for 1.5 hours at 37 ℃ with stirring. After filtration of the supernatant, the cells were washed once in 1 × PBS, the beads were resuspended in FACS buffer, and flow cytometric analysis was performed on them.

Immunophenotyping and cytokine analysis by flow cytometry for fluorescent staining of a subset of immune cells, 4-6 × 10⁵The individual cells were incubated with the following fluorescent dye-binding primary mouse-specific antibodies for 20 minutes, anti-CD 3PE-Cy5 clone strain 145-2C11 (eBioscience of San Diego, Calif.), anti-CD 4 PE-Cy7 clone strain GK1.5(eBioscience), anti-CD 4 APC clone strain RM4-5 and anti-CD 25 biotin clone 7D4(BDbiosciences), cells were washed with FACS buffer (1 × PBS supplemented with 5% FBS and 0.09% sodium azide), for intracellular staining of transcription factors and cytokines, cells were fixed and permeabilized using a commercial kit according to the manufacturer's instructions (Bioscience.) briefly, cells were permeabilized for 20 minutes with fixed cells, cells were permeabilized with mouse anti-CD 16/CD32 FcBlots (BD) until blocking the receptor, and the following fluorescent dye-binding of primary mouse-specific antibodies were used for the following fluorescent dye-binding of mouse-Cy 5 clone strain, anti-Cy 7 clone strain GK1.5(eBioscience), anti-CD 4-APC clone RM4-5 and anti-CD 25 BioFACS clone 7D4 (BDBioFACS). The buffer was applied to the intracellular staining of transcription factors until all murine cells were stained with fluorescent dye-BioFACS buffer (BioFACS) and anti-CD 11) until all samples were collected on a sample-CD 3, CD32 FcBlocks were applied to the mouse monoclonal antibody-CD 19, BioFACS, CD3, CD-CD 3-CD^TMData analysis was performed (BD Biosciences) and Flow Jo (Tree Star Inc).

Quantitative real-time PCR. Total RNA was isolated from mouse colon using RNEASY PLUS Mini kit (Qiagen) according to the manufacturer's instructions. Total RNA (1. mu.g) for use with ISCRIPT^TMcDNA Synthesis kit (Bio-Rad) to generate cDNA templates. The total reaction volume was 20. mu.L, where in MJ MINI^TMThe reactions were incubated in a thermal cycler (Bio-Rad) as follows: 5 minutes at 25 ℃, 30 minutes at 52 ℃,5 minutes at 85 ℃ and maintained at 4 ℃. PCR was performed on the cDNA using Taq DNA polymerase (Invitrogen). Each gene amplicon was purified using the MINELUTE PCR purification kit (Qiagen) and in agaroseQuantification was performed on the gel by using a DNA mass ladder (Promega corporation) and using nanodroplets. These purified amplicons are used to optimize real-time PCR conditions and generate standard curves in real-time PCR assays. Primers were designed using Oligo 6 software. For each primer set, primer concentration and binding temperature were optimized for ICYCLER IQ using a systematic gradient scheme^TMThe system (Bio-Rad) maintained PCR efficiencies between 92% and 105% and correlation coefficients for each primer set during optimization and also during sample DNA real-time PCR>0.98. ICYCLER IQ use by real-time qPCR^TMSystem and IQ^TM Green super mixes (Bio-Rad) were examined for cDNA concentration of the genes of interest. A standard curve for each gene was generated using a 10-fold dilution of the purified amplicon starting with 5pg cDNA and later used to calculate the initial amount of target cDNA in an unknown sample.Green I is a generic double-stranded DNA insertion dye and thus non-specific products and primers/dimers can be detected in addition to the amplicon of interest. To determine the number of products synthesized during real-time PCR, a melting curve analysis was performed on each product. Real-time PCR was used to measure the initial amount of nucleic acid in each unknown cDNA sample on the same 96-well plate.

And (5) carrying out statistical analysis. Parameter data were analyzed using ANOVA followed by multiple comparisons of snow costs. Nonparametric data were analyzed by using the mann-whitney U test followed by dunne multiple comparison test. ANOVA was performed by using the general linear model program of SAS version 6.0.3(SAS Institute). Statistical significance was assessed as P.ltoreq.0.05.

Results

BT-11 improves disease activity in a chronic IL-10-/-model of IBD. In view of the fact that IL-10 is known to suppress the secretion of many proinflammatory cytokines [21], various animal studies investigating the chronic nature of Crohn's disease have employed an interleukin-10 deficient mouse (IL-10-/-) mouse model. To assess the efficacy of BT-11 not only in acute but also in chronic models of colitis, we set up a colitis IL-10 knockout mouse model study and treated the mice with increasing doses of BT-11(20, 40 and 80 mg/Kg). In treated mice, treatment with BT-11 significantly reduced disease activity index scores compared to their vehicle-treated littermates (fig. 18). Furthermore, mice treated with the highest dose of BT-11(80mg/Kg) significantly reduced scores compared to those treated with 20 or 40mg/Kg BT-11 compound starting at week 13 and until the end of the experiment.

BT-11 reduced macroscopic lesions in spleen, MLN and colon in IL 10-/-chronic model of IBD. To initially determine clinical efficacy, we assessed macroscopic tissue lesions after treatment with BT-11 and subsequent activation of the LANCL2 pathway. At 19 weeks after study initiation, we performed macroscopic scoring of spleen (fig. 19, panel a), MLN (fig. 19, panel B) and colon (fig. 19, panel C) immediately after euthanasia and tissue collection. Treatment with BT-11 at concentrations as low as 20mg/Kg greatly and significantly reduced macroscopic scores in the three tissues, exhibiting its potent efficacy.

BT-11 ameliorates histopathological changes and inflammation in the IL-10-/-chronic model of IBD. To assess histopathological lesions and general pathology in the intestinal mucosa, sections of colon were stained with H & E and observed under a microscope. Our results show how treatment with BT-11 significantly reduced inflammation based on reduction of leukocyte infiltration (fig. 20, panel a), epithelial cell erosion (fig. 20, panel B), and mucosal thickening (fig. 20, panel C). We also observed a dose-dependent mechanism of the amount of infiltration in the intestinal mucosa associated with mucosal thickening.

Treatment with BT-11 induced a potent anti-inflammatory response in colonic lamina propria, spleen and MLN and reduced the pro-inflammatory subset. To determine the effect of BT-11 on immune cell subsets, we phenotypically characterized cells isolated from colon, spleen and MLN. Our analysis indicated that BT-11 significantly reduced the percentage of pro-inflammatory F4/80+ macrophages (fig. 21, panel a), MHC-II + CD11c + dendritic cells (fig. 21, panel B), and effector Th1 cells (fig. 21, panel D) in the colonic lamina propria. Furthermore, BT-11 exerted anti-inflammatory properties via upregulation of FOXP 3-expressing CD4+ T cells in colonic LP (fig. 21, panel C).

Upregulation of FOXP 3-expressing CD4+ T cells was also noted in MLN (fig. 22, panel B) and spleen (fig. 22, panel C), also showing and demonstrating how BT-11 has a systemic effect. Downregulation of the pro-inflammatory Th1 cells was also observed in the spleen in a dose-responsive manner (figure 22, panel D). Finally, effector Th17 cells, characterized by their ROR γ t expression, were down-regulated in MLN (fig. 22, panel a).

Furthermore, gene expression analysis confirmed that treatment with BT-11 up-regulated colonic LANCL2 expression (fig. 23, panel a) and down-regulated TNF α expression (fig. 23, panel B). These expression effects are dose-dependent on the amount of BT-11 administered.

BT-11 exhibits improvement in disease activity in CD4+ T cell induction in models of IBD colitis. To further validate the efficacy of BT-11 in another chronic model of IBD, we adoptively transferred untreated CD4+ T cells from wild-type and LANCL 2-/-mice into the RAG 2-/-recipient. RAG 2-/-mice were treated with vehicle or BT-11 based on experimental design. In treated mice, treatment with our optimized compound BT-11 significantly reduced disease activity index scores when compared to their wild type littermates (fig. 24). We found that these results were LANCL2 dependent (FIG. 25) since the effects of BT-11 were completely eliminated with the loss of LANCL 2.

Interestingly, weight loss was significantly improved in BT-11 treated mice when compared to vehicle treated mice starting at 7 weeks until the end of the experiment (fig. 26).

BT-11 reduces macroscopic lesions in spleen, MLN and colon in a chronic model of adoptive transfer of IBD. To confirm clinical efficacy in the second model of chronic colitis, we assessed macroscopic tissue lesions in mice adoptively transferred with wild-type or LANCL 2-/-cells and treated with vehicle or BT-11 after treatment with BT-11 and subsequent activation of the LANCL2 pathway. At 11 weeks after study initiation, we performed macroscopic scoring of spleen (fig. 27, panel a), MLN (fig. 27, panel B) and colon (fig. 27, panel C) and ileum (fig. 27, panel D) immediately after euthanasia and tissue collection. Treatment with BT-11 at a concentration of 80mg/Kg greatly and significantly reduced the macroscopic score in the four tissues, exhibiting its potent efficacy. We found that these observations were LANCL2 dependent, and that loss of LANCL2 completely abolished the effect of BT-11 (fig. 28).

BT-11 also ameliorates histopathological changes and inflammation in models of adoptive transfer of chronic colitis. Similar to the IL-10-/-induced colitis experiment and in order to confirm histopathological lesions and general pathology in the intestinal mucosa with the second mouse model of IBD, colon sections were stained with H & E and observed under a microscope. Our results confirmed how treatment with BT-11 significantly reduced inflammation based on the reduction of leukocyte infiltration (fig. 29, panels a and B) and mucosal thickening (fig. 29, panels E and F) in the colon and ileum. Notably, epithelial erosion of the ileum was less affected (fig. 29, panel D), but erosion in the colon was found to be significantly lower in mice treated with our optimized compound BT-11 (fig. 29, panel C). To confirm the BT-11 dependence on LANCL2, we performed adoptive transfer studies and transferred CD4+ T cells from LANCL 2-/-donors. Our results show how the reduction of leukocyte infiltration, epithelial erosion and mucosal thickening is greatly eliminated in recipients of LANCL 2-/-metastases (fig. 30).

BT-11 constantly induces a tremendous anti-inflammatory response in mice and down regulates pro-inflammatory mediators. To characterize the immune cell profile in mice treated with BT-11 contrast media, we performed flow cytometry analysis in cells isolated from colon, spleen and mesenteric lymph nodes. We confirmed that recipient mice treated with BT-11 for a period of 11 weeks had significantly lower levels of infiltrating F4/80+ CD11B + pro-inflammatory macrophages in the colon (fig. 31, panel a) and decreased levels of IFN γ based on analysis of total CD45+ leukocytes (fig. 31, panel B) in the second chronic mouse model of IBD. Furthermore, treatment with BT-11 consistently upregulated regulatory CD4+ T cells by promoting expression of FOXP3 (fig. 31, panel C) and the potent anti-inflammatory cytokine IL-10 (fig. 31, panel D) at the site of local inflammation (in this case, the colonic mucosa).

Similar to the profile observed in colon lamina propria cells, we characterized these populations in the induction sites (e.g. spleen and MLN). Immunophenotypic results show how treatment with BT-11 also increased FOXP3 and IL-10 levels in induction sites (e.g., spleen and MLN) (FIG. 32, FIG. A, B, D and E). Notably, BT-11 treatment reduced IFN γ expression in both MLN and spleen in CD45+ populations (fig. 32, panels C and F).

The effect of BT-11 targeting LANCL2 was independent of PPAR γ. Based on our experimental results, activation of LANCL2 activates a plethora of pathways that ultimately regulate the anti-inflammatory response based on IL-10, which regulates inflammation at the systemic level. One activated LANCL2 downstream pathway is the PPAR γ pathway. To help overcome the potential toxicological problems of secondary activation of this nuclear and transcription factor, we also transferred RAG 2-/-mice with CD4+ T cells from PPAR γ -/-donors. We then treated these mice with vehicle or 80mg/Kg BT-11. Our results clearly demonstrate that the beneficial effects of BT-11 on disease activity and histopathology via LANCL2 activation proceed in a PPAR γ -independent manner (fig. 33, panels a-D). These results demonstrate that activation of LANCL2 also modulates other pathways that modulate the anti-inflammatory effects of LANCL2 activation.

Discussion of the related Art

Current therapies directed against Inflammatory Bowel Disease (IBD) are moderately successful and have significant adverse side effects on long-term control of the disease [17 ]. The plant compound abscisic acid (ABA) exerts potent anti-inflammatory effects in mouse models of colitis [22, 23 ]. Lanthionine synthase component C-like protein 2(LANCL2) is a target for ABA binding and signaling [15, 19, 24 ]. Thus, LANCL2 has emerged as a promising novel therapeutic target against inflammation [18 ]. Compound 61610 (bis (benzimidazolyl) terephthalanilide, BTT) was identified as binding with highest affinity to LANCL2 in libraries with millions of chemicals. In addition, 61610 exerts potent anti-inflammatory effects in a mouse model of intestinal inflammation [25 ]. A 20-topical library of 61610-derived BTTs was generated and BT-11 was identified as the best exemplified compound. BT-11 binds to LANCL2, is orally active, has demonstrated anti-inflammatory efficacy and a prominent safety profile in 3 colitis mouse models.

According to the united states foundation of crohn's disease and colitis, IBD afflicts more than 1 million people in north america and 4 million people worldwide. This widespread and debilitating disease results in a reduction in quality of life and a significant health care related cost [26 ]. In the United states, the average medical cost for treating a single episode of IBD exceeds $55,000[27] per patient, and the total cost exceeds $150 billion per year. In addition, indirect costs include the cost of treating recurrent pancreatitis [28] or other complications of IBD, such as abscesses, ileus, anemia, thrombosis, perianal lesions, arthritis, uveitis, iritis, or skin lesions [29 ]. IBD places a great burden on the patient, often isolating him from society, affecting family relations and limiting his professional opportunities [17 ]. In this regard, IBD patients are not involved in labor at a high rate; this high ratio persists over time [30 ]. In addition, intestinal inflammation (ulcerative colitis (UC) and Crohn's Disease (CD)) increases the risk of developing colon cancer, especially at early age (< 30 years of age) [31 ]. According to the new reports of global industry analysis companies, the global IBD therapeutic market is expected to reach $43 billion by 2015.

Although current treatments for IBD have improved [17, 32], they have been only moderately successful for chronic disease control and result in significant side effects, including a diminished ability of the immune system to mount a protective immune response against pathogens or malignant diseases. Treatment options for patients include addressing inflammatory symptoms. Most pharmacological treatments used on the market today include aminosalicylates, corticosteroids, immunomodulators, antibiotics, biologicals (anti-tumor necrosis factor-alpha antibodies). Aminosalicylates are extremely effective and generally well tolerated. However, patients with recurrent or more moderate disease may require more aggressive treatment, which includes a short-term dose of corticosteroid for a shorter period of time to control symptoms. This type of fast acting therapy cannot be tolerated for a long period of time. Immunomodulators are also commonly used in CD and UC in order to maintain the pathology, but they have a slower onset of action (3 to 6 months for full effect). These agents have potentially significant adverse side effects ranging from pancreatitis to diabetes to scarred liver and inflamed lung. For moderate to severe disease cases that fail to be controlled with other therapies, anti-TNF-alpha will be administered to the patient intravenously every 6-8 weeks in a controlled setting. This extremely expensive therapy, while effective, is difficult to use due to the need for administration by skilled technicians and clinical settings. In addition, there are significant side effects such as cushing's syndrome, bipolar disorder, insomnia, hypertension, hyperglycemia, osteoporosis, malignant disease, infection, and avascular necrosis of long bones.

Exemplary compound BT-11 has demonstrated an extremely safe toxicological profile. Our efficacy data in chronic models of IBD show how BT-11 treatment improves disease activity scores (figures 18 and 24) and weight loss (figure 26) in two chronic IBD models. Our data show how these effects are LANCL2 dependent (fig. 25) our efficacy data also show that activation of the LANCL2 pathway by BT-11 promotes anti-inflammatory responses characterized primarily by: IL-10 production and FOXP 3-expressing CD4+ T cells (fig. 21, 22, 31 and 32) and significant reduction in inflammatory macrophages, dendritic cells and proinflammatory factors (such as IFN γ) (fig. 22, 23, 31 and 32). Furthermore, gene expression analysis confirmed these cell-based findings by demonstrating how treatment with BT-11 reduced TNF α levels in the colon (fig. 23). All these findings together resulted in a dramatic LANCL 2-dependent improvement in colonic mucosa in terms of leukocyte infiltration, epithelial erosion and mucosal thickening in two chronic IBD models (figures 20, 29 and 30). We have also demonstrated that the effect of BT-11 upon binding to LANCL2 is PPAR γ independent (fig. 33). These results confirm that activation of LANCL2 activates a plethora of downstream activators that regulate inflammation via PPAR γ independent mechanisms. Together, these results strongly support the following facts: LANCL2 is a novel therapeutic target for inflammatory diseases, and BT-11 is useful as a novel drug.

Example 22: use of BT-11 for treating type 1 diabetes (T1D)

Introduction to

Diabetes Mellitus (DM), also referred to simply as diabetes mellitus (diabetes), is a group of metabolic diseases in which high blood glucose levels are present over a long period of time. Two types of diabetes are referred to as type 1 and type 2. The previous names for these conditions are insulin-dependent and non-insulin-dependent diabetes, or juvenile-onset and adult-onset diabetes. In T1D, the body does not produce insulin. With respect to T2D, T1D is not as common at all as T2D. In fact, approximately 10% of all diabetes cases are type 1. T1D plagues 3 million Americans. Each year, over 15,000 children and 15,000 adults in the united states are diagnosed with T1D. The incidence of T1D among children under the age of 14 worldwide is estimated to increase by 3% annually. Patients with T1D require insulin injections to remain viable, but they do not cure the disease or prevent its serious side effects.

Current antidiabetic agents are effective in improving insulin sensitivity but have significant side effects such as cardiovascular complications, hepatotoxicity, weight gain, fluid retention and bladder tumors upon chronic administration. The lanthionine synthase component C-like 2(LANCL2) pathway exerts an antidiabetic effect without side effects [18 ]. BT-11 binds to LANCL2, is orally active, has demonstrated anti-diabetic efficacy and a prominent safety profile in mice.

Method of producing a composite material

A mouse. NOD mice were purchased from Jackson Laboratory and housed in ventilated racks under specific pathogen-free conditions. The mice were maintained in an animal facility. All experimental protocols were approved by the institutional animal care and use committee and met or exceeded guidelines for national institutes of health laboratory animal welfare and public health services office policies.

Assessment of body weight and glucose tolerance. All mice were determined to be normoglycemic (fasting blood glucose levels below 250mg/dl) and of similar weight (20 ± 1.5g) before the study began. Mice were weighed weekly and examined for clinical signs of disease by blinded observers. After a standard 12-hour fast, useA blood glucose meter (Indianapolis, IN) measures glucose. Blood was collected via the lateral tail vein and placed onto a capillary blood collection tube.

Histopathology. Pancreatic sections from NOD studies in mice were fixed in 10% buffered neutral formalin, later embedded in paraffin, and then sectioned (5 μm) and stained with H & E stain for histological examination. Sections were graded with a score of 0-4 depending on lymphocyte infiltration, cell damage and tissue erosion, and data were analyzed as normalized mixed scores.

And (5) carrying out statistical analysis. Parameter data were analyzed using ANOVA followed by multiple comparisons of snow costs. Nonparametric data were analyzed by using the mann-whitney U test followed by dunne multiple comparison test. ANOVA was performed by using the general linear model program of SAS version 6.0.3(SAS Institute). Statistical significance was assessed as P.ltoreq.0.05.

Results

BT-11 decreased fasting blood glucose levels and increased insulin in a type 1 diabetic mouse model.

To determine the effect of BT-11 in modulating blood glucose levels in the T1D mouse model, we performed fasting blood glucose tests at weeks 0, 1,3, 4,5, 10, and 11 after the start of the study. Our results show how mice treated with our compound BT-11 had significantly lower blood glucose levels after a 12 hour period of fasting (fig. 34, panel a). In parallel, we assessed insulin levels at week 5, and our results show how mice treated with BT-11 had significantly elevated plasma insulin levels (fig. 34, panel B).

BT-11 ameliorates clinical histopathological pancreatic lesions and inflammation in the mouse NOD model. To assess histopathological lesions in the T1D mouse model, pancreas was collected and fixed with 10% formalin. The pancreatic sections were then stained with H & E and observed under a microscope. Our results show how treatment with BT-11 significantly reduced clinical histopathological lesions in the pancreas when compared to vehicle-treated mice (fig. 35).

Discussion of the related Art

There is a need for an effective and safe oral agent for type 1 diabetes (T1D), said T1D being a disease that afflicts more than 3 million americans. ABA treatment exerts an antidiabetic effect [2 ]. Lanthionine synthase component C-like protein 2(LANCL2) is a target for ABA binding and signaling [15, 19, 24 ]. Thus, LANCL2 has emerged as a promising novel therapeutic target against inflammation [18 ]. ABA is effective in ameliorating diabetes [2, 33] and immune-mediated diseases such as Inflammatory Bowel Disease (IBD) [22, 23 ]. Compound 61610 (bis (benzimidazolyl) terephthalanilide, BTT) binds with highest affinity to LANCL2 in libraries with millions of chemicals. In addition, 61610 exerts potent immunomodulatory effects in mouse models of intestinal inflammation [25 ]. BT-11 exerts an anti-diabetic effect in NOD mice (FIGS. 34 and 35). In addition, ABA increases insulin secretion in human pancreatic beta-cells [34], suggesting a potential use of ABA as a treatment for type 1 diabetes (T1D).

In immune cells, ABA is recognized by LANCL2, which LANCL2 is a G-protein coupled receptor associated with the cell membrane after myristoylation [19, 35 ]. ABA bound to LANCL2 increases cAMP and triggers signaling through PKA, and modulates immune responses in macrophages and T cells [8 ]. We performed homology modeling by using the crystal structure of LANCL1 as a template to construct the three-dimensional structure of LANCL 2. Using molecular docking, first in silico simulated and then demonstrated ABA binding to LANCL2 in vitro. This computational prediction was validated by SPR results and binding analysis with human LANCL2 [35 ]. We performed virtual screening based on LANCL2 using the LANCL2 structure obtained via homology modeling to find a new LANCL2 ligand. Compounds from NCI Diversity Set II, chemcridge and ZINC natural products databases were docked into the LANCL2 model with Auto Dock and ranked by affinity calculations. While ABA has a high affinity for LANCL2, other diene-containing natural compounds (e.g., 61610) are also predicted to bind in the same region and may also be considered LANCL 2-binding drugs [12 ]. BT-11 also exhibited strong binding to LANCL2 and therapeutic efficacy in NOD mouse model of T1D (fig. 34). This data provides some validation that the LANCL2 pathway and other compounds of the invention are useful as immunomodulatory drugs for T1D. Further evidence supporting the role of the LANCL2 pathway as a means of modulating immune responses and ameliorating autoimmune disease includes the LANCL2 binding and protective effects of ABA [22, 23], 61610[12, 18] and BT-11 in a mouse model of Inflammatory Bowel Disease (IBD).

The incidence of T1D increases worldwide at an estimated rate of 3% per year [36-38 ]. Although successful transplantation of pancreatic islets can treat T1D, the lack of sufficient islets, ongoing immune-mediated destruction of transplanted islets, and the side effects of immunosuppressive drugs greatly limit the widespread use of this approach [39 ]. Thus, therapies that safely combine the ability to promote pancreatic β -cell function with immune modulation are the basic strategy for treatment of T1D. Our data demonstrate that activation of LANCL2 by BT-11 not only improved the glucose level in blood, but also improved its normalization after glucose challenge (fig. 34). In addition, treatment with BT-11 during the T1d episode improved histopathology in the pancreas (fig. 35). Indeed, ABA suppresses inflammation and improves glucose tolerance both prophylactically and therapeutically [2, 3 ]. Thus, natural activation of LANCL2 leads to immunomodulation [12, 18, 22, 23] and glucose homeostasis as illustrated by its therapeutic role in IBD, which is attributed to inflammation suppression and enhanced insulin sensitivity [2, 3 ]. Based on this background and the data presented in fig. 34 and 35, the study of the role of LANCL2 as a T1D therapeutic target is crucial.

Example 23: use of BT-11 for treating type 2diabetes (T2D)

Introduction to

Diabetes Mellitus (DM) is a chronic condition that occurs when the body fails to produce sufficient insulin or fails to use insulin effectively, and is induced by a coupling of genetic predisposition to environmental factors. Unlike people with type 1 diabetes, type 2diabetes is capable of producing insulin. However, the pancreas of such patients does not produce sufficient insulin, or the body does not use insulin sufficiently. This phenomenon is called insulin resistance. Glucose cannot be processed and used when there is not enough insulin or insulin is not used in the way it should be used. Thus, when glucose accumulates in the bloodstream rather than entering a cell and being metabolized, other cells in the system are unable to function properly. Indeed, hyperglycemia and diabetes are important causes of morbidity and mortality due to cardiovascular disease (CVD), nephropathy, neuropathy, foot ulcers and retinopathy.

About 2830 million americans have type 2diabetes (T2D), and over 40.1% of middle-aged people have prediabetes, a condition characterized by impaired glucose tolerance, systemic inflammation, and insulin resistance. The world health organization estimates that the number of people with T2D will increase to 3 hundred million 6600 ten thousand by 2030.

As stated above, current anti-diabetic agents are effective in improving insulin sensitivity, but their chronic administration has significant side effects such as cardiovascular complications, hepatotoxicity, weight gain, fluid retention, and bladder tumors. The lanthionine synthase component C-like 2(LANCL2) pathway exerts an antidiabetic effect without side effects [18 ]. BT-11 binds to LANCL2, is orally active, has demonstrated anti-diabetic efficacy and a prominent safety profile in mice.

Method of producing a composite material

Mice and dietary treatments. C57BL/6 and db/db mice were purchased from Jackson Laboratory and housed in ventilated shelves under specific pathogen-free conditions. Mice in the diet induced obesity diabetes model (DIO) were fed a high fat diet (40 Kcal% fat). The mice were maintained in an animal facility. All experimental protocols were approved by the institutional animal care and use committee and met or exceeded guidelines for national institutes of health laboratory animal welfare and public health services office policies.

Assessment of body weight and glucose tolerance. All mice were determined to be normoglycemic (fasting blood glucose levels below 250mg/dl) and of similar weight (weight ± 1.5g) before the study began. Mice were weighed weekly and examined for clinical signs of disease by blinded observers. Glucose was measured on different days after a standard 12-hour fast. Briefly, blood was collected via the lateral tail vein and placed onto a capillary blood collection tube. The mice were then administered a glucose tolerance test by intraperitoneal injection of D-glucose (2g per kg body weight) and blood samples were collected prior to injection (time 0) (corresponding to baseline FBG levels after a 12 hour fasting starting at 6 am) and at 15, 60 and 90 minutes (db/db model) or 15, 30, 60, 90, 120, 180, 220 and 265 minutes (DIO model) after glucose injection. Abdominal (epididymal) White Adipose Tissue (WAT), subcutaneous WAT and liver were then excised and weighed. Abdominal (epididymal) WAT was then digested and fractionated.

Digestion of white adipose tissue. Abdominal WAT was excised, weighed, chopped into <10mg pieces, and placed into digestion medium (1 × hbss (Mediatech) containing collagen type II protease (0.2%, sigma-aldrich) supplemented with 2.5% HEPES (Mediatech, Herndon, VA) and 10% fetal bovine serum). The samples were incubated in an incubator at 37 ℃ for 30 minutes, filtered through a 100 μm nylon cell filter to remove undigested particles, and centrifuged at 1000 × g for 10 minutes at 4 ℃. The pellet consisting of Stromal Vascular Cells (SVC) was washed with 1 × HBSS and centrifuged at 1000 × g for 10 min at 4 ℃. The supernatant was discarded, and erythrocytes were lysed by incubating SVC in 2mL of erythrocyte lysis buffer for 2 minutes, followed by stopping the reaction with 9mL of 1 × PBS. The cells were then centrifuged again at 1000 Xg for 10 minutes at 4 ℃, suspended in 1mL of 1 XPBS, and counted using a Coulter counter (Beckman Coulter, Fullerton, Calif.).

For immunophenotyping, SVC was seeded at 2 × 105 cells/well into 96-well plates (Costar.) after an initial 20 min incubation with FcBlock (20. mu.g/mL; BD Biosciences-Pharmingen) to inhibit non-specific binding, cells were washed in PBS (FACS buffer) containing 5% serum and 0.09% sodium azide and stained with specific primary anti-mouse antibodies flow cytometry results were calculated with a FacsAria flow cytometer and FACSDIVA was used^TM(BD Biosciences) and FlowJo (TreeStar) performed data analysis. .

And (5) real-time quantitative PCR. Total RNA was isolated from adipose tissue using the RNEASY lipid mini kit (Qiagen) and from cells using the RNEASY mini kit (Qiagen) according to the manufacturer's instructions. Total RNA for use with QSCRIPT^TMcDNA synthesis kit (Gaithersburg, M. in Maryland)D) Quanta Biosciences) to generate complementary dna (cdna) templates. The total reaction volume was 20. mu.L, where in MJ MINI^TMThe reactions were incubated in a thermal cycler (Bio-Rad) as follows: 5 minutes at 25 ℃, 30 minutes at 52 ℃,5 minutes at 85 ℃, held at 4 ℃. Each gene amplicon was purified using the MINELUTE PCR purification kit (Qiagen) and quantified on an agarose gel by using a DNA quality ladder (plomega). These purified amplicons are used to optimize real-time PCR conditions in real-time PCR assays. For each primer set, the primer concentration and the binding temperature were optimized using a system gradient protocol for the CFX system (Bio-Rad) maintaining PCR efficiency between 92% and 105% and a correlation coefficient above 0.98 for each primer set during optimization and also during sample DNA real-time PCR. Data are presented using the Δ Δ Ct quantification method.

Results

BT-11 reduced fasting blood glucose levels in the DIO model of T2D mice. To assess the efficacy of exemplary compound BT-11 in the T2D model, we fed a high fat diet (DIO model) to C57BL/6 mice. At week 12 of high fat feeding, oral BT-11 administration significantly reduced blood glucose levels in BT-11 treated mice when compared to their vehicle-treated littermates (fig. 36, panel a). Furthermore, after 12 hours of fasting and glucose challenge at 2g per kg body weight via IP, mice treated with BT-11 were able to normalize blood glucose levels much faster than untreated mice (fig. 36, panel B).

BT-11 treatment reduced proinflammatory macrophage infiltration and proinflammatory granulocytes in white adipose tissue. To characterize the cells infiltrating white adipose tissue, abdominal WAT was collected and digested as specified in the methods section. Flow cytometry analysis was performed to assess different proinflammatory populations in WAT. Our results show how treatment with BT-11 significantly reduced the levels of F4/80+ CD11B + pro-inflammatory macrophages (fig. 37, panel a) and the number of pro-inflammatory granulocytes with high levels of Ly6c (GR1+ Ly6c high) (fig. 37, panel B).

BT-11 reduced fasting blood glucose levels in the db/db model of T2D mice. To evaluate the therapeutic efficacy of oral BT-11 treatment in two diabetic mouse models, we also used db/db mice, which developed self-developed T2D due to mutations in the leptin receptor. BT-11 was administered to db/db mice by oral gavage at a daily dose of 80 mg/Kg. We determined the effect of BT-11 on glucose homeostasis by measuring fasting blood glucose concentration. Treatment with BT-11 significantly reduced blood glucose levels as early as one week compared to littermates treated with its vehicle, making the difference over time more prominent at week 3 (fig. 38, panel a). To determine whether oral BT-11 treatment modulates how animals induce glucose homeostasis, we administered an intraperitoneal glucose challenge to experimental animals and evaluated plasma glucose kinetics from 0 to 265 minutes post glucose injection. Blood samples were collected prior to injection (time 0), corresponding to baseline FBG levels after a 12 hour fast. Our results show how oral treatment with BT-11 significantly reduced glucose levels prior to IP glucose challenge (time 0, fig. 38, panel B). After glucose challenge in the db/db model, our results show how glucose levels in mice treated with our optimal compound BT-11 fall to normal levels more rapidly than vehicle-treated mice (fig. 38, panel B).

BT-11 reduced the mRNA levels of TNF α and MCP-1 and up-regulated LANCL 2. To further confirm the anti-inflammatory efficacy of BT-11, we assessed gene expression on WAT as indicated in the methods section. Our results show how BT-11 treated mice had higher expression levels of LANCL2 and significantly reduced mRNA levels of the pro-inflammatory factors TNF α and MCP-1 when compared to untreated mice (figure 39).

Discussion of the related Art

As the rate of obesity and type 2diabetes (T2D) continues to rise in the united states, more and more people become dependent on oral antidiabetic drugs. About 2830 million (8.3% of the population) Americans have T2D, and more than 40.1% middle-aged have prediabetes, a condition characterized by impaired glucose tolerance and insulin resistance [40]. In the United states, the total direct and indirect costs attributable to T2D exceed $1320 billion [40 $ [, N]. Despite this growthHowever, pharmaceutical manufacturers have not been able to develop safe and effective agents. One of the most popular and effective oral antidiabetic agents is the insulin sensitiser of the Thiazolidinedione (TZD) class. Although TZD enhances insulin sensitivity, it has significant adverse side effects that limit its availability, including weight gain, congestive heart failure, bladder cancer, hepatotoxicity and fluid retention [41, 42]. For example, approximately 10% -15% of patients using TZDs are forced to stop treatment due to edema, and the increase in extracellular volume from excessive fluid retention is also a major problem in individuals with pre-existing congestive heart failure. In 2000, troglitazoneAfter the first 3 years, it was removed from the market due to severe liver damage and death reports [43]. Security concerns with other TZDs create mandatory black box markers and subsequent use restrictions.

LANCL2 is a second member of the LanC-like protein family to be identified. The first member, LANCL1, was isolated from human erythrocyte membranes [44 ]. LANCL2 was subsequently identified and expressed throughout the body [1, 18], including immune cells, pancreas, lung and intestine [1, 44 ]. The lanthionine synthase C-like 2(LANCL2) pathway emerged as a novel therapeutic target for T2D [18 ]. A number of preclinical tests provide ample evidence for the therapeutic potential of LANCL2 ligands such as abscisic acid (ABA) in diabetes and chronic inflammatory diseases [2, 3, 22, 23, 45 ]. Compound 61610 (bis (benzimidazolyl) terephthalanilide, BTT) binds with highest affinity to LANCL2 in libraries with millions of chemicals.

In view of the fact that the current drugs used for T2D fail to meet the first needs of patients (i.e. glucose control) without side effects, BT-11 represents a very attractive potential replacement. Our results show how administration of BT-11 in a different T2D mouse model significantly reduced the glucose level in blood after the fasting period (fig. 36 and 38). In addition, administration of this compound also helped to normalize glucose levels after glucose challenge (fig. 36 and 38). The anti-inflammatory properties of BT-11 are also reflected in our immunophenotypic results. Indeed, administration of BT-11 caused less infiltration of pro-inflammatory macrophages and pro-inflammatory granulocytes in abdominal WAT (fig. 37). These results are supported by gene expression data for two very important proinflammatory factors, TNF α and MCP-1, which were found to be significantly reduced in mice treated with BT-11 (fig. 39).

Example 24: use of BT-11 during influenza infection

Introduction to

Respiratory pathogens responsible for pneumonia are the leading cause of infectious disease-related death in industrialized countries. The absence of effective vaccines and antiviral agents coupled with the growing concern of emergence of antiviral resistance underscores the need to develop immunotherapeutic approaches that target the host. The pulmonary pathogenesis and clinical disease associated with respiratory infections often results from a combination of the cytopathic effects of the virus and the host immune response. In this regard, therapies directed to modulating the innate immune response are contemplated for the treatment of influenza [46 ].

Influenza remains a major public health problem worldwide. Seasonal influenza is associated with upper respiratory tract processes that are often disabling and require several days of restricted activity. It is estimated that the annual influenza pandemic alone in the united states results in 3000 million outpatient visits and 300,000 hospitalizations. Certain populations (e.g., young children, the elderly, and people with a predisposition to medical conditions) have a higher risk of developing viral pneumonia. Experts have estimated that 25,000 to 35,000 people die annually in the united states due to seasonal influenza, and the global financial burden has been calculated to be billions of dollars [47 ]. Influenza pandemic cycles occur every 30-50 years with increased complexity due to their unpredictable behavior and lack of pre-existing immunity, and are associated with high mortality rates [48 ]. Influenza is associated with higher morbidity and mortality, but lacks effective and safe medications.

The data indicate that lanthionine synthase component C-like protein 2(LANCL2) is a target for ABA binding and signaling [15, 19, 24 ]. Thus, LANCL2 has emerged as a promising novel therapeutic target for immunomodulation. Using molecular modeling and Surface Plasmon Resonance (SPR), BTI identified compound BT-11 (bis (benzimidazolyl) terephthalanilide, BTT), which binds with high affinity to LANCL 2. In addition, BT-11 exerts a potent pro-repercussive (pro-repertoire) effect in the lungs and reduces mortality and morbidity in influenza mouse models.

Method of producing a composite material

A mouse. C57BL/6 mice were purchased from Jackson Laboratory and housed in ventilated racks under specific pathogen-free conditions. All experimental protocols were approved by the institutional animal care and use committee and met or exceeded guidelines for national institutes of health laboratory animal welfare and public health services office policies.

Mice were infected intranasally with influenza virus. Mice were anesthetized with 2% -5% isoflurane using a vaporizer station and 50 μ L10 was administered through the nostril³Viral dilutions of TCID50 (25 μ L each). The mice were then placed in their cages and monitored for anesthesia recovery.

BT-11 was administered orally by orogastric tube feeding. BT-11 was administered to mice via oral gastric tube feeding using commercially available safety balloon tipped feeding needles (18-24 gauge, depending on animal weight). This procedure does not cause pain or suffering. Mice were treated with BT-11 at a dose of 80mg/Kg for the duration of the experiment once every 24 hours.

Mice and disease activity were monitored and weighed. Mice were monitored once daily after infection (or every 4 hours if they developed severe clinical signs of disease equivalent to disease score 2) and euthanized prior to the planned endpoint if they developed overt signs as measured by weight loss (i.e., gradual loss of 25% of initial body weight), dehydration, loss of mobility, protection/protection of painful areas, frizzy hair (piloerection). Mice were weighed once a day for the duration of the experiment.

Results

Oral administration of BT-11 reduced clinical scores and morbidity in mice with influenza virus.

To evaluate the therapeutic efficacy of BT-11, we used an influenza infection mouse model in mice. Briefly, mice were infected intranasally after anesthetizing with 5% isoflurane. Mice were treated daily with BT-11 oral suspension at 80 or 40 mg/Kg. Mice were weighed and scored for the duration of the experiment (16 days). The results show how administration of BT-11 started on day 3 and significantly reduced activity throughout the experiment (figure 40, panel a). Furthermore, the clinical score of physical appearance was significantly reduced in mice receiving 40 and 80mg/Kg BT-11 treatment (figure 40, panel B).

To assess the role of treatment in disease incidence, we calculated the percent weight loss and further assessed the number of mice lost more than 15% within each experimental group. Treatment with 80mg/Kg BT-11 caused less morbidity when compared to the vehicle group starting on day 6 post infection. The differences began to stand out on day 10 and continued through day 12 (fig. 40, panel C).

Discussion of the related Art

Traditional methods of controlling influenza spread and disease are centered on the viral side via vaccination and antiviral treatment. Vaccines must be formulated annually based on the epidemic strains of the previous season. However, whether the novel vaccine is for seasonal influenza or pandemic influenza, it takes about 4 to 6 months to produce, license and test its efficacy [49 ]. The main disadvantage of antiviral agents is the very frequent occurrence and selection of resistant strains. In addition to virus-centered treatment, therapies based on controlling an exacerbated host response have been developed with a high potential for supplementing antimicrobial and prophylactic strategies. Therapeutic agents that target the host have the advantage of providing cross-protection under different hybridization conditions and are therefore effective quarterly, can be produced and stockpiled, and can be used to treat disease following viral exposure [46, 50, 51 ].

The identification of LANCL2 as a novel therapeutic target for influenza opens a new avenue for targeting host therapeutics. We show that activation of LANCL2 by BT-11 not only improves activity and clinical score, but also reduces morbidity caused by influenza virus and promotes recovery from influenza infection (figure 33). These results strongly support that LANCL2 is a novel therapeutic target for influenza and BT-11 is a potential new drug targeting the host.

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