Small organic molecules for the treatment of neuroinflammatory disorders
阅读说明：本技术 用于治疗神经炎性紊乱的有机小分子 (Small organic molecules for the treatment of neuroinflammatory disorders ) 是由 阿尔农·卡尔尼 卡琳·伯纳特·法因伯格 于 2018-04-26 设计创作，主要内容包括：本发明提供了可用作神经退行性疾病的治疗剂的有机小分子。充当骨形态发生蛋白(BMP)的抑制剂的有机小分子可用于治疗神经炎性紊乱,特别是多发性硬化。(The present invention provides small organic molecules useful as therapeutic agents for neurodegenerative diseases. Small organic molecules that act as inhibitors of Bone Morphogenic Proteins (BMPs) are useful in the treatment of neuroinflammatory disorders, particularly multiple sclerosis.)
1. A pharmaceutical composition for treating a neuroinflammatory disorder, the pharmaceutical composition comprising an inhibitor of bone morphogenetic protein 2(BMP2), wherein the BMP2 inhibitor is a small molecule, wherein the small molecule is selected from the group consisting of: SM1, SM2, SM3, SM4, SM5, SM6, SM7, SM8, SM9
2. The pharmaceutical composition of claim 1, wherein the treatment further comprises an additional therapeutic agent.
3. The pharmaceutical composition of any one of the preceding claims, wherein the neuroinflammatory disease is multiple sclerosis.
4. The pharmaceutical composition of claim 3, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
5. A pharmaceutical composition comprising a compound having the general formula (I):
wherein
X1And X2Each independently is S, O or N-R4Wherein R is4Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
X3selected from the group consisting of: CN, halogen, NO2、CO-X9And SO2X9Wherein X is9Selected from the group consisting of: OH, O-And NH2；
X4、X5、X6、X7And X8Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, NO2、CN、CO-X10And SO2X10Wherein X is10Selected from the group consisting of: OH, O-And NH2；
R1And R2Is absent, and R1And R2Is selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X11And SO2X11Wherein X is11Selected from the group consisting of: OH, O-And NH2；
R3Selected from the group consisting of: OR (OR)5、O-And NR6R7Wherein R is5、R6And R7Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
Wherein each dotted line independently represents a single or double bond.
6. The pharmaceutical composition of claim 5, for use in treating a neuroinflammatory disorder.
7. The pharmaceutical composition of claim 6, wherein the treatment further comprises an additional therapeutic agent.
8. The pharmaceutical composition of any one of claims 6-7, wherein the neuroinflammatory disease is multiple sclerosis.
9. The pharmaceutical composition of claim 8, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
10. The pharmaceutical composition of any one of claims 5-9, wherein the compound is SM 1:
11. a pharmaceutical composition comprising a compound having the general formula (IX) or a salt thereof:
wherein
X12、X13、X15And X16Each independently selected from the group consisting of: H. alkyl, cycloalkylAryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X19And SO2X19Wherein X is19Selected from the group consisting of: OH, O-And NH2；
X14And X18Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X20And SO2X20Wherein X is20Selected from the group consisting of: OH, O-And NH2；
X17Is S, O or N-R9Wherein R is9Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
n is 1 or 2; and is
R8Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and aryl;
the pharmaceutical composition is used for treating neuroinflammatory diseases.
12. The pharmaceutical composition of claim 11, wherein the treatment further comprises an additional therapeutic agent.
13. The pharmaceutical composition of any one of claims 11 and 12, wherein the neuroinflammatory disease is multiple sclerosis.
14. The pharmaceutical composition of claim 13, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
15. The pharmaceutical composition of any one of claims 11-14, wherein the compound is SM 9:
16. a pharmaceutical composition comprising a compound having the general formula (VII):
wherein
X21And R10Each independently selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X23And SO2X23Wherein X is23Selected from the group consisting of: OH, O-And NH2；
X22Selected from the group consisting of: OR (OR)12、O-And NR13R14Wherein R is12、R13And R14Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
R11Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X24And SO2X24Wherein X is24Selected from the group consisting of: OH, O-And NH2；
The pharmaceutical composition is used for treating neuroinflammatory diseases.
17. The pharmaceutical composition of claim 16, wherein the treatment further comprises an additional therapeutic agent.
18. The pharmaceutical composition of any one of claims 16 and 17, wherein the neuroinflammatory disease is multiple sclerosis.
19. The pharmaceutical composition of claim 18, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
20. The pharmaceutical composition of any one of claims 16-19, wherein the compound is SM 7:
21. a pharmaceutical composition comprising a compound having the general formula (VI):
wherein
X24Is O or S;
R15and X25Each independently selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X26And SO2X26Wherein X is26Selected from the group consisting of: OH, O-And NH2(ii) a And is
R16Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X24And SO2X24Wherein X is24Selected from the group consisting of: OH, O-And NH2。
22. The pharmaceutical composition of claim 21, for use in treating a neuroinflammatory disorder.
23. The pharmaceutical composition of claim 22, wherein the treatment further comprises an additional therapeutic agent.
24. The pharmaceutical composition of any one of claims 22-23, wherein the neuroinflammatory disease is multiple sclerosis.
25. The pharmaceutical composition of claim 24, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
26. A pharmaceutical composition comprising a compound having the general formula (II):
wherein
R17And R18Each independently is an optionally substituted phenyl ring;
R19selected from the group consisting of: H. alkyl, cycloalkyl and aryl;
R20and R21Is absent, and R20Or R21Is selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
Each dotted line independently represents a single or double bond;
the pharmaceutical composition is used for treating neuroinflammatory diseases.
27. The pharmaceutical composition of claim 26, wherein the treatment further comprises an additional therapeutic agent.
28. The pharmaceutical composition of any one of claims 26 and 27, wherein the neuroinflammatory disease is multiple sclerosis.
29. The pharmaceutical composition of claim 28, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
30. The pharmaceutical composition of any one of claims 26-29, wherein the compound is SM 2:
31. a pharmaceutical composition comprising a compound having the general formula (III):
wherein
R22And R23Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
X27Selected from the group consisting of: haloalkyl, amine, halogen, alkoxy, hydroxy, CN and NO2。
32. The pharmaceutical composition of claim 31, for use in treating a neuroinflammatory disorder.
33. The pharmaceutical composition of claim 32, wherein the treatment further comprises an additional therapeutic agent.
34. The pharmaceutical composition of any one of claims 32 and 33, wherein the neuroinflammatory disease is multiple sclerosis.
35. The pharmaceutical composition of claim 24, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
36. The pharmaceutical composition of any one of claims 31-35, wherein the compound is SM 3:
37. a pharmaceutical composition comprising a compound having the general formula (IV):
wherein
X28Selected from the group consisting of: haloalkyl, amine, halogen, alkoxy, hydroxy, CN and NO2；
R24Selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
R25Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN and NO2。
38. The pharmaceutical composition of claim 37, for use in treating a neuroinflammatory disorder.
39. The pharmaceutical composition of claim 38, wherein the treatment further comprises an additional therapeutic agent.
40. The pharmaceutical composition of any one of claims 38-39, wherein the neuroinflammatory disease is multiple sclerosis.
41. The pharmaceutical composition of claim 40, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
42. The pharmaceutical composition of any one of claims 37-41, wherein the compound is SM 4:
43. a pharmaceutical composition comprising a compound having the general formula (V):
wherein
X29Selected from the group consisting of: OR (OR)26、O-And NR27R28Wherein R is26、R27And R28Each independently selected from the group consisting of: H. COR (continuous operating reference)29Alkyl, cycloalkyl and aryl, wherein R29Selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
X30Selected from the group consisting of: OR (OR)30、O-And NR31R32Wherein R is30、R31And R32Each independently selected from the group consisting of: H. COR (continuous operating reference)33Alkyl, cycloalkyl and aryl, wherein R33Selected from the group consisting of: H. alkyl, cycloalkyl and aryl.
44. The pharmaceutical composition of claim 43, for use in treating a neuroinflammatory disorder.
45. The pharmaceutical composition of claim 44, wherein the treatment further comprises an additional therapeutic agent.
46. The pharmaceutical composition of any one of claims 44 and 45, wherein the neuroinflammatory disease is multiple sclerosis.
47. The pharmaceutical composition of claim 46, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
48. The pharmaceutical composition of any one of claims 43-46, wherein the compound is SM 5:
49. a pharmaceutical composition comprising a compound having the general formula (VIII):
wherein
X31Selected from S and O;
R34selected from the group consisting of halogenated aryl and halogenated heteroaryl; and is
R35And R36Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl.
50. The pharmaceutical composition of claim 49, for use in treating a neuroinflammatory disorder.
51. The pharmaceutical composition of claim 50, wherein the treatment further comprises an additional therapeutic agent.
52. The pharmaceutical composition of any one of claims 50 and 51, wherein the neuroinflammatory disease is multiple sclerosis.
53. The pharmaceutical composition of claim 52, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
54. The pharmaceutical composition of any one of claims 49-53, wherein the compound is SM 8:
55. a method of treating a neuroinflammatory disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a bone morphogenetic protein 2(BMP2) inhibitor, wherein the BMP2 inhibitor is a small molecule selected from the group consisting of: SM1, SM2, SM3, SM4, SM5, SM6, SM7, SM8, SM9
56. The method of claim 55, wherein the neuroinflammatory disease is multiple sclerosis.
57. The method of claim 56, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.
58. The method of any one of claims 55-57, further comprising the step of administering to the subject an additional therapeutic agent.
Technical Field
The present invention relates to the field of therapeutic agents for neurodegenerative and neuroinflammatory diseases. Small organic molecules are disclosed that can act as inhibitors of Bone Morphogenetic Proteins (BMPs) and are useful in the treatment of neuroinflammatory disorders, particularly multiple sclerosis.
Background
Multiple Sclerosis (MS) is a well-known demyelinating disease in which myelin (an insulating covering of nerve cells) in the brain and spinal cord is damaged [1 ]. Bone Morphogenetic Proteins (BMPs) are considered (administered) as inhibitors of myelination during development and disease states (reviewed in [2 ]).
WO 2013/186777[17] discloses a pharmaceutical composition for the treatment of neuroinflammatory or neurodegenerative diseases, which comprises a single blocker of BMP signaling or a combination of several blockers.
US 2015/139983[3] discloses a method for treating neuroinflammatory or neurodegenerative diseases, which method comprises a single antibody or a combination of several antibodies against BMP-2 and BMP-4.
US 2008/0249038[4] discloses methods of using BMP2A inhibitors, particularly siRNA molecules or antisense molecules, to reduce or reduce symptoms and signs associated with damaged neuronal tissue, whether caused by tissue trauma or chronic or acute degenerative changes.
Simonini et al [5] examined the effect of the PPAR delta (peroxisome proliferator activated receptor) agonist GW0742 on OPC (oligodendrocyte progenitor cells) and showed that GW0742 reduced BMP2 and BMP4 mRNA levels in OPC with less effect on astrocytes. Simonini et al concluded that PPAR δ plays a role in OPC maturation mediated in part by modulation of BMP and BMP antagonists.
Li et al [6] evaluated hippocampal cell proliferation and BMP4 mRNA levels in the DG granule sub-region (DG-SGZ) of APPSwe/PS1 Δ E9 transgenic mice (a mouse model of Alzheimer's disease). Researchers found a significant correlation between increased BMP4 mRNA expression and decreased BrdU-labelled cell numbers and suggested that an increase in BMP4 mRNA expression within hippocampus' DG might contribute to decreased cell proliferation in APPswe/PS1 Δ E9 transgenic mice.
Mabie et al [7] reported that BMPs promote selective, dose-dependent differentiation of oligodendrocyte-astrocyte progenitor cells (O-2A) into astrocytes while inhibiting oligodendrocyte differentiation.
Gross et al [8] demonstrated that BMP causes selective, dose-dependent refinement of astrocytic lineages from murine embryonic subventricular zone (SVZ) multipotent progenitors.
Gomes et al [9] constructed transgenic
Lim et al [10] showed that the BMP antagonist Noggin was expressed by ependymal cells adjacent the subventricular zone (SVZ) of the brain. SVZ cells were found to express BMP and its cognate receptor. Purified mouse Noggin protein promotes neurogenesis in vitro and inhibits glial cell differentiation, and ectopic Noggin promotes neuronal differentiation of SVZ cells introduced into the striatum. Thus, researchers have suggested that the production of ependymal Noggin creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling.
WO 11/019678[11] discloses the use of isothiazoles for the treatment of ocular conditions such as age-related macular degeneration, US 2006/217390[12] discloses cycloalkyl isothiazoles, aryl isothiazoles and heteroaryl amino isothiazoles for the treatment of hepatitis C WO 03/105857[13] discloses pharmaceutical compositions containing active compounds which inhibit the activity of chemokines (MIP-1 α and RANTES). The methods of using these pharmaceutical compositions for the treatment of inflammatory diseases US 2004/039037[14] discloses substituted isothiazole compounds for the inhibition of various protein kinases, particularly MEK and/or ERK.A method of treating diseases associated with abnormal MEK and/or ERK function is also disclosed.
There remains a need for improved treatments for multiple sclerosis.
Summary of The Invention
It is now disclosed for the first time that small organic molecules can act as inhibitors of Bone Morphogenic Proteins (BMPs), including but not limited to
The present invention is based in part on the discovery that: small organic compounds of formula (I) -formula (IX), including the specific compounds SM1, SM6, SM7 and SM9 specified herein inhibit the activity of bone morphogenic protein 2(BMP2) in cell-based assays. In addition, these molecules inhibit disease progression in an animal model of experimental autoimmune encephalomyelitis of relapsing/remitting type (RR-EAE), a well-established model that mimics Multiple Sclerosis (MS). Without wishing to be bound by any particular theory of mechanism of action, these small organic molecules have been shown to induce neurogenesis and oligodendropoiesis (oligodendrogenesis) and maintain the amount of myelin by inhibiting demyelination, inducing remyelination, or both.
According to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (I):
wherein
X1And X2Each independently is S, O or N-R4Wherein R is4Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
X3selected from the group consisting of: CN, halogen, nitro, CO-X9And SO2X9Wherein X is9Selected from the group consisting of: OH, O-And NH2；
X4、X5、X6、X7And X8Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, nitro, CO-X10And SO2X10Wherein X is10Selected from the group consisting of: OH, O-And NH2；
R1And R2Is absent, and R1And R2Is selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X11And SO2X11Wherein X is11Selected from the group consisting of: OH, O-And NH2；
R3Selected from the group consisting of: OR (OR)5、O-And NR6R7Wherein R is5、R6And R7Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
Wherein each dotted line independently represents a single or double bond.
According to some embodiments, the pharmaceutical composition comprises a compound of formula (Ia):
according to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (I) or a salt thereof, wherein X1、X2、X3、X4、X5、X6、X7、X8、R1、R2And R3Each as described herein.
According to some embodiments, there is provided a method of treating a neuroinflammatory disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (Ia) or a salt thereof, wherein X1、X2、X3、X4、X5、X6、X7、X8、R1And R3Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (I) or a salt thereof, wherein X is1、X2、X3、X4、X5、X6、X7、X8、R1、R2And R3As already described.
According to some embodiments, X1And X2Are both S.
According to some embodiments, X3Is CN.
According to some embodiments, X4、X5、X6、X7And X8Each of which is H.
According to some embodiments, R is1Is H.
According to some embodimentsWherein R is3Is OR5。
According to some embodiments, R is5Is methyl or ethyl.
According to some embodiments, the compound has formula SM 1:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula (IX):
wherein
X12、X13、X15And X16Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X19And SO2X19Wherein X is19Selected from the group consisting of: OH, O-And NH2；
X14And X18Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X20And SO2X20Wherein X is20Selected from the group consisting of: OH, O-And NH2；
X17Is S, O or N-R9Wherein R is9Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
n is 1 or 2; and is
R8Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and aryl;
the pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, there is provided a method of treating neuroinflammatory disordersA method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (IX) or a salt thereof, wherein X12、X13、X14、X15、X16、X17、X18And R8Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (IX) or a salt thereof, wherein X is X, and a pharmaceutically acceptable carrier for the manufacture of a pharmaceutical composition for treating a subject suffering from a neuroinflammatory disease12、X13、X14、X15、X16、X17、X18And R8Each as described herein.
According to some embodiments, X12、X13、X15And X16Each of which is H.
According to some embodiments, X14Is a halogen.
According to some embodiments, X14Is C1.
According to some embodiments, X18Is CN.
According to some embodiments, X17Is O.
According to some embodiments, R is8Is methyl.
According to some embodiments, n is 2.
According to some embodiments, the compound has formula SM 9:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM7, an ester or a salt thereof:
the pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM6 or a salt thereof:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM2 or a salt thereof:
the pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM3 or a salt thereof:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM4 or a salt thereof:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM5 or a salt thereof:
according to some embodiments, there is provided a pharmaceutical composition comprising a compound having the general formula SM8 or a salt thereof:
according to some embodiments, the pharmaceutical composition is for use in the treatment of a neuroinflammatory disease.
According to some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.
According to some embodiments, the neuroinflammatory disease is multiple sclerosis.
According to some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis.
According to some embodiments, the method further comprises administering to the subject an additional therapeutic agent.
According to some embodiments, the additional therapeutic agent is administered prior to, simultaneously with or after the administration of the at least one compound or the pharmaceutical composition comprising the at least one compound.
Brief Description of Drawings
In order to better understand the subject matter disclosed herein and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
figure 1-High Throughput Screening (HTS) bioassay evaluation. (A) ALP Activity in the Presence or absence of BMP-2 ("with BMP-2" -Square, "without BMP-2" -Diamond)Chemiluminescent substrate determination, expressed as o.d405nmA hole. (B) Cell viability in the Presence or absence of BMP-2 ("BMP-2" -Square, "BMP-2" -Diamond-free "), by CellLuminescent substrate determination, expressed as o.d405nmA hole. (C) ALP/cell ratio per well in the presence or absence of BMP-2 ("with BMP-2" -squares, "without BMP-2" -diamonds).
FIG. 2-effect of SM1-SM9 on ALP stimulation of BPM-2 and on cell viability. The percentage (%) ALP and percentage (%) cell viability (squares) are shown for different concentrations of SM1-SM9 molecules (0.62. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M).
FIGS. 3-SM 1-SM9 roles in RR-EAE. EAE clinical scores of RR-EAE mice, each group (n ═ 7) treated with small molecules (SM 1-SM9, 10 mg/kg/day, respectively) for 30 days from day 9 post-immunization compared to vehicle alone (5% DMSO in PBS, 200 μ g/mouse). SM-diamonds, vehicle-squares.
FIGS. 4-
Figure 5-effect of SM1, SM6, SM7 and SM9 on the number of mice with moderate-severe EAE. Number of mice with clinical score greater than or equal to 2 per group per day (10 mg/kg/day-square, 20 mg/kg/day-triangle, vehicle-diamond).
Fig. 6-effect of SM1, SM6, SM7 and SM9 on demyelination. (A) Representative images of LFB (anterolateral chordae) stained lumbar spinal cord sections of mice treated with vehicle, SM 110 mg/kg/day, SM120 mg/kg/day, SM 710 mg/kg/day, SM 910 mg/kg/day, SM 920 mg/kg/day, SM 610 mg/kg/day, and SM620 mg/kg/day. (B) Quantification of myelinated areas. The graph shows the percentage (%) of LFB stained area to total spinal cord sections at day 48 post-immunization. Quantification was performed on 6 mice/group and 3 sections/mouse using Image J software.
Figure 7-effect of SM1, SM7, and SM9 on neuronal phenotype in P19 cells. (A) Representative images of P19 cells stained with MAP-2 and Hoechst on day 8. Images were obtained using an Olympus BX 81 inverted fluorescence microscope. (B) Analysis of% MAP-2 positive cells. Analysis was performed by Image J software.
Figure 8-effect of SM1, SM7, and SM9 on SMAD1/5/8 signaling as detected by western blot. (A) Phosphorylated SMAD (p-SMAD), total SMAD, and tubulin were western blotted in response to no stimulation ("control") or stimulation with BMP-2, BMP-2+ anti-BMP-2/4 Ab, BMP-2+ SM1 or SM7 or SM9 at 2.5 μ M and 5 μ M as indicated. (B) Quantification of p-SMAD/tubulin as performed by Image J software.
FIGS. 9-role of SM1, SM7, SM9 and SM6 in BMP-4/BMP-2 induced ATDC5 bioassay. ALP induction% and percent cell viability for 2.5. mu.M and 5. mu.M SM1, SM7, SM9, and SM6 in the presence of BMP-4 stimulation and BMP-2 stimulation.
FIG. 10-the effect of SM1, SM7, SM9, and SM6 on de novo expression of the neuroblast marker biscortin in SVZ. (A) Immunofluorescence images showing labeling of BrdU and Double Cortin (DCX) in SVZ. Images of coronal sections were obtained using a Zeiss 710 confocal microscope. LV, lateral ventricle, SVZ, subventricular zone. Image: a. scale bar of c, e, g, i, k, m, o is 100 μm (magnification × 10), and image: b. the scale bar for d, f, h, j, l, n, and p is 20 μm (magnification × 63). (B) For BrdU in SVZ+DCX+Quantification of cells. Analysis was performed on 3 sections from each mouse (3 mice per group, total 9) using image J software. Values are given as mean ± SEM, and the results of student's t-test as p<And 0.05.
FIG. 11-Effect of SM1, SM7, SM9 and SM6 on de novo expression of double cortins in SGZ. (A) Is an immunohistochemical image showing the targeting of BrdU in SGZ+DCX+Labeling of the cells. Images were obtained using an Olympus8.1 microscope (magnification × 10). (B) For BrdU in SGZ+DCX+Quantification of cells. Analysis was performed on 3 sections of each mouse (3 mice per group, total 9) using image J software. Values are given as mean ± SEM, and the results of student's t-test as p<And 0.05.
Figure 12-effect of SM1, SM7, SM9 and SM6 on de novo expression of the mature neuronal marker NeuN in SGZ. (A) Immunohistochemistry image showing targeting of BrdU in SGZ+NeuN+Labeling of the cells. Images were taken using an Olympus8.1 microscope(magnification × 10). (B) For BrdU in SGZ+NeuN+And (4) quantifying. Analysis was performed on 3 sections of each mouse (3 mice per group, total 9) using image J software. Values are given as mean ± SEM, and the results of student's t-test as p<And 0.05.
Detailed Description
The present invention provides small organic molecules capable of inhibiting BMP that show therapeutic utility in inhibiting EAE, particularly relapsing forms of EAE, which are models that mimic relapsing-remitting multiple sclerosis.
The present invention is based in part on the discovery that: small organic compounds of formula (I) -formula (IX), including but not limited to the specific compounds SM1, SM6, SM7 and SM9 specified herein, inhibit the activity of bone morphogenic protein 2(BMP2) in cell-based assays. These molecules were shown to inhibit disease progression in a relapsing/remitting experimental autoimmune encephalomyelitis (RR-EAE) animal model, a well-established model that mimics Multiple Sclerosis (MS). Without wishing to be bound by any particular theory of mechanism of action, these molecules have been shown to induce neurogenesis and oligodendrocyte formation and maintain the amount of myelin by inhibiting demyelination, inducing remyelination, or both.
Thus, according to some embodiments, there is provided a pharmaceutical composition comprising an inhibitor of bone morphogenetic protein 2(BMP2), wherein the BMP2 inhibitor is a small molecule. According to some embodiments, the pharmaceutical composition is for use in the treatment of a neuroinflammatory disease, such as multiple sclerosis.
According to some embodiments, there is provided a method for treating a neuroinflammatory disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of bone morphogenetic protein 2(BMP2), wherein the BMP2 inhibitor is a small molecule.
According to some embodiments, there is provided a use of an inhibitor of bone morphogenetic protein 2(BMP2) in the manufacture of a pharmaceutical composition for treating a subject having a neuroinflammatory disorder, wherein the BMP2 inhibitor is a small molecule.
According to some embodiments, there is provided a use of an inhibitor of bone morphogenetic protein 2(BMP2) for treating a subject having a neuroinflammatory disorder, wherein the BMP2 inhibitor is a small molecule.
According to some embodiments, the small molecule has a molecular weight of no more than 1000 gr/mol.
According to some embodiments, the small molecule has a formula selected from the group consisting of: formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX) and salts thereof. According to some embodiments, the small molecule has a formula selected from the group consisting of: formula (I), formula (VI), formula (VII), formula (IX) and salts thereof. According to some embodiments, the small molecule has a formula selected from the group consisting of: formula (I), formula (IX) and salts thereof. According to some embodiments, the small molecule has a formula selected from the group consisting of: SM1, SM2, SM3, SM4, SM5, SM6, SM7, SM8, SM9 and salts thereof. According to some embodiments, the small molecule has a formula selected from the group consisting of: SM1, SM6, SM7, SM9 and salts thereof. According to some embodiments, the small molecule has a formula selected from the group consisting of: SM1, SM9 and salts thereof.
According to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (I):
wherein
X1And X2Each independently is S, O or N-R4Wherein R is4Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
X3selected from the group consisting of: CN, halogen, nitro, CO-X9And SO2X9Wherein X is9Selected from the group consisting of: OH, O-And NH2；
X4、X5、X6、X7And X8Each independently selected from the group consisting ofGroup (c): H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, nitro, CO-X10And SO2X10Wherein X is10Selected from the group consisting of: OH, O-And NH2；
R1And R2Is absent, and R1And R2Is selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X11And SO2X11Wherein X is11Selected from the group consisting of: OH, O-And NH2；
R3Selected from the group consisting of: OR (OR)5、O-And NR6R7Wherein R is5、R6And R7Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
Wherein each dotted line independently represents a single or double bond.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (I) or a salt thereof, wherein X1、X2、X3、X4、X5、X6、X7、X8、R1、R2And R3Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (I) or a salt thereof, wherein X is1、X2、X3、X4、X5、X6、X7、X8、R1、R2And R3Each as described herein.
According to some embodiments, the pharmaceutical composition comprises a compound of formula (Ia):
according to some embodiments, there is provided a method of treating a neuroinflammatory disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (Ia) or a salt thereof, wherein X1、X2、X3、X4、X5、X6、X7、X8、R1And R3Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (Ia) or a salt thereof, wherein X is for treating a subject having a neuroinflammatory disorder1、X2、X3、X4、X5、X6、X7、X8、R1And R3Each as described herein.
According to some embodiments, X1And X2Each independently is S or O. According to some embodiments, X1Is S or O. According to some embodiments, X2Is S or O. According to some embodiments, X1Is S. According to some embodiments, X2Is S. According to some embodiments, X1And X2Are both S.
According to some embodiments, X3Selected from the group consisting of: CN, halogen and nitro. According to some embodiments, X3Is CN.
According to some embodiments, X4、X5、X6、X7And X8Each independently selected from the group consisting of: H. alkyl, halogen, hydroxy and alkoxy. According to some embodiments, X4、X5、X6、X7And X8Each independently is H or halogen. According to some embodiments, X4Is H. According to some embodiments, X5Is H. According to some embodiments, X6Is H. According to some embodiments, X7Is H. According to some embodiments, X8Is H. According to some embodimentsEmbodiment (I) X4、X5、X6、X7And X8Is H. According to some embodiments, X4、X5、X6、X7And X8Is H. According to some embodiments, X4、X5、X6、X7And X8Is H. According to some embodiments, X4、X5、X6、X7And X8Is H. According to some embodiments, X4、X5、X6、X7And X8Each independently is H.
According to some embodiments, R is1And R2Is absent, and R1And R2Is selected from the group consisting of: H. alkyl, cycloalkyl, aryl, CO-X11And SO2X11. According to some embodiments, R is2Is absent, and R1Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, CO-X11And SO2X11. According to some embodiments, R is1Is H.
According to some embodiments, R is3Selected from the group consisting of: OR (OR)5And O-. According to some embodiments, R is3Is OR5. According to some embodiments, R is5Is an alkyl chain. According to some embodiments, R is5Selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, butyl, n-butyl, sec-butyl, and isobutyl. According to some embodiments, R is5Is methyl or ethyl. According to some embodiments, R is5Is methyl. According to some embodiments, R is3Is OMe or OEt. According to some embodiments, R is3Is OMe.
According to some embodiments, the compound has formula SM 1:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (IX):
wherein
X12、X13、X15And X16Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X19And SO2X19Wherein X is19Selected from the group consisting of: OH, O-And NH2；
X14And X18Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X20And SO2X20Wherein X is20Selected from the group consisting of: OH, O-And NH2；
X17Is S, O or N-R9Wherein R is9Selected from the group consisting of: H. alkyl, cycloalkyl, aryl and heteroaryl;
n is 1 or 2; and is
R8Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and aryl;
the pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (IX) or a salt thereof, wherein X12、X13、X14、X15、X16、X17、X18And R8Each as described herein.
According to some embodiments, there is provided a compound having the general formula (IX) orUse of a salt thereof for treating a subject having a neuroinflammatory disorder, wherein X12、X13、X14、X15、X16、X17、X18And R8Each as described herein.
According to some embodiments, X12、X13、X15And X16Each independently selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, CN and NO2. According to some embodiments, X12、X13、X15And X16Each independently selected from the group consisting of: H. alkyl, halogen, hydroxy and alkoxy. According to some embodiments, X12、X13、X15And X16Each independently is H or halogen. According to some embodiments, X12Is H. According to some embodiments, X13Is H. According to some embodiments, X15Is H. According to some embodiments, X16Is H. According to some embodiments, X12、X13、X15And X16Is H. According to some embodiments, X12、X13、X15And X16Is H. According to some embodiments, X12、X13、X15And X16Is H. According to some embodiments, X4、X5、X6、X7And X8Each independently is H.
According to some embodiments, X14Selected from the group consisting of: H. amine, halogen, alkoxy, hydroxy, CN and NO2. According to some embodiments, X14Selected from the group consisting of: NH (NH)2Halogen, alkoxy, hydroxy, CN and NO2. According to some embodiments, X14Selected from the group consisting of: halogen and hydroxyl. According to some embodiments, X14Is a halogen. According to some embodiments, X14Selected from the group consisting of: f and Cl. According to some embodiments, X14Is Cl.
According to some embodiments, X18Selected from the group consisting of: halogen, CN, NO2、CO-X20And SO2X20Wherein X is20Selected from the group consisting of: OH, O-And NH2. According to some embodiments, X18Is CN.
According to some embodiments, n is 2.
According to some embodiments, R is8Selected from the group consisting of: alkyl groups and haloalkyl groups. According to some embodiments, R is8Is an alkyl group. According to some embodiments, R is8Is C1-4An alkyl group. According to some embodiments, R is8Selected from the group consisting of: me, Et and CF3. According to some embodiments, R is8Is Me.
According to some embodiments, the compound has formula SM 9:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (VII):
wherein
X21And R10Each independently selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X23And SO2X23Wherein X is23Selected from the group consisting of: OH, O-And NH2；
X22Selected from the group consisting of: OR (OR)12、O-And NR13R14Wherein R is12、R13And R14Each independently selected from the group consisting ofGroup (2): H. alkyl, cycloalkyl and aryl; and is
R11Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X24And SO2X24Wherein X is24Selected from the group consisting of: OH, O-And NH2；
The pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (VII) or a salt thereof, wherein X21、X22、R10And R11Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (VII) or a salt thereof, wherein X is21、X22、R10And R11Each as described herein.
According to some embodiments, X21Selected from the group consisting of: halogen, CN, NO2、CO-X23And SO2X23. According to some embodiments, X21Is CN.
According to some embodiments, X22Is OR12. According to some embodiments, R is12Is H or alkyl. According to some embodiments, R is12Is H. According to some embodiments, X22Is OH.
According to some embodiments, R is10Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and halogen. According to some embodiments, R is10Is a haloalkyl group. According to some embodiments, R is10Is a fluorinated alkyl group. According to some embodiments, R is10Is CF3。
According to some embodiments, R is11Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroarylRadical, CN, CO-X24And SO2X24. According to some embodiments, R is11Is H or CO-X24. According to some embodiments, R is11Is H.
According to some embodiments, the compound has formula SM 7:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (VI):
wherein
X24Is O or S;
R15and X25Each independently selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN, NO2、CO-X26And SO2X26Wherein X is26Selected from the group consisting of: OH, O-And NH2(ii) a And is
R16Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X24And SO2X24Wherein X is24Selected from the group consisting of: OH, O-And NH2。
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (VI) or a salt thereof, wherein X24、X25、R15And R16Each as described herein。
According to some embodiments, there is provided the use of a compound having the general formula (VI) or a salt thereof, wherein X is for treating a subject having a neuroinflammatory disorder24、X25、R15And R16Each as described herein.
According to some embodiments, X24Is O.
According to some embodiments, X25Selected from the group consisting of: amine, halogen, CN, NO2、CO-X26And SO2X26. According to some embodiments, X25Containing a nitrogen atom. According to some embodiments, X25Is NO2。
According to some embodiments, R is15Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and halogen. According to some embodiments, R is15Is a haloalkyl group. According to some embodiments, R is15Is a fluorinated alkyl group. According to some embodiments, R is15Is CF3。
According to some embodiments, R is16Selected from the group consisting of: H. alkyl, cycloalkyl, aryl, amine, halogen, alkoxy, heteroaryl, CN, CO-X24And SO2X24. According to some embodiments, R is16Is H or CO-X24. According to some embodiments, R is16Is H.
According to some embodiments, the compound has formula SM 6:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (II):
wherein
R17And R18Each independently is an optionally substituted phenyl ring;
R19selected from the group consisting of: H. alkyl, cycloalkyl and aryl;
R20and R21Is absent, and R20Or R21Is selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
Each dotted line independently represents a single or double bond;
the pharmaceutical composition is used for treating neuroinflammatory diseases.
According to some embodiments, the compound has the general formula (IIa):
wherein R is17、R18、R19And R20Each as described herein.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (II) or a salt thereof, wherein R17、R18、R19And R20Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (II) or a salt thereof, wherein R is for treating a subject having a neuroinflammatory disorder17、R18、R19And R20Each as described herein.
According to some embodiments, R is17Is C6H5. According to some embodiments, R is18Is C6H5. According to some embodiments, R is17And R18Are all C6H5。
According to some embodiments, R is19Is H.
According to some embodiments, R is20Is H.
According to some embodiments, R is21Is absent.
According to some embodiments, the compound has formula SM 2:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (III):
wherein
R22And R23Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
X27Selected from the group consisting of: haloalkyl, amine, halogen, alkoxy, hydroxy, CN and NO2。
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (III) or a salt thereof, wherein X27、R22And R23Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (III) or a salt thereof, wherein X is for treating a subject having a neuroinflammatory disorder27、R22And R23Each as described herein.
According to some embodiments, X27Containing a nitrogen atom. According to some embodiments, X27Is NO2。
According to some embodiments, R is22Is an alkyl chain. According to some embodiments, R is22Selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, butyl, n-butyl, sec-butyl, and isobutyl. According to some embodiments, R is22Is methyl or ethyl. According to some embodiments, R is22Is ethyl.
According to some embodiments, R is23Is H.
According to some embodiments, the compound has formula SM 3:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (IV):
wherein
X28Selected from the group consisting of: haloalkyl, amine, halogen, alkoxy, hydroxy, CN and NO2；
R24Selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
R25Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl, aryl, amine, halogen, alkoxy, hydroxy, heteroaryl, CN and NO2。
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (IV) or a salt thereof, wherein X28、R24And R25Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (IV) or a salt thereof, wherein X is for treating a subject having a neuroinflammatory disorder28、R24And R25Each as described herein.
According to some embodiments, X28Containing a nitrogen atom. According to some embodiments, X28Is NO2。
According toSome embodiments, R24Is H.
According to some embodiments, R is25Selected from the group consisting of: alkyl, haloalkyl, cycloalkyl and halogen. According to some embodiments, R is25Is a haloalkyl group. According to some embodiments, R is25Is a fluoroalkyl group. According to some embodiments, R is25Is CF3。
According to some embodiments, the compound has formula SM 4:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (V):
wherein
X29Selected from the group consisting of: OR (OR)26、O-And NR27R28Wherein R is26、R27And R28Each independently selected from the group consisting of: H. COR (continuous operating reference)29Alkyl, cycloalkyl and aryl, wherein R29Selected from the group consisting of: H. alkyl, cycloalkyl and aryl; and is
X30Selected from the group consisting of: OR (OR)30、O-And NR31R32Wherein R is30、R31And R32Each independently selected from the group consisting of: H. COR (continuous operating reference)33Alkyl, cycloalkyl and aryl, wherein R33Selected from the group consisting of: H. alkyl, cycloalkyl and aryl.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (V) or a salt thereof, wherein X29And X30In (1)Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (V) or a salt thereof, wherein X is29And X30Each as described herein.
According to some embodiments, X29Is NR27R28. According to some embodiments, R is27Is H. According to some embodiments, R is28Is COR29. According to some embodiments, R is29Is an alkyl group. According to some embodiments, R is29Is C1-4An alkyl group. According to some embodiments, R is29Is methyl. According to some embodiments, X29Is NHAc.
According to some embodiments, X30Is NR31R32. According to some embodiments, R is31Is H. According to some embodiments, R is32Is COR33. According to some embodiments, R is33Is an alkyl group. According to some embodiments, R is33Is C1-4An alkyl group. According to some embodiments, R is33Is methyl. According to some embodiments, X33Is NHAc.
According to some embodiments, the compound has formula SM 5:
according to some embodiments, the present invention provides a pharmaceutical composition comprising a compound having the general formula (VIII):
wherein
X31Selected from S and O;
R34selected from the group consisting of halogenated aryl and halogenated heteroaryl; and is
R35And R36Each independently selected from the group consisting of: H. alkyl, cycloalkyl and aryl.
According to some embodiments, there is provided a method of treating neuroinflammatory disorders, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula (VIII) or a salt thereof, wherein X31、R34、R35And R36Each as described herein.
According to some embodiments, there is provided the use of a compound having the general formula (VIII) or a salt thereof, wherein X is for treating a subject having a neuroinflammatory disorder31、R34、R35And R36Each as described herein.
According to some embodiments, X31Is S.
According to some embodiments, R is34Is a halogenated heteroaryl group. According to some embodiments, R is34Comprising at least two halogen atoms. According to some embodiments, R is34Containing two halogen atoms. According to some embodiments, the halogen atom is a chlorine atom. According to some embodiments, R is34Is a halogenated pyridine. According to some embodiments, R is34The method comprises the following steps:
according to some embodiments, R is35Is H. According to some embodiments, R is36Is H. According to some embodiments, R is35And R36Are all H.
According to some embodiments, the compound has formula SM 8:
according to some embodiments, the pharmaceutical composition is for use in the treatment of a neuroinflammatory disease.
According to some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.
According to some embodiments, the neuroinflammatory disease is multiple sclerosis.
According to some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis.
According to some embodiments, the method further comprises administering to the subject an additional therapeutic agent.
According to some embodiments, the additional therapeutic agent is administered prior to, simultaneously with or after the administration of the at least one compound or the pharmaceutical composition comprising the at least one compound.
In the compounds used according to the invention:
the terms "small molecule" and "organic small molecule" as used herein are interchangeable and refer to an organic molecule having a molecular weight of no more than 2000 gr/mol. According to some embodiments, the small molecule has a molecular weight of no more than 1000 gr/mol. According to some embodiments, the small molecule has a molecular weight of no more than 750 gr/mol. According to some embodiments, the small molecule has a molecular weight of no more than 500 gr/mol.
The term "carbocyclyl" as used herein refers to a ring structure comprising a plurality of carbon atoms and one or more heteroatoms selected from N, S and O. The carbocyclyl group may be a 5-or 6-membered ring comprising a single ring structure or a ring structure comprising two or more rings, each of which may be a 5-or 6-membered ring. Two or more rings of a ring structure may be fused or connected to each other via a covalent bond.
The terms "alkyl", "alkenyl" and "alkynyl" carbon chains, if not specifically designated, refer to carbon chains each containing from 1 to 20 carbons or from 1 or 2 to 16 carbons and are straight or branched. Each such group may be substituted. In some embodiments, the carbon chain comprises 1 to 10 carbon atoms. In some embodiments, the carbon chain comprises 1 to 6 carbon atoms. In some embodiments, the carbon chain comprises 2 to 6 carbon atoms. In some embodiments, the alkyl group is a haloalkyl group. The term haloalkyl refers to any alkyl chain covalently attached to at least one halogen atom, such as, but not limited to, trifluoromethyl. The alkenyl carbon chain may comprise from 2 to 20 carbons, or 2 to 18 carbons, or 2 to 16 carbons, or 2 to 14 carbons, or 2 to 12 carbons, or 2 to 10 carbons, or 2 to 8 carbons, or 2 to 6 carbons, or 2 to 4 carbons. The alkenyl carbon chain may likewise comprise 1 to 8 double bonds, or 1 to 7 double bonds, or 1 to 6 double bonds, or 1 to 5 double bonds, or 1 to 4 double bonds, or 1 to 3 double bonds, or 1 double bond, or 2 double bonds. Alkynyl carbon chains contain from 2 to 20 carbons, or 2 to 18 carbons, or 2 to 16 carbons, or 2 to 14 carbons, or 2 to 12 carbons, or 2 to 10 carbons, or 2 to 8 carbons, or 2 to 6 carbons, or 2 to 4 carbons. Alkynyl carbon chains may likewise contain 1 to 8 triple bonds, or 1 to 7 triple bonds, or 1 to 6 triple bonds, or 1 to 5 triple bonds, or 1 to 4 triple bonds, or 1 to 3 triple bonds, or 1 triple bond, or 2 triple bonds. Exemplary alkyl, alkenyl, and alkynyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isohexyl, allyl (propenyl), and propargyl (propynyl). The term "haloalkyl" refers to an alkyl group having at least one halogen covalently attached thereto. Exemplary haloalkyl groups include, but are not limited to, trifluoromethyl, chloromethyl, and the like.
Is named as "-C1-C5The radical-alkylene- "is alkylene having between 1 and 5 carbon atoms. In some embodiments, the group is selected from methylene, ethylene, propylene, butylene and pentylene or any other alkyl group having between 1 and 5 carbon atoms, such as isopropylene, among others. This group may be substituted.
Is named as "-C2-C5A group-alkenylene- "is a carbon group comprising at least two carbon atoms and one or more C ═ C bonds (double bonds). This group may be substituted.
"cycloalkyl" refers to a saturated monocyclic or polycyclic ring system, in certain embodiments having 3 to 10 carbon atoms, in other embodiments having 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to monocyclic or polycyclic ring systems which each contain at least one double bond and at least one triple bond. Cycloalkenyl groups and cycloalkynyl groups may contain between 3 and 10 carbon atoms in some embodiments, between 4 and 7 carbon atoms in other embodiments, and cycloalkynyl groups between 8 and 10 carbon atoms in other embodiments. The ring systems of the cycloalkyl groups, cycloalkenyl groups, and cycloalkynyl groups can comprise one ring or two or more rings that can be joined together in a fused, bridged, or spiro-linked manner.
"aryl" means an aromatic monocyclic or polycyclic group containing from 6 to 10 carbon atoms. Aryl groups include, but are not limited to, groups such as unsubstituted or substituted fluorenyl, unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl. radical-C6-arylene- "is a phenyl group or a phenyl-substituted group.
In certain embodiments, "heteroaryl" refers to about 5-to about 15-membered monocyclic or polycyclic aromatic ring systems, wherein one or more (in some
"heterocyclyl" refers in one embodiment to a 3-to 10-membered, in another embodiment to 4-to 7-membered, and in yet another embodiment to 5-to 6-membered saturated monocyclic or polycyclic ring system, wherein one or more (in
The terms "halide", "halogen (halo)" and "halogen (halo)" describe fluorine, chlorine, bromine or iodine.
The term "alkoxy" refers to — O-alkyl. Thus, an alkoxy group is an alkyl (carbon and hydrogen chain) group that is bound solely to oxygen. Non-limiting examples include methoxy (OMe) and ethoxy (OEt).
Group "-O-And O-"refers to a negatively charged oxygen atom. It is understood that the negatively charged oxygen atom is coupled to a cation, such as a metal cation, including but not limited to Na+、K+、Li+(ii) a Or organic cations such as NR "" R '"R'+Wherein R ', R ' and R ' may each independently be a hydrogen atom or an alkyl group.
The term "amine" refers to the group-NR' R2A "group. The group "-NR' R" "refers to an amine group, where R is1And R2Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, ester and carbonyl, each as defined herein or alternatively as known in the art.
As used herein, the term "salt" or "salt thereof" refers to a pharmaceutically acceptable salt of a compound disclosed herein. Non-limiting examples include acid addition cation salts and anion salts. An "acid addition cation salt" is typically formed when a compound having a basic atom is exposed to an acidic environment. As non-limiting examples, these include ammonium ions, such as those formed by protonation of nitrogen-containing compounds. An "anionic salt" is typically formed when a compound having a hydrogen atom is exposed to an alkaline environment. These include, as non-limiting examples, carboxylate salts that may be formed upon deprotonation of carboxylic acids or saponification of esters; and compounds having deprotonated nitrogen atoms.
When referring to possible substitutions, any group recited as "optionally substituted" or "substituted" is said to have one or more atoms or groups of atoms replacing the natural atom or group on any atom thereof (such as a hydrogen atom). In some embodiments, the substituents are selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, alkylene-COOH, ester, -OH, -SH, and-NH. In some embodiments, the number of substituents on a certain ligand is 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 substituents.
The invention also provides the use of any one of the compounds designated herein as SM1, SM6, SM7 or SM 9.
The present invention also provides compositions, e.g., pharmaceutical compositions, comprising at least one compound of formula (I), e.g., a compound designated herein as "
By another aspect, the present invention provides a pharmaceutical composition comprising at least one compound designated "
The invention also encompasses enantiomers, stereoisomers, or any other isomer of any of the compounds listed herein.
The therapeutic efficacy of a compound of the invention, or any enantiomer, stereoisomer or isomer thereof, may be tested in vitro or in animal models (e.g., EAE animal models).
All of these compounds are available from commercial sources, such as Thermo Fisher Scientific Inc.
The compounds of the present invention may be modified and administered as prodrugs.
As used herein, the term "pharmaceutical composition" refers to a preparation of at least one small organic molecule as described herein. In addition, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier, diluent or excipient.
The term "pharmaceutically acceptable carrier, diluent or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, and refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the small organic compound being administered. The term includes adjuvants. Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient.
Non-limiting examples of excipients or nontoxic carriers include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, glucose, sucrose, sodium saccharin, mannitol, lactose, cellulose derivatives, gelatin, vegetable oils, magnesium stearate, magnesium carbonate, talc, polyalkylene glycols, and polyethylene glycols.
The pharmaceutical compositions according to the invention may be prepared by processes well known in the art using one or more pharmaceutically acceptable carriers, for example by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. The appropriate formulation depends on the route of administration chosen. The pharmaceutical compositions may be formulated in solid dosage forms such as powders, pills, tablets and the like, or as solutions, emulsions, suspensions, aerosols, syrups or elixirs.
The term "treatment" or "method of treatment" as defined herein refers to clinical intervention in an attempt to alter the natural disease process of the subject being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable therapeutic effects include preventing the onset or recurrence of disease, reducing, alleviating, or eliminating symptoms, reducing the rate of disease progression, ameliorating or reducing the severity or state of disease, improving prognosis, delaying the onset of disease symptoms, delaying recurrence, and inducing neurogenesis and myelination.
In some embodiments, the effect of a treatment according to the invention on a subject with MS may be monitored by the "extended disability status scale" (EDSS), which is a scale representing the level of disability in 0.5 unit increments over the range from 0 to 10. The scoring is based on examination by a neurologist. EDSS steps (step)1.0 to 4.5 refer to persons with MS who are able to walk without any help and are based on measurements of impairment of the eight Functional Systems (FS) as follows: pyramidal, cerebellum, brainstem, sensation, bowel & bladder, vision, brain and other functional systems (walking) [15 ]. Each functional system is scored on a scale of 0 (normal) to 5 or 6 (maximum damage). EDSS steps 5.0 to 9.5 are defined by walking disorders.
The terms "neuroinflammatory disorder," "neuroinflammatory disease," or "neuroinflammatory condition" are used interchangeably herein and refer to a condition in which an immune response damages a component of the nervous system. Common examples are Multiple Sclerosis (MS) and neuromyelitis optica (NMO), which are characterized by inflammatory demyelination of the central nervous system and subsequent damage to nerve cells and axons. Inflammatory mechanisms have also been implicated in the pathogenesis of a number of other CNS disorders, including systemic inflammatory conditions with central nervous system involvement, such as vasculitis, sarcoidosis and Behcet disease, as well as neurodegenerative diseases, psychiatric disorders, neurotoxic conditions, stroke, and brain injury.
The term "neurodegenerative disease, condition or disorder" as defined herein is a gradual loss of structure or function of neurons in the brain or spinal cord, including the death of neurons. Neurodegeneration is observed after viral damage and mainly in various so-called ' neurodegenerative diseases ' commonly observed in the elderly, such as Alzheimer's Disease (AD), Parkinson's Disease (PD), and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease).
In some embodiments, the pharmaceutical composition according to the invention is for use in the treatment of a "demyelinating disease". The term "demyelinating disease" as defined herein is any neurological disease in which the myelin sheath of neurons is damaged or removed resulting in a loss of function of neuronal cells.
In some embodiments, the neurodegenerative disease according to the invention is multiple sclerosis. In a further specific embodiment, the pharmaceutical composition according to the invention is for use in the treatment of multiple sclerosis.
The term "multiple sclerosis" (MS) as defined herein is a chronic inflammatory neurodegenerative disease of the central nervous system that destroys myelin sheaths, oligodendrocytes, and axons. MS is the most common neurological disorder in young adults, usually occurring between the ages of 20 and 40. The symptoms of MS are the same, ranging from the appearance of visual disorders such as visual loss of one eye, double vision to muscle weakness, fatigue, pain, numbness, stiffness and instability, loss of coordination, and other symptoms such as tremor, dizziness, slurred mouth, dysphagia, and mood disorders. As the disease progresses, patients may lose their ability to walk, may experience cognitive decline, loss of self-management of daily activities, and may become severely disabled and dependent.
MS symptoms develop because immune system elements attack brain cells (glia and/or neurons) and damage the protective myelin sheath of axons. The area in which these attacks occur is called the lesion, which disrupts the information transfer through the brain.
Multiple sclerosis is characterized by disease progression and is divided into four types: (1) relapsing-remitting ms (rrms) characterized by relapses (sudden outbreaks of episodes (attacks-up)) followed by remissions (periods of stable and possibly recovery; while there is complete recovery in some remissions, there is partial or no recovery in others). Symptoms of RRMS can vary from mild to severe, and recurrence may last days or months. More than 80% of patients with MS begin with a relapsing-remitting cycle; (2) secondary progressive MS (spms) develops in patients with relapsing remitting MS. In SPMS, relapse may occur, but there is no remission (stabilization) for a meaningful period of time, and disability gradually worsens; (3) primary progressive MS (ppms), which progresses slowly and steadily from onset and accounts for less than 20% of MS cases. There is no remission, and symptoms are usually not reduced in intensity; and (4) progressive relapsing ms (prms). In this type of MS, people experience both symptoms and attacks that steadily worsen during the remission period.
Known agents for treating MS are corticosteroids, which are primarily used to reduce peak-to-peak inflammation during relapse, β interferon, which slows progression of multiple sclerosis, reduces the number of episodes, and reduces the severity of episodes, glatiramer acetate
Diagnosis of multiple sclerosis can be performed by any method known in the art and includes lumbar puncture (spinal puncture) for cerebrospinal fluid testing, including CSF oligoclonal strips, MRI scanning of the brain and MRI scanning of the spine (spinal cord), and neuronal pathway function studies (evoked potential testing).
As shown in the examples below (e.g., examples 2 and 3), intraperitoneal administration of the small organic compounds SM1, SM7, and SM9 of the present invention improved clinical symptoms in a relapsing/remitting experimental autoimmune encephalomyelitis (RR-EAE) animal model, a well-established model that mimics Multiple Sclerosis (MS).
The term "experimental autoimmune encephalomyelitis" (EAE, or experimental allergic encephalomyelitis) as defined herein generally refers to an induced inflammatory demyelinating disease of the Central Nervous System (CNS), which is widely accepted as an animal model of human CNS demyelinating diseases including, but not limited to, Multiple Sclerosis (MS) and Acute Disseminated Encephalomyelitis (ADEM).
EAE can be induced in many species, including mice, rats, guinea pigs, rabbits, and primates. Disease induction is typically performed by exposing the animal to various antigens. The most commonly used antigens are Spinal Cord Homogenate (SCH), purified myelin, myelin proteins such as Myelin Basic Protein (MBP), myelin proteolipid protein (PLP or lipophilin), and Myelin Oligodendrocyte Glycoprotein (MOG) or peptides of these proteins, all of which lead to different models with different disease characteristics in both immunology and pathology.
Depending on the antigen used and the genetic makeup of the animal, rodents may exhibit monophasic attack of EAE (monophasic bout), relapsing remitting forms, or chronic EAE. A typical susceptible rodent will first develop clinical symptoms about two weeks after immunization and will exhibit symptoms of relapsing remitting disease.
Modeling of multiple sclerosis can be performed with SJL/J mice. The EAE model was induced by proteolipid protein (PLP) fragments (along with pertussis toxin) in 8-week-old SJL/J female mice. This model shows a relapsing-remitting (RR) disease process similar to that observed in MS patients.
Modeling of multiple sclerosis can also be performed using C57BL/6 female mice, where the disease is induced by myelin-oligodendrocyte glycoprotein peptide (MOG). This model represents a progressive (also called chronic) form of the disease.
As is known in the art, the disease activity of a model animal is typically scored using the following scoring index (referred to as "disease activity index", DAI): 0, normal mice, no obvious signs of disease; "1", a soft tail or hind limb weakness, but not both; "2", soft tail and hind limb weakness; 3, paralysis of partial hind limbs; 4, complete hind limb paralysis; and "5", death or sacrifice for humane reasons. Other DAIs also exist, such as DIA using the following scores: 1, tail weakness; "2", mild to moderate hind limb weakness; 3, severe hind limb weakness; "4", complete paralysis of one or more limbs; "5" dying.
The scoring index above can therefore be used to monitor the severity of the disease and the onset of recurrence in order to determine the therapeutic efficacy of the small organic molecules of the invention.
Thus, in one embodiment, the compounds of the invention may be used to treat RR-MS.
In other embodiments, the compounds of the invention may be used to treat secondary progressive ms (spms), or primary progressive ms (ppms), or progressive relapsing ms (prms).
In another aspect, the present invention provides a method of treating multiple sclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of at least one compound of formula (la) selected from the group consisting of: "
For example, the step of administering a compound of the invention or a pharmaceutical composition of the invention may be performed by, but is not limited to, the following routes of administration: oral, rectal, transmucosal, nasal, intestinal, or parenteral delivery (including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections). Alternatively, one may administer a pharmaceutical composition according to the invention in a local rather than systemic manner, e.g. via direct injection of the pharmaceutical composition into a tissue region of the subject, e.g. into an affected CNS region.
In some embodiments, the small organic molecules of the present invention are administered in combination with an additional therapeutic agent.
The additional therapeutic agent may be any therapeutic agent suitable for or known to be useful in the treatment of multiple sclerosis or for reducing the rate of relapse and affecting the progression of disability non-limiting examples include interferon- β 1 (such as
The additional therapeutic agent may be a cell therapy comprising administration of cells including, but not limited to, Mesenchymal Stem Cells (MSCs), MSC-like cells, neural progenitor cells, CD34+Cells, CD133 from all available sources+Cells, induced pluripotent stem cells (ipscs), differentiated pluripotent stem cells, and the like.
In one embodiment, the additional therapeutic agent is an agent that mediates BBB opening, such as mannitol, which may be injected intravenously.
The additional therapeutic agents may be formulated with the small organic compounds of the present invention or be part of a separate composition. Additional agents may be administered with the compounds of the present invention or separately. It may be administered prior to, simultaneously with or after administration of the compound or pharmaceutical composition of the invention.
As known in the art, the term "Blood Brain Barrier (BBB)" relates to the separation of the central nervous system from the membrane structure of the circulating blood.
The term "therapeutically effective amount" (or amount) of at least one small organic molecule according to the present invention is determined by considerations known in the art to cure, arrest, or at least alleviate a medical condition. The precise dosage and frequency of administration depends on the severity of the disease in the patient, the route of administration and the pharmacokinetics of the compound. Determining dosages is a routine procedure and is well known to physicians and others of ordinary skill in the art. For any preparation used in the methods of the invention, the dosage or therapeutically effective amount may be initially assessed by in vitro assays, cell culture assays, and in vivo experiments in animal models (e.g., EAE).
For example, the doses provided below were evaluated based on the EAE mouse model of multiple sclerosis. As shown in examples 2 and 3 below, treatment with each of SM1, SM7, or SM9 at 10mg/kg or 20mg/kg improved EAE. Clearly, the symptoms of the treated EAE mice were less severe than those of the controls at each stage of the study. This effect is also manifested by the fact that: in the group of treated EAE mice, fewer mice exhibited a more severe form of the disease (i.e., a clinical score of 2-5). Furthermore, it has been shown that treatment results in an increased amount of myelin in the spinal cord of EAE mice, indicating either increased remyelination processes or decreased demyelination, or both, in response to these SM treatments.
Non-limiting examples of therapeutic doses include, but are not limited to, 400mg-800mg per about 70kg human.
It should be noted that the amount of small organic molecules to be administered may vary by about 5% -25% taking into account the molecular weight and other characteristics of the particular agent. Thus, the term "about" as defined herein refers to a fluctuation of 5% -25% of the amount as defined herein. Preferably, the term "about" encompasses a variation of +/-10%, more preferably +/-5%, even more preferably +/-1%, and still more preferably +/-0.1% from the specified value.
The small organic molecules according to the invention or any pharmaceutical composition comprising said small organic molecules may be administered to a patient in single or multiple administrations. The small organic molecule or pharmaceutical composition comprising the small organic molecule may be administered to a patient continuously or for discrete periods of time, as determined by considerations known to those skilled in the art, in order to cure, arrest or at least alleviate the medical condition.
Toxicity and therapeutic efficacy of the small organic molecules described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture, or in animal experiments. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage may vary depending on the dosage form employed and the route of administration.
The term "subject" as used herein means a warm-blooded animal such as, for example, rats, mice, dogs, cats, guinea pigs, primates, and humans. Although the methods of the invention are particularly intended for treating human subjects suffering from neurodegenerative diseases, other mammalian subjects are also included. The terms subject and patient are used interchangeably herein.
The invention also discloses a compound of formula "
Also disclosed is the use of at least one compound of formula "
The compounds of the present invention were initially selected based on their ability to inhibit BMP-2 signaling in vitro. As shown in example 1, small organic molecules SM1, SM6, SM7 and SM9 were screened from large libraries for their ability to inhibit BMP-2 in vitro without generating cytotoxicity in mouse ATDC5 cells. Furthermore, as shown in example 4, example 7 and example 8, these compounds show an increase in several markers indicative of neural differentiation in vitro.
The term "bone morphogenic protein" (BMP) as defined herein refers to a group of growth factors also known as cytokines or morphogens (metablogens) that induce bone and cartilage formation and have multiple roles in embryonic brain development to
"bone
In some embodiments, bone
"bone
In some embodiments, bone
BMPs interact with specific receptors on the cell surface, known as Bone Morphogenetic Protein Receptors (BMPR). Signal transduction by BMPR leads to mobilization of SMAD protein family members (mobilisation). As used herein, the term "BMP signaling" refers to the signaling pathway initiated by the binding of BMP to its receptor, and subsequent cellular processing induced by this binding, such as mobilization of members of the SMAD protein family.
It is to be understood that this invention is not limited to the particular embodiments, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be defined only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of the present invention as described herein, the following terms are defined.
Examples
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way whatsoever.
Standard molecular biology protocols known in the art, not specifically described herein, generally substantially follow Sambrook & Russell, 2001.
Abbreviations:
small molecule-SM
Experimental autoimmune encephalomyelitis-EAE;
relapsed and remitted-RR;
multiple sclerosis-MS;
recurrent and remitting experimental autoimmune encephalomyelitis-RR-EAE;
proteolipid protein peptide-PLP;
myelin-oligodendrocyte glycoprotein peptide-MOG;
phosphate buffered saline-PBS;
complete freund's adjuvant-CFA;
pertussis toxin-PTX;
bone morphogenetic protein-BMP;
blood brain barrier-BBB;
central nervous system-CNS;
h-hr, h;
min-min.
Experimental procedures
High Throughput Screening (HTS) bioassays
poly-L-lysine (Sigma-Aldrich) was added to 384 well plates and removed after 30min of RT incubation. Assay medium containing DMEM/F12, 2% FBS, 100 units/ml penicillin, 100mg/ml streptomycin (Biologicalindinduries) was added to each well. Then recombinant human BMP-2(rhBMP-2, R)&D Systems) was added to each well to a final concentration of 2 μ g/ml. ATDC5 cells (Sigma-Aldrich) were harvested, resuspended in assay medium and added to a final concentration of 2000 cells/well. Finally, heparin (Sigma-Aldrich) was added to a final concentration of 2. mu.g/ml. Cells were incubated with 5% CO at 37 ℃ in the presence or absence of a potential small molecule inhibitor of BPM-22And incubated in a humidified chamber for 48 hours. After incubation, ALP levels and cell viability were determined using a fully automated protocol. Cells were washed with PBS, lysed with lysis buffer containing 0.2% Triton X-100 in PBS supplemented with protease inhibitor cocktail (Sigma-Aldrich), and incubated for 25min at RT. Then by adding
Induction of Experimental Autoimmune Encephalomyelitis (EAE) in mice
100 μ g/mouse proteolipid protein peptide (PLP) in 0.1ml PBS by subcutaneous immunization (day 0) in SJL female mice (6-8 weeks old)139-151Synthesized by Sigma-Aldrich) to induce RR-EAE. The peptide was emulsified in an equal volume of complete Freund's adjuvant (CFA, from DIFCO) containing 500. mu.g M.tuberculosis (Mycobacterium tuberculosis) H37RA (MT, from DIFCO). Mice also received intraperitoneal injections of 300ng pertussis toxin (PTX, from Sigma-Aldrich) in 0.2ml PBS. A second injection of PTX (300 ng/mouse) was given 48h later.
Mice were monitored for RR-EAE symptoms and scored as follows: "0", no disease; 1, paralysis of tail; 2, weakness of hind limbs; 3, hind limb paralysis; "4", weakness/paralysis of hind and fore limbs; "5" dying.
All procedures involving mice were performed according to the guidelines of the animal ethics committee of the Sourasky medical center.
In vitro differentiation assay of P19 cells
P19 cells were grown at 37 ℃ in 5% CO2 in α -minimal essential medium (α MEM) (Gibco) containing 7.5% calf serum, 2.5% fetal bovine serum, and 0.4. mu.l/ml penicillin-streptomycin (Gibco). The cells were supplemented with fresh medium every 48h for differentiation studies, P19 cells were grown at
1)5×10-7m all-trans Retinoic Acid (RA) (Sigma)
2)RA+5ng/ml rhBMP2(R&D systems)
3) RA + rhBMP-2+500ng/ml mouse anti-human BMP-2/4mAb (R & D systems)
4)RA+rhBMP-2+SM1/SM7/SM9(Maybridge(HitFinderTMCollection)), the concentration of SM1/SM7/SM9 is: 0.625. mu.M, 1.25. mu.M and 2.5. mu.M.
Media containing RA, BMP supplements and SM were supplemented after 48 h. After 4 days, aggregates formed during RA treatment were passed through the enzyme(0.05% v/v trypsin-0.02% v/v EDTA) and mechanical means, and they were plated in tissue culture grade dishes. At this stage, cells were cultured in medium without RA, BMP and SM, which was refreshed every 48 h. MAP-2 positive neurons were examined by immunofluorescence on day 8 (i.e.,
Determination of SMAD1/5/8 Signaling by Western blot analysis
P19 cells were seeded in 6-well plates (3.5X 10)5Individual cells/well). On the following day, the cells were not treated, or treated with 5X 10-7M RA, 5ng/ml BMP-2 or SM for 4 h. Cells were washed with ice cold PBS, harvested and lysed with ice cold RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail and sodium orthovanadate (Na3VO4) as phosphatase inhibitor (Sigma-Aldrich). Protein concentration was determined using BCA protein assay kit (Pierce, Rockford, IL). Cell lysates (40 μ g-60 μ g protein) were separated by electrophoresis on 4% -15% SDS-PAGE gels and then transferred to 0.2A nitrocellulose membranes for 2 h. The membranes were blocked in 5% (w/v) skim milk powder for 1h at room temperature and then incubated with
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