Treatment of demyelinating diseases

Abstract

Described herein are methods of promoting remyelination in a subject having a demyelinating disease by administering to the subject a combination of a steroid hormone and a Hedgehog signaling pathway modulator. Also described are methods of administering a combination of pharmaceutical products, wherein the combination of pharmaceutical products is in a composition suitable for nasal administration.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application 62 / 616,173, filed January 11, 2018, pursuant to 35 U.S.SC §119, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This article describes compositions and methods for treating demyelinating diseases using steroid hormones and sonic hedgehog signal transduction pathway modulators. Background Technology

[0004] Myelin is a fatty, white substance that forms an electrical insulating layer around the axons of certain nerve cells. In the human brain, approximately 40% of the brain contains white matter, which consists of densely packed fibers, of which myelin is the main component (50-60% of the dry weight of white matter). Myelin is synthesized and maintained by oligodendrocytes (OLs) in the central nervous system (CNS). Oligodendrocytes are a type of glial cell that provides support and insulation for axons in the CNS. Oligodendrocytes are produced by oligodendrocyte precursor cells (OPCs) and are found only in the CNS.

[0005] In demyelinating diseases, the myelin sheath of neurons in the nervous system is damaged. This damage can impair signal transmission in the affected nerves, leading to deficits in functions such as sensation, movement, cognition, and others, depending on the nerve involved. Among various demyelinating diseases, multiple sclerosis (MS) is the most prevalent disabling neurological condition among young adults worldwide. The Multiple Sclerosis Foundation estimates that more than 400,000 people in the United States and approximately 2.5 million worldwide suffer from MS. In the United States, approximately 200 new cases are diagnosed every week. It is an expensive disease to treat, and in the United States, direct and indirect medical costs range from $8,528 to $54,244 per patient per year.

[0006] Multiple sclerosis (MS) impairs the ability of some parts of the nervous system to communicate, leading to a range of physical, psychological, and sometimes psychiatric problems. There is no known cure for MS, but current treatments attempt to improve function after an attack and prevent new attacks. Most drugs used to treat MS may be effective in relapsing-remitting forms of the disease, but are generally ineffective in the progressive form characterized by chronic demyelination of the axons. Although the immunomodulatory agent ocrelizumab has recently shown efficacy in the progressive form, it is associated with major potential side effects and is poorly tolerable. See Montalban X. et al., “Ocrelizumab vs. placebo in primary progressive multiple sclerosis,” New England Journal of Medicine 376 209-220 (2017).

[0007] In another approach, US 2013 / 0226133 describes a method for restoring the myelin sheath of nerve fibers using sclerotinia sulfate. In yet another approach, US 2004 / 0141947 discloses a method for treating demyelinating CNS diseases using colony-stimulating factors or colony-stimulating factor-like ligands, such as sargramostim (a type 1 interferon homolog), and at least one additional therapeutic agent. In yet another approach, US 2004 / 0053850 describes a method for treating demyelinating CNS diseases by co-administering a tripeptide gly-pro-glu and an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) / algaeine antagonist compound. In yet another approach, US Patent No. 4,760,092 claims a method for treating demyelinating diseases such as multiple sclerosis using colchicine or colchicine. In another approach, US 2013 / 0302410 describes a method for neuroprotection using dimethyl fumarate or monomethyl fumarate in demyelinating diseases. In yet another approach, US 2013 / 0108643 describes a method for treating autoimmune or inflammatory diseases using an inhibitor of macrophage scavenger receptor class 1 MSR1. In yet another approach, EP 0423943 describes the use of inhibitors of members of the mammalian collagenase family for the treatment of demyelinating diseases.

[0008] Despite these proposed methods, there is still a need for ways to promote myelin regeneration in subjects with demyelinating diseases such as MS. Summary of the Invention

[0009] According to some embodiments, a method for promoting myelin regeneration in a subject in need is provided, the method comprising administering to the subject an effective amount of a steroid hormone and a hedgehog signaling pathway modulator. In some embodiments, the steroid hormone is an androgen receptor ligand such as testosterone, a progesterone receptor ligand such as progesterone or allogeneic estradiolone, an estrogen receptor ligand such as estradiol, or dehydroepiandrosterone, or a selective hormone receptor modulator such as a selective androgen receptor modulator, a selective estrogen receptor modulator, or a selective progesterone receptor modulator. In some embodiments, the hedgehog signaling pathway modulator is a smoothed (Smo) agonist, such as 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide (SAG). In some embodiments, the hedgehog signaling pathway modulator is a hedgehog signaling pathway antagonist, such as a Gli antagonist. In some embodiments, the hedgehog signaling pathway antagonist is 2,2'-[[dihydro-2-(4-pyridyl)-1,3(2H,4H)-pyrimidinidyl]bis(methylene)]bis[N,N-dimethylaniline] (GANT-61). In some embodiments, the method comprises administering both a Smo agonist and a Gli antagonist, such as SAG as the Smo agonist and GANT-61 as the Gli antagonist.

[0010] In some embodiments, the steroid hormone and the hedgehog signaling pathway modulator are administered substantially simultaneously or sequentially as separate compositions. In other embodiments, the steroid hormone and the hedgehog signaling pathway modulator are administered as the same composition.

[0011] In some embodiments, one or both of the steroid hormone and the hedgehog signaling pathway modulator are administered intranasally as an intranasal pharmaceutical composition, the intranasal pharmaceutical composition further comprising: (a) at least one lipophilic or partially lipophilic carrier present in an amount of about 60% to about 98% by weight of the formulation; (b) at least one compound having surface tension reducing activity present in an amount of about 1% to about 20% by weight of the formulation; and (c) at least one viscosity modifier present in an amount of about 0.5% to about 10% by weight of the formulation. In some embodiments, the intranasal pharmaceutical composition comprises the steroid hormone. In some embodiments, the intranasal pharmaceutical composition comprises the hedgehog signaling pathway modulator. In some embodiments, the intranasal pharmaceutical composition comprises the steroid hormone and the hedgehog signaling pathway modulator. In some embodiments, the intranasal pharmaceutical composition comprises the steroid hormone, a Smo agonist, and a Gli antagonist.

[0012] In some embodiments, one or both of the steroid hormone and the hedgehog signaling pathway modulator are administered intranasally as an intranasal pharmaceutical composition comprising a porous excipient, wherein the steroid hormone and / or the hedgehog signaling pathway modulator is loaded onto a surface of the porous excipient located within the pores of the porous excipient. In some embodiments, the steroid hormone is loaded onto a surface of the porous excipient located within the pores of the porous excipient. In some embodiments, the hedgehog signaling pathway modulator is loaded onto a surface of the porous excipient located within the pores of the porous excipient. In some embodiments, both the steroid hormone and the hedgehog signaling pathway modulator are loaded onto a surface of the porous excipient located within the pores of the porous excipient. In some embodiments, the steroid hormone, Smo agonist, and Gli antagonist are loaded onto a surface of the porous excipient located within the pores of the porous excipient.

[0013] According to any embodiment, the subject may be a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.

[0014] According to any embodiment, the subject may suffer from a demyelinating disease, such as a central nervous system demyelinating disease selected from multiple sclerosis, amyotrophic lateral sclerosis, Devic's disease, inflammatory demyelinating diseases, central nervous system neuropathy, central pontine myelinolysis, spinal cord lesions, tabes dorsalis, syphilitic spinal cord lesions, white matter lesions such as progressive multifocal leukoencephalopathy, leukodystrophy, and Alzheimer's disease; or a peripheral nervous system demyelinating disease selected from Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie Tooth disease, hereditary neuropathy prone to compressive paralysis, peripheral neuropathy, spinal cord disease, optic neuropathy, and progressive inflammatory neuropathy.

[0015] Also provided is a steroid hormone as described herein and a hedgehog signaling pathway modulator as described herein, for use in a method as described herein for promoting myelin regeneration in subjects in need.

[0016] Also provided is the use of a steroid hormone as described herein and / or a hedgehog signaling pathway modulator as described herein in the preparation of a medicament for treating demyelinating subjects in need, wherein the method comprises administering the steroid hormone and the hedgehog signaling pathway modulator to the subject as described herein.

[0017] According to other embodiments, a method for generating oligodendrocytes is provided, the method comprising culturing primary mixed glial cells in a culture medium comprising a steroid hormone as described herein and a Smoothened agonist as described herein.

[0018] According to other embodiments, a method for differentiating oligodendrocytes into myelin-producing cells is provided, the method comprising incubating oligodendrocytes in a culture medium comprising steroid hormones as described herein and hedgehog signaling pathway antagonists as described herein. Attached Figure Description

[0019] Figure 1 A-1D shows the dynamic transfection of hedgehog and androgen signaling components during the final oligodendrogenesis wave and myelination process in the early postnatal dorsal medulla oblongata. Figure 1 A shows the relative expression of the transcript encoding myelin basic protein (Mbp). Figure 1 B shows the relative expression of Shh ligands in hedgehog signaling. Figure 1 C shows the relative expression of the hedgehog signaling component transcription factor Gli1. Figure 1 D shows the relative expression of the major receptor mediating androgen signaling (AR). Expression is reported relative to GAPDH as determined by quantitative RT-PCR performed on the dorsal telebrain of male (grey bars) or female (black bars) mouse pups aged 0, 3, 8, or 15 days. Sexual bimorphism in AR expression was detected only. Reported values ​​are mean ± SEM from 3 pups of each sex at each age. *, p ≤ 0.05.

[0020] Figure 2 A-2B illustrates the functional interactions between hedgehog and testosterone signaling pathways in vitro regarding the control of OPC proliferation and differentiation. Figure 2 A shows Olign2 incorporating the proliferation marker BrdU 2 hours before the end of the culture. + Cell quantification was performed and the synergistic effect of SAG (0.1 μM) and testosterone (1 μM) was demonstrated. Figure 2 A also showed that the Smo antagonist SANT-1 (SANT-1, 0.1 μM) blocked testosterone-induced Olig2 + BrdU + Increase in cells. Figure 2 B shows the Plp of myelin marker Mbp, which is differentiated from primary mixed glial cells and therefore co-expresses the marker assessed in the absence (Ctrl) or presence (of SAG, SANT-1, or testosterone (T)). + GFP + Number of oligodendrocytes. Reported values ​​are mean ± SEM from 3–4 independent cultures. Compared with Ctrl, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Comparisons between different drugs are as indicated: # p≤0.05; ## p≤0.01; ### p≤0.001; $$$ p≤0.001.

[0021] Figure 3 A-3D demonstrates that blocking hedgehog signaling during the final oligodendrocyte generation wave in the dorsal forebrain enhances testosterone-induced OPC differentiation in vivo. Male pups 10 days postpartum were treated with the Smo agonist SAG, the Smo antagonist SANT-1, the steroid hormone testosterone (T), SAG and T, SANT-1 and T, or as a control (Ctr). Figure 3 A shows the quantification of the number of oligodendrocytes and astrocytes in brain slices of treated mice, as indicated by immunostaining based on the transcription factor Olign2. Figure 3 B shows the quantification of oligodendrocytes (OPCs) in brain slices of treated mice, as indicated by immunostaining based on platelet-derived growth factor receptor A (PDGFRα). Figure 3 C shows the quantification of mature oligodendrocytes (OLs) in brain slices of treated mice as indicated by immunostaining based on colonic adenomatous polyposis (APC). Figure 3 D shows the quantification of myelin OLs expressing myelin basic protein (MBP) in brain slices from treated mice, as indicated. Significantly, MBP is required for maturation into axonal wrapping and testosterone-induced segment elongation. + OL was enhanced by blocking Hedgehog signaling. Reported values ​​are mean ± SEM from 3–5 animals for each condition. Compared with Ctrl, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Comparisons between different drugs are as indicated: # p≤0.05.

[0022] Figure 4A-4E demonstrates that in a mouse model of central nervous system demyelination, Smo's pharmacological activity enhances OPC / mature OL density and specifically promotes the transition to a precocious puberty phenotype towards a pro-regenerative phenotype in microglia activation. Figure 4 A shows a histogram based on immunostaining of brain slices from adult male mice 10 days after injection of lysophosphatidylcholine (LPC) into the corpus callosum, with SAG present (Ctrl, white bar) or absent (black bar) to visualize the quantitative distribution of PDGFRα+OPC on the surface of each cell. Figure 4 B shows a histogram derived from immunostaining of brain slices from adult male mice 10 days after injection of lysophosphatidylcholine (LPC) into the corpus callosum, with SAG present (Ctrl, white bars) or absent (black bars), representing Olig2 per unit surface area. + / APC + Quantification of mature oligodendrocytes (OLs) and visualization of the percentage of Oligodendrocyte lineages co-expressing the APC marker. Reported values ​​are mean ± SEM data from n = 4–6 animals for each disease condition. Figure 4 C shows a histogram based on immunostaining of brain slices from adult male mice 2 days after injection of lysophosphatidylcholine (LPC) into the corpus callosum in the presence of SAG (Ctrl, white bars) or in the absence of SAG (black bars) to quantify Ki67+ / PDGFRα+ OPC per unit surface area. Figure 4 C, based on Ki67 and PDGFRα immunostaining (left panel), further shows that a higher number of proliferating OPCs was observed in the symptom after SAG treatment, but the number of PDGFRα+ cells was not changed by SAG treatment at this early time point (right panel). Figure 4 D shows a histogram that visualizes the quantification of GFAP+ astrocytes as a percentage of the total area. Figure 4 E shows a histogram derived from immunostaining of brain slices from adult male mice 2 days after injection of lysophosphatidylcholine (LPC) into the corpus callosum in the presence of SAG (Ctrl, white bar) or the absence of SAG (black bar), visualizing Iba1+, Arg1+ cells (left panel) and Arg1+ cells (right panel) per surface unit as the percentage of Iba1+ cells. Figure 4 E further demonstrated the detection of Arg-1 in lesions of animals treated with SAG. + It promotes the regeneration of microglia by a much higher number.

[0023] Figure 5A-5F demonstrates that a combination therapy based on Smo-mediated simultaneous drug activation of Hh and androgen signaling highly alleviates the progression of experimental autoimmune encephalomyelitis (EAE). Figure 5 A shows the EAE clinical score after therapeutic administration of SAG and testosterone, either alone or in combination, compared to the mediator administration. Figure 5 B shows electron micrographs of the lumbar spinal cord from EAE mice treated with catarrhal agent (Ctrl), testosterone (T), SAG, and SAG+T. In addition to normal myelinated axons (rightmost arrow), demyelinated axons (bottom arrow) and abnormal axons (leftmost arrow) were observed at higher levels in the control symptom. Figure 5 C shows an analysis of the g-ratio (axon diameter / axon + myelin diameter) and indicates significantly lower values ​​compared to the control when testosterone and SAG are used alone or together. SAG alone or with testosterone showed a higher effect on the g-ratio than testosterone alone. Figure 5 D illustrates the quantification of aberrant axons, which include axons exhibiting compacted but detached myelin sheaths, double myelin sheaths, or multilayered myelin sheaths with medial obstruction. The percentage of axons with abnormalities was significantly reduced in the treated condition compared to the control. Figure 5 F-5F shows immunostaining of Iba and Arg1 in the lumbar spinal cord of EAE animals treated with a catalytic agent (Ctrl) or with the drugs testosterone and SAG, alone or in combination, based on Iba1. + ( Figure 5 E) and Arg1 + ( Figure 5 F) Quantification of the area occupied by cells as a percentage of the total lesion area in the image. Figure 5 G shows the quantification of the GFAP-positive area in the spinal cord of EAE animals treated with testosterone and SAG alone or simultaneously as a percentage of the lesion area. Figure 5 H shows the quantification of the area positive for sealing protein as a percentage of the lesion area in EAE animals treated with testosterone and SAG alone or simultaneously. Data reported are mean ± SEM (n = 10 mice for each symptom). *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, ##, p = 0.001, ****, p < 0.0001, one-way ANOVA versus Tukey's multiple comparison test. Detailed Implementation

[0024] This article describes a method for promoting myelin regeneration in subjects in need, the method comprising administering to the subject an effective amount of a steroid hormone and a hedgehog signaling pathway modulator. In some embodiments, the method is used to treat demyelinating diseases such as MS peak. Related compositions and uses of the steroid hormone and hedgehog signaling pathway modulator are also described. Further descriptions include the use of steroid hormones, smoothed agonists, and hedgehog signaling pathway antagonists. An in vitro method for generating proliferating oligodendrocytes is also described, the in vitro method involving culturing primary mixed glial cells in a culture medium comprising a steroid hormone and a Smo agonist. An in vitro method for differentiating oligodendrocytes into myelin-producing cells is also described, the in vitro method involving incubating oligodendrocytes in a culture medium comprising a steroid hormone and a Smo antagonist.

[0025] definition

[0026] The technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art as per the understanding of the terms involved in this invention, unless otherwise defined. Unless otherwise stated, the materials, reagents, etc., referred to in the following description and examples are commercially available.

[0027] As used herein, the singular forms “a / an” and “the” refer to both the singular and plural forms, unless explicitly stated otherwise.

[0028] The term "about" means that the number is not limited to the exact number set forth herein, and is intended to refer substantially around the number without departing from the scope of the invention. As used herein, "about" will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If there is a use of terminology that is not clear to those skilled in the art, then, taking into account the context in which the term is used, "about" means up to ±10% of the particular term.

[0029] As used in this article, “Smoothened” or “Smo” refers to a 7-transmembrane GPCR-like receptor located primarily in the membrane of intracellular vesicles or at their activated plasma membrane. Smo is the Shh signaling pathway. The Shh pathway plays a role in controlling oligodendrocyte generation during embryonic development. See, for example, Traiffort E. et al., “Hedgehog: A key signaling in the development of theoligodendrocyte lineage,” *Dev.Biol.* 4:28 (2016); Ferent and Traiffort, “Hedgehog: Multiple Paths for Multiple Roles in Shaping the Brain and Spinal Cord,” *Neuroscientist* 21:356-71 (2015).

[0030] As used herein, “subject” means any mammal, including humans, that requires treatment for a demyelinating disease or condition or requires promotion of myelin regeneration. For example, a subject may have a demyelinating disease or condition or be at risk of having a demyelinating disease or condition.

[0031] As used herein, the term “application” includes direct application to another, self-application, and application of prescribed or directed pharmaceutical agents as disclosed herein.

[0032] As used herein, the phrases “effective amount” and “therapeutic effective amount” refer to the dose of the active agent in a subject or the plasma concentration that provides for the specific pharmacological effect of administering the active agent in a subject requiring such treatment. It should be emphasized that an effective amount of the active agent is not always effective in treating the symptoms / diseases described herein, even if such a dose is considered therapeutically effective by those skilled in the art.

[0033] As used herein, the term "pharmaceutical composition" refers to one or more active agents formulated using a pharmaceutically acceptable carrier, excipient, or diluent.

[0034] The phrase "pharmaceutically acceptable" is used here to refer to compounds, materials, compositions, and / or dosage forms that are within the bounds of reasonable medical judgment, suitable for use in vivo without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0035] Methods to promote myelin regeneration

[0036] The method described in this article is based on the surprising discovery that treatment with steroid hormones and hedgehog signaling pathway modulators dramatically increased the number of oligodendrocytes and myelin-producing cells. Although therapies using steroid hormones alone or hedgehog signaling pathway modulators alone have been described (see, for example, El-Etr et al., "Hormonal influences in multiple sclerosis: new therapeutic benefits for steroids," *Maturitas* 68:47-51 (2011); Bielecki et al., "Unexpected central role of the androgen receptor in the spontaneous regeneration of myelin," *Proceedings of the National Academy of Sciences* 113:14829-14834 (2016); Samanta et al., "Inhibition of Gli1 mobilizes endogenous neural stem cells for myelin regeneration") "Sonic Hedgehog signaling is a positive oligodendrocyte regulator during demyelination" (J. Neuroscience) 33:1759-72 (2013). However, the inventors found that using steroid hormones in conjunction with hedgehog signaling pathway regulators synergistically enhances the production of oligodendrocytes and myelin-producing cells, thereby leading to improved promotion of myelin regeneration and providing more effective treatment for demyelinating diseases.

[0037] In this regard, the inventors discovered an overlapping expression pattern of Shh signaling components and androgen receptors during the early development of oligodendrocytes. See Example 1. Figure 1These expression patterns appear consistent with the first evidence of a functional interaction between Shh signaling and steroid hormones during myelination. Indeed, the inventors found that treatment with androgens (such as testosterone), at higher levels in the presence of Smo agonists compared to the absence of Smo agonists, promoted oligodendrocyte precursor cell proliferation. Furthermore, the concomitant use of Smo antagonists and testosterone was found to promote the synergistic differentiation of myelin-producing cells. See Example 1. Figure 2 And Example 1, Figure 3 These results support the method described in this paper.

[0038] The inventors also made the surprising discovery that, in the context of myelin repair, Smo agonists promote the conversion of microglia activation toward a precocious puberty phenotype that promotes regeneration, and can synergize with steroid hormones (such as testosterone) to promote myelin regeneration, as illustrated in the examples reported below. These results also support the methods described herein.

[0039] Steroid hormones

[0040] Steroid hormones used in the compositions and methods described herein include, but are not limited to, hormones of the progesterone, estrogen, and androgen families, synthetic steroid hormones, and selective hormone receptor modulators.

[0041] In some embodiments, steroid hormones are androgen receptor ligands (e.g., androgens). Androgens are a group of steroid hormones whose effects are mediated by the binding and activation of androgen receptors (ARs). As used herein, androgens include testosterone and dihydrotestosterone. In a specific embodiment, the steroid hormone is the androgen testosterone.

[0042] In some embodiments, the steroid hormone is a progestin. Progestins are a group of steroid hormones whose effects are mediated by the binding and activation of the progesterone receptor (PR). In some specific embodiments, the progestin is progesterone or allogestrinone, which is derived from progesterone and activates the γ-aminobutyric acid (GABA) receptor.

[0043] In some embodiments, steroid hormones are estrogen receptor ligands (e.g., estrogens). Estrogens are a group of steroid hormones whose effects are mediated by the binding and activation of estrogen receptors (ER). As used herein, estrogens include estradiol.

[0044] In some embodiments, the steroid hormone is dehydroepiandrosterone (DHEA). DHEA can act as a precursor to both androgenic steroids and estrogen steroids.

[0045] In some embodiments, the steroid hormone is a synthetic steroid.

[0046] In some embodiments, selective hormone receptor modulators are used as “steroid hormones” in the methods described herein. Selective hormone receptor modulators act similarly to steroid hormones, but are generally more selective than steroids themselves. As used herein, “selective hormone receptor modulator” includes, but is not limited to, selective androgen receptor modulators, selective estrogen receptor modulators, and selective progesterone receptor modulators.

[0047] Hedgehog signal transduction pathway modulator

[0048] Hedgehog signaling pathway modulators used in the compositions and methods described herein comprise a Smo agonist that increases Shh signaling, a Smo antagonist that decreases Shh signaling, and a Gli antagonist.

[0049] Therefore, in some embodiments, the hedgehog signaling pathway regulator is a smoothed (Smo) agonist. The Smo agonist can interact with the Smo receptor to activate downstream Gli transcription factors. See, for example, Hadden et al., “Hedgehog Pathway Agonism: Therapeutic Potential and Small-Molecule Development,” *Chem. Med. Chem.* 9:27–37 (2014); Chen et al., “Small molecule modulation of smoothed activity,” *Proceedings of the National Academy of Sciences* 99:14071-14076 (2002). Examples of smoothed agonists include 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide (SAG) and 3-chloro-4,7-difluoro-N-(4-(carboxamide)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide (Hh Ag-1.5), glucocorticoids, 9H-purine-6-amino, 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthoxy), propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroazanaphthalene-3-carboxamide) benzoate (GSA-10), cholesterol, and osteogenic (1H)-quinolone-based compounds, such as GSA-10-like compounds 20 and 25 (Manetti et al., “Design, synthesis and biological characterization of a new class of osteogenic (1H)-quinolone derivatives”, Eur. J. Med. Chem. 121:747-757 (2016)). In a specific embodiment, the hedgehog signal transduction pathway modulator is the Smo agonist 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide (SAG).

[0050] In some embodiments, the hedgehog signaling pathway modulator is a Gli antagonist. Gli antagonists include, but are not limited to, small molecule Gli1 antagonists disclosed in Lauth et al., “Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists,” Proceedings of the National Academy of Sciences 104:8455-60 (2007), such as 2,2'-[[dihydro-2-(4-pyridyl)-1,3(2H,4H)-pyrimidinidyl]bis(methylene)]bis[N,N-dimethylaniline] (GANT61) and 2,3,4,5-tetra(4-pyridyl)thiophene, 4,4′,4″,4″′-thiophene-2,3,4,5-dimethylpyrimidine (GANT58). Both GANT61 and GANT58 are believed to function in the nucleus to block Gli function, and GANT61 is believed to interfere with Gli1 DNA binding. In a specific embodiment, the hedgehog regulator is the Gli antagonist GANT61.

[0051] Other modulators of the hedgehog signaling pathway are Smoothened antagonists, such as Chen (2002) (ibid.) and Rimkus et al., “Targeting the Sonic Hedgehog signaling pathway: Review of Smoothened and Gli inhibitors”. The inhibitors disclosed in Cancer (8:pii:E22 (2016)) comprise (4-benzyl-piperazin-1-yl)-(3,5-dimethyl-1-phenyl-1H-pyrazole-4-ylmethylene)-amino (SANT-1), N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-triethoxybenzamide SANT-2 and (4-benzyl-piperazin-1-yl)-(3,5-dimethyl-1-phenyl-1H-pyrazole-4-ylmethylene)-amino (SANT-3) and SANT-4, having the following structures:

[0052]

[0053] In some embodiments, both a Smo agonist and a Gli antagonist are used as modulators of the hedgehog signaling pathway. That is, in some embodiments, the hedgehog signaling pathway modulator comprises both a Smo agonist and a Gli antagonist. Thus, in some embodiments, the steroid hormone, Smo agonist, and Gli antagonist are used or administered as described herein.

[0054] Pharmaceutical Composition

[0055] Steroid hormones and hedgehog signaling pathway modulators can be administered as separate compositions (essentially simultaneously or sequentially), or as a single composition.

[0056] One or more compositions can be any pharmaceutical composition formulated for any route of administration suitable for the administration of steroid hormones and / or hedgehog signaling pathway modulators. Suitable routes of administration may include, for example, oral, rectal, transmucosal, especially intranasal, enteric or parenteral delivery, including intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular, intracardiac to, for example, right or left ventricular cavity, common coronary artery injection, intravenous, intraperitoneal, intranasal or intraocular injection. Some embodiments relate to oral administration. Some embodiments relate to intranasal administration.

[0057] In some embodiments, steroid hormones and / or hedgehog signaling pathway modulators are administered in the form of intranasal pharmaceutical compositions. As used herein, "intranasal composition" means a composition suitable for intranasal delivery. Such embodiments may provide enhanced uptake of steroid hormones and / or hedgehog signaling pathway modulators.

[0058] Exemplary oleogel-type intranasal pharmaceutical compositions targeting testosterone have been described, for example, in U.S. Patent 8,574,622, which is incorporated herein by reference. In some embodiments, one or both of the steroid hormone and the hedgehog signaling pathway are formulated into the form of an intranasal pharmaceutical composition as described in U.S. Patent 8,574,622, such as comprising one or more active agents and further comprising: (a) at least one lipophilic or partially lipophilic carrier present in an amount of about 60% to about 98% by weight of the formulation; (b) at least one compound having surface tension reducing activity present in an amount of about 1% to about 20% by weight of the formulation; and (c) at least one viscosity modifier present in an amount of about 0.5% to about 10% by weight of the formulation.

[0059] In such oleogel embodiments, the lipophilic or partially lipophilic carrier may be any such carrier or mediator suitable for use in nasal pharmaceutical compositions, such as oils, such as vegetable oils, such as castor oil, hydrogenated castor oil, soybean oil, sesame oil, or peanut oil, or any other lipophilic or partially lipophilic carrier discussed below, or any other suitable lipophilic or partially lipophilic carrier.

[0060] In such oleogel embodiments, one or more compounds with surface tension-reducing activity may be one or more surfactants, such as lecithin, polyols, sorbitan, polyoxyethylene sorbitan, polyoxyethylene, sucrose, fatty acid esters of polyglycerol; and / or a humectant, such as sorbitol, glycerol, polyethylene glycol, and polyethylene glycol glycerol fatty acid esters, or one or more oleoyl polyethylene glycol glycerol esters (such as those available from Gattefosse (France)). M 1944CS); or any surfactant discussed below or any other suitable surfactant.

[0061] In such oleogel embodiments, one or more viscosity modifiers may be selected from thickeners and gelling agents, such as cellulose and cellulose derivatives, polysaccharides, carbomer, polyvinyl alcohol, povidone, colloidal silica, cetyl alcohol, stearic acid, beeswax, petrolatum, triglycerides and lanolin, or any viscosity modifier discussed below or any other suitable surfactant.

[0062] Other exemplary intranasal pharmaceutical compositions comprise the intranasal pharmaceutical compositions described in U.S. Patent Application 15 / 612,454, the entire contents of which are incorporated herein by reference. U.S. Patent Application 15 / 612,454 describes intranasal pharmaceutical compositions in which an active agent is loaded onto a porous agent. Therefore, in some embodiments, one or both of a steroid hormone and a hedgehog signaling pathway modulator are formulated as in the form of an intranasal pharmaceutical composition as described in U.S. Patent Application 15 / 612,454, such as compositions comprising a porous agent in which the steroid hormone and / or hedgehog signaling pathway modulator is loaded onto a surface of the porous agent located within the pores of the porous agent. As described in US 15 / 612,454, the active agent-loaded porous agent itself can be formulated as an oleogel composition, such as those described in U.S. Patent 8,574,622.

[0063] In such embodiments of porous agents, the porous agent may include inorganic porous materials, such as colloidal silica, microporous silica, mesoporous silica, macroporous silica, polyorganosiloxanes, pharmaceutical clays, silica nanotubes, silica gels, and magnesium aluminosilicates (e.g., but not limited to, from Vanderbilt Minerals, LLC). ), activated carbon, anhydrous calcium phosphate, calcium carbonate, alumina, and any combination of two or more thereof. Exemplary inorganic porous materials may contain... The brand purchased porous silica (such as, but not limited to, from WR Grace & Co.) from Grace Company. 244FP, 72FP, XDP6035 (also known as SILSOL) TM (6035), XDP3050, XDP3150, AL-1FP, and any combination of two or more thereof), can The brand obtains porous silica (such as, but not limited to:) from Evonik Industries, Corp. 300, with a surface area of ​​approximately 260m². 2 / g to 320m 2 / g (approximately 300mg) 2 Silica from Millipore (EMD), with pore volumes of approximately 1.5 ml / g to 1.9 ml / g and average particle sizes of approximately 20 μm to approximately 60 μm. SLC, from Fuji Chemical Industry (Synthetic, amorphous form of magnesium aluminum silicate), zeolite Socony Mobil-5, Mobil 41, SBA-15, FDU-11, OMS-7, OMS-Lemon-7, and IITM-56. In some embodiments, the porousing agent comprises a silicon-based powder, which may be hydrophobic or hydrophilic, depending on the groups chemically bonded to its surface.

[0064] In some embodiments, the porous agent comprises an organic-inorganic hybrid, such as a metal-organic framework (MOF). Exemplary hybrid materials can be formed by the self-assembly of multidentate bridging ligands and metal junctions.

[0065] In some embodiments, the porous agent comprises an organic polymer, such as a microporous organic polymer, polystyrene, cellulose, and / or poly(methyl methacrylate). In some embodiments, the microporous organic polymer is formed via a carbon-carbon coupling reaction and comprises non-metallic elements such as carbon, hydrogen, oxygen, nitrogen, and / or boron. In some embodiments, the organic polymer is generated via emulsion polymerization and hypercrosslinking, followed by chemical etching to sacrifice the SiO2 core. In some embodiments, the organic polymer network is composed of small organic structural units.

[0066] In some embodiments, the porous agent includes a complexing agent-based porous material, such as an ion exchange resin (e.g., but not limited to cross-linked polystyrene) or an adsorbent (e.g., but not limited to β-cyclodextrin-based porous silica, α-cyclodextrin-based porous silica, hydroxypropyl-β-cyclodextrin-based porous silica, and porous materials based on other adsorbent resins).

[0067] In some embodiments, the surface of the porous agent—including the pore surface—is functionalized to bind one or more active agents after a certain period of time or in response to a stimulus and / or control the release of one or more active agents.

[0068] The active agent-loaded porous agent can be formulated into any mediator form suitable as a mediator in a nasal pharmaceutical composition. In some embodiments, the mediator of the porous agent is a hydrophilic mediator. In some embodiments, the mediator is a lipophilic or partially lipophilic mediator, such as a mediator comprising one or more fats, oils, waxes, phospholipids, steroids (e.g., cholesterol), sphingolipids, ceramides, sphingosine, prostaglandins, and / or fat-oil vitamins. In some embodiments, the mediator comprises: oils or mixtures of oils, such as vegetable oils, castor oil, hydrogenated castor oil, soybean oil, sesame oil, or peanut oil; fatty acid esters, such as ethyl oleate and oleyl alcohol oleate, isopropyl myristate; medium-chain triglycerides; glycerol fatty acid esters; polyethylene glycol; phospholipids; leucocele; or any combination of two or more thereof.

[0069] The mediator may be present in any suitable amount, such as an amount effective for providing the desired properties, desired physical properties, desired release properties, desired pharmacokinetics, etc., for nasal administration. In some embodiments, the composition comprises a mediator in an amount based on the total weight of the composition of: about 15% to about 98% by weight, about 30% to about 98% by weight, about 50% to about 95% by weight, about 75% to about 95% by weight, about 80% by weight, or about 90% by weight. In some embodiments, the composition comprises a mediator in an amount based on the total weight of the composition of: 15% to 98% by weight, 30% to 98% by weight, 50% to 95% by weight, 75% to 95% by weight, 80% by weight, or 90% by weight.

[0070] The active agent-loaded porous agent can utilize one or more compounds having surface-reducing activity, such as surfactant formulations. If present, the surfactant can be any surfactant suitable for use in nasal pharmaceutical compositions. In some embodiments, the surfactant is selected from anionic, cationic, amphoteric, and nonionic surfactants, including but not limited to lecithin, fatty acid esters of polyols, fatty acid esters of sorbitol, fatty acid esters of polyoxyethylene sorbitol, fatty acid esters of fatty polyoxyethylene, fatty acid esters of sucrose, fatty acid esters of polyglycerol, oleoyl polyoxyethylene glycerol esters (such as, but not limited to, almond oil PEG-6-ester), oleoyl polyethylene glycol glycerol esters, and / or humectants such as sorbitol, glycerin, polyethylene glycol, polyethylene glycol glycerol fatty acid esters, and any combination of two or more thereof. In some embodiments, the surfactant includes oleoyl polyethylene glycol glycerol esters (… M 1944CS (Gafals, Saint-Priest, France) or a mixture of oleoyl polyethylene glycol glycerol esters.

[0071] The active agent-loaded porous agent can be formulated with one or more viscosity modifiers, which can be any viscosity modifier suitable for use as a viscosity modifier in a nasal pharmaceutical composition. In some embodiments, the viscosity modifier comprises mesoporous silica (which may be loaded with or unloaded with an active agent). In some embodiments, the viscosity modifier comprises cellulose, cellulose-containing substances, polysaccharides, carbomer, polyvinyl alcohol, povidone, colloidal silica, cetyl alcohol, stearic acid, beeswax, petrolatum, triglycerides, lanolin, or any combination of two or more. In some embodiments, the viscosity modifier comprises colloidal silica (such as, but not limited to: 200 (Evonik) and / or CAB-O- M5 (Cabot). In some embodiments, the viscosity modifier includes synthetic silica, such as that from Grace Company. (precipitated silica, which has a density of approximately 110 kg / m³) 3 The compacted bulk density is approximately 190 m³. 2 (specific surface area / g and average particle size of approximately 18μm) or Silica (porous silica gel having a pore volume of about 1.6 ml / g and an average particle size of about 3 μm). In some embodiments, the viscosity modifier comprises hydrophilic calcined silica, such as... 200 and / or lipophilic silica, such as R972 (which is calcined silicon dioxide after treatment with dimethyldichlorosilane, and has a surface area of ​​approximately 90 m²) 2 / g to approximately 130m 2 / g). Unbound by theory, hydrophilic calcined silica is believed to be able to be used to prepare thixotropic gel compositions with high-temperature stability, compared to comparable gels produced with other viscosity modifiers.

[0072] If present, the viscosity modifier may be present in an amount effective for adjusting the viscosity of the composition to the desired level. In some embodiments, the composition comprises the following viscosity modifiers based on the total weight of the composition: about 0.5 wt% to about 20 wt%, about 0.5 wt% to about 10 wt%, about 0.5 wt% to about 7 wt%, about 1 wt% to about 4 wt%, about 4 wt%, or about 2 wt%. In some embodiments, the composition comprises the following viscosity modifiers based on the total weight of the composition: 0.5 wt% to 20 wt%, 0.5 wt% to 10 wt%, 0.5 wt% to 7 wt%, 1 wt% to 4 wt%, 4 wt%, or 2 wt%.

[0073] Regardless of the specific formulation used, steroid hormones and hedgehog signaling pathway modulators are formulated to provide a therapeutically effective amount of the active agent at a dose appropriate for the route of administration, such as the volume of the composition for administration into one or both nostrils, the volume for oral administration, or the volume for intravenous, subcutaneous, or intramuscular administration.

[0074] How to use

[0075] As noted above, steroid hormones and hedgehog signaling pathway modulators are administered to subjects in need, such as those requiring promotion of myelin regeneration and / or those requiring treatment of demyelinating diseases or conditions, in accordance with the methods and uses described herein. Subjects may be any mammal, such as humans, non-human primates, dogs, cats, cattle, sheep, horses, rabbits, mice, or rats.

[0076] Demyelinating diseases can be divided into those affecting the central nervous system and those affecting the peripheral nervous system, presenting with different demyelinating symptoms. In some embodiments, subjects have CNS demyelinating diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Dweck's disease, inflammatory demyelinating diseases, central nervous system neuropathy, central pontine myelinolysis, spinal cord lesions, tabes dorsalis, syphilitic myelopathy, leukoencephalopathy (including progressive multifocal leukoencephalopathy), leukodystrophy, and Alzheimer's disease. In some embodiments, subjects have peripheral nervous system demyelinating diseases such as Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, antimyelin-associated glycoprotein peripheral neuropathy, Charcot-Marie-Tooth disease, hereditary neuropathy prone to compression paralysis, peripheral neuropathy, myelopathy, optic neuropathy, and progressive inflammatory neuropathy.

[0077] As indicated above, the steroid hormone and the hedgehog signaling pathway modulator can be administered as a single composition (substantially or in any sequential order) or as the same composition. Also as indicated above, in any embodiment, the steroid hormone and the hedgehog signaling pathway modulator can be administered via any suitable route of administration, including intranasal, oral, intravenous, subcutaneous, and intramuscular. When the steroid hormone and the hedgehog signaling pathway modulator are administered as different compositions, they can be administered via the same or different routes of administration (e.g., oral or intranasal). In a specific embodiment, the steroid hormone is administered orally or intranasally. Independently, in a specific embodiment, the hedgehog signaling pathway modulator is administered orally or intranasally.

[0078] As indicated above, steroid hormones and hedgehog signaling pathway modulators are administered in amounts sufficient to promote myelin regeneration. As used herein, “myelin regeneration” refers to the generation of new myelin sheaths. Myelin regeneration can be assessed by methods that directly determine the state of myelin in a subject, such as measuring white matter quality using magnetic resonance imaging (MRI), measuring myelin fiber thickness using magnetic resonance spectroscopy (MRS) brain scans, or any other direct measurement (e.g., positron emission tomography (PET), diffusion-weighted imaging (DW-I or DW-MRI), diffusion tensor imaging, myelography, magnetization transfer, etc.). Alternatively, myelin regeneration can be assessed by detecting a reduction in the size or number of inflammatory lesions (i.e., sclerosis) present in the patient; monitoring the patient's cerebrospinal fluid (which can be obtained, for example, by lumbar puncture) for a reduction in, for example, the following: (i) abnormal proteins, such as small fragments of myelin; (ii) elevated levels or specific types of lymphocytes; and / or (iii) abnormal levels of immunoglobulin (IgG) molecules; monitoring the patient for positive changes in neuropsychology (e.g., states of various abilities such as memory, arithmetic, attention, judgment, and reasoning); and / or monitoring the patient's urine for a decrease in levels of myelin basic protein-like material (MBPLM). Myelin regeneration can be assessed using any one or more of these methods, or alternative methods can be used. The methods described herein are not limited to these or other specific methods for assessing myelin regeneration.

[0079] In some embodiments, the steroid hormone is testosterone and is administered at a dose of about 0.05 mg / day to about 0.5 mg / day, including about 0.1 mg / day to about 0.3 mg / day, including about 0.2 mg / day. In embodiments using different androgen receptor ligands, corresponding molar amounts of androgen receptor ligands may be used.

[0080] In some embodiments, the hedgehog signaling pathway modulator is SAG and is administered at a dose of about 5 mg / kg to about 25 mg / kg of the subject's body weight, comprising about 10 mg / kg to about 20 mg / kg, comprising about 15 mg / kg. In embodiments using different Smo agonists, corresponding molar amounts of the Smo agonist may be used.

[0081] In some embodiments, the hedgehog signaling pathway modulator is GANT-61 and is administered at a dose of about 5 mg / kg to about 25 mg / kg of the subject's body weight, comprising about 10 mg / kg to about 20 mg / kg, comprising about 15 mg / kg. In embodiments using different hedgehog signaling pathway antagonists, corresponding molar amounts of the hedgehog signaling pathway antagonist may be used.

[0082] In some embodiments, the amount of one or both of the steroid hormone and the hedgehog signaling pathway modulator applied is effective in enhancing the myelin regeneration-promoting activity of the other. Therefore, in some embodiments, a given amount of steroid hormone applied together with a given amount of hedgehog signaling pathway modulator is more effective in promoting myelin regeneration than the same amount of hedgehog signaling pathway modulator alone. Alternatively or additionally, in some embodiments, a given amount of hedgehog signaling pathway modulator applied together with a given amount of steroid hormone is more effective in promoting myelin regeneration than the same amount of steroid hormone alone.

[0083] Also provided are steroid hormones and hedgehog signaling pathway modulators as described herein, for use in methods of promoting myelin regeneration in subjects in need, or for the treatment of demyelinating diseases or conditions as discussed above.

[0084] Also provided is the use of steroid hormones and hedgehog signaling pathway modulators as described herein in the preparation of medicaments as described herein, said steroid hormones and said hedgehog signaling pathway modulators for use in methods of promoting myelin regeneration in subjects in need, or for treating demyelinating diseases or conditions as discussed above.

[0085] As indicated above and as illustrated in the examples, the inventors have found that the use of steroid hormones (such as testosterone) and Smo agonists (such as SAG) synergistically promotes the production of oligodendrocytes and alleviates the progression of autoimmune encephalomyelitis in experimental models. Furthermore, the use of steroid hormones (such as testosterone) and Gli antagonists (such as GANT61) can synergistically promote the production of myelin-producing cells.

[0086] The following examples are provided to illustrate the present invention, but it should be understood that the present invention is not limited to the specific symptoms or details of these examples.

[0087] Example

[0088] Materials and methods

[0089] Animal handling. Wild-type male C57Bl / 6 mice, aged 8 to 12 weeks, with intact gonads or castrated, were purchased from the Janvier Labs Breeding Center (France). For in vitro experiments, litter was obtained from timely mated C57Bl / 6 females purchased from the Janvier Labs Breeding Center, or said litter was bred in the laboratory and mated with Plp-EGFP mice obtained from Dr. Wendy Macklin (University of Colorado, USA). See Mallon et al., Journal of Neuroscience 22:876-885 (2002). All animals were housed under standard conditions, including a 12-week light-dark cycle, with free access to food and water. All procedures for the care and use of laboratory animals are carried out in accordance with the European Communities Council Directive (86 / 806 / EEC) and approved by the Regional Ethics Committee (CEEA26, Ministry of Education Nationale, de l'Enseignement et de la Recherche).

[0090] Preparation of activator formulations. The Smo agonist (SAG) and antagonist (SANT) used were those described in Chen et al., “Small molecule modulation of smoothed activity,” Proceedings of the National Academy of Sciences 99:14071-14076 (2002), and were purchased from D&C Chemicals (China). (SAG, product number: DC-8225; SANT-1, product number: DC-8327). The activator was dissolved in dimethyl sulfoxide (10 mM) and then diluted in culture medium or 0.9% NaCl to achieve the appropriate concentration. Testosterone was supplied by Sigma-Aldrich. Testosterone was dissolved in sesame oil (1 mg / ml) and then diluted to obtain the desired steroid hormone concentration.

[0091] Immunostaining experiments. The primary antibodies used for immunostaining are as follows: Oligosaccharide transcription factor 2 (Olig2) (rabbit, Millibor; mouse, Millibor), Myelin Basic Protein (MBP), (rabbit, Millibor); Anti-NG2 (rabbit, Millibor); Colonic Adenomatous Polyposis (APC / CC1) (mouse, Calbiochem), BrdU antibody (rat, Abcam); Glial Fibrinolytic Acidic Protein (GFAP) (rabbit, Dako; mouse, Sigma); Ionized Calcium Binding Adaptor 1 (Iba1, rabbit, Wako); Arginase-1 (goat, Santa Cruz), Protein Lipoprotein (PLP), (mouse, Millibor); Platelet-Derived Growth Factor Receptor α (PDGFRa), (mouse, Millibor); Neurofilament 200 (NF200), (chicken, Neuromics); Ki67 (mouse monoclonal; BD Pharmingen). The secondary antibodies used were: goat anti-rabbit anthocyanin 3 conjugate (Jackson Immunoresearch); goat anti-mouse Alexa 488, anti-rabbit Alexa 633, anti-chicken Alexa 546, and donkey anti-goat Alexa 546 (Thermo Fisher Scientific).

[0092] Image acquisition and analysis. Images were acquired using an Axiovision 4.2 microscopy analysis system (Carl Zeiss, Inc.), a confocal Zeiss LSM 510-Meta Confocor 2, and a scanner imager equipped with CaseViewer software. Analysis was performed using ImageJ software. At least 10 sections from each mouse were analyzed, and data were the mean of 3–5 mice. For brains derived from animals injected with lysophosphatidylcholine (LPC), immunofluorescence-positive cells or regions were identified every 5 sections throughout the demyelinating lesion in each mouse, and the mean was calculated for each animal. The lesion surface was determined by measuring the area of ​​nuclear compaction (corresponding to myelin loss visualized by MBP or PLP staining) every 5 sections throughout the demyelinating lesion.

[0093] Electron microscopy. Ultrathin sections of the lumbar spinal cord were examined using a transmission electron microscope (1011JEOL) equipped with a Gatan digital camera. The g-ratio (the ratio between axonal diameter and fiber diameter corresponding to myelin sheath + axonal diameter) was estimated by measuring the minimum and maximum axonal diameter and fiber diameter for each axon using ImageJ software. At least 300 randomly selected myelinated axons were evaluated for each animal.

[0094] RT-qPCR analysis. At least four animals of each sex and age were euthanized by decapitation. The dorsal mesobranch was dissected and frozen in liquid nitrogen for further processing. Total RNA was isolated using Trizol technology (Thermo Fisher Scientific) and the RNeasy mini kit (Qiagen). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR was performed using TaqMan gene expression premix (Thermo Fisher Scientific), and gene expression normalized relative to the reference gene GAPDH was analyzed using 7300 System SDS software (Applied Biosystems). The TaqMan probes were as follows: GAPDH, Mm99999915_m1; MBP, Mm01266402_m1; Shh, Mm00436528_m1; Gli1, Mm00494654; AR, Mm00442688.

[0095] Statistical analysis. Data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 6.0. One-way ANOVA was used to assess statistical significance. Significance levels were defined as *p<0.05, **p<0.01, and ***p<0.001.

[0096] Example 1:

[0097] Functional interactions between hedgehogs and androgen signaling during early postnatal myelination.

[0098] The inventors identified a functional interaction between hedgehog (Hh) and androgen signaling during the early stages of postnatal myelination in the telencephalon. In this regard, the expression profiles of transcripts encoding myelin basic protein (Mbp), Hh signaling components (Shh, Gli1), and androgen receptor (AR) were investigated using the dorsal forebrain of male and female mice from birth to 15 days postpartum (P15). This stage encompasses the nascent oligodendrogenesis wave, the maturation of the resulting oligodendrocyte precursor cells (OLPs), and the physiological processes of myelination in the dorsal forebrain. Kessaris et al., *Nature Neuroscience* 9:173-179 (2006). Mbp transcription was detected at very low levels at P3 and then... Figure 1 Figure A shows increases of approximately 10-fold and 60-fold at P8 and P15, respectively. (See figure.) Figure 1 As shown in B, Shh mRNA increases slightly before reaching the plateau phase, but only significantly at P8. Unexpectedly, as... Figure 1As shown in C, Gli1 gradually decreases between P0 and P15. Conversely, as... Figure 1 As shown in D, androgen receptor (AR) transcription was detected at low levels at birth, but increased sharply only at P15, reaching levels 10 to 24 times higher in both males and females. Transcription of Shh, Gli1, and Mbp did not differ significantly between sexes at the study time points. Conversely, while AR expression was significantly higher in males than females at birth, AR transcription was comparable regardless of sex at P3 and P8, and AR expression at P15 unexpectedly showed a slightly but significantly higher level in females than males. Figure 1 As shown in Figure D. These results indicate that androgens and hedgehog signaling pathways can communicate to regulate the myelination process.

[0099] SAG+T promotes the proliferation of oligodendrocytes during developmental myelination.

[0100] SANT+T promotes myelin-producing oligodendrocytes

[0101] Differentiation occurs during developmental myelination.

[0102] As previously described in Feutz et al., "Isolation and characterization of defective jimpy oligodendrocytes in culture," 24:865-877 (2001), primary glial cell cultures were prepared from the dorsal mesobrain of newborn (P1) male mice. Briefly, the meninges were removed, and the dorsal mesobrain was microanalyzed and mechanically dissected in DMEM supplemented with 10% fetal bovine serum, penicillin (50 U / ml), and streptomycin (50 μg / ml) (Thermo Fisher Scientific, France). The cell suspension was plated in 24-well plates containing 0.5 ml of cultured medium coated with 30 μg / ml poly-L-lysine (Sigma-Aldrich). Cultures containing astrocytes, oligodendrocytes (OLs), and microglia were then incubated at 37°C in a humidified atmosphere (90%) with 5% CO2 and 95% air.

[0103] At day 5 in vitro (DIV), the culture medium containing the primary glial cells was replaced with fresh medium supplemented with one of the following: (i) Smo agonist SAG (0.1 or 1 μM), (ii) SANT-1 (0.1 μM), (iii) testosterone (T, 1 μM), (iv) SAG (0.1 or 1 μM) and testosterone (1 μM), (v) SANT-1 (0.1 μM) and testosterone (1 μM), or (vi) as a control drug carrier. The supplemented culture medium was replaced with fresh solution every other day. At 12 DIV, cells were fixed in PBS with 4% paraformaldehyde (PFA) for 20 minutes, then permeabilized with 0.025% Triton X-100 for 10 minutes, and blocked with seawater blocking buffer (Thermo Fisher Scientific) for 1 hour.

[0104] Cells were immunostained for BrdU and Olig2. For immunostaining, after washing three times in PBS, cells were incubated overnight at 4°C with the primary antibody, followed by incubation with an appropriate secondary antibody for 2 hours. Cells were then washed with PBS and mounted with fluorescent mounting medium (Vector, Clinicscients, France). Images were acquired using immunofluorescence microscopy as described above (data not shown). The quantification of Olig2+ and BrdU+ was assessed by analyzing 3-4 independent cultures for each test symptom, and these results were... Figure 2 This was reported in A-2B.

[0105] Figure 2 A shows Olign2 incorporating the proliferation marker BrdU 2 hours before the end of the culture. + Cell quantification and indicated the synergistic effect of SAG (0.1 μM) and testosterone (1 μM). Figure 2 A also shows that Olig2 in the absence of surfactants + BrdU + Proliferating glial cells represent Olig2 + The total number of cells was 2.0 ± 0.4%, while the percentage of proliferating cells was significantly increased compared to the control with SAG (1 μM) or testosterone (1 μM), reaching 8.4 ± 0.5% (p = 6.93E-09) and 3.8 ± 0.6% (p = 0.04) of total Olig2 cells, respectively.

[0106] To analyze the differentiation of myelin-producing cells, primary mixed glial cells were derived from Plp-EGP mice and cultured as described above. Detection of PLP+ cells co-expressing MBP was performed using primary mixed glial cells derived from Plp-EGP mice and immunostaining targeting Mbp. Immunofluorescence images were acquired as described above, and PLP+ cells co-expressing MBP were evaluated. + The number of cells. Figure 2 B shows that co-administration of SANT and testosterone in primary mixed glial cells induced significantly higher levels of MBP expression compared to the levels induced by each active agent used alone (p = 0.001). Immunostaining data (not shown) revealed that testosterone (1 μM) and SANT (0.1 μM) highly increased the number of co-expressing MBP plc. + GFP + Cell number. The Smo agonist SAG (0.1 μM or 1 μM) did not modify Mbp expression when used alone. However, both SAG concentrations induced a slightly but significantly reduced testosterone-mediated OL differentiation. Significantly, the differentiation effects of testosterone and SANT appeared to be additive when the drugs were used together. Inhibition of Smo by SANT (0.1 μM) or testosterone (T, 1 μM) induced PLPs co-expressing MBP. + The percentage of cells increased fourfold compared to the control group (12.4 ± 0.8 for SANT1, and 11.7 ± 1.5 for testosterone, p = 6.09E-07 and p = 0.0005).

[0107] Figure 2 B further shows that when SANT and testosterone were used together, the percentage of mature OLs was observed to increase 7-fold compared to the control (22.3 ± 2.1 vs. 3.0 ± 0.9; p = 2.22E-06). On the other hand, the Smo agonist SAG (0.1 μM or 1 μM) did not modify Mbp expression when used alone. However, both concentrations of SAG induced a slightly but significantly reduced testosterone-mediated OL differentiation. Significantly, the differentiation effects of testosterone and SANT appear to be additive when the drugs are used together. Although not bound by theory, these results suggest that Hedgehog signaling blockade enhances the maturation of oligodendrocytes (OPCs) into MBP+myelinated OLs by testosterone-induced maturation.

[0108] To further investigate whether SANT+T promotes the differentiation of myelin-producing oligodendrocytes during developmental myelination, male P3 pups were subcutaneously treated with SANT and / or testosterone every other day from day 3 to day 10 postpartum (n = 3–5 animals per group). SANT was administered at a concentration of 20 μg / g pup weight, while testosterone was administered at 20 μg / g for each dose. The drugs were injected subcutaneously every other day from day 3 postpartum. At P10, pups were deeply anesthetized and perfused with 4% PFA. The brain was removed, postfixed in 4% PFA for 1 hour, and cryopreserved in 30% sucrose, followed by freezing and sectioning (14 μm). Subsequently, immunostaining of Olig2, Olig2 / APC, Olig2 / PDGFRα, and MBP / NF200 was performed on brain slices at the subventricular region and adjacent corpus callosum (cc) levels in male pups 10 days postpartum treated with the Smo agonist SAG, the Smo antagonist SANT, and the steroid hormone testosterone (T). Images were acquired using immunofluorescence microscopy as described above, and as... Figure 3 The A-3D diagram illustrates the quantification of different cell populations.

[0109] Figure 3 D shows that combined treatment with testosterone and SANT-1 promotes myelin sheath production in male pups at P10. In the absence of the active agent, the area occupied by MBP (the area of ​​myelinated sheath) corresponds to 62.4 ± 2.2% of the total area occupied by axons expressing neurofilament protein NF200. See also Figure 3 D. In the presence of SANT or testosterone, the percentage of myelinated area reached significantly higher values: 75.1 ± 4.3% (SANT) (p = 0.04) and 78.0 ± 4.1% (T) (p = 0.01). See also Figure 3 D. During the concurrent injection of SANT and testosterone into the pups, the myelinated NF200... + MBP + The area occupied by axons reached 88.9 ± 2.7%, which was significantly higher than the values ​​obtained using SANT or testosterone alone (p = 0.03 for SANT, p = 0.05 for testosterone).

[0110] Therefore, these data suggest that hedgehog and androgen signaling pathways functionally interact during wave generation in nascent oligodendrocyte precursor cells and during the subsequent differentiation of these cells into myelinated oligodendrocytes.

[0111] Overall, the results indicate that the combination of SAG and testosterone promotes the proliferation of oligodendrocyte precursor cells, but SAG inhibits the testosterone-induced maturation of these cells into myelin-producing oligodendrocytes.

[0112] Example 2:

[0113] Smo activity promotes the production of newly matured oligodendrocytes and increases precocious puberty in a demyelinating LPC model by promoting the production of regenerative microglia via SAG.

[0114] The effects of Smo activation on demyelination via the Smo agonist SAG were investigated as follows. LPC-induced demyelination was performed in young male adult mice with or without SAG, as previously described by Ferent et al., *Journal of Neuroscience* 33:1759-1772 (2013). In short, unilateral demyelinating lesions were induced by stereotactic injection of 2 μl of a solution containing 1% LPC (Sigma-Aldrich) along with SAG (0.2 μM) or a corresponding mediator into the right corpus callosum using a Hamilton syringe (NH BIO, France) specifically designed for neurosurgery. Injections were performed at the following coordinates (to the anterior fontanelle): anterior-posterior (AP) +1 mm, lateral +1 mm, dorsoventral (DV) -2.2 mm. The brain was removed from deeply anesthetized mice, and the heart was perfused with 4% PFA. Tissues were postfixed in fresh 4% PFA solution for 4 hours, then cryopreserved in 30% sucrose, frozen in liquid nitrogen, and cryosectioned (14 μm). Four to five mice were used at each time point for each treatment symptom. Mice were sacrificed at 2 and 10 days post-lesion (dpl) and prepared for immunostaining of PDGFRα, Olig2 / APC, Ki67 / PDGFRα, GFAP, and Iba1 / Arg1. Immunostaining images (data not shown) were acquired using immunofluorescence microscopy and for PDGFRα (… Figure 4 A) Olig2 / APC ( Figure 4 B), Ki67 / PDGFRα ( Figure 4 C), GFAP Figure 4 D), Iba1 / Arg1 Figure 4 E) The results were quantified by assessing the number of cells per surface unit.

[0115] Notably, at 10 dpl, animals treated with SAG showed a two-fold increase in PDGFRα+ cells compared to controls (121±11 vs 65±5, p = 0.008). Figure 4 A). Furthermore, the density of Olig2+APC+ mature OL was found to be significantly higher in the presence of SAG compared to the control (83±7 vs. 59±1, p = 0.027). Figure 4 B). Since the proportion of mature OLs did not differ significantly between SAG-treated animals and control animals, it appears that SAG could not promote OPC differentiation.

[0116] At an earlier time point (2 dpl), new OPCs began to be recruited and proliferated highly in the lesions, while the tissue already showed a high level of inflammation. SAG treatment was found to induce an increase in the density of proliferating Ki67+PDGFRα+ OPCs compared to the control group (39±2 vs. 28±2, p = 0.007). Figure 4 (C, left side panel). Interestingly, the total number of PDGFRα+OPCs remained unchanged, indicating that SAG induces OPCs to enter the cell cycle. Figure 4 C, the diagram on the right.

[0117] To investigate potential effects on inflammatory cells, astrocytes and microglia were analyzed at the same time point. The area occupied by GFAP+ astrocytes tended to decrease in the presence of SAG, but in a non-significant manner. See also Figure 4 D. It was found that Iba1+ microglia were also unaffected by the presence of SAG. However, the density of a subset of microglia termed "pro-regenerative" and characterized by arginase-1 (Arg1) expression was found to be 2-fold increased in SAG-treated mice (103±7 vs 54±8, p = 0.035). Figure 4 E, left panel). Furthermore, the proportion of Iba1 microglia co-expressing Arg1 increased twofold by SAG treatment (58±5 vs. 25±2, p = 0.017). Figure 4 E (Right side figure). These results indicate that hedgehog signaling activation promotes the regenerative potential of activated microglia through Smo agonists.

[0118] Example 3:

[0119] SAG+T alleviated experimental autoimmune encephalomyelitis.

[0120] Castrated male mice aged 9–10 weeks were kept for one week to acclimatize prior to experimental autoimmune encephalomyelitis (EAE) (n = 10 animals per symptom). Pathogenesis was induced according to guidelines from the provider (Hooke Laboratories, MA, USA). Briefly, mice were immunized by subcutaneous injection of MOG in complete Freund's adjuvant. 35-55An emulsion of the peptide (myelin oligodendrocyte glycoprotein / MBP fragment 35-55) (0.1 ml of pretreated mixture at two sites for each site) was administered, followed by the first intraperitoneal injection of pertussis toxin in the form of PBS on the same day (day 0) and a second injection (250 ng / dose) on day 1. From day 7 post-immunization until day 30, mice were blindly scored once daily according to the following ratios: 0.0 = No significant change in motor function; 0.5 = Weakness at the tip of the tail; 1.0 = Weak tail; 1.5 = Weak tail and hind leg inhibition; 2.0 = Weak tail and signs of hind leg weakness or head tilt; 2.5 = Weak tail and signs of hind leg dragging or head tilt; 3.0 = Weak tail and complete paralysis of the hind legs or tail of all four limbs, including one foreleg and one hind leg; 3.5 = Weak tail and complete hind leg paralysis, and the animal is unable to stand upright when placed in a horizontal position; 4.0 = Weak tail, complete hind leg and partial foreleg paralysis, with little movement and feeding; 4.5 = Complete hind leg and partial foreleg paralysis, no movement around the cage, and the animal appears no longer alert; 5.0 = Extreme paralysis, requiring euthanasia.

[0121] Mice with EAE were randomly assigned to treatment groups of mediator, SAG, testosterone, or SAG+testosterone to form groups with similar EAE onset time and similar disease scores (N = 10 animals per group). The active agent was administered before the onset of clinical symptoms until day 30 post-immunization. Testosterone was administered intranasally (0.2 mg / day, 2.5 μl volume per nostril) in the form of the oleogel composition described above. SAG was administered orally every other day via intensive feeding (15 μg / g mouse weight).

[0122] Animals were euthanized by ketamine overdose. The spinal cord / vertebrae were removed, and lumbar spinal cord / vertebrae samples were processed according to the requirements of various histological procedures. For immunostaining, lumbar spinal cord / vertebrae fragments were post-fixed in 4% PFA for 24 hours. The spinal cord was removed from the spine, treated in an ethanol / xylene bath, and embedded in paraffin blocks. 7-μm sections were then obtained using a microtome (Leica) and allowed to dry overnight on slides at 37°C. For electron microscopy, the spinal cord / vertebrae fragments were post-fixed in a mixture of 2% PFA and 2% glutaraldehyde for 5 days. The spinal cord was removed from the spine, post-fixed in 1% osmium tetroxide buffered with carboxylate at 4°C for 1 hour, and then fixed in 2% uranyl acetate at room temperature for 1 hour, followed by dehydration with a continuously diluted ethanol solution and embedding in epoxy resin. Ultrathin sections were compared with saturated uranyl acetate solution.

[0123] Figure 5A shows the improved clinical scores observed after treatment with both SAG and testosterone compared to treatment with each active agent alone. Animals in all three groups achieved a score of 1.0 on days 18–19, maintaining this score until day 21 of each treatment. Between day 21 and day 30, animals treated with testosterone showed relapse and clinical scores approaching 2.0. Conversely, animals treated with SAG stabilized at scores significantly below 1.0. Significantly, the drug combination fluctuated between a minimum clinical score of 0.5 and 1.0, indicating an improvement in clinical scores with the combination of SAG and testosterone compared to using these drugs alone. See also Figure 5 A.

[0124] To investigate the mechanisms involved in alleviating EAE, myelin levels and axonal pathology were examined 30 days post-EAE induction for each condition. Electron microscopy images showed that the spinal cords of animals treated with SAG and testosterone alone or in combination had significantly higher myelin axonal density, with aberrant structures observed only occasionally. See also Figure 5 B. Next, the g-ratio (axon diameter / total outer diameter of myelin fibers) of at least 300 small-diameter axons (≤2.5 μm) per animal (n = 3 for each symptom) was determined and shown in [the table / ... Figure 5 In C, the g-ratio value (0.834 ± 0.004) in the spinal cord derived from control EAE animals was significantly higher than that for the control EAE animals (0.773 ± 0.003, p < 10). -10 SAG (0.737±0.005, p<10) -10 () or drug combination (0.743±0.005, p<10) -10 The value of the treated animals. Interestingly, Figure 5 C further showed that administration of SAG alone or in combination with testosterone resulted in a significantly lower g ratio compared to testosterone alone (p<10). -5 However, the effects of SAG alone were not significantly different from those induced by the drug combination. The number of aberrant structures (as described above) was then assessed. See also Figure 5 D. A higher percentage of abnormal axons were detected in the spinal cord of control EAE animals (36.4 ± 4.0) compared to mice treated with testosterone (15.8 ± 1.4, p = 0.0004), SAG (14.0 ± 1.7, p = 0.0002), or SAG + testosterone (11.8 ± 2.6, p = 0.0001).

[0125] In summary, administration of SAG and testosterone, alone or substantially simultaneously, promotes functional recovery associated with myelin regeneration and neuroprotection.

[0126] Because the observed effects of combination therapy tended to be greater than those of SAG alone, the effects of the active agent on microglia were investigated. Spinal cord sections from EAE animals were immunostained with Iba1 and Arg1 antibodies, respectively, allowing visualization of the entire activated microglia and their pro-regenerative phenotype. Images were acquired using immunofluorescence microscopy (data not shown), and the area of ​​activated microglia was quantified and visualized. Figure 5 Histogram in EF. Numerous activated Iba1+ microglia were found in the spinal cord of control EAE mice, but these cells did not polarize toward their pro-regenerative phenotype. (See image below.) Figure 5 As shown in E, no significant alteration was found in the total density of activated microglia in the spinal cord by testosterone or SAG. However, as with Figure 5 Compared to the control (4.8 ± 0.5) shown in F, SAG (10.9 ± 1.7, p = 0.03) but not testosterone (6.8 ± 1.4) promoted the polarization of microglia toward an Arg1+ anti-inflammatory and pro-regenerative phenotype. Unexpectedly, compared to the control, the overall number of activated microglia collapsed (1.8 ± 0.2, p = 0.03) when the active agent was used concurrently. Figure 5 F), which suggests that the combination appears to resolve pathological microglial cell activation.

[0127] To evaluate the effects of testosterone and SAG alone or in combination on astrocytes, immunostaining of spinal cord from EAE animals was performed using GFAP antibodies. Quantification performed on immunofluorescence microscopy images showed that testosterone and SAG tended to increase or decrease GFAP-positive area, respectively. The GFAP-positive area in EAE animals treated with testosterone, SAG, or a combination of testosterone and SAG was not significantly different from that in the control group. However, testosterone (38.8 ± 1.8) induced significantly higher GFAP staining than SAG (25.1 ± 2.2, p = 0.001). Co-administration of testosterone and SAG resulted in a GFAP-positive area comparable to that of control animals. Figure 5 The GFAP-positive area shown in G is compared with that of the control group, suggesting that testosterone and SAG can modulate different subsets of GFAP-positive astrocytes potentially involved in the beneficial effects of the combination therapy.

[0128] Since the recruitment of lymphocytes across the blood-brain barrier (BBB) ​​to vascular endothelial cells into the brain represents a crucial event in the pathogenesis of both the EAE model and multiple sclerosis itself, the effects of testosterone and SAG, alone or in combination, on BBB permeability were evaluated. To assess BBB permeability, an antibody against the tight junction protein Claudin 5 was used, as proteins in this family confer the ability of endothelial cells to tightly regulate the passage of soluble and cytokine elements between the blood and the central nervous system. Quantification performed on immunofluorescence microscopy images showed that, compared to control symptom (0.65 ± 0.11), which showed no significant difference between treatments, testosterone (2.53 ± 0.09, p < 0.0001), SAG (2.35 ± 0.27, p < 0.0001), and combination therapy (2.03 ± 0.15, p < 0.0003) induced a significant increase in Claudin 5 expression. Figure 5 As shown in H. Therefore, testosterone and SAG, used alone or in combination, appear to exhibit beneficial activity in restoring the blood-brain barrier efficiency.

[0129] In summary, the results indicate that combination therapy with Smo agonists and corticosteroids has a synergistic therapeutic effect on MS in one of the most relevant models.

Claims

1. Use of an androgen receptor ligand and a Smo agonist in the preparation of a medicament for promoting myelin regeneration in a subject with multiple sclerosis, wherein the use comprises administering to the subject an effective amount of an androgen receptor ligand and a Smo agonist, wherein the androgen receptor ligand is testosterone, and wherein the Smo agonist is 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide (SAG).

2. The use according to claim 1, wherein the androgen receptor ligand and the Smo agonist are administered substantially simultaneously or sequentially in the form of individual compositions.

3. The use according to claim 1 or 2, wherein the androgen receptor ligand and the Smo agonist are administered in the form of the same composition.

4. The use according to any one of the preceding claims, wherein one or both of the androgen receptor ligand and the Smo agonist are administered intranasally as an intranasal pharmaceutical composition, the intranasal pharmaceutical composition further comprising: (a) at least one lipophilic or partially lipophilic carrier present in an amount of about 60% to about 98% by weight of the formulation; (b) at least one compound having surface tension reducing activity present in an amount of about 1% to about 20% by weight of the formulation; and (c) at least one viscosity modifier present in an amount of about 0.5% to about 10% by weight of the formulation.

5. The use according to claim 4, wherein the intranasal pharmaceutical composition comprises the androgen receptor ligand.

6. The use according to claim 4, wherein the intranasal pharmaceutical composition comprises the Smo agonist.

7. The use according to claim 4, wherein the intranasal pharmaceutical composition comprises the androgen receptor ligand and the Smo agonist.

8. The use according to any one of claims 1 to 7, wherein the androgen receptor ligand and the Smo agonist are administered intranasally as an intranasal pharmaceutical composition comprising a porous excipient wherein the androgen receptor ligand and / or the Smo agonist are loaded onto a surface of the porous excipient located within the pores of the porous excipient.

9. The use according to claim 8, wherein the androgen receptor ligand is loaded onto the surface of the porous excipient located inside the pores of the porous excipient.

10. The use according to claim 8, wherein the Smo agonist is loaded onto the surface of the porous excipient located inside the pores of the porous excipient.

11. The use according to claim 8, wherein both the androgen receptor ligand and the Smo agonist are loaded onto the surface of the porous excipient located inside the pores of the porous excipient.

12. The use according to any one of the preceding claims, wherein the subject is a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.

13. Use of an androgen receptor ligand and a Smo agonist in the preparation of a medicament for treating demyelination in a subject with multiple sclerosis, wherein the use comprises administering the androgen receptor ligand and the Smo agonist to the subject, wherein the androgen receptor ligand is testosterone, and wherein the Smo agonist is 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide (SAG).

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