WO2024143309A1 - Prophylactic/therapeutic agent for neuronal gaucher disease - Google Patents

Abstract

A prophylactic/therapeutic agent for neuronal Gaucher disease according to the present invention contains as an active ingredient a microglial activation inhibitor, a tumor necrosis factor inhibitor or a combination thereof (preferably minocycline and etanercept).

Description

Preventive and therapeutic agents for neurological Gaucher disease

The present invention relates to a preventive and/or therapeutic agent for neuronal Gaucher disease (hereinafter, sometimes abbreviated as "prophylactic/therapeutic agent"). More specifically, the present invention relates to a preventive/therapeutic agent for neuronal Gaucher disease, which is a combination of a microglial activation inhibitor (e.g., minocycline) and a tumor necrosis factor (TNF) inhibitor (e.g., etanercept).

Gaucher disease (GD) is a congenital metabolic disorder in which β-glucosylceramide (GlcCer) accumulates throughout the body due to reduced or absent activity of glucocerebrosidase (GCase) caused by a mutation in the GBA gene (Non-Patent Document 1). In addition to hepatosplenomegaly, hematological abnormalities, and bone lesions, approximately 40% of patients in Japan develop neurological GD, which presents with severe central nervous system lesions such as delayed psychomotor development, convulsions, neck retroversion, and swallowing disorders (Non-Patent Document 2).

However, the detailed molecular mechanism by which β-GlcCer accumulation causes neurological symptoms has not been clarified. In addition, enzyme replacement therapy (ERT), the first-choice drug for GD, has not been shown to be effective against central nervous system lesions because GCase does not cross the blood-brain barrier (BBB), and the prognosis for patients with neurological GD is extremely poor (Non-Patent Document 3). GlcCer synthesis inhibitors that can cross the BBB, GCase chaperone therapy, and gene therapy are all in the research and development stage (Non-Patent Document 4), and there is a need for rapid development of treatments based on a detailed understanding of the pathology.

The object of the present invention is therefore to elucidate the pathological mechanism of neuronal GD and to provide a new and effective agent for preventing and treating neuronal GD by suppressing said mechanism.

To achieve the above-mentioned objective, the present inventors investigated the pathological mechanism using a neuronal GD model mouse (Gba ^flox/flox × Nestin-Cre; Gba ^ΔNes ). As a result, we found that β-GlcCer accumulated in the brain directly activates microglia, which produces tumor necrosis factor (TNF), which causes necroptotic stress in neurons, inducing the exposure of phagocytosis signal phosphatidylserine (PS) on the neuronal cell surface, and that microglia that recognize PS phagocytose neurons, promoting neuronal cell death. Furthermore, microglial activation and the resulting TNF production were observed in the brain tissue of patients with neuronal GD. These results suggest that activated microglia and TNF may contribute to the deterioration of the pathology in neuronal GD.

Therefore, the present inventors attempted to evaluate the therapeutic effects of FDA (Food and Drug Administration) approved drugs targeting these. Minocycline (Mino) was used to suppress microglial activation. Mino is a tetracycline antibiotic preparation that is applicable to bacterial infections, but it is said to have anti-inflammatory effects in addition to antibacterial effects. It can also pass through the BBB and is widely used in experimental medicine to suppress microglial activation (Griffin, Pharmacal. Res. 2011). Etanercept (ETN) was used to neutralize TNF. ETN is a fully human soluble TNF receptor preparation that is applicable to rheumatoid arthritis and also has a neutralizing effect on mouse TNF (Mohler, J. Immunol. 1993). When Mino or ETN was administered alone or in combination to neuronal GD model mice, neuronal cell death was reduced, neuronal loss was improved, and the progression of motor symptoms was alleviated in both cases. The combined treatment group showed an increased survival time. These results suggest that the pathology of neuronal GD can be improved by suppressing microglial activation and/or inhibiting TNF.
On the other hand, since it has been reported that proper activation of microglia and TNF signaling during development are necessary for normal brain and nerve development (Ueno, Nat. Neurosci. 2013; Marchetti, J. Biol.Chem. 2004), we investigated the effects of combined use of Mino and ETN on motor and sensory functions during development, but no abnormalities were observed in either area.

Based on these findings, the present inventors concluded that microglial activation inhibitors and/or TNF inhibitors can be used safely and effectively for the prevention and treatment of neuronal GD, and thus completed the present invention.
That is, the present invention provides the following.

[Item 1]
A preventive and/or therapeutic agent for neuronal Gaucher disease, comprising as an active ingredient a microglial activation inhibitor, a tumor necrosis factor (TNF) inhibitor, or a combination thereof.
[Item 2]
Item 2. The agent according to Item 1, comprising a combination of a microglia activation inhibitor and a TNF inhibitor.
[Item 3]
Item 3. The agent according to Item 1 or 2, wherein the microglia activation inhibitor is a tetracycline antibiotic or a derivative thereof having an anti-inflammatory effect.
[Item 4]
Item 4. The agent according to any one of Items 1 to 3, wherein the TNF inhibitor is selected from the group consisting of a soluble TNF receptor (sTNFR), an antibody against TNF, an antagonist that binds to TNF, a mutant or fragment of TNF, an antibody against the TNF receptor, and a substance that inhibits the expression of TNF.
[Item 5]
Item 3. The agent according to Item 1 or 2, wherein the microglia activation inhibitor is minocycline and the TNF inhibitor is etanercept.
[Item 6]
A method for preventing and/or treating neuronal Gaucher disease in a subject, comprising administering to the subject an effective amount of a microglia activation inhibitor and/or a TNF inhibitor.
[Item 7]
Use of a microglia activation inhibitor, a tumor necrosis factor (TNF) inhibitor, or a combination thereof in the manufacture of an agent for the prevention and/or treatment of neuronal Gaucher disease.
[Item 8]
A microglial activation inhibitor, a tumor necrosis factor (TNF) inhibitor or a combination thereof for use in the prevention and/or treatment of neuronal Gaucher disease.

The present invention can suppress microglial activation caused by β-GlcCer and can also suppress necroptotic stress in neurons induced by TNF, which is highly expressed in activated microglia, thereby improving the neurological symptoms in neuronal GD.

Figure 1. Microglia are highly activated in GD mice. Sections from brain tissues of wild-type (WT) and GD (Gba ^ΔNes ) mice at postnatal day 21 were stained with NeuN (blue), FJC (green), and Iba1 (red) at the indicated areas. Iba1-positive cells were prominently detected in brain tissues of GD mice, indicating that microglia were activated. MOp: primary motor cortex; CP: caudate putamen; HIP: hippocampus; TH: thalamus; MB: midbrain; P: pons; MY: medulla; CB: cerebellum; DH: dorsal horn; VH: ventral horn. Sections from three mice (three sections per animal) were analyzed for each genotype, and representative staining images are shown. Figure 1: β-GlcCer directly activates microglia. Left: Primary microglia from WT and Mincle ^-/- mice were stimulated with β-GlcCer d18:1/18:0 (10 nmol/well) or LPS (1 μg/mL) and immunostained for Iba1 (red). Representative staining images from three independent experiments are shown. Right: The size of Iba1-positive cells was calculated. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer test. *p < 0.05 Figure 1. Activated microglia produce TNF. Tnf gene expression levels in brain cells from WT and GD mice at postnatal day 14 are shown as a heat map in a UMAP plot (left panel). Clusters with the highest microglial activation scores are outlined in a solid line. Also shown are TNF production levels in brains from WT (n=4) and GD (n=6) mice at postnatal day 21 (right panel). Bar graphs show the mean values for each group. Statistical analysis was performed using an unpaired two-tailed Student's t-test. *p<0.05 This figure shows that neurons undergo necroptotic stress and expose phagocytosis signals (PS) on the cell surface. Left: Western blot showing expression of phosphorylated and non-phosphorylated RIPK1, RIPK3 and MLKL in neurons isolated from WT and GD mice at 1, 2 and 3 weeks after birth. Right: Percentage of PI negative (membrane intact) and PS positive cells in neurons isolated from WT and GD mice at 21 days after birth. Statistical analysis was performed using unpaired two-tailed Student's t-test. ***p < 0.0001 Microglia phagocytosing live neurons. Layer V cortex from postnatal day 21 WT and GD mice was immunostained for NeuN (blue), DAPI (cyan), FJC (green) and Iba1 (red). Arrowheads and arrows indicate live neurons (FJC ^- NeuN ⁺ ) and degenerated neurons (FJC ⁺ ), respectively, that are being phagocytosed by microglia. Figure 1 shows the neuroprotective effect of minocycline and etanercept, either alone or in combination, in layer V of the cerebral cortex of 3-week-old GD mice. Top: The number of degenerated neurons (FJC positive) (left) and the number of surviving neurons (FJC negative) (right) per ^mm2 in the minocycline-treated group (n=6) and the non-treated group (n=5). Center: The number of degenerated neurons (FJC positive) (left) and the number of surviving neurons (FJC negative) (right) per ^mm2 in the etanercept-treated group (n=6) and the non-treated group (n=5). Bottom: The number of degenerated neurons (FJC positive) (left) and the number of surviving neurons (FJC negative) (right) per ^mm2 in the minocycline and etanercept-treated group (n=6) and the non-treated group (n=5). Statistical analysis was performed using unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01 Figure showing the effect of administration of minocycline and etanercept alone or in combination on improving motor symptoms in GD mice. Top: Percentage of loss of righting reflex in the minocycline-treated group (n=7) and non-treated group (n=11). Middle: Percentage of loss of righting reflex in the etanercept-treated group (n=12) and non-treated group (n=11). Bottom: Percentage of loss of righting reflex in the minocycline and etanercept combined treatment group (n=4) and non-treated group (n=4). Data were analyzed using the log-rank test. *p < 0.05, **p < 0.01 This figure shows the effect of combined administration of minocycline and etanercept on extending the survival time of GD mice. Survival curves are shown for the minocycline and etanercept combined administration group (n=5) and the non-administration group (n=5). *p<0.05 This figure shows that no side effects were observed with the combined administration of minocycline and etanercept. WT mice (combined treatment group: n=3; non-treatment group: n=3) on the 21st day of life were placed on a wire mesh and turned over, and the time it took for the mouse to fall was measured. Three experiments were performed for each mouse, and the longest time was recorded (left). In addition, a pain test was performed using von Frey filaments, and the threshold was calculated until the mouse withdrew its hind paw (right). Bar graphs show the average values. Data were analyzed using the log-rank test. ns: no significant difference This figure shows that microglia phagocytose neurons and produce TNF in the brains of patients with neuronal GD. Brain tissue from patients with neuronal GD and control donors was immunostained for NeuN (blue), FJC (green) and Iba1 (red) (top panel). Cortical layer V from patients with neuronal GD and control donors was immunostained for NeuN (blue), TNF (green) and Iba1 (red) (bottom panel). Arrows indicate TNF-positive microglia.

The present invention provides a preventive and/or therapeutic agent for neuronal Gaucher disease (GD) (hereinafter also referred to as the "preventive and therapeutic agent of the present invention"), which contains as an active ingredient a microglial activation inhibitor, a tumor necrosis factor (TNF) inhibitor, or a combination thereof.

In this specification, "neuropathic Gaucher disease (GD)" refers to GD that is accompanied by primary central nervous system symptoms, and of the three main clinical types, this includes type 2 (acute/infantile) and type 3 (subacute/juvenile), but may also include cardiovascular GD accompanied by oculomotor apraxia.

In this specification, the term "prevention and treatment of neurological GD" is used to refer to the prevention or delay of the onset of neurological GD, improvement of the pathology of neurological GD, and prevention or delay of progression.

(1) Microglia Activation Inhibitor In this specification, "activation of microglia" means that quiescent (M0) microglia are stimulated by β-GlcCer to become M1 type microglia that produce toxic molecules such as TNF. Therefore, in the present invention, "microglia activation inhibitor" refers to an agent that inhibits the transformation (activation) of microglia into TNF-producing M1 type microglia.
The present invention is based, at least in part, on the discovery that β-GlcCer directly stimulates microglia, and the activated microglia produce high levels of TNF, causing necroptosis stress in neurons, exposing the phagocytosis signal PS to neurons, and microglia that recognize PS phagocytose neurons to promote neuronal death. Therefore, although it is desirable to simultaneously reduce the phagocytic and TNF-producing abilities of microglia, it is also possible to suppress neuronal death by changing microglia to the M2 type, which produces protective molecules such as IL-4, IL-10, and arginase 1, thereby reducing TNF production and reducing necroptosis stress on neurons.

Microglial activation inhibitors that can be used in the preventive and therapeutic agents of the present invention are not particularly limited as long as they can inhibit the transformation (activation) of microglia into the TNF-producing M1 type, and may also include drugs that can transform M0 microglia into the M2 type that produces protective molecules such as IL-4, IL-10, and arginase 1, and drugs that can shift M1 microglia to the M2 type.

In a preferred embodiment, the microglial activation inhibitor may be a tetracycline antibiotic having an anti-inflammatory effect, such as minocycline, doxycycline, tetracycline, oxytetracycline, or tigecycline. Minocycline or doxycycline is preferable, and minocycline is more preferable. In addition, these anti-inflammatory tetracycline antibiotics may be derivatives thereof as long as they retain the effect of suppressing microglial activation, and the derivatives may not have antibacterial activity. Examples of such derivatives include, but are not limited to, incyclinide (COL-3). In one embodiment, the tetracycline antibiotic or its derivative that can be used in the present invention may be a compound represented by the following general formula:

(In the formula, the carbons at positions 4, 5, 6, 7 and 9 may have one or two substituents, and when they have a substituent, each substituent may be independently selected from hydroxy, C _1-3 alkyl (e.g., methyl, ethyl, propyl, isopropyl), C _1-3 alkoxy (e.g., methoxy, ethoxy, propyloxy, isopropynoxy), monoalkylamino (e.g., methylamino, ethylamino), dialkylamino (e.g., dimethylamino, diethylamino), halogen (e.g., fluorine, chlorine, bromine, iodine).)

These drugs may be in the free form or in the form of a salt (unless otherwise specified in this specification). Such salts include salts with physiologically acceptable acids (e.g., inorganic acids, organic acids) and bases (e.g., alkali metals, alkaline earth metals), and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). Alternatively, these drugs may be in the form of a hydrate.

Tetracycline antibiotics such as minocycline and doxycycline are known to cross the BBB and can act on the central nervous system when administered systemically (e.g., orally).

Tetracycline antibiotics such as minocycline and doxycycline can be produced by known methods. These drugs are also commercially available.

In another embodiment, examples of microglial activation inhibitors that can be used in the preventive and therapeutic agents of the present invention include existing antipsychotics and antidepressants. These drugs have been believed to act exclusively on the nervous and synaptic systems, but in recent years, it has been reported that they may act directly on microglia and have a brain-protecting effect. Examples of such antipsychotics and antidepressants include, but are not limited to, COX-2 inhibitors (e.g., celecoxib), risperidone, fluvoxamine, reboxetine, imipramine, perospirone, ziprasidone, quetiapine, paroxetine, sertraline, and aripiprazole.

These drugs may be in the free form or as a salt. Alternatively, these drugs may be in the form of a hydrate.

These existing antipsychotics and antidepressants can cross the BBB and therefore act on the central nervous system when administered systemically (e.g., orally).

These antipsychotics and antidepressants can be manufactured by known methods. In addition, these drugs are commercially available.

(2) TNF Inhibitors TNF, another target molecule of the prophylactic and therapeutic agent of the present invention, is a member of the TNF protein family, and three types of TNF, TNF-α, TNF-β (LT-α), and LT-β, are known that share a common receptor and exhibit similar physiological activities. As used herein, the term "TNF inhibitor" refers to a drug that can at least inhibit the function or expression of TNF-α via the receptor.

TNF, especially TNF-α produced by microglia activated by β-GlcCer accumulated in neurons lacking the GBA gene (GD neurons), stimulates the TNF receptor on neurons, activating receptor-interacting kinase 1 (RIK1), which in turn phosphorylates and activates RIPK3, which in turn phosphorylates mixed lineage kinase domain-like (MLKL) by activated RIPK3, which then trimers and migrates to the cell membrane, exposing PS on the cell surface and inducing phagocytosis of neurons by microglia. Therefore, by inhibiting the function or expression of TNF, it is possible to suppress necroptotic stress on neurons and inhibit neuronal death.

(Substance that inhibits the function of TNF)
In the present invention, a "substance that inhibits the function of TNF" may be any substance as long as it suppresses the function of TNF (necroptosis stress) on nerve cells once it has been functionally produced, and examples of such substances include substances that bind to TNF and inhibit the function, and substances that bind to TNF receptors (e.g., TNFR1, TNFR2) and inhibit the binding of TNF to the receptor.

Specifically, examples of substances that bind to TNF and inhibit its function include soluble TNF receptors. In this specification, "soluble TNF receptor (sTNFR)" refers to a protein that contains all or part of a TNF receptor (e.g., TNFR1, TNFR2), preferably the extracellular domain of TNFR2 (e.g., human TNFR2 consists of 129 amino acids (see UniprotKB Q9NP84), with positions 28-80 being the extracellular domain), and that retains binding activity to TNF. sTNFR may be derived from any mammal, but is preferably derived from the same mammal as the subject of administration. sTNFR may be a monomer or a polymer (e.g., dimer, trimer). sTNFR may be terminally modified, such as by PEGylation, to impart functionality such as improved stability in the body, or may be a fusion protein with the Fc domain of IgG.

In a preferred embodiment, etanercept can be used as the sTNFR. Etanercept is a dimer of a recombinant protein that fuses the extracellular domain of human TNFR2 with the Fc domain of human IgG, and is a fully human sTNFR. Etanercept can be recombinantly produced by a method known per se, and pharmaceuticals containing it as an active ingredient are commercially available (e.g., product name: Enbrel).

In another embodiment, the substance that binds to TNF and inhibits its function includes, for example, an antibody against TNF. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a known method for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM or IgA, and particularly preferably _IgG . The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to TNF, and may be a complete antibody molecule, or a derivative thereof modified with a molecule having a protein stabilizing effect such as polyethylene glycol (PEG), or the like.

In a preferred embodiment, the antibody against TNF is used as a pharmaceutical intended for administration to humans, and therefore the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of exhibiting antigenicity when administered to humans, specifically a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and is particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to standard methods. Although fully human antibodies can also be produced from human-human (or mouse) hybridomas, in order to stably provide large amounts of antibodies at low cost, it is desirable to produce them using human antibody-producing mice or phage display methods.
Alternatively, pharmaceuticals containing anti-human TNF antibodies as the active ingredient are commercially available (e.g., infliximab, adamlimab, golimumab, and certolizumab), and these can also be used.

The substance that binds to TNF and inhibits its function may also be an antagonist that binds to TNF competitively with the TNF receptor, similar to sTNFR. Such an antagonist can be obtained by constructing a competitive assay system using TNF and the TNF receptor and screening a compound library.

On the other hand, examples of substances that bind to TNF receptors and inhibit the binding of TNF to the receptor include mutants and fragments of TNF that retain receptor binding ability but do not have receptor activation (signal transduction) ability, antibodies against TNF receptors, and antagonists that competitively bind to TNF receptors with TNF. The above TNF mutants can be obtained, for example, by creating a TNF mutant library using a phage display method, selecting mutants with strong affinity to TNF receptors, and selecting from among them mutants in which the biological activity of TNF (e.g., necroptotic stress on nerve cells) has disappeared or been reduced. Antibodies against TNF receptors can be produced by a method known per se, in the same manner as anti-TNF antibodies. Antagonists that competitively bind to TNF receptors with TNF can be obtained, for example, by selecting compounds with strong affinity to TNF receptors from a compound library, and selecting from among them compounds that do not have TNF-like biological activity (e.g., necroptotic stress on nerve cells).

(Substance that inhibits the expression of TNF)
In the present invention, the term "substance that inhibits the expression of TNF" may be one that acts at any stage, such as the transcription level of the TNF gene, the level of post-transcriptional regulation, the level of translation into protein, or the level of post-translational modification. Therefore, examples of substances that inhibit the expression of TNF include substances that inhibit the transcription of the TNF gene (e.g., antigene), substances that inhibit the processing of an initial transcription product into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acid, miRNA) or that degrade mRNA (e.g., siRNA, ribozyme, miRNA), and substances that inhibit the post-translational modification of an initial translation product. Although substances that act at any stage can be used, substances that bind complementarily to mRNA to inhibit translation into protein or degrade mRNA are preferred.

(3) Medicines containing microglia activation inhibitors and/or TNF inhibitors as active ingredients As described above, in neuronal GD, β-GlcCer accumulated in the brain directly activates microglia, and TNF produced by microglia exerts necroptosis stress on neurons, inducing exposure of PS, a phagocytosis signal, on the surface of neuronal membranes, and microglia recognize PS and phagocytose neurons, promoting neuronal death. Therefore, when microglia activation inhibitors are administered to subjects suffering from neuronal GD or subjects with a genetic mutation that is likely to develop neuronal GD, they have the effect of reducing the phagocytic ability of microglia and/or reducing TNF production from microglia, and thus can be used as a preventive and therapeutic drug for neuronal GD by suppressing neuronal death. In addition, TNF inhibitors, when administered to subjects suffering from neuronal GD or subjects at high risk of developing neuronal GD, suppress necroptotic stress on neurons caused by TNF, and suppress activation of the necroptotic pathway and exposure of the phagocytic signal PS on the surface of the neuronal membrane, thus preventing phagocytosis by microglia and suppressing neuronal death, making them useful as preventive and therapeutic agents for neuronal GD. Furthermore, microglial activation inhibitors and TNF inhibitors can block the progression cycle of neuronal death (microglial activation by β-GlcCer → TNF production from microglia → necroptotic stress on neurons → expression of phagocytic signals in neurons → phagocytosis of neurons by microglia) through different mechanisms, and therefore can synergistically suppress neuronal death, making the combined administration of these drugs more effective for the prevention and treatment of neuronal GD.

(Medicine containing a microglial activation inhibitor)
Only one type of microglia activation inhibitor may be used, or two or more types may be used in combination. Two or more types of microglia activation inhibitors may be formulated as separate pharmaceuticals, or may be combined in the same pharmaceutical composition. When two or more types of microglia activation inhibitors are formulated as separate pharmaceuticals, the respective preparations may be administered simultaneously or at different times. The administration route may be the same or different. The dosage described below indicates the dosage of one type of microglia activation inhibitor, but even when two or more substances are used in combination, the same dosage can be used for each drug within a range that does not have an undesirable effect on the subject.

Medicines containing microglia activation inhibitors can be administered orally or parenterally (e.g., intravenously, intraarterially, intraperitoneally, intramuscularly, intradermally, subcutaneously, intraventricularly, intrathecally, etc.) to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cows, pigs, monkeys, etc.) either as liquids or as pharmaceutical compositions in an appropriate dosage form.

The microglial activation inhibitor may be administered as it is, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the microglial activation inhibitor and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

Compositions for parenteral administration include, for example, injections, suppositories, intranasal preparations, etc., and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying the antibody or low molecular weight compound of the present invention or a salt thereof in a sterile aqueous or oily liquid that is usually used for injections. As aqueous liquids for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., can be used, and may be used in combination with appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into an appropriate ampule. Suppositories for rectal administration may be prepared by mixing the antibody or its salt with a normal suppository base.

The above-mentioned oral or parenteral pharmaceutical compositions are advantageously prepared in dosage unit forms that correspond to the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The microglial activation inhibitor is preferably contained in an amount of usually 0.1 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned medicine containing a microglia activation inhibitor varies depending on the subject of administration, the clinical type of GD, symptoms, administration route, etc., but for example, a single dose of the microglia activation inhibitor is usually about 0.0001 to 200 mg/kg body weight, and is conveniently administered orally or parenterally about 1 to 5 times a day. When symptoms are particularly severe, the dosage may be increased according to the symptoms.
As described above, in a preferred embodiment, the microglial activation inhibitor is an existing anti-inflammatory tetracycline antibiotic such as minocycline or doxycycline, and its safety has been confirmed in the dosage and administration as an antibiotic. Therefore, when used as a microglial activation inhibitor, it can be administered in the same dosage and administration as when used as an antibiotic. Furthermore, as shown in the examples below, it has been confirmed in animal experiments that even when used in a higher dose, for example, when administered once a day at a dose of 100 mg/kg, no abnormalities occur in motor function or sensory function.
In another embodiment, the microglial activation inhibitor is an existing antipsychotic/antidepressant drug, and the safety of the dosage and administration thereof in medical use has been confirmed. Therefore, the microglial activation inhibitor can be administered in the same dosage and administration as the antipsychotic/antidepressant drug.

The pharmaceutical of the present invention may contain other active ingredients as long as they do not cause undesirable interactions when combined with the microglia activation inhibitor. Examples of other active ingredients include various compounds that have an effect of improving or alleviating any pathological condition of GD, but are not limited to these.

(Medicine containing TNF inhibitor)
Only one type of TNF inhibitor may be used, or two or more types may be used in combination. Two or more types of TNF inhibitors may be formulated as separate pharmaceuticals, or may be combined in the same pharmaceutical composition. When two or more types of TNF inhibitors are formulated as separate pharmaceuticals, the respective preparations may be administered simultaneously or at different times. The administration route may be the same or different. The dosage described below indicates the dosage of one type of TNF inhibitor, but even when two or more substances are used in combination, the same dosage can be used for each drug within the range that does not have an undesirable effect on the subject.

Medicines containing TNF inhibitors can be administered orally or parenterally (e.g., intravascularly, subcutaneously, etc.) to humans or other warm-blooded animals (e.g., mice, rats, rabbits, dogs, cats, cows, pigs, monkeys, chickens, etc.) either as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form.

The TNF inhibitor may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the TNF inhibitor and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for parenteral or oral administration.

Compositions for parenteral administration include, for example, injections, suppositories, intranasal preparations, etc., and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying the above-mentioned TNF inhibitors in a sterile aqueous or oily liquid that is usually used for injections. As aqueous liquids for injection, for example, saline, isotonic solutions containing glucose and other adjuvants, etc., can be used, and may be used in combination with appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into a suitable ampule. Suppositories for rectal administration may be prepared by mixing the above-mentioned TNF inhibitor with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

The above-mentioned parenteral or oral pharmaceutical compositions are conveniently prepared in a dosage unit form that matches the dosage of the active ingredient. Examples of such dosage unit forms include injections (ampoules), tablets, pills, capsules, and suppositories. The TNF inhibitor is preferably contained in an amount of usually 0.1 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned medicines containing TNF inhibitors varies depending on the recipient, the clinical type of GD, symptoms, route of administration, etc., but for example, a single dose of TNF inhibitor is usually about 0.0001-100 mg/kg body weight, conveniently administered by intracerebroventricular or intrathecal injection once a day to once a month. Similar amounts can also be administered for other parenteral and oral administrations. If symptoms are particularly severe, the dosage may be increased according to the symptoms. As described above, in a preferred embodiment, the TNF inhibitor is a soluble TNF receptor or an anti-TNF antibody, which do not inherently cross the BBB; however, they can be made to cross the BBB by using BBB-crossing technologies such as LRP-1 affinity peptide (Angiopep; Angiochem), insulin receptor antibody (ArmaGen), glutathione-PEG-liposomes (2-BBB Medicines BV), melanotransferrin, melanotransferrin peptide (both from bioasis), transferrin receptor antibody (Roche (Genentech)), and J-Brain-Cargo (registered trademark; JCR Pharma), so administration routes other than intraventricular or intrathecal administration are not excluded.

The pharmaceutical of the present invention may contain other active ingredients as long as they do not cause undesirable interactions when combined with the TNF inhibitor. Examples of other active ingredients include various compounds that have an effect of improving or alleviating any pathological condition of GD. For example, other active ingredients include other active ingredients that can be used in combination with the microglia activation inhibitors described above.

In a more preferred embodiment, the preventive/therapeutic agent of the present invention is a medicine comprising a combination of a microglia activation inhibitor and a TNF inhibitor. The microglia activation inhibitor and the TNF inhibitor may be formulated as separate medicines, or may be combined in a single pharmaceutical composition as long as they can be formulated and the same administration route can be selected. When the microglia activation inhibitor and the TNF inhibitor are formulated as separate medicines, the respective preparations may be administered simultaneously or at different times. The administration routes may be the same or different. Even when the microglia activation inhibitor and the TNF inhibitor are used in combination, the same dosages as described above can be used for each drug, as long as they do not have an undesirable effect on the subject.

In a particularly preferred embodiment, the preventive and therapeutic agent of the present invention is a medicine comprising a combination of minocycline and etanercept. Minocycline and etanercept can each be formulated as separate pharmaceutical compositions, and the former can be administered orally, intravenously, intraperitoneally, intramuscularly, intradermally, subcutaneously, etc., while the latter can be administered intraventricularly, intrathecally, etc.

The present invention will be explained in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these.

Materials and Methods
1. Mice Mincle-deficient mice were backcrossed with C57BL/6J (WT) mice (purchased from CLEA Japan, Inc.) for at least 23 generations. Nestin-Cre mice were purchased from The Jackson Laboratory. Both male and female mice were used in all experiments. Gba-floxed mice were generated by gene targeting. Gba ^flox/flox mice were crossed with Gba ^flox/+ × Nestin-Cre mice to generate Gba ^flox/flox × Nestin-Cre (Gba ^ΔNes ) mice. All mice were maintained in laminar flow cages under SPF conditions with filtered air, fed standard laboratory chow, and allowed free access to water. All animal experiments were performed with the approval of the Ethics Committee for Animal Experiments at the Institute for Microbial Diseases, Osaka University.

2. Humans This study was conducted with the approval of the ethical committees of the Research Institute for Microbial Diseases, Osaka University (RIMD-2021-10) and the National Center of Neurology and Psychiatry (A2022-006). Written informed consent was obtained from the patient. Formalin-fixed temporal lobe tissue samples from a patient with GD3 (female, 24 years old) and a control donor without neurological disease (male, 25 years old) were obtained from the National Center of Neurology and Psychiatry.

3. Immunohistochemistry After cardiac perfusion with PBS, whole brains were fixed overnight in 4% (w/v) paraformaldehyde (PFA; Nacalai Tesque, 09154-85) in PBS. For fluorescence imaging, brains were transferred to 30% (w/v) sucrose (Nacalai Tesque, 30404-45) in PBS and left to settle until the tissues sank. Cryoprotected tissues were then embedded in OCT compound (Sakura Finetech Japan, 4583) and 12-μm-thick sections were prepared using a Cryostar NX50 cryostat (Thermo Fisher Scientific) and mounted on MAS-coated glass slides (Matsunami Glass Co., Ltd., MAS-01). Brain tissue sections were permeabilized with 0.1% Triton X-100 (Nacalai Tesque, 28229-25) and blocked with Protein Block (Dako, X090930-2) for 10 min at room temperature, then incubated overnight at 4°C with the following antibodies diluted in 1% (w/v) BSA PBS solution:
Rabbit anti-Iba1 (1:1,000, EPR16588; Abcam, ab178846)
Mouse anti-NeuN (1:1,000, 1B7; Abcam, ab104224)
The sections were then incubated with the following secondary antibodies diluted in 1% (w/v) BSA PBS solution for 2 h at 37°C.
Goat anti-rabbit IgG H&L Alexa Fluor 594 (1:500; Abcam, ab150080)
Goat anti-mouse IgG H&L Alexa Fluor 647 (1:500; Abcam, ab150115)
Additionally, FJC staining was performed according to the manufacturer's instructions with some modifications. Briefly, sections were incubated with 0.0001% (v/v) FJC working solution (Biosensis, TR-100-FJT) for 10 min at room temperature and mounted in FluorSave (Calbiochem, 345789). Sections were visualized with an FV3000 confocal laser scanning microscope (Olympus).

For staining of human brain, postmortem brains were embedded in paraffin and sections were mounted on glass slides. Sections were deparaffinized and antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) at 98°C for 20 minutes. After cooling, brain tissue sections were permeabilized with 0.1% Triton X-100 and blocked with Protein Block for 10 minutes at room temperature. Sections were then incubated overnight at 4°C with rabbit anti-Iba1 (1:1,000, EPR16588), guinea pig anti-IBA1 (1:500; Synaptic Systems, 234 004), mouse anti-NeuN (1:1,000, 1B7) and/or rabbit anti-TNF (1:500; Cell Signaling Technology, 3707) diluted in 1% (w/v) BSA in PBS. Sections were then incubated with goat anti-rabbit IgG H&L Alexa Fluor 488 (1:500; Abcam, ab150077), goat anti-rabbit IgG H&L Alexa Fluor 594 (1:500; Abcam, ab150080), goat anti-mouse IgG H&L Alexa Fluor 647 (1:500; Abcam, ab150115) and/or goat anti-guinea pig IgG H&L Alexa Fluor 594 (1:500; Abcam, ab175678) diluted in 1% (w/v) BSA in PBS for 2 hours at 37°C. Sections were then incubated with 0.0001% (v/v) FJC working solution for 10 minutes at room temperature and mounted in FluorSave. Sections were visualized with an FV3000 confocal laser scanning microscope.

4. Primary microglial culture Primary microglial cultures were prepared as previously described with some modifications. Briefly, minced cerebral cortex from postnatal day 0-2 male and female WT mice was dissociated in 5 ml of PBS containing 1 μM EDTA (Nacalai Tesque, 15130-95) for 10 min at 180 rpm on a shaker and treated with 1 μg/ml DNase I (Roche, 11284932001) for 30 s at 37°C. After centrifugation at 400×g for 3 min at 4°C, the dissociated cells were suspended in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, D5796) supplemented with 0.1% penicillin (Sigma-Aldrich, P3032)/streptomycin (MP Biochemicals, 194541) and 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, 175012). The cells were then seeded onto poly-D-lysine-coated T-75 flasks (Corning, 354537) and cultured at 37°C in a 5% _CO2 incubator for 2–3 weeks. Finally, microglia were harvested by shaking the flasks at 180 rpm for 2 h.

5. Immunocytochemistry Primary microglia were fixed with 4% (w/v) PFA in PBS for 15 min at room temperature, permeabilized with 0.1% TritonX-100, and blocked with 3% (w/v) BSA for 1 h at room temperature. Next, the cells were incubated with rabbit anti-Iba1 (1:1,000, EPR16588) diluted in 1% (w/v) BSA in PBS overnight at 4°C, and then incubated with goat anti-rabbit IgG H&L Alexa Fluor 594 (1:500; Abcam, ab150080) diluted in 1% (w/v) BSA in PBS for 2 h at 37°C. Finally, the cells were visualized using a BZ-9000 fluorescence microscope (Keyence).

6. Dissociation of brain tissue Single cell suspensions of brain tissue were prepared as previously described. Briefly, mice were killed by _CO2 asphyxiation and then cardiac perfused with PBS. Whole brains were minced with scissors and enzymatically dissociated using a papain dissociation system (Worthington Biochemical Corp., LK003150) containing papain (20 U/mL), L-cysteine (1 mM), DNase I (400 U/mL), and EDTA (0.5 mM) in Eagle's balanced salt solution. Before use, papain was equilibrated with 5% _CO2 at 37°C for 30 min. After the treatment, the tissue was washed with papain inhibitor buffer containing 1 mg/mL BSA and 1 mg/mL ovomucoid, and then the tissue was physically dissociated by gentle pipetting. Finally, dissociated cells were resuspended in 20% (v/v) Percoll (Cytiva, 17089101) in PBS and centrifuged at 400 × g for 25 min at 4 °C to remove myelin and debris.

7. Magnetic-activated cell sorting. Isolated microglia and neurons were prepared as previously described with some modifications. Briefly, single-cell suspensions of brain tissue were incubated with anti-mouse CD16/CD32 (1:100, 2.4G2) for 20 min at 4°C. Next, cells were labeled with anti-CD11b APC (1:200, M1/70; BioLegend, 101212) for 10 min at 4°C, and further incubated with anti-APC microbeads (1:5; Miltenyi Biotec, 130-090-855) for 10 min at 4°C. Cells were then loaded into an MS column (Miltenyi Biotec, 130-042-201) and placed on a MACS separator (Miltenyi Biotec, 130-042-501). The CD11b-labeled cell fraction was collected as microglia. The unlabeled (negative) cell fraction was further incubated with non-neuronal cell biotin antibody cocktail for 5 min at 4°C and labeled with anti-biotin microbeads (Miltenyi Biotec, 130-090-485) for 10 min at 4°C. The cells were then loaded into the MS column and placed in the MACS separation device. The unlabeled flow-through cell fraction was collected as neurons.

8. Single-cell RNA sequencing Single-cell suspensions (containing approximately 1 × ¹⁰⁴ cells) from postnatal day 14 mouse brains were loaded onto the Chromium Next GEM Chip G Single Cell Kit (10x Genomics, PN-1000127) and barcoding and cDNA synthesis were performed using the Chromium Controller. cDNA was amplified using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, PN-1000269) to construct cDNA libraries that were sequenced in paired-end mode (read 1, 28 bp; read 2, 91 bp) on an Illumina NovaSeq 6000 System. Data analysis and visualization were handled by BBrowser v2.10.48 software (BioTuring). Briefly, cells with mitochondrial content >10%, <200 genes, and >4,000 genes were considered as dead cells, empty droplets, and doublets, respectively, and were excluded. Differential cell types were identified by uniform manifold approximation and projection (UMAP) based on differentially expressed genes (DEGs) in each cluster. A signature score of microglial activation was generated based on reported microglial activation genes (Cst7, Spp1, Ccl3, Igf1, Gpnmb, Itgax, and Lgals3) and displayed as a heatmap in the UMAP plot using BBrowser.

9. Cytokine Measurements TNF levels in brain tissue lysates were quantified using the LEGENDplex Mouse Inflammation Panel (13-plex) (BioLegend, 740150) according to the manufacturer's instructions. After analysis using a Gallios flow cytometer, the data were analyzed using LEGENDplex v8.0 software (BioLegend).

Neurons isolated from mice were sonicated and suspended in lysis buffer containing 1% Nonidet P-40 (Nacalai Tesque, 23640-94), 1 mM phenylmethylsulfonyl fluoride (Nacalai Tesque, 27327-52), and a protease inhibitor cocktail containing 10 μg/mL aprotinin (Sigma-Aldrich, A6012), 12.5 μg/mL antipain dihydrochloride (Sigma-Aldrich, A6191), 12.5 μg/mL chymostatin (Sigma-Aldrich, C7268), 50 μg/mL leupeptin trifluoroacetate (Sigma-Aldrich, L2023), and 25 μg/mL pepstatin A (Sigma-Aldrich, P4265). After centrifugation at 20,000 × g for 60 min at 4°C, proteins in the supernatant were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The lysates were mixed with sodium dodecyl sulfate (SDS) sample buffer and boiled at 95°C for 5 min. Protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon-P PVDF membranes (Merck Millipore, IPVH20200) blocked with Block Ace (KAC Co. Ltd., UKB40), and incubated overnight at 4°C with the following primary antibodies:
Rabbit anti-RIPK1 (1:1,000, D94C12; Cell Signaling Technology, 3493)
Rabbit anti-RIP3 (1:1,000; ProSci Inc., 2283)
Rabbit anti-MLKL (1:1000, D6W1K; Cell Signaling Technology, 37705)
Rabbit anti-phosphorylated MLKL (1:1000, D6E3G; Cell Signaling Technology, 37333)
Rabbit anti-β-actin (1:3,000; 13E5, Cell Signaling Technology, 4970)
The membrane was then incubated with sheep anti-rabbit IgG (1:10,000; Cytiva, NA935) for 40 min at room temperature. Immunoreactive bands were stained with Chemi-Lumi One Super (Nacalai Tesque, 02230-30) and analyzed using a LAS 1000 luminescence image analyzer (Fujifilm).

11. PS labeling In vivo PS labeling was performed as previously described. Briefly, 1 μL of 1 mM PSVue 643 (Molecular Targeting Technologies, P-1006) was injected into the right lateral ventricle of mice using a 33-gauge Neuros syringe (Hamilton Company, 4015-63014). Mice were then killed by _CO2 asphyxiation 24 h after injection and cardiac perfusion with PBS. Brain cells from the left cerebral cortex were stained with a non-neuronal biotin antibody cocktail and streptavidin PE, then resuspended in PI and analyzed on a Gallios flow cytometer. The obtained data were evaluated using FlowJo software.

Minocycline (Sigma-Aldrich, M9511) was dissolved in PBS and administered intraperitoneally to mice at 100 mg/kg daily from postnatal day 10. Etanercept (Pfizer) was dissolved in PBS and administered intracerebroventricularly at 5 mg/kg (2 μL) using a 33-gauge Neuros syringe every other day from postnatal day 10.

13. Phenotypic Evaluation Righting reflex was measured as the time it took for mice to return to a standing position after being placed on their back. Loss of righting reflex was defined as the inability to right themselves within 60 s.
To evaluate the motor function, the mouse was placed on the wire mesh of a Wire Hanging Test Chamber (O'HARA & CO., LTD. WH-3002) and turned over. The time it took to fall was recorded three times, and the longest time was used.
To assess sensory function, mice were placed on a wire mesh and stimulated with von Frey filaments (Neuroscience, inc. 514000-20C) on the midplantar surface of the hind paw until the mouse withdrew its hind paw in response to the physical stimulus. The 50% threshold was determined using the up-down stimulation method.

14. Cell quantification. Three images from each mouse from stereotaxically identified areas of the motor cortex, binarized by applying an automatic threshold, were used to quantify NeuN ⁺ and FJC ⁺ cells and were calculated using the Analyze Particle function in ImageJ. Phagocytosis of neurons by microglia was analyzed using Z-stack images acquired at 0.4 μm intervals, visualized using Orthogonal View or the 3D Viewer plugin (https://imagej.net/plugins/3d-viewer/).

15. Statistical Analysis Data were analyzed using GraphPad Prism v9.1.0 software (GraphPad Software). Mean values between two groups were compared using unpaired two-tailed Student's t-test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer test. Time-to-event distributions between two groups were compared using the log-rank test. A p-value <0.05 was considered statistically significant.

(result)
<Microglia are highly activated in GD mice>
We established a conditional knockout mouse (Gba ^ΔNes ) that lacks the Gba gene specifically in neural cells as a Gaucher disease (GD) model mouse, and examined the expression of NeuN (neurons), FJC (dead neurons), and Iba1 (microglia) in brain tissue sections taken 21 days after birth by immunohistochemical staining. The results are shown in Figure 1. Compared to wild-type (WT) mice, it was revealed that microglia, which are immune cells in the brain, were highly activated in GD mice.

<β-GlcCer directly activates microglia>
To investigate whether β-GlcCer, which accumulates due to enzyme deficiency in GD, directly activates microglia, primary microglia prepared from WT mice and mice lacking Mincle, an immunoreceptor of β-GlcCer in microglia (Mincle ^-/- ), were stimulated with β-GlcCer and their size was measured by visualization using Iba1 positivity as an indicator. The results are shown in Figure 2. The increase in microglia size was Mincle-dependent, demonstrating that β-GlcCer directly activates microglia.

<Microglia produce TNF>
Next, we examined the expression levels of the inflammatory cytokine TNF in the brains of WT and GD mice by single-cell RNA analysis of brain cells. The results are shown in Figure 3. In GD mice, activated microglia highly expressed TNF, and the TNF concentration in the brain was actually increased.

<Stressed neurons expose phagocytosis signals>
Activation (phosphorylation) of proteins involved in necroptosis (RIPK1, RIPK3, MLKL) in neurons isolated from 1-, 2-, and 3-week-old WT and GD mice was analyzed by Western blot. In addition, flow cytometry analysis of the left hemisphere of each mouse at 3 weeks of age detected neurons exposing the phagocytosis signal phosphatidylserine (PS) on the cell membrane surface. As a result, it was revealed that as the disease progresses, intracellular stress (necroptotic signature) increases in neurons of GD mice, which in turn exposes the phagocytosis signal PS on the cell surface (Fig. 4).

<Microglia phagocytose living nerve cells>
To examine whether activated microglia phagocytose GD neurons that express phagocytic signals, we performed immunohistochemical staining of brain tissue sections from 3-week-old WT and GD mice for the microglial marker Iba1, neuronal marker NeuN, and dead neuron marker FJC. As a result, we observed that neurons in GD mice that express phagocytic signals are phagocytosed by activated microglia (Figure 5). Notably, not only dead cells (FJC positive) but also live neurons were phagocytosed.

　From the above, it has been thought that the intracellular toxicity caused by β-GlcCer accumulated in nerve cells leads to nerve cell death, which then leads to neurological symptoms. However, this invention has revealed that there exists an inflammatory condition in which β-GlcCer directly activates microglia, the TNF produced stresses neurons, and the stressed neurons are phagocytosed by microglia while still alive, ultimately leading to neurological symptoms. At the same time, the possibility of a new treatment method different from conventional enzyme replacement therapy has been suggested.

<Minocycline and etanercept are effective in GD mice>
Therefore, aiming for rapid clinical application, we selected drugs targeting inflammatory pathology from commercially available drugs and investigated their effects. Minocycline is an antibacterial drug used for various infections and is known to suppress the activation of microglia. Etanercept is a TNF inhibitor that is applicable to rheumatoid arthritis.
When GD mice were administered minocycline or etanercept, either alone or in combination, neuronal degeneration was significantly reduced and the number of surviving neurons was increased in all treatment groups compared to the control group (Figure 6).

<The combination of minocycline and etanercept is particularly effective in GD mice>
The effects of minocycline and etanercept, either alone or in combination, were examined using a righting reflex test. As a result, motor symptoms improved significantly in both groups compared to the control group, but the combination group tended to show greater improvement compared to the single group (Figure 7). The combination group showed a significant increase in survival time compared to the control group (Figure 8).

<No adverse effects on neurological function were observed with the combined administration>
Since microglial activation and TNF signaling are known to be necessary for normal brain development during development, we examined the effects of combined administration of minocycline and etanercept on motor and sensory functions in WT mice, and found no abnormalities in either motor or sensory functions (Figure 9).

<Microglia phagocytosis and TNF production observed in GD patients>
Finally, to confirm whether the novel treatment for neuronal GD targeting microglial activation and TNF production can be applied to humans, we performed immunohistochemical staining of brain tissue sections from patients with neuronal GD, and found that microglial activation and TNF production were observed in the brain tissue of patients with neuronal GD.
Based on the above, minocycline and etanercept, either alone or in combination, are considered to be a treatment for neurological GD that can be rapidly applied clinically.

This application is based on Patent Application No. 2022-208678 filed in Japan on December 26, 2022, the entire contents of which are incorporated herein by reference.

The medicine using the microglia activation inhibitor and/or TNF inhibitor of the present invention is useful for improving the neurological symptoms of neurological GD, which has had no effective treatment and a very poor prognosis until now. In particular, the use of existing drugs minocycline and etanercept will enable early clinical application.

Claims (5)

1. A preventive and/or therapeutic agent for neuronal Gaucher disease, the active ingredient of which is a microglial activation inhibitor, a tumor necrosis factor (TNF) inhibitor, or a combination thereof.
2. The agent according to claim 1, which is a combination of a microglial activation inhibitor and a TNF inhibitor.
3. The agent according to claim 1 or 2, wherein the microglial activation inhibitor is a tetracycline antibiotic or a derivative thereof having an anti-inflammatory effect.
4. The agent according to claim 1 or 2, wherein the TNF inhibitor is selected from the group consisting of a soluble TNF receptor (sTNFR), an antibody against TNF, an antagonist that binds to TNF, a mutant or fragment of TNF, an antibody against the TNF receptor, and a substance that inhibits the expression of TNF.
5. The agent according to claim 1 or 2, wherein the microglial activation inhibitor is minocycline and the TNF inhibitor is etanercept.