Pharmaceutical composition for promoting migration of substance to central nervous system

Abstract

Provided is a composition for promoting migration of a target substance to the central nervous system, the composition containing a substance having an inhibitory activity against sphingomyelin synthase 1 (SMS1). Examples of the target substance include a compound, a peptide, a nucleic acid, a protein, an antibody, and a nucleic acid-containing vector, which are useful for preventing or treating central nervous system diseases.

Description

Pharmaceutical compositions for promoting the transfer of substances to the central nervous system

[0001] This disclosure relates to pharmaceutical compositions for assisting the passage of substances across the blood-brain barrier and facilitating their transfer to the central nervous system.

[0002] The brain contains a mechanism called the blood-brain barrier (BBB), which selectively restricts the passage of substances from the bloodstream into the brain. This mechanism protects the brain from harmful substances, but at the same time, it also acts as a barrier to the delivery of drugs to the brain.

[0003] There is a need to develop substances that can help substances in the blood cross the blood-brain barrier and promote their transfer to the central nervous system, such as the brain and spinal cord.

[0004] This disclosure aims to provide a pharmaceutical composition that can help substances in the blood cross the blood-brain barrier and promote their transfer to the central nervous system, such as the brain and spinal cord.

[0005] The inventors noted that sphingomyelin (SM) is abundantly expressed in cerebral vascular endothelial cells. Sphingomyelin has a structure in which phosphocholine is bound to ceramide and makes up the majority of sphingolipids that form the lipid bilayer. Sphingomyelin synthase (SMS) consists of two isoforms, SMS1 and SMS2, which have different amino acid sequences.

[0006] The inventors discovered that SMS1 is highly expressed in cerebral vascular endothelial cells. The SMS1 enzyme catalyzes the transfer of phosphocholine from phosphatidylcholine to ceramide, producing sphingomyelin as a product (Figure 1). The inventors used SMS1 as a target to study the blood-brain barrier (BBB) ​​permeability of substances and completed the present invention.

[0007] In other words, the present invention comprises the following: (1) A composition that promotes the central nervous system penetration of a target substance, comprising a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1). (2) The central nervous system penetration promoting composition according to (1), wherein the target substance is an effective component for treating central nervous system diseases. (3) The central nervous system penetration promoting composition according to (1) or (2), wherein the target substance is selected from the group consisting of compounds, peptides, nucleic acids, proteins, antibodies, and vectors containing nucleic acids. (4) The central nervous system penetration promoting composition according to (3), wherein the target substance is an antibody. (5) The central nervous system penetration promoting composition according to any one of (1) to (4), wherein the substance having inhibitory activity against SMS1 is provided as a composition separate from the target substance. (6) The central nervous system penetration promoting composition according to (5), which is administered before the administration of the target substance. (7) The central nervous system penetration promoting composition according to (5), which is administered simultaneously with the target substance. (8) A central nervous system penetration promoting composition according to (5), to be administered after administration of the target substance. (9) A central nervous system penetration promoting composition according to any one of (1) to (4), comprising a substance having inhibitory activity against SMS1 together with the target substance. (10) A combination for the treatment of central nervous system diseases of a pharmaceutical composition containing a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1), and a pharmaceutical composition containing an ingredient effective for the treatment of central nervous system diseases. (11) A screening method for central nervous system penetration promoting agents, comprising the steps of examining the inhibitory activity of candidate substances against sphingomyelin synthase 1, and selecting candidate substances that have been found to have inhibitory activity against sphingomyelin synthase 1 as central nervous system penetration promoting agents. (12) A method for treating central nervous system diseases, comprising administering a combination of an active ingredient of a pharmaceutical for the treatment of central nervous system diseases and a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) to a subject requiring treatment of central nervous system diseases. (13) Use of a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) for the manufacture of a pharmaceutical product for promoting the transfer of a target substance to the central nervous system. (14) A substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) for use in promoting the transfer of a target substance to the central nervous system.(15) A pharmaceutical composition containing a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) and the target substance, and further containing a pharmaceutically acceptable carrier.

[0008] A diagram showing the function of sphingomyelin synthase 1. A diagram showing the results of Example 1 (leaking of autoantibodies into the brain). A diagram showing the results of Example 1 (leaking of albumin-Evans blue into the brain). A diagram showing the effect of the SMS1 inhibitor SMS1-IN-1 on albumin permeability in Example 2, an in vitro human BBB model. A diagram showing the effect of SMS1-targeting siRNA on albumin permeability in Example 2, an in vitro human BBB model. A diagram showing the concentration-dependent knockdown efficiency of the SMS1-targeting siRNA used in Example 2. A diagram showing the results of Example 4 (leaking of exogenous antibodies into the brain).

[0009] Unless otherwise specified, terms used in this disclosure have the meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Some definitions of terms used in this disclosure are given below, but these definitions take precedence over general understandings in this disclosure.

[0010] This disclosure provides a composition that promotes the central nervous system transport of a target substance, comprising a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1).

[0011] Substances having inhibitory activity against SMS1 are not particularly limited as long as they block, eliminate, inhibit, or weaken the activity of human SMS1 in transferring phosphocholine from phosphatidylcholine to ceramide to produce sphingomyelin. Examples include small molecule compounds, antibodies, peptides, and nucleic acids that target the SMS1 gene.

[0012] Examples of small molecule compounds include, but are not limited to, commercially available SMS1-IN-1 (MedChemexpress Co., limited, CAS: 1807943-38-9). The IC of SMS1-IN-1 relative to SMS1 is also mentioned. 50The reported concentration is 2.1 μM (Li et al., Bioorganic & Medicinal Chemistry Volume 23, Issue 18, 15 September 2015, Pages 6173-6184).

[0013] Nucleic acids that target the SMS1 gene include those containing a sequence complementary to a portion of the mRNA sequence of the SMS1 gene. Examples of nucleic acids containing a sequence complementary to a portion of the mRNA sequence of the SMS1 gene include nucleic acids that cause RNA interference and antisense oligonucleotides. Examples of nucleic acids that cause RNA interference include siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), and nucleic acids that produce said RNA. Nucleic acids that produce said RNA include, for example, DNA corresponding to said RNA (DNA encoding said RNA), precursor RNA of said RNA, and DNA corresponding to said precursor RNA. Precursor RNA of said RNA is, for example, a long double-stranded or single-stranded RNA molecule that is processed in a cell to produce short RNA, such as siRNA, miRNA, etc. Antisense nucleotides are single-stranded DNA or RNA and may be appropriately modified based on known techniques in the art.

[0014] The nucleic acid sequence of human SMS1 is known, and the siRNA for human SMS1 can be prepared using a standard method based on the known sequence.

[0015] Furthermore, as a substance that inhibits the activity of SMS1, a substance that has been confirmed to inhibit the activity of SMS1 through screening as shown in Example 3 below can be used.

[0016] Inhibiting the activity of SMS1 increases the permeability of the BBB, promoting the transfer of the target substance to the central nervous system. In this disclosure, the central nervous system consists of the brain and spinal cord and, together with the peripheral nervous system, constitutes the nervous system. The brain includes the cerebrum (cerebral cortex [frontal lobe, parietal lobe, temporal lobe, occipital lobe], cerebral white matter, basal ganglia [striatum (putamen, caudate nucleus), globus pallidus, substantia nigra, subthalamic nucleus, amygdala], hippocampus, cingulate gyrus, etc.), diencephalon (thalamus, hypothalamus, subthalamic nucleus, hyperthalamus, thalamic relay nucleus, pineal gland), cerebellum (cerebellar cortex, cerebellar nuclei, vermis, cerebellar hemispheres), brainstem (midbrain, substantia nigra, pons, medulla oblongata, red nucleus, reticular formation, superior colliculus, inferior colliculus), ventricular system (lateral ventricles, third ventricle, fourth ventricle, periventricular organs), and corpus callosum, etc. The spinal cord also includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cords. The central nervous system transfer-promoting compositions of this disclosure can be used in particular to promote the transfer of the target substance to the brain.

[0017] Examples of target substances include active ingredients in pharmaceuticals that target the central nervous system and are used to treat central nervous system diseases. These active ingredients may include not only substances already in practical use, but also substances under development and those to be discovered in the future; they are not particularly limited.

[0018] The central nervous system penetration-promoting compositions of this disclosure are useful for treating central nervous system diseases caused by target substances by promoting the transfer of the target substance to the target central nervous system.

[0019] In this disclosure, "subjects" means mammals, including, but not limited to, humans or non-human mammals such as cattle, horses, dogs, sheep, or cats. It is particularly preferred for use with human subjects.

[0020] Examples of central nervous system disorders that can be treated with the central nervous system penetration-promoting compositions of this disclosure include, but are not limited to, Alzheimer's disease (hereinafter referred to as AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (hereinafter referred to as ALS), multiple sclerosis, cerebrovascular disorders (e.g., stroke, subarachnoid hemorrhage, cerebral hemorrhage, cerebral infarction, transient ischemic attack), lysosomal storage diseases (e.g., Gaucher disease, Tay-Sachs disease, Fabry disease, Niemann-Pick disease), haploinsufficiency disorders (e.g., Rett syndrome, Charcot-Marie-Tooth disease), mental disorders (e.g., depression, schizophrenia), and pain disorders (e.g., neuropathic pain, migraine, fibromyalgia).

[0021] Examples of active ingredients in pharmaceuticals for the treatment of central nervous system disorders include, but are not limited to, compounds, bioactive substances (e.g., dopamine), proteins (e.g., imiglucerase, veraglucerase alfa, taliglucerase alfa, agalsidase alfa, agalsidase beta, idursulfase, laronidase, alglucosidase alfa), antibodies (e.g., aducanumab, donanemab, lecanemab, natalizumab, eculizumab, inebilizumab, rituximab, ocrelizumab, ofatumumab, mepolizumab, satratizumab, tocilizumab, mogamulizumab, bevacizumab, galcanezumab, fremanezumab, erenumab, idarucizumab), nucleic acids (e.g., patisiran, lucinersen, viltolarsen, tofersen), and vectors containing these nucleic acids.

[0022] Antibodies include not only monoclonal antibodies but also binding fragments that retain the antigen-binding properties of antibodies. Examples of binding fragments that retain antigen-binding properties of antibodies include Fab, Fab', and F(ab'). ２ , Fab ３ Fab-sc-Fv (bibody), Fab-sc-Fv ２ Examples include (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), TriFabs, Fab-scFv-Fc, etc.

[0023] Nucleic acids encoding the aforementioned proteins, antibodies, or fragments thereof that maintain their antigen-binding properties are also exemplified as target substances of this disclosure. Vectors containing such nucleic acids are also exemplified. Furthermore, siRNA, shRNA, miRNA, and antisense oligonucleotides targeting genes involved in the onset and progression of central nervous system diseases are also exemplified.

[0024] In this disclosure, the term “treatment” means reducing or relieving the target disease and / or symptoms associated therewith. It will be understood that this does not require the complete elimination of the disorder, symptoms, or symptoms associated therewith.

[0025] The central nervous system penetration-promoting compositions of this disclosure are administered to the target in an effective amount via an administration route suitable for the substance having inhibitory activity against SMS1 contained herein. The administration regimen may be determined by the combination of the target substance and the substance of the central nervous system penetration-promoting composition of this disclosure that has inhibitory activity against SMS1. The central nervous system penetration-promoting compositions of this disclosure may be administered simultaneously with a pharmaceutical product containing the target substance, before the administration of said pharmaceutical product, or after the administration of said pharmaceutical product.

[0026] Examples of methods for administering the central nervous system penetration-promoting compositions of this disclosure include oral administration and parenteral administration (e.g., intravenous, intramuscular, subcutaneous, transdermal, transnasal, or transpulmonary administration). The compositions may be in dosage forms suitable for each route of administration. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered by subcutaneous injection.

[0027] In this disclosure, the effective amount of a substance having inhibitory activity against SMS1 means the amount of a substance having inhibitory activity against SMS1 required to achieve enhanced central nervous system penetration of the target substance. The amount of a substance having inhibitory activity against SMS1 when the central nervous system penetration-enhancing composition of this disclosure is actually used may be determined as appropriate depending on the administration method, the age, weight, and overall health of the subject, as well as the properties of the target substance.

[0028] The central nervous system penetration-promoting compositions of this disclosure may be provided as formulations independent of the target substance, as a combination with a pharmaceutical composition containing the target substance, or as a combination further containing the target substance.

[0029] The central nervous system penetration-promoting compositions of this disclosure are formulated with a pharmaceutically acceptable carrier in a dosage form suitable for the route of administration. Examples of dosage forms include, but are not limited to, tablets, pills, capsules, granules, powders, oral solutions (e.g., elixirs, suspensions, and emulsions), syrups, inhalants, suppositories, injections, and patches. Examples of injections include liquid injections and solid injections that are dissolved before use (e.g., lyophilized injections).

[0030] In this disclosure, the term "pharmaceutically acceptable carrier" means one or more miscible solid or liquid extenders, diluents, or encapsulants suitable for administration to humans or other vertebrates, and includes materials and combinations thereof known to those skilled in the art, such as all aqueous solvents (e.g., water, alcoholic solutions / aqueous solutions, saline and Ringer's solution), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate), dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, pigments, fluids and nutritional solutions.

[0031] The present disclosure will be further described below with reference to examples. The following examples are for illustrative purposes only and do not limit the invention of the present disclosure in any way.

[0032] Leakage of endogenous antibodies into the brain in vascular endothelial-specific SMS1-deficient mice: Sgms1 flox mice (derived from the C57BL / 6N-A tm1Brd Sgms1 tm1a(EUCOMM)Wtsi / WtsiH strain obtained from EUCOMM and generated by mating with flippase-expressing mice) in which the gene encoding SMS1, Sgms1, can be deleted in a Cre recombinase-dependent manner were mated with Cdh5-Cre mice in which Cre recombinase is activated specifically in vascular endothelial cells by tamoxifen ERT2 to generate tamoxifen-inducible vascular endothelial-specific deficient mice (Sgms1 flox / flox ; Cdh5-Cre ERT2 , hereinafter referred to as iECKO mice).

[0033] In male mice of this strain, tamoxifen was intraperitoneally administered from 12 weeks of age (100 mg / kg of body weight, using corn oil as a solvent, administered once a day for 3 consecutive days) to specifically delete SMS1 in vascular endothelial cells. Siblings without Cdh5-Cre ERT2 were Sgms1 flox / floxMale mice were similarly administered tamoxifen and used as control mice. One week after the start of tamoxifen administration, iECKO and control mice were anesthetized with pentobarbital, perfused systemically through the heart with 15 mL of PBS, then perfused systemically through the heart with 30 mL of 4% paraformaldehyde, and the brains were collected. The collected brains were fixed by immersion in 30 mL of 4% paraformaldehyde overnight (4°C), and then washed with 30 mL of PBS at room temperature for 30 minutes, repeating this process three times. Brain tissue sections of 50 μm were then prepared using a vibratome (LEICA VT1200S). Brain tissue sections were permeabilized with 0.5% Triton X-100 / PBS at room temperature for 30 minutes with shaking, then blocked with 2% Donkey Serum (Jackson ImmunoResearch) / 0.5% Triton X-100 / PBS (blocking buffer) at room temperature for 30 minutes with shaking, followed by immersion in anti-mouse donkey IgG Alexa Fluor 647 (diluted 400-fold in blocking buffer) at 4°C overnight. Afterward, the sections were washed three times with 1 mL of 0.5% Triton X-100 / PBS at room temperature for 30 minutes each with shaking, transferred to glass slides, and mounted using a mounting agent (SouthernBiotech Fluoromount-G). Images were acquired using a fluorescence microscope (Axio Observer, Zeiss). Fluorescence intensity was quantified using Image J, with three images obtained from each mouse's brain tissue section, and the average fluorescence intensity was considered the value for one individual. The results are shown in Figure 2.

[0034] Evaluation of albumin permeability to the brain in vascular endothelium-specific SMS1-deficient mice: iECKO and Control mice (1 week post-administration) administered tamoxifen in the same manner as above were given Evans Blue, which strongly binds to albumin, by tail vein injection (100 mg / kg body weight, using PBS as the solvent). Sixteen hours after Evans Blue administration, the mice were anesthetized with pentobarbital, perfused systemically through the heart with 30 mL of PBS, and the brains were collected and weighed. Next, the brains were homogenized in a 1:1 mixture of trichloroacetic acid and PBS (8 mL / g of brain weight), incubated overnight at 4°C, and the supernatant was collected the following day by centrifugation (12,000 xg, 20 min). Evans Blue in the supernatant was measured using a SpectraMax microplate reader (Molecular Devices, San Jose, CA, USA) with the excitation / emission wavelength (nm) set to 620 / 680. Brains were collected from male mice of the same age that had not been administered Evans Blue, after systemic perfusion, similar to the sample. Extracts were then collected using a 1:1 mixture of trichloroacetic acid and PBS. A dilution series of Evans Blue was created using this extract, and this was used as a calibration curve to calculate the concentration of Evans Blue in the sample. The results are shown in Figure 3.

[0035] [Results] Figure 2 shows the results of verifying brain leakage of endogenous IgG (molecular weight approximately 150,000) in control and iECKO mice. Anti-mouse IgG staining revealed that endogenous IgG leaked into the brain parenchyma by genetically inducible deletion of SMS1 (Figure 2A, scale bar is 100 μm). Figure 2B shows the results of measuring the degree of leakage in four control and four iECKO mice by fluorescence intensity quantification.

[0036] Figure 3 shows the quantitative results of Evans Blue brain leakage in control (3 mice) and iECKO mice (3 mice). It was confirmed that inducible deficiency of SMS1 enhances the leakage of endogenous IgG and Evans Blue, i.e., albumin, into the brain.

[0037] Evaluation of the effect of SMS1 inhibitors on albumin permeability in an in vitro human blood-brain barrier (BBB) model An in vitro human BBB model was constructed by co-culturing immortalized human cells in a Transwell system (Cat.: 353095, Corning).

[0038] As SMS1 inhibitors, a commercially available SMS1 inhibitory compound, SMS1-IN-1 (MedChemexpress Co., limited, CAS: 1807943-38-9), and siRNA against SMS1 (Accell Human SGMS1) manufactured by Horizon Limited were used.

[0039] On the day after the start of co-culture, SMS1-IN-1 was added at final concentrations of 1, 3, and 10 μM, and an albumin permeability test was performed 72 hours after compound treatment. On the day after the start of co-culture, siRNA against commercially available SMS1 was added at final concentrations of 0.1, 0.3, and 1 μM, and an albumin permeability test was performed 72 hours after substance treatment.

[0040] In the BBB permeability test of albumin, the insert seeded with cells was washed once with PBS (+) (Invitrogen), the medium was then exchanged with Hank’s balanced salt solution with calcium and magnecium (HBSS), and pre-incubated at 37°C for 10 minutes. Subsequently, FITC-labeled albumin (Albumin Fluorescein isothiocyanate Conjugate bovine, Sigma) was added to the apical side of the insert to initiate the reaction. The final added concentration of the test substance was set at 250 μg / mL. After incubating for 30, 60, and 90 minutes after the start of the reaction, the solution was collected from the basolateral side of each insert at each time point, and the compound concentration in the solution was measured. Fluorescence measurement of FITC-albumin was performed using a SpectraMax microplate reader (Molecular Devices, San Jose, CA, USA), with the excitation / emission wavelength (nm) set at 492 / 5184 for measurement.

[0041] [Results] The change in albumin permeability upon treatment of the in vitro human BBB model with SMS1-IN-1, a commercially available SMS1 inhibitor as an SMS1 inhibitor, was observed. The IC 50 of SMS1-IN-1 for SMS1 is 2.1 μM (Li et al., Bioorganic & Medicinal Chemistry Volume 23, Issue 18, 15 September 2015, Pages 6173-6184). Based on this, the treatment concentrations were set at 1, 3, and 10 μM. The results are shown in Figure 4. SMS1-IN-1 was confirmed to enhance the BBB permeability of albumin permeability in a concentration-dependent manner in the in vitro human BBB model.

[0042] The change in albumin permeability upon treatment of the in vitro human BBB model with siRNA against commercially available SMS1 as an SMS1 inhibitor was observed. The results are shown in Figure 5.

[0043] Also, the results of the mRNA knockdown efficiency of SMS1 siRNA are shown in Figure 6. SMS1 siRNA was confirmed to suppress the expression of SMS1 in a concentration-dependent manner in the in vitro human BBB model, enhance the BBB permeability in a concentration-dependent manner, and allow albumin to permeate.

[0044] From these results, it became clear that by inhibiting or suppressing the activity of SMS1, the BBB permeability is enhanced, and the permeability of relatively large molecules such as proteins and antibodies is enhanced.

[0045] SMS1 Inhibition Activity Assay The RapidFire / MS assay is used to measure the SMS1 inhibition activity. In this assay, mass spectrometry is performed using an Agilent RapidFire microfluidic solid-phase extraction system (RF) and an Agilent 6460 triple quadrupole mass spectrometer (MS). The assay conditions are shown in Tables 1 and 2.

[0046]

[0047]

[0048] 50 μL of a microsomal fraction (50 μg protein / mL) prepared by forced expression of SMS1 in insect cells, and 1 μL of a test compound solution at 100 times the evaluation concentration were added to a 96-well plate (Corning) and allowed to stand at room temperature for 5 minutes. 1 μL of the test compound medium was added to the control group. Next, 50 μL of the substrate, 10 μM C2 ceramide (Cer), was added and the mixture was reacted at room temperature for 10 minutes. After the reaction, 100 μL of 5% TFA / 50% MtOH containing 10 μM C6 sphingomyelin (SM, internal standard) was added to stop the reaction. The mixture was centrifuged at 1,500 rpm for 10 minutes to precipitate denatured proteins, and the C2 SM in the supernatant was measured using RapidFire 365 high-throughput MS. If the amount of C2 sphingomyelin in the supernatant decreases compared to the control group, the test compound is considered to have inhibitory activity against SMS1.

[0049] Leakage of exogenous antibodies into the brain in vascular endothelium-specific SMS1-deficient mice: Sgms1 flox mice (created by crossing the C57BL / 6N-A tm1Brd Sgms1 tm1a(EUCOMM)Wtsi / WtsiH strain obtained from EUCOMM with flippase-expressing mice), in which the Sgms1 gene encoding SMS1 can be deleted in a Cre recombinase-dependent manner, were crossed with Cdh5-CreERT2 mice in which Cre recombinase is activated specifically in the vascular endothelium by tamoxifen, thereby creating tamoxifen-induced vascular endothelium-specific deficiency mice (Sgms1 flox / flox; Cdh5-CreERT2, hereafter referred to as iECKO mice). In female mice of the strain in question, tamoxifen was administered intraperitoneally from 8 weeks of age (50 mg / kg of body weight, using corn oil as a solvent, once a day for 3 consecutive days) to specifically deficiency SMS1 in the vascular endothelium. As a control group (hereinafter referred to as Control mice), female mice of the same strain and litter that were not administered tamoxifen were prepared. Six days after the start of tamoxifen administration, human IgG1 isotype control antibody (BioXcell, #BP0297, molecular weight approximately 150,000) (hereinafter referred to as human IgG) was administered intraperitoneally at a dose of 10 mg / kg. Two days after antibody administration, iECKO and Control mice were anesthetized with pentobarbital, and blood was collected from the abdominal vena cava and collected in a blood collection tube (Capiject II, Terumo Corporation, #CJ-2DK) supplemented with EDTA. After systemic perfusion from the heart with 30 mL of PBS, the brain was collected. The obtained blood was separated into plasma by centrifugation. The resulting plasma and brain were stored in a -80°C freezer until used for human IgG concentration measurement experiments.

[0050] Human IgG concentrations in the obtained brain and plasma were measured using the Human IgG ELISA Kit (abcam, #ab195215). Brain extracts were prepared using the Cell Extraction Buffer provided with the kit, and protein concentrations were measured using BCA protein assay kits (Thermo Fisher Scientific, #23225). After diluting the brain extracts and plasma with the diluents provided with the kits, the human IgG concentrations were measured according to the kit instructions. From the measurement results, the brain concentration (ng / mg protein) relative to the plasma concentration (μg / mL) of human IgG in each individual was calculated, and the brain transfer rate of exogenous antibodies was compared.

[0051] [Results] Figure 7 shows the ratio of brain human IgG (ng / mg protein) concentration to plasma human IgG concentration (μg / mL) in control and iECKO mice. It was revealed that genetically inducible deletion of SMS1 increased the leakage of exogenous IgG into the brain parenchyma.

Claims

1. A composition that promotes the central nervous system transport of a target substance, comprising a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1).

2. The central nervous system penetration promoting composition according to claim 1, wherein the target substance is an effective component for treating central nervous system diseases.

3. The central nervous system penetration promoting composition according to claim 1 or 2, wherein the target substance is selected from the group consisting of compounds, peptides, nucleic acids, proteins, antibodies, and vectors containing nucleic acids.

4. The central nervous system penetration-promoting composition according to claim 3, wherein the target substance is an antibody.

5. The central nervous system penetration promoting composition according to any one of claims 1 to 4, wherein a substance having inhibitory activity against SMS1 is provided as a composition separate from the target substance.

6. The central nervous system penetration promoting composition according to claim 5, which is administered before administering the target substance.

7. The central nervous system penetration promoting composition according to claim 5, which is administered simultaneously with the target substance.

8. The central nervous system penetration promoting composition according to claim 5, which is administered after the administration of the target substance.

9. A central nervous system penetration promoting composition according to any one of claims 1 to 4, comprising a substance having inhibitory activity against SMS1 together with the target substance.

10. A pharmaceutical composition containing a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1), and a combination of a pharmaceutical composition containing an ingredient effective for the treatment of central nervous system disorders for the treatment of central nervous system disorders.

11. A screening method for central nervous system translocation agents, comprising the steps of: examining the inhibitory activity of candidate substances against sphingomyelin synthase 1; and selecting candidate substances that have shown inhibitory activity against sphingomyelin synthase 1 as central nervous system translocation agents.

12. A method for treating a central nervous system disorder, comprising administering to a subject requiring treatment of a central nervous system disorder a combination of the active ingredient of a pharmaceutical product for the treatment of a central nervous system disorder and a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1).

13. Use of a substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) for the manufacture of a pharmaceutical product to promote the transfer of a target substance to the central nervous system.

14. A substance having inhibitory activity against sphingomyelin synthase 1 (SMS1) for use in promoting the transfer of target substances to the central nervous system.

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