Application of amitriptyline in acidic sphingomyelinase deficiency, Gaucher disease, spherical cell white matter dystrophy and Niemann-Pick type C

Abstract

The invention provides application of amitriptyline in treatment of acid sphingomyelinase deficiency, Gaucher disease, spherical cell white matter dystrophy and Niemann-Pick type C.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of amitriptyline in the treatment of acid sphingomyelinase deficiency, Gaucher disease, globoid cell leukodystrophy and Niemann-Pick type C. Background Art

[0002] Treatments for acid sphingomyelinase deficiency and Gaucher disease include enzyme replacement therapy and hematopoietic stem cell transplantation; for globular cell leukodystrophy, hematopoietic stem cell transplantation is available. However, enzyme replacement therapy requires two weeks of intravenous infusion, is expensive, cannot cross the blood-brain barrier, and has no therapeutic effect on neurological symptoms. Hematopoietic stem cell transplantation can be associated with multiple complications, carries significant risks, and does not improve neurological symptoms.

[0003] Niemann-Pick type C is mainly treated symptomatically. Miglustat is currently the only drug approved in China for the treatment of progressive neurological symptoms in adults and children with NPC, but its therapeutic effect is limited, and some patients have serious adverse reactions to the drug. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned drawbacks and provide a new treatment method for acid sphingomyelinase deficiency, Gaucher disease, globular cell leukodystrophy and Niemann-Pick type C. Specifically:

[0005] The present invention provides application of amitriptyline in preparing medicine for treating acid sphingomyelinase deficiency.

[0006] The present invention provides application of amitriptyline in preparing medicine for treating Gaucher disease.

[0007] The present invention provides application of amitriptyline in preparing medicine for treating globoid cell leukodystrophy.

[0008] The present invention provides application of amitriptyline in preparing medicine for treating Niemann-Pick type C.

[0009] The medicine provided by the present invention is characterized in that: the medicine is in the form of powder, capsule, tablet, or solvent.

[0010] The present invention provides the use of amitriptyline in preparing a preparation for regulating the content of sphingomyelin deacetylation product SPC.

[0011] The present invention provides application of amitriptyline in preparing a preparation for regulating the content of glucose sphingosine.

[0012] The present invention provides application of amitriptyline in preparing a preparation for regulating psychosine content.

[0013] The present invention provides the use of amitriptyline in preparing a preparation for regulating the expression amount of NPC1 protein.

[0014] The present invention provides the use of amitriptyline in preparing a preparation for regulating the content of lysosphingomyelin-509.

[0015] The effects and effects of the present invention:

[0016] Amitriptyline is a tricyclic antidepressant commonly used to treat various depression and other diseases. From the perspective of clinical application, it is safe. Amitriptyline is a small molecule drug that can cross the blood-brain barrier and is effective for neurological symptoms. Amitriptyline is low in price and has high accessibility compared to current treatment drugs for diseases, which lowers the threshold for treatment. The present invention has found through a large number of experiments that amitriptyline can effectively treat acid sphingomyelinase deficiency, Gaucher disease, globular cell leukodystrophy and Niemann-Pick type C, including reducing the storage of abnormal metabolic substrates in acid sphingomyelinase deficiency, Gaucher disease and globular cell leukodystrophy, increasing NPC1 protein expression, and reducing the storage of abnormal metabolic substrates in Niemann-Pick type C. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 .SPC content in HEK293T-SMPD1-KO cells;

[0018] Figure 2 SPC content of B lymphocytes in ASMD patients. (A) SPC content of B lymphocytes in healthy subjects and ASMD patients. (B) SPC content of B lymphocytes in ASMD patients after amitriptyline administration.

[0019] Figure 3 .LyGb-1 content of B lymphocytes in GD patients;

[0020] Figure 4 .PSY content of B lymphocytes in KD patients;

[0021] Figure 5 NPC1 protein expression in fibroblasts from NPC patients;

[0022] Figure 6 Abnormal metabolic substrate content in NPC fibroblasts. (A) LySM-509; (B) SPC;

[0023] Figure 7 .Toxicity testing. DETAILED DESCRIPTION

[0024] The present invention is susceptible to various modifications and embodiments, and thus specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the present invention to specific embodiments, but rather should be understood to encompass all modifications, equivalents, and even substitutes that fall within the spirit and technical scope of the present invention.

[0025] Amitriptyline powder is made into capsules, tablets, or solutions to treat acid sphingomyelinase deficiency, Gaucher disease, globoid cell leukodystrophy, and Niemann-Pick type C.

[0026] 1. Establishment of experimental cell model

[0027] 1.1. Acid sphingomyelinase deficiency cell model:

[0028] 1.1.1.KO cell line: HEK293T cells were used as vectors to construct an acid sphingomyelinase deficiency cell line with SMPD1 gene knockout. The acid sphingomyelinase activity in HEK293T and HEK293T-SMPD1-KO cells was detected using an artificial fluorescent substrate method. The activity was significantly lower than that in normal 293T cells, confirming the success of the model.

[0029] 1.1.2. B Lymphocyte Cell Lines: Lymphocytes were isolated from peripheral blood of healthy individuals and children with acid sphingomyelinase deficiency using lymphocyte separation medium. These cells were then cultured in medium containing Epstein-Barr virus and cyclosporine to generate B lymphocyte cell lines from healthy individuals and children with acid sphingomyelinase deficiency. After establishing the cell model, acid sphingomyelinase activity in cells from healthy individuals and patients with acid sphingomyelinase deficiency was measured using an artificial fluorescent substrate assay. Significantly lower activity compared to healthy individuals confirmed successful modeling.

[0030] 1.2. Gaucher disease cell model:

[0031] Using peripheral blood from healthy individuals and children with Gaucher disease as raw material, lymphocytes were isolated using lymphocyte separation medium and cultured in medium containing Epstein-Barr virus and cyclosporine to obtain B lymphocyte cell lines from healthy individuals and children with Gaucher disease. After establishing the cell model, glucocerebrosidase activity in healthy individuals and patients with Gaucher disease was measured using an artificial fluorescent substrate method. Significantly lower activity compared to healthy individuals confirmed the successful modeling.

[0032] 1.3 Globular cell leukodystrophy cell model:

[0033] Peripheral blood from healthy individuals and children with globular cell leukodystrophy was used as the starting material. Lymphocytes were isolated using lymphocyte separation medium and cultured in a medium containing Epstein-Barr virus and cyclosporine to generate B lymphocyte cell lines from healthy individuals and children with globular cell leukodystrophy. After establishing the cell model, galactocerebrosidase activity in healthy individuals and patients with globular cell leukodystrophy was measured using an artificial fluorescent substrate method. Significantly lower activity compared to healthy individuals confirmed the successful modeling.

[0034] 1.4 Niemann-Pick C-cell model:

[0035] A primary skin fibroblast model was established using 3mm skin of normal subjects and children with Niemann-Pick type C as raw materials. The expression level of NPC1 protein in cells of normal subjects and patients with Niemann-Pick type C was detected by immunoblotting. The protein expression was significantly lower than that of normal subjects, verifying the success of the model.

[0036] 2. Treatment methods

[0037] 2.1 Acid sphingomyelinase deficiency (ASMD)

[0038] 2.1.1.KO cell model:

[0039] Control group (N=3): normal human HEK293T cells were cultured normally without any treatment;

[0040] ASMD model group (N=3): acid sphingomyelinase deficiency HEK293T-SMPD1-KO cells were cultured normally without any treatment;

[0041] ASMD modeling group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=3): Acid sphingomyelinase deficiency HEK293T-SMPD1-KO cells were cultured normally and administered once a day for 3 days, with each dose ranging from 0.1 to 10 μmol / L.

[0042] 2.1.2. B lymphocyte model

[0043] Control group (N=3): normal human B lymphocytes were cultured normally without any treatment;

[0044] ASMD modeling group (N=3): B lymphocytes from patients with acid sphingomyelinase deficiency were cultured normally without any treatment;

[0045] ASMD modeling group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=3): B lymphocytes from patients with acid sphingomyelinase deficiency were cultured normally and administered once a day for 3 days, with each dose being 10 μmol / L.

[0046] 2.2 Gaucher disease (GD)

[0047] Control group (N=3): normal human B lymphocytes were cultured normally without any treatment;

[0048] GD modeling group (N=4): B lymphocytes from patients with Gaucher disease were cultured normally without any treatment;

[0049] GD modeling group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=4): B lymphocytes from patients with Gaucher disease were cultured normally and administered once a day for 3 days, with each dose being 10 μmol / L.

[0050] 2.3 Globular cell leukodystrophy (KD)

[0051] Control group (N=3): normal human B lymphocytes were cultured normally without any treatment;

[0052] KD modeling group (N=3): B lymphocytes from patients with Gaucher disease were cultured normally without any treatment;

[0053] KD modeling group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=3): B lymphocytes from patients with Gaucher disease were cultured normally and administered once a day for 3 days, with each dose being 10 μmol / L.

[0054] 2.4 Niemann-Pick C (NPC)

[0055] Control group (N=3): normal human primary skin fibroblasts were cultured normally without any treatment;

[0056] NPC model group (N=3): Primary skin fibroblasts from NPC patients were cultured normally without any treatment;

[0057] NPC model group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=3): Primary skin fibroblasts from NPC patients were cultured normally and administered once a day for 3 days, with each dose being 10 μmol / L.

[0058] 3. Results

[0059] 3.1. Acid sphingomyelinase deficiency (ASMD)

[0060] 3.1.1. Content of sphingosylphosphorylcholine (orlysosphingomyelin), corresponding to the deacylated form of sphingomyelin

[0061] 3.1.1.1.KO cell model: Figure 1As shown,

[0062] Control group (N=3): normal HEK293T cells with low SPC content;

[0063] ASMD model group (N=3): The SPC content in acid sphingomyelinase deficiency HEK293T-SMPD1-KO cells was significantly increased compared with normal control cells;

[0064] ASMD model group + amitriptyline treatment group (N=3): After treatment with amitriptyline at a concentration of 0.1-10 μmol / L, the cell SPC content was significantly lower than that in the ASMD model group and there was a statistical difference (p<0.05).

[0065] 3.1.1.2 B lymphocyte model: Figure 2 As shown,

[0066] Control group (N=3): normal human B lymphocytes with low SPC content;

[0067] ASMD model group (N=3): The SPC content in B lymphocytes of patients with acid sphingomyelinase deficiency was significantly increased compared with that in normal control cells;

[0068] ASMD model group + amitriptyline treatment group (N=3): After treatment with amitriptyline at a concentration of 10 μmol / L, the cell SPC content was significantly lower than that in the ASMD model group and the difference was statistically significant (p<0.05).

[0069] 3.2 Gaucher disease (GD)

[0070] 3.2.1 LyGb-1 content: Figure 3 As shown,

[0071] Control group (N=3): normal human cells with low LyGb-1 content;

[0072] GD model group (N=3): LyGb-1 content in Gaucher disease patient cells was significantly increased compared with normal control cells;

[0073] GD model group + amitriptyline treatment group (N=3): After treatment with amitriptyline at a concentration of 10 μmol / L, the cell LyGb-1 content was significantly lower than that in the GD model group and the difference was statistically significant (p<0.05).

[0074] 3.3 Globular cell leukodystrophy (KD)

[0075] 3.3.1 Sphingosine galactosidase (PSY), such as Figure 4 As shown,

[0076] Control group (N=3): normal human cells with low PSY content;

[0077] KD model group (N=3): PSY content in Gaucher disease patient cells was significantly increased compared with normal control cells;

[0078] KD modeling group + amitriptyline treatment group (N=3): After treatment with amitriptyline at a concentration of 10 μmol / L, the cell PSY content was significantly lower than that in the KD modeling group and the difference was statistically significant (p<0.05).

[0079] 3.4 Niemann-Pick C (NPC)

[0080] 3.4.1NPC1 protein expression: Figure 5 As shown,

[0081] Control group (N=5): normal subjects with high NPC1 expression;

[0082] NPC model group (N=5): NPC1 protein expression in NPC1 patient cells was significantly lower than that in normal control cells;

[0083] NPC model group + amitriptyline treatment group (N=5): After treatment with amitriptyline at a concentration of 10 μmol / L, the expression of NPC1 protein in cells was significantly higher than that in the NPC model group and the difference was statistically significant (p<0.05).

[0084] 3.4.2 Lyso-sphingomyelin-509 (LySM-509) and sphingomyelin deacetylation product SPC content: Figure 6 As shown,

[0085] Control group (N=3): normal people with low lySM-509 and SPC levels;

[0086] NPC model group (N=3): The levels of lySM-509 and SPC in NPC patient cells were significantly increased compared with normal control cells;

[0087] NPC model group + amitriptyline treatment group (N=3): After treatment with amitriptyline at a concentration of 10 μmol / L, the cell lySM-509 and SPC contents were significantly lower than those in the NPC model group and the difference was statistically significant (p<0.05).

[0088] 3.5. Toxicity test, such as Figure 7 As shown,

[0089] Control group (N=6): Normal human cells have high viability;

[0090] NPC model group (N=6): The cell viability of NPC patients was significantly lower than that of normal control cells;

[0091] NPC model group + amitriptyline treatment group (N=6): After treatment with amitriptyline at a concentration of 10 μmol / L, there was no significant change in cell viability.

[0092] While the above description focuses on the embodiments, this is merely illustrative and does not limit the present invention. Persons skilled in the art will readily appreciate that various modifications and applications not illustrated above are possible without departing from the essential characteristics of the embodiments. For example, the various components specifically illustrated in the embodiments may be implemented with modifications. Furthermore, any differences associated with such modifications and applications should be construed as being within the scope of the present invention as defined in the appended claims.

Claims

1. Use of amitriptyline in the preparation of a drug for treating acid sphingomyelinase deficiency.

2. Use of amitriptyline in the preparation of drugs for treating Gaucher disease.

3. Use of amitriptyline in the preparation of drugs for treating globoid cell leukodystrophy.

4. Use of amitriptyline in the preparation of drugs for treating Niemann-Pick type C.

5. The drug according to any one of claims 1 to 4, characterized in that: The medicine is in the form of powder, capsule, tablet, or solvent.

6. Application of amitriptyline in the preparation of preparations for regulating the content of sphingomyelin deacetylation product SPC.

7. Use of amitriptyline in the preparation of preparations for regulating the content of glucose sphingosine.

8. Use of amitriptyline in the preparation of a preparation for regulating psychosine content.

9. Application of amitriptyline in the preparation of preparations for regulating NPC1 protein expression.

10. Use of amitriptyline in the preparation of a preparation for regulating the content of lysosphingomyelin-509.

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