Use of amitriptyline in acid sphingomyelinase deficiency, Gaucher disease, globoid cell leukodystrophy and Niemann-Pick type C

Abstract

The present application provides the application of amitriptyline in the treatment of acid sphingomyelinase deficiency, Gaucher disease, globoid cell leukodystrophy and Niemann Pick type C.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of amitriptyline in the treatment of acid sphingomyelinase deficiency, Gaucher disease, spherocyte leukodystrophy, and Niemann-Pick type C. Background Technology

[0002] Treatments for acid sphingomyelinase deficiency and Gaucher disease include enzyme replacement therapy and hematopoietic stem cell transplantation; hematopoietic stem cell transplantation is also a treatment for spherocytic leukodystrophy. 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 may cause various complications, carries significant risks, and does not improve neurological symptoms.

[0003] Niemann-Pick C is mainly for symptomatic treatment. Miglucopy is currently the only drug approved in China for treating progressive neurological symptoms in adult and pediatric NPC patients, but its therapeutic effect is limited and some patients experience serious adverse reactions to the drug. Summary of the Invention

[0004] This invention aims to overcome the above-mentioned deficiencies and provide new treatment methods for acid sphingomyelinase deficiency, Gaucher disease, spherocytic leukodystrophy, and Niemann-Pick type C, specifically:

[0005] This invention provides the use of amitriptyline in the preparation of a medicament for treating acid sphingomyelinase deficiency.

[0006] This invention provides the use of amitriptyline in the preparation of medicaments for treating Gaucher disease.

[0007] This invention provides the application of amitriptyline in the preparation of a medicament for treating spherocyte leukodystrophy.

[0008] This invention provides the use of amitriptyline in the preparation of a medicament for treating Niemann-Pick type C.

[0009] The present invention provides a drug characterized in that the drug is a powder, capsule, tablet, or solvent.

[0010] This invention provides the application of amitriptyline in the preparation of formulations that regulate the content of sphingomyelin deacetylated products (SPC).

[0011] This invention provides the application of amitriptyline in the preparation of formulations that regulate glucosamine content.

[0012] This invention provides the application of amitriptyline in the preparation of formulations that regulate the content of sphingosine galactoside.

[0013] This invention provides the application of amitriptyline in the preparation of formulations that regulate the expression level of NPC1 protein.

[0014] This invention provides the application of amitriptyline in the preparation of formulations that regulate the content of lysophospholipid-509.

[0015] The function and effects of this invention:

[0016] Amitriptyline is a tricyclic antidepressant commonly used to treat various types of depression and other disorders, and is considered safe from a clinical perspective. As a small molecule drug, amitriptyline can cross the blood-brain barrier and is effective for neurological symptoms. Its low price and high accessibility compared to current treatments lower the barrier to treatment. This invention, through extensive experimental research, has found that amitriptyline is effective in treating acid sphingomyelinase deficiency, Gaucher disease, spherocytic leukodystrophy, and Niemann-Pick type C, including reducing the accumulation of abnormal metabolic substrates in these conditions, increasing NPC1 protein expression, and decreasing the accumulation of abnormal metabolic substrates in Niemann-Pick type C. Attached Figure Description

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

[0018] Figure 2 SPC levels of B lymphocytes in ASMD patients. (A) SPC levels of B lymphocytes in normal individuals and ASMD patients. (B) SPC levels of B lymphocytes in ASMD patients after amitriptyline administration;

[0019] Figure 3 LyGb-1 levels in B lymphocytes of patients with Gram-D disease;

[0020] Figure 4 The PSY level of B lymphocytes in KD patients;

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

[0022] Figure 6 Content of abnormal metabolic substrates in NPC fibroblasts. (A) LySM-509; (B) SPC;

[0023] Figure 7 Toxicity testing. Detailed Implementation

[0024] This invention is capable of various modifications and embodiments, and therefore specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the invention to specific implementations, but should be understood to include all modifications, equivalents, and even substitutions that fall within the spirit and scope of this invention.

[0025] Amitriptyline powder, when formulated into capsules, tablets, or solutions, is used to treat acid sphingomyelinase deficiency, Gaucher disease, spherocytic leukodystrophy, and Niemann-Pick type C.

[0026] 1. Establishment of experimental cell models

[0027] 1.1. Cellular model of acid sphingomyelinase deficiency:

[0028] 1.1.1. KO cell line: Using HEK293T as a vector, an acid sphingomyelinase deficiency cell line with SMPD1 gene knockout was constructed. The acid sphingomyelinase activity of HEK293T and HEK293T-SMPD1-KO cells was detected using the artificial fluorescent substrate method. The activity was significantly reduced compared with normal 293T cells to verify the successful modeling.

[0029] 1.1.2. B Lymphocyte Lines: Peripheral blood from healthy individuals and children with acid sphingomyelinase deficiency was used as the primary material. Lymphocytes were separated using lymphocyte separation medium and cultured in a medium containing EB virus and cyclosporine to obtain B lymphocyte lines from healthy individuals and children with sphingomyelinase deficiency. After establishing the cell model, the acid sphingomyelinase activity in cells from healthy individuals and patients with sphingomyelinase deficiency was detected using an artificial fluorescent substrate method. A significant decrease in activity compared to healthy individuals confirmed successful model establishment.

[0030] 1.2. Gaucher disease cell model:

[0031] Peripheral blood from healthy individuals and children with Gaucher disease was used as the primary source. Lymphocytes were separated using lymphocyte separation medium and cultured in a medium containing Epstein-Barr virus and cyclosporine to obtain B lymphocyte lines from these individuals and children with Gaucher disease. After establishing the cell model, the activity of glucocerebrosidase in healthy individuals and patients with Gaucher disease was detected using an artificial fluorescent substrate method. The activity was significantly lower than that in healthy individuals, confirming the successful establishment of the model.

[0032] 1.3 Spherical cell model of leukodystrophy in the brain:

[0033] Peripheral blood from healthy individuals and children with spherocytic leukodystrophy was used as the primary source. Lymphocytes were separated using lymphocyte separation medium and cultured in a medium containing Epstein-Barr virus (EBV) and cyclosporine to obtain B lymphocyte lines from both individuals. After establishing the cell model, the activity of galactocerebrosidase in both individuals and those with spherocytic leukodystrophy was detected using an artificial fluorescent substrate method. The activity was significantly lower than that in healthy individuals, confirming successful model establishment.

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

[0035] Using 3mm skin from normal individuals and children with Niemann-Pick type C as raw materials, a primary skin fibroblast model was established. The expression level of NPC1 protein in cells from normal individuals and Niemann-Pick type C patients was detected by immunoblotting. The protein expression was significantly lower than that in normal individuals to verify the successful establishment of the model.

[0036] 2. Handling methods

[0037] 2.1 Acid sphingomyelinase deficiency (ASMD)

[0038] 2.1.1. KO cell model:

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

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

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

[0046] 2.2 Gaucher disease (GD)

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

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

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

[0050] 2.3 Kleptotrophic dystrophy (KD)

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

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

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

[0054] 2.4 Niemann-Pick Type C (NPC)

[0055] Control group (N=3): Normal primary skin fibroblasts from healthy individuals 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 modeling group + amitriptyline (amitriptyline dissolved in aqueous solution) treatment group (N=3): Primary skin fibroblasts of NPC patients were cultured normally and administered the drug for 3 days, once a day, 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 (SPC)

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

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

[0063] ASMD model group (N=3): SPC content in HEK293T-SMPD1-KO cells with acid sphingomyelinase deficiency 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 SPC content of cells was significantly lower than that of the ASMD model group and the difference was statistically significant (p<0.05).

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

[0066] Control group (N=3): Low SPC content in B lymphocytes in normal individuals;

[0067] ASMD model group (N=3): The content of SPC 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 SPC content of cells was significantly lower than that of the ASMD model group and the difference was statistically significant (p<0.05).

[0069] 3.2 Gaucher disease (GD)

[0070] 3.2.1 Glucosaminoglycine (LyGb-1) content: such as Figure 3 As shown,

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

[0072] GD model group (N=3): LyGb-1 content in cells of Gaucher disease patients 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 LyGb-1 content in cells was significantly lower than that in the GD model group and the difference was statistically significant (p<0.05).

[0074] 3.3 Kleptotrophic dystrophy (KD)

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

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

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

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

[0079] 3.4 Niemann-Pick Type C (NPC)

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

[0081] Control group (N=5): NPC1 expression was high in normal individuals;

[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 increased compared with that in the NPC model group, and the difference was statistically significant (p<0.05).

[0084] 3.4.2 Content of lysophospholipid-509 (LySM-509) and sphingomyelin deacetylation product SPC: (e.g.) Figure 6 As shown,

[0085] Control group (N=3): Normal individuals had low levels of lySM-509 and SPC;

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

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

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

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

[0090] NPC model group (N=6): 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, cell viability was not significantly changed.

[0092] While the foregoing has focused on embodiments, these are merely illustrative and do not limit the invention. Those skilled in the art will understand that various modifications and applications not illustrated above can be made without departing from the essential characteristics of these embodiments. For example, the constituent elements specifically shown in the embodiments can be implemented through modifications. Furthermore, various differences related to such modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims.

Claims

1. Application of amitriptyline in the preparation of drugs for the treatment of spherocytic leukodystrophy and Niemann-Pick type C.

2. Use according to claim 1, characterized in that: The drug is in the form of powder, capsules, tablets, or solvent.

3. Use according to claim 1, characterized in that: The drug regulates the content of sphingomyelin deacetylation product SPC.

4. The use according to claim 1, characterized in that: The drug regulates the glucosamine content.

5. The use according to claim 1, characterized in that: The drug regulates the content of sphingosine galactoside.

6. The use according to claim 1, characterized in that: The drug regulates the expression level of NPC1 protein.

7. The use according to claim 1, characterized in that: The drug regulates the content of lysophospholipid-509.

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