Use of nikkomycin for the preparation of a medicament for the treatment of a lipid storage myopathy

Abstract

This invention belongs to the pharmaceutical field and relates to the application of nikkomycin in the preparation of drugs for treating lipid storage myopathy. This invention discloses the use of nikkomycin in the treatment of lipid storage myopathy, particularly in the treatment of simple myopathic neutral lipopathy caused by PNPLA2 gene mutations. Simple myopathic neutral lipopathy is an autosomal recessive, progressive lipid storage myopathy affecting multiple systems throughout the body. Currently, there is no treatment available, and patients develop fatal symptoms such as generalized muscle weakness and cardiomyopathy in the late stages. This invention discovers that nikkomycin can significantly reverse lipid droplet deposition in fibroblasts of patients with simple myopathic neutral lipopathy, reduce inflammatory responses, improve autophagy inhibition, and increase energy production. It can serve as an effective ingredient for treating this disease and has clear application prospects in patients with untreatable simple myopathic neutral lipopathy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals and relates to the use of nicotinic acid in the preparation of drugs for treating lipid deposition myopathy. Background Technology

[0002] Lipid storage myopathy (LSM) is a group of inherited metabolic myopathies caused by abnormalities in lipid metabolism-related enzymes, leading to lipid accumulation in muscle cells. It is an autosomal recessive genetic disorder and includes simple myopathy-type neutral fat storage disease, primary carnitine deficiency, carnitine palmitoyltransferase deficiency, and various acyl-CoA dehydrogenase deficiencies.

[0003] Among them, neutral lipid storage disease with myopathy (NLSDM) is caused by autosomal dominant neutrophils. PNPLA2 Mutations in the PNPLA2 gene (Patatin-like phospholipase domain-containing protein 2) lead to dysfunction of adipose triglyceride lipase (ATGL), resulting in adipose metabolic myopathy. ATGL is a key enzyme in the first step of triglyceride breakdown in the lipocatabolism pathway. Therefore, mutations in the PNPLA2 gene encoding ATGL in NLSDM inevitably impair the first step of ATGL breakdown, leading to a large accumulation of triglycerides in the cytoplasm, and pathologically, the observation of numerous lipid droplet deposits within the muscle fibers.

[0004] The clinical characteristics of NLSDM include late-onset, insidious onset (onset in young to middle-aged adults), with a prominent feature of asymmetrical muscle weakness throughout the body, especially in the distal limbs, and may include muscle atrophy and winged scapula [1-3]. About one-third of patients also have cardiomyopathy, mostly with congestive heart failure as the main symptom, which may require heart transplantation in severe cases. The liver may also be involved [4-6]. NLSDM is the second leading cause of lipid deposition diseases in Chinese people.

[0005] Currently, there are no effective drugs for treating NLSDM. Hormones, L-carnitine, coenzyme Q10, and riboflavin treatments have all shown no significant effect. NLSDM can manifest as multi-system involvement, causing muscle weakness in the skeletal muscle system, and can also present as fatal cardiomyopathy, requiring heart transplantation in severe cases, which is extremely costly. Therefore, effective treatment is of paramount importance in relieving patients' suffering. As an enzyme deficiency disease, the ideal treatment for NLSDM is enzyme replacement therapy, which involves the exogenous introduction of functional enzymes. However, as a rare disease affecting a small population, the enormous time and financial costs, as well as the unknown risks associated with introducing enzymes, make this ideal approach difficult to implement. Therefore, there is an urgent need for economical, safe, and effective treatments to significantly improve patients' quality of life, reduce the burden on families and society, and promote progress in clinical medicine.

[0006] [1] W. Zhang, B. Wen, J. Lu, Y. Zhao, D. Hong, Z. Zhao, et al. Neutral lipid storage disease with myopathy in China: a large multicentriccohort study. Orphanet J Rare Dis 2019; 14: 234.

[0007] [2] J. Tan, H. Yang, J. Fan, Y. Fan, and F. Xiao. Patients with neutral lipid storage disease with myopathy (NLSDM) in Southwestern China. Clin Neurol Neurosurg 2018; 168: 102-7.

[0008] [3] J. Zhang, J. Han, Y. Wang, Y. Wu, X. Song, and G. Ji. Neutrallipid storage disease with myopathy presenting asymmetrical muscle weakness: a case report. Int J Clin Exp Pathol 2020; 13: 559-62.

[0009] [4] MB Pasanisi, S. Missaglia, D. Cassandrini, F. Salerno, S.Farina, D. Andreini, et al. Severe cardiomyopathy in a young patient with complete deficiency of adipose triglyceride lipase due to a novel mutation inPNPLA2 gene. Int J Cardiol 2016; 207: 165-7.

[0010] [5] M. Samukawa, N. Nakamura, M. Hirano, M. Morikawa, H. Sakata, I.Nishino, et al. Neutral Lipid Storage Disease Associated with the PNPLA2Gene: Case Report and Literature Review. Eur Neurol 2020; 83: 317-22.

[0011] [6] M. Rao, G. Guo, M. Li, S. Chen, K. Chen, X. Chen, et al. Thehomozygous variant c.245G > A / p.G82D in PNPLA2 is associated with arrhythmogenic cardiomyopathy phenotypic manifestations. Clin Genet 2019; 96:532-40. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention innovatively proposes and demonstrates that nikkomycin can significantly reverse lipid droplet deposition in fibroblasts of NLSDM patients, reduce inflammatory responses, improve autophagy inhibition, and increase energy production, thus serving as a treatment for this disease. Furthermore, this application develops a pharmacological effect of nikkomycin that differs from its previous antifungal uses, clarifying the therapeutic effect and mechanism of nikkomycin on NLSDM.

[0013] Specifically, the technical solution of the present invention is as follows:

[0014] Application of nicotinic acid in the preparation of drugs for treating lipid storage myopathy.

[0015] On the other hand, this application protects the use of nicotinic acid in the preparation of a medicament for the prevention of lipid deposition myopathy.

[0016] Furthermore, the nicotinic acid described in this application is a nucleoside peptide antibiotic produced by microorganisms such as *Streptomyces chromogenicus*. Its chemical structure is highly similar to that of UDP-N-acetylglucosamine, the natural substrate of chitin synthase, and it contains more than 20 active single components, including but not limited to Nikomycin Z, Nikomycin X, Nikomycin I, and Nikomycin J, as shown in Formula 1.

[0017]

[0018] Formula 1

[0019]

[0020] Preferably, the nicotinic acid is Nikomycin Z, CAS number 59456-70-1, molecular formula C 20 H 25 N5O 10 The structure is shown in Equation 2:

[0021]

[0022] Equation 2.

[0023] The lipid deposition myopathy includes simple myopathy-type neutral fat deposition disease, primary carnitine deficiency, carnitine palmitoyltransferase deficiency, and multiple acyl-CoA dehydrogenase deficiencies.

[0024] Preferably, the lipid deposition myopathy is simple myopathy-type neutral lipid deposition disease, that is, the use of nicotinic acid in the preparation of a drug for treating simple myopathy-type neutral lipid deposition disease is protected in this application.

[0025] Furthermore, the nicotinic acid or its active single component is the only active ingredient in the drug.

[0026] Furthermore, the nicotinic acid also includes Nikomycin Z, Nikomycin X, Nikomycin I, Nikomycin J, and a pharmaceutically acceptable salt of any of the active single components.

[0027] The medicament for treating simple myopathy-type neutral fat storage disease includes pharmaceutically acceptable excipients. Further, the acceptable excipients are selected from one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchangers, flavoring agents, or antioxidants.

[0028] The beneficial effects of this invention are as follows:

[0029] Nicotinic acid is a highly safe antifungal drug with well-defined pharmacological effects and pharmacokinetics. Extensive clinical data and clinical trial results exist. Therefore, using nicotinic acid as the active ingredient avoids the lengthy time and enormous economic costs of developing new drugs, while also offering good safety and a relatively low economic burden with long-term use. This invention innovatively demonstrates that nicotinic acid can significantly reverse lipid droplet deposition in fibroblasts of NLSDM patients, reduce inflammatory responses, improve autophagy inhibition, and increase energy production, making it a potential treatment for this disease. This invention discovers a new use for nicotinic acid, which is of great significance for advancing the treatment of NLSDM and reducing the social burden of the disease. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 These are pathological images of muscle tissue from patients with NLSDM. In image A, ORO staining indicates significant lipid droplet deposition within muscle fibers; in image B, MGT staining shows numerous bordered vacuoles and active autophagy within muscle fibers; and in images C and D, HE staining indicates muscle fiber necrosis and inflammatory cell infiltration. These results suggest that NLSDM patients exhibit abnormal lipid metabolism, autophagy, and inflammatory responses in their muscles.

[0032] Figure 2 These are comparative images of muscle pathology from two patients with NLSDM. A shows HE staining in patient 1, and B shows HE staining in patient 2. C shows MGT staining in patient 1, and D shows MGT staining in patient 2. Patient 2 shows more rimmed vacuoles in their muscle fibers. These results suggest that the severity of autophagy and inflammation is correlated with the degree of skeletal muscle damage, with patient 2 showing significantly more severe damage than patient 1.

[0033] Figure 3Immunohistochemical pathological images of muscle in NLSDM patients (inflammatory response and inflammatory cell subclass staining); Among them, A, C and E are HE staining, B is CD68 immunohistochemical staining, indicating that macrophages are involved in muscle necrosis, D is CD3 immunohistochemical staining, indicating that T lymphocytes are involved in the inflammatory response of muscle necrosis, and F is CD4-labeled T lymphocytes, indicating that CD4+ T lymphocytes are involved in the inflammatory response of muscle necrosis.

[0034] Figure 4 The images show the immunofluorescence results (autophagy markers) of muscle pathology in NLSDM. A, D, and G are HE staining; B, E, and H are LC3 immunofluorescence staining; C, F, and I are P62 immunofluorescence staining; A, B, and C are normal controls; D, E, and F are patient 1; and G, H, and I are patient 2. It can be seen that the LC3 green immunofluorescence signal in the muscle specimens of patients 1 and 2 (E and H) is significantly higher than that of the normal control (B), and the P62 green immunofluorescence signal in the muscle specimens of patients 1 and 2 (F and I) is significantly higher than that of the normal control (C). These results suggest the presence of a significantly abnormally upregulated autophagy response in the patients' muscles.

[0035] Figure 5 The results of immunoblotting of muscle specimens from NLSDM patients (autophagy markers) are shown in the figure. As can be seen from the figure, the protein expression level of LAMP1 in the muscles of the two patients on the right (P1, P2) is significantly higher than that in the two controls on the left (NC1, NC2). GAPDH is used as an internal control.

[0036] Figure 6 This image shows the comparison of lipid droplets in fibroblasts after nikochondrial treatment. A, B, and C represent fibroblasts from patients without intervention; D, E, and F represent fibroblasts from patients treated with nikochondrial at 1 μg / ml; and G, H, and I represent fibroblasts from patients treated with nikochondrial at 6 μg / ml. A, D, and G show DAPI staining to label cell nuclei (blue fluorescence); B, E, and H show BODIPY staining to label lipid droplets (green fluorescence); and C, F, and I show the fusion of blue cell nuclei and green lipid droplets. The results indicate that after nikochondrial intervention, lipid droplets in the patient's fibroblasts (…) Figure 6 China E and Figure 6 The concentration of H in the middle liposome was significantly reduced, and 6 μg / ml ( Figure 6 (H) is better than 1ug / ml ( Figure 6 Group E)

[0037] Figure 7 The results show the ATP production of skin fibroblasts in patients after nicotinic acid treatment. As can be seen from the figure, after intervention with 6 ug / ml nicotinic acid, the oxygen consumption rate (OCR) of fibroblasts in patients was significantly better than before the intervention, with a statistically significant difference (p < 0.01).

[0038] Figure 8 Before and after nicotinic acid intervention PNPLA2 Real-time quantitative PCR results of gene mRNA transcription; as can be seen from the figure, the patient... PNPLA2 The mRNA transcription level of the gene was significantly lower than that of the normal control, but after intervention with nicotinic acid, the patient's mRNA transcription level was significantly lower than that of the normal control. PNPLA2 Gene transcription levels were significantly upregulated, with a statistically significant difference (p < 0.01).

[0039] Figure 9 The results of the immunoblotting experiment confirmed that nikoxam upregulated ATGL protein expression. As shown in the figure, the ATGL protein expression level in patients was significantly lower than that in normal controls, while after nikoxam intervention, the ATGL protein expression level in patients was significantly upregulated.

[0040] Figure 10 This study demonstrates that nicotinic acid reduces lipid droplet deposition in fibroblasts via ATGL, and that inhibiting ATGL function can reverse the lipid droplet reduction effect of nicotinic acid. A, B, and C represent fibroblasts from patients without intervention; D, E, and F represent fibroblasts from patients treated with nicotinic acid 6 μg / ml; G, H, and I represent fibroblasts from patients treated with nicotinic acid 6 μg / ml + ATGL inhibitor 40 μM; J, K, and L represent fibroblasts from patients treated with ATGL inhibitor 40 μM. A, D, G, and J show DAPI staining to label cell nuclei (blue fluorescence); B, E, H, and K show BODIPY staining to label lipid droplets (green fluorescence); C, F, I, and L show the fusion of blue cell nuclei and green lipid droplets. The results show that after nicotinic acid 6 μg / ml intervention, lipid droplets in patient fibroblasts (… Figure 10 (E) compared to before intervention ( Figure 10 The number of lipid droplets in fibroblasts was significantly reduced, but in the presence of ATGL inhibitors, even after intervention with nicotinic acid 6ug / ml, the number of lipid droplets in fibroblasts was significantly reduced. Figure 10 H) compared to no ATGL inhibitor ( Figure 10 The level of E in the middle jiao (e.g.) increased significantly, even exceeding the level before nicotinic acid intervention (e.g., E.g.). Figure 10 In Group B, there were statistically significant differences among the groups (p < 0.05 or p < 0.001).

[0041] Figure 11This experiment aimed to confirm the activation of autophagy in fibroblasts from NLSDM patients using immunofluorescence. In the diagram, A, B, G, and H are DAPI staining (labeling cell nuclei, blue fluorescence), C and D are LAMP2 staining (green fluorescence), I and J are BODIPY staining (labeling lipid droplets, green fluorescence), E and F are P62 staining (red fluorescence), and K and L are LC3 staining (red fluorescence). A, C, E, G, I, and K represent patients, while B, D, F, H, J, and L are normal controls. The figure shows that lipid droplets in patient cells (…) Figure 11 The content of LAMP2 (I) increased significantly, and LAMP2 (I) also increased significantly. Figure 11 (C), P62 ( Figure 11 (E) and LC3 ( Figure 11 Immunofluorescence signals of markers for autophagy (K) were significantly upregulated.

[0042] Figure 12 This study aimed to confirm the activation of autophagy in skin fibroblasts of NLSDM patients using Western blotting. The figure shows that the expression of autophagy markers LAMP1, P62, and LC3B proteins in fibroblasts from the three patients was significantly upregulated, with ACTIN used as an internal control.

[0043] Figure 13 This study aimed to confirm the results of immunoblotting in downregulating abnormally elevated lipopyphary markers in skin fibroblasts of NLSDM patients with nikkomycin. As shown in the figure, in the absence of autophagy induction or activation, intervention with nikkomycin at 6 μg / ml significantly downregulated the expression of autophagy markers LAMP1, P62, and LC3B proteins in fibroblasts (lane 5), a result superior to that of intervention with the autophagy inducer EBSS (lane 3). ACTIN was used as an internal control. Detailed Implementation

[0044] To enable those skilled in the art to better understand this application, this application will now be further described in conjunction with specific embodiments.

[0045] In some embodiments of the present invention, the therapeutic effect of nicotinic acid in simple myopathy-type neutral fat storage disease was demonstrated, and the excellent effect of nicotinic acid in cell models was also verified.

[0046] Unless otherwise specified, all raw materials or reagents used in the embodiments of the present invention are commercially available products.

[0047] Unless otherwise specified, all percentages used in the embodiments of the present invention are mass percentages.

[0048] In some embodiments, Nikomycin Z of the present invention is a pharmaceutical preparation that is provided in a single dose, or in multiple doses over a period of time, with the dose being implemented as a total volume concentration, such that the dose is 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml or higher, depending on the therapeutic effect. In other embodiments, administration is carried out in a ratio of the mass of Nikomycin Z to the mass of the individual being treated, such that the dose is 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg or higher, depending on the therapeutic effect. In embodiments with multiple dosing, the dosing regimen may be 1 dose / day, 2 doses / day, 3 doses / day or more, and may continue for the necessary duration, such that administration may continue for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, or perpetually throughout the lifespan of the organism.

[0049] In the embodiments of this application, the relevant data were statistically analyzed using SPSS 26.0 software. Normally distributed data are expressed as mean ± standard error (SEM). Two-tailed t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons of three or more groups; and Tukey's method was used for pairwise comparisons among multiple groups. P < 0.05 was considered statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05).

[0050] Example 1

[0051] Experiment on muscle pathological characteristics of NLSDM patients.

[0052] 1. Obtaining skeletal muscle biopsy specimens

[0053] The biopsy site is selected based on the location and severity of the patient's muscle weakness, usually the biceps brachii or quadriceps femoris. Fresh skeletal muscle specimens are fixed to a wooden frame with xanthophyll glue, then rapidly frozen in isopentane cooled with liquid nitrogen. The frozen skeletal muscle specimens are then stored at -80°C.

[0054] 2. Pathological staining and immunofluorescence staining of muscle tissue

[0055] 2.1 Materials needed: tweezers, pipettes, beakers, glass slides, coverslips, hematoxylin and eosin (H&E) staining solution, Oil Red O (ORO) staining solution, succinate dehydrogenase (SDH) staining solution, cytochrome c oxidase (COX) staining solution, waste container, ethanol, neutral resin, glycerol gelatin, cryostat, optical microscope, hair dryer, fume hood;

[0056] 2.2 Experimental Methods:

[0057] 1) After removing the skeletal muscle specimen from the -80℃ freezer, immediately place it into a -20℃ cryostat.

[0058] 2) Fix the skeletal muscle onto the microtome tray using a wooden support, adjust the slice thickness to 8μm / 5μm, and then slice continuously.

[0059] 3) After attaching the tissue to a glass slide, perform HE, ORO, MGT, and various immunofluorescence and histochemical staining.

[0060] 4) Seal the slide with neutral resin / glycerin and cover it with a coverslip.

[0061] 5) Observe the prepared pathological specimens under an optical microscope and take photos for preservation.

[0062] 3. Conclusion: Pathological staining and immunofluorescence staining of muscle tissue from NLSDM patients.

[0063] 3.1 After enzyme-linked chemical staining of skeletal muscle biopsy specimens from NLSDM patients, such as... Figure 1 Numerous lipid droplet deposits can be observed in the muscle pathology of NLSDM patients. Figure 1 (A), rimmed bubble ( Figure 1 (B) and inflammatory cell infiltration ( Figure 1 C in the middle Figure 1 (Middle D), which suggests that NLSDM patients have abnormal lipid metabolism, autophagy, and inflammatory responses in their muscles.

[0064] 3.2 The skeletal muscle biopsy staining images of two NLSDM patients were analyzed. Figure 2 A represents HE staining of patient 1. Figure 2 In the middle section, HE staining of patient 2 shows that patient 2 has more severe myofiber necrosis and regeneration, more atrophied small fibers, and more severe myofiber interstitial hyperplasia. Figure 2 C represents the MGT staining of patient 1. Figure 2MGT staining in patient 2 (D) shows that patient 2 has more rimmed vacuoles in its muscle fibers. These results suggest that the severity of autophagy and inflammation in NLSDM patients is correlated with the degree of skeletal muscle damage, and patient 2 ( Figure 2 China B and Figure 2 The severity of the disease in patient D was greater than that in patient 1 ( Figure 2 China A and Figure 2 (C)

[0065] 3.3 Immunohistochemical staining was performed on NLSDM skeletal muscle biopsy specimens to observe inflammatory responses and inflammatory cell subclasses. Figure 3 China A, Figure 3 C and Figure 3 E in the middle is stained with HE. Figure 3 In the middle section, CD68 immunohistochemical staining suggests that macrophages are involved in muscle necrosis. Figure 3 The D-staining pattern represents CD3 immunohistochemical staining, suggesting that T lymphocytes are involved in the inflammatory response of muscle necrosis. Figure 3 F in the middle represents CD4-labeled T lymphocytes, suggesting that CD4+ T lymphocytes are involved in the inflammatory response of muscle necrosis.

[0066] 3.4 Immunohistofluorescence staining was performed on NLSDM skeletal muscle biopsy specimens to observe autophagy markers. Figure 4 China A, Figure 4 China D and Figure 4 G in the middle is stained with HE. Figure 4 B, Figure 4 China E and Figure 4 H in the middle represents LC3 immunofluorescence staining. Figure 4 C in the middle Figure 4 China F and Figure 4 I represents P62 immunofluorescence staining. Figure 4 China A, Figure 4 China B and Figure 4 C represents the normal control group. Figure 4 D, Figure 4 China E and Figure 4 In the middle, F represents patient 1. Figure 4 China G, Figure 4 H and Figure 4 In the middle, I represents patient 2. It can be seen that the muscle specimens from patients 1 and 2 ( Figure 4 China E and Figure 4 The green immunofluorescence signal of LC3 in H1N1 was significantly higher than that in the normal control. Figure 4 (Middle B), muscle specimens from patients 1 and 2 ( Figure 4 China F and Figure 4 The green immunofluorescence signal of P62 in the middle I) was significantly higher than that in the normal control ( Figure 4 (C) The results indicated that there was a significantly abnormally upregulated autophagy response in the patient's muscles.

[0067] Example 2

[0068] Western blot protein immunoblotting

[0069] 1. Sample preparation

[0070] Materials needed: RIPA lysis buffer, protease, phosphatase inhibitor, Thermo Fisher BCA protein quantification kit, centrifuge, and loading buffer.

[0071] 2. Experimental methods:

[0072] 1) Take out the skeletal muscle that was placed in a -80℃ freezer, cut about 50 specimens with a thickness of about 8μm in a cryostat, and place them in pre-cooled EP tubes.

[0073] 2) Add 150 μl of RIPA lysis buffer containing protease and phosphatase inhibitors, pipette thoroughly, and lyse on ice for 30 min.

[0074] 3) Centrifuge at 4℃ and 12000rpm for 10min, then transfer the supernatant to a new EP tube, which is the sample to be tested.

[0075] 4) Protein concentration determination. Perform protein concentration determination according to the instructions:

[0076] A. Add protein standards to a 96-well plate: perform tests in 3 replicates for each well.

[0077]

[0078] B. Add the protein to be tested. Add 10 μl of sample protein (1 μl of the protein to be tested + 9 μl of ddH2O) to each well and perform 3 replicates for each well.

[0079] C. Add 200 μl of a mixture of solution A and solution B (1:50) to each well.

[0080] D. After mixing, incubate at room temperature for 30 minutes.

[0081] E. The optical density (OD) of the protein at 562 nm was detected using a multi-functional microplate reader.

[0082] F. Construct a protein concentration standard curve based on the OD values ​​of the standards, and calculate the concentration of the sample to be tested based on the regression equation of the standard curve.

[0083] 5) Add 1 / 3 volume of 4× loading buffer to the protein sample. Do not heat. Store in a -80℃ freezer for a long time, avoiding repeated freeze-thaw cycles.

[0084] 3. Preparation of experimental reagents

[0085] 1) 10× transfer buffer: 30.3g Tris, 144g Glycine, add water to a final volume of 1000mL.

[0086] 2) 1× Transfer Buffer: Add 200 mL of methanol to 100 mL of 10× transfer buffer, then add water to bring the volume to 1000 mL.

[0087] 3) 10× Electrophoresis Buffer: 30.3g Tris, 144g Glycine, 10g SDS, add water to a final volume of 1000mL. Dilute to 1× before use for electrophoresis.

[0088] 4) 10×TBS: 24.2 gTris, 87.66 gNaCl, add water to make up to 1000 mL.

[0089] 5) 1×TBST: 10×TBS 100mL, Tween-20 1mL, add water to make up to 1000mL.

[0090] 4 Electrophoresis

[0091] 4.1 Materials to be prepared: Vertical gel electrophoresis system: including electrophoresis apparatus, electrophoresis tank, glass plates, combs, etc., protein marker, isopropanol, 1× electrophoresis buffer, Beyotime SDS-PAGE gel preparation kit.

[0092] 4.2 Experimental Methods:

[0093] 1) Clean the glass plate and comb in advance, and assemble them after they are dry.

[0094] 2) Glue pouring: Add the separating glue to the glass plate until the top edge is about 2-3 cm from the short plate. Add isopropyl alcohol to press the line and let it stand at room temperature until the separating glue solidifies. Pour off the isopropyl alcohol, rinse the glue surface with double-distilled water, and then absorb the water with absorbent paper. Add the stacking glue, insert the comb, and let it stand at room temperature until the stacking glue solidifies.

[0095] 3) Sample loading: Place the glass plate in the electrophoresis tank and pour in the electrophoresis buffer. Add the protein sample and protein marker to the gel wells in sequence.

[0096] 4) Electrophoresis: Add electrophoresis buffer, connect the power supply, and adjust the voltage to 80V. After the proteins are separated according to their molecular weight in the separating gel, adjust the voltage to 120V for constant voltage electrophoresis. Stop electrophoresis when the bromophenol blue reaches the bottom edge of the gel.

[0097] 4.3 Transfer, antibody incubation and scanning: Materials needed: 1× transfer buffer, 1×TBST, PVDF membrane, sponge pad, transfer filter paper, transfer apparatus, skim milk powder, primary antibody, horseradish peroxidase (HRP) labeled secondary antibody, scanning imaging system, shaker.

[0098] 4.4 Experimental Methods:

[0099] 1) After electrophoresis stops, remove the gel and cut off the stacking gel portion.

[0100] 2) Place the sponge pad, transfer filter paper, PVDF membrane, gel, transfer filter paper, and sponge pad in sequence from the white side to the black side of the transfer clamp. Remove all air bubbles and clamp tightly.

[0101] 3) Fix the transfer clamp in the transfer tank, add the transfer solution, and transfer at 110V for 60 minutes.

[0102] 4) After the transfer is complete, remove the PVDF membrane, place it in a plastic box, and seal it with 0.5% skim milk dissolved in TBST for more than 1 hour.

[0103] 5) Add the primary antibody (1:1000 dilution) and incubate overnight on a shaker at 4°C.

[0104] 6) Recover the primary antibody and wash the membrane three times with TBST for 10 minutes each time.

[0105] 7) Incubate with the corresponding secondary antibody dilution of HRP (1:5000) on a shaker at room temperature for 1 hour.

[0106] 8) Recover the secondary antibody TBST and wash the membrane 3 times, 10 min each time.

[0107] 9) Add ECL luminescent solution evenly to the PVDF film and let it stand for about 1 minute to develop.

[0108] 5. Conclusion:

[0109] Muscle specimens from NLSDM patients were confirmed by immunoblotting to show abnormal autophagy at the protein level. Figure 5 (NC1 / NC2: normal controls; P1 / P2: patients) It can be seen that the protein expression level of LAMP1 in the muscles of the two patients on the right side is significantly higher than that in the two controls on the left side. GAPDH is used as an internal reference.

[0110] Example 3

[0111] Cellular experiments demonstrating the therapeutic effect of nicotinic acid on NLSDM:

[0112] 1. Fibroblast Culture

[0113] (1) Cell resuscitation

[0114] Preheat the water bath to 37°C and prepare T25 or 6-well plates, adding 2 ml of complete culture medium beforehand. Remove the cell cryopreservation tubes from the liquid nitrogen container and quickly place them in the water bath. After 1-2 minutes in the water bath, when the cryopreservation solution is almost completely thawed, quickly transfer them to a clean bench and add them to a centrifuge tube containing 2 ml of complete culture medium. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in 1 ml of complete culture medium, and add the resuspended cells to the T25 or 6-well plates containing the culture medium. Mix well with a cross-shaped wedge and observe. Place the plates in an incubator for incubation.

[0115] (2) Cell medium exchange and passage

[0116] After cell resuscitation, observe the culture medium. Once the medium turns yellow, change the medium, aspirate the original medium, wash once with PBS, aspirate the PBS again, add fresh complete culture medium, and incubate in a 5% CO2, 37°C incubator. Once the cells have reached confluence, they can be passaged. Aspirate the supernatant from the cells to be passaged, wash once with PBS to remove residual culture medium and prevent residual serum from affecting digestion, add 1 ml of trypsin, and when under a microscope the cells become rounded, loosely connected, and about to fall off with gentle shaking, immediately add 2 ml of complete culture medium to stop digestion and prevent over-digestion that could worsen cell condition. Gently pipette to detach the cells, being careful not to damage them excessively. Transfer the collected cells to centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in 1 ml of complete culture medium, and add to new culture flasks or well plates at a ratio of 1:3 to 1:5 for culture.

[0117] (3) Cell cryopreservation

[0118] Prepare the cryopreservation solution (FBS:DMSO=9:1) in advance and pre-cool it at 4°C. Aspirate the culture medium from the cells to be cryopreserved, wash them once with PBS, add 1 ml of trypsin (1 ml for T25 culture flasks; if using a six-well plate, reduce the amount to 500 ml), and digest for 2-3 minutes. When the cells become rounded and loosely connected under the microscope, and show a tendency to detach after moving or shaking the culture flask, add twice the amount of complete culture medium containing FBS to stop digestion. Collect the cells by pipetting and transfer them to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, resuspend them in the pre-cooled cryopreservation solution, add them to the cryopreservation tube, and label the wall with the cell name, passage number, type, and cryopreservation time. Quickly place the tube in a cryopreservation box for gradient cooling to prevent damage to the cells from room temperature DMSO. Place the cryopreservation box in an ultra-low temperature freezer at -80°C, and transfer it to a liquid nitrogen tank after 1-2 days.

[0119] 2. Drug treatment of fibroblasts in patients

[0120] Healthy fibroblasts were seeded into 6-well or 24-well plates. The seeding density of fibroblasts was adjusted so that the cell density was about 60-70% after 24 hours. Nicotinic acid zolpidem was added alone or at 1 μg / ml or 6 μg / ml, or together with the ATGL inhibitor Atglistatin (40 μM). After 24 hours of treatment, the cells were harvested for subsequent experiments, such as immunofluorescence staining in Example 1 and Western blotting in Example 2.

[0121] 3. Cellular mitochondrial pressure measurement

[0122] (1) Preparation before the experiment

[0123] A. Cell seeding: Patient and control cells were seeded into Seahorse XFe24 cell culture microplates. A blank control group was prepared. After cell counting, the density of cells in each well was adjusted to ensure that the cell density was the same. The cell culture plates were placed in a clean bench for 1 hour and then placed in an incubator. When the cell density reached 60-70% under a microscope, nicotinic acid Z was added for treatment. The cells were then analyzed by machine after 24 hours.

[0124] B. Hydration probe plate: Add 1 ml of XF calibration solution (XF Calibrant) to each well of the hydration plate and incubate overnight in a CO2-free incubator at 37 °C.

[0125] C. Machine preheating: Turn on the machine one day in advance and open the software to allow it to preheat automatically to 37°C.

[0126] (2) Formal Experiment

[0127] A. Preparation of Seahorse Detection Solution: Prepare 20 mL of Seahorse detection solution. Add 10 mM glucose and 2 mM glutamine to Seahorse XF medium, i.e., according to the ratio of DMEM medium: glucose: glutamine = 20 mL: 200 μL: 200 μL. Add 200 μL of glucose and glutamine to 20 mL of DMEM medium to prepare the Seahorse detection solution.

[0128] B. Cell medium replacement: Discard the original culture medium, add 500ul of Seahorse detection solution to each well, place the microplate in a 37 °C CO2-free incubator, and wait for the drug addition to be completed before starting the instrument. The time is 45 minutes to 1 hour.

[0129] C. Reagent Preparation: Prepare 1.5 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone / antimycin A using Seahorse assay buffer preheated to 37 °C. Add the diluted reagents to reagent bins A, B, and C on the test plate, respectively. Add 56 μL to each well in reagent bin A, 62 μL to each well in reagent bin B, and 69 μL to each well in reagent bin C.

[0130] D. Instrumental Testing: After running the program, combine the test plate with the added medication and the hydration plate containing the calibration solution and place them into the instrument to calibrate the probe parameters. After calibration, replace the hydration plate with a cell culture plate.

[0131] E. Data Analysis: Analyze the data after the test is completed.

[0132] 4. Conclusion

[0133] 4.1 From Figure 6 The results showed that after nicotinic acid treatment of fibroblasts in NLSDM patients, lipid droplets in the fibroblasts decreased. Figure 6 China E and Figure 6 H) compared to before intervention ( Figure 6 The expression of green fluorescent lipid droplets in (B) was significantly reduced, and at 6 ug / ml ( Figure 6 (H) is better than 1ug / ml ( Figure 6 Group E) Figure 6 China A Figure 6 China B and Figure 6 C represents fibroblasts from patients without intervention. Figure 6 D, Figure 6 China E and Figure 6 In the middle F group, fibroblasts were treated with nicotinic acid at a concentration of 1 μg / ml. Figure 6 China G, Figure 6 H and Figure 6 In the middle section, fibroblasts were treated with nicotinic acid at a concentration of 6 μg / ml. Figure 6 China A Figure 6 China D and Figure 6 G in the middle represents DAPI staining, used to label cell nuclei; it exhibits blue fluorescence. Figure 6 B, Figure 6 China E and Figure 6 The middle H is a BODIPY staining agent used to label lipid droplets; it exhibits green fluorescence. Figure 6 C, Figure 6 China F and Figure 6 In the middle, I represents the fusion of a blue cell nucleus and a green lipid droplet.

[0134] 4.2 Niacinamide treatment significantly improved ATP production in the skin fibroblasts of patients. From Figure 7As can be seen, after 24 hours of intervention with 6ug / ml nicotinic acid, the oxygen consumption rate of fibroblasts in patients was significantly better than before the intervention, with a statistically significant difference (p<0.01). (Note: OCR: oxygen consumption rate)

[0135] Time (minutes): Time (minutes)

[0136] 4.3 from Figure 8 PNPLA2 The results of real-time quantitative PCR of gene mRNA transcription show that the patient PNPLA2 The mRNA transcription level of the gene (encoding the ATGL protein) was significantly lower than that of the normal control, but after intervention with 6 μg / ml nicotinic acid, the patient's... PNPLA2 Gene transcription levels were significantly upregulated, with a statistically significant difference (p < 0.01).

[0137] 4.4 From Figure 9 The results show that nicotinic acid significantly upregulated ATGL protein expression in patients. Patients' ATGL protein expression levels were significantly lower than those of normal controls, but after intervention with 6 μg / ml nicotinic acid, ATGL protein expression levels were significantly upregulated.

[0138] 4.5 From Figure 10 It can be seen that nicotinic acid reduces lipid droplet deposition in fibroblasts via ATGL, and inhibiting ATGL function can reverse the lipid droplet-reducing effect of nicotinic acid. After intervention with nicotinic acid at 6 μg / ml, the lipid droplets in the fibroblasts of patients ( Figure 10 (E) compared to before intervention ( Figure 10 The number of lipid droplets in fibroblasts was significantly reduced, but in the presence of ATGL inhibitors, even after intervention with nicotinic acid 6ug / ml, the number of lipid droplets in fibroblasts was significantly reduced. Figure 10 H) compared to no ATGL inhibitor ( Figure 10 The level of E in the middle jiao (e.g.) increased significantly, even exceeding the level before nicotinic acid intervention (e.g., E.g.). Figure 10 In group B, all groups showed statistically significant differences (p < 0.05 or p < 0.001). However, the addition of only an ATGL inhibitor (…) Figure 10 When K is present, lipid droplets increase significantly. Figure 10 China A, Figure 10 China B and Figure 10 C represents fibroblasts from patients without intervention. Figure 10 D, Figure 10 China E and Figure 10 In the middle F group, fibroblasts were treated with nicotinic acid at a concentration of 6 μg / ml. Figure 10 China G, Figure 10 H and Figure 10 In patient I, fibroblasts were treated with nicotinic acid 6 μg / ml and ATGL inhibitor 40 μM. Figure 10 J, Figure 10 K and Figure 10 L represents fibroblasts from patients treated with 40 μM of the ATGL inhibitor. Figure 10 China A, Figure 10 D, Figure 10 China G and Figure 10 J represents DAPI staining to label cell nuclei; it exhibits blue fluorescence. Figure 10 B, Figure 10 E, Figure 10 H and Figure 10 K is BODIPY stained to label lipid droplets, exhibiting green fluorescence. Figure 10 C in the middle Figure 10 China F, Figure 10 I and Figure 10 In the middle L, the blue cell nucleus and green lipid droplets are fused together.

[0139] 4.6 Immunofluorescence confirmed a significant upregulation of autophagy in fibroblasts of NLSDM patients. From Figure 11 It can be seen from the patient's intracellular lipid droplets ( Figure 11 The content of LAMP2 (I) increased significantly, and LAMP2 (I) also increased significantly. Figure 11 (C), P62 ( Figure 11 (E) and LC3 ( Figure 11 The immunofluorescence signal of the marker K (representing autophagy) was significantly higher than that of the normal control. Figure 11 China A Figure 11 B, Figure 11 China G and Figure 11 H in the middle represents DAPI staining to label cell nuclei; it produces blue fluorescence. Figure 11 C and Figure 11 D in the middle is stained with LAMP2 and exhibits green fluorescence. Figure 11 I and Figure 11 J in the middle is BODIPY staining, labeling lipid droplets, green fluorescence. Figure 11 China E and Figure 11 Middle F is P62 stained and exhibits red fluorescence. Figure 11 K and Figure 11 L in the middle is LC3 stained and exhibits red fluorescence. Figure 11 China A Figure 11 C, Figure 11 E, Figure 11 China G, Figure 11 Patients I and K in group 11 are patients. Figure 11 B, Figure 11 D, Figure 11 China F, Figure 11 H, Figure 11 J and L in 11 are normal controls. (Note: DAPI: labeling cell nuclear staining, blue fluorescence; BODIPY: labeling lipid droplet staining, green fluorescence)

[0140] 4.7 Immunoblotting confirmed that autophagy was significantly upregulated in the skin fibroblasts of NLSDM patients. Figure 12 As can be seen, the expression of autophagy markers LAMP1, P62 and LC3B proteins in the fibroblasts of the three patients was significantly higher than that in the three controls, with ACTIN as an internal control.

[0141] 4.8 Immunoblotting confirmed that nicotinic acid downregulated abnormally elevated lipopyphatypic markers in skin fibroblasts of NLSDM patients. From Figure 13 As can be seen, in the absence of autophagy induction or activation, intervention with nicotinic acid at 6 μg / ml significantly downregulated the expression of autophagy markers LAMP1, P62, and LC3B proteins in patient fibroblasts (lane 5), which was superior to the results of intervention with the autophagy inducer EBSS (lane 3). ACTIN was used as an internal control. (Note: EBSS induces autophagy, BAFA1 inhibits autophagy)

[0142] In summary, nicotinic acid has a good therapeutic effect on NLSDM.

Claims

1. The use of nicotinic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment of lipid storage myopathy; The nicotinic acid is Nikomycin Z, and its structure is shown in Formula 2: ； Formula 2; The lipid deposition myopathy is a simple myopathy-type neutral lipid deposition disease.

2. The application as described in claim 1, characterized in that, The nicotinic acid is the only active ingredient in the drug.

3. The application as described in claim 1, characterized in that, The medication for treating lipid deposition myopathy includes pharmaceutically acceptable excipients.

4. The application as described in claim 3, characterized in that, The acceptable excipients are selected from one or more of the following: diluents, disintegrants, flow aids, binders, dispersants, isotonic agents, thickeners, stabilizers, hydrating agents, ion exchangers, and flavoring agents.

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