Use of protein polymer in treatment of amyotrophic lateral sclerosis

Abstract

Use of a protein polymer in the treatment of amyotrophic lateral sclerosis. The protein polymer is obtained by stimulating and subsequently lysing mesenchymal stem cells, followed by isolation and purification. In SOD1 mutant mouse experiments, the protein polymer can significantly increase the number of motor neurons in the spinal anterior horn, ameliorate muscle atrophy, improve the motor ability of mice, and prolong the survival time of mice. In clinical trials, the protein polymer exhibits the effect of blocking the progression of amyotrophic lateral sclerosis, can reduce the concentration of TDP-43 protein in cerebrospinal fluid, and can increase the concentration of TDP-43 protein in serum.

Description

Application of a protein polymer in the treatment of ALS

[0001] This disclosure claims priority to Chinese Patent Application No. 202410945391.8, filed on July 15, 2024, entitled "Application of a Protein Polymer in the Treatment of ALS", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to the application of a protein polymer in the treatment of ALS (Amyotrophic Lateral Sclerosis). Background Technology

[0003] Amyotrophic lateral sclerosis (ALS) is the most common type of motor neuron disease. It generally affects middle-aged and elderly individuals, with the peak age of onset in my country around 50 years old, and there is a trend towards younger onset, with a small number of patients developing the disease around age 20. Clinically, it is characterized by progressive skeletal muscle weakness and atrophy, fasciculations, bulbar palsy, and pyramidal tract signs. Some ALS patients may also exhibit varying degrees of cognitive and / or behavioral impairments related to frontotemporal lobe involvement. Approximately 10% of ALS cases are familial, and several genes have been identified as associated with it. Early clinical manifestations of ALS are diverse, and specific biological diagnostic indicators are lacking. Determining the extent of upper and lower motor neuron involvement is a crucial step in clinical diagnosis. Based on the anatomical location of the patient's symptoms and signs, the affected area is typically divided into four regions: brainstem, cervical, thoracic, and lumbosacral.

[0004] Influenced by genetic factors, lifestyle, work pressure and other factors, the number of patients with ALS is showing an increasing trend and the onset of the disease at a younger age. However, there is currently no satisfactory treatment plan. There is an urgent need in this field to develop new drugs and methods to treat or alleviate ALS. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an application of a protein polymer in the treatment of ALS.

[0006] The technical solution adopted in this invention is:

[0007] The application of a protein polymer in the treatment of ALS, wherein the protein polymer is used to reduce the concentration of TDP-43 protein in cerebrospinal fluid and increase the concentration of TDP-43 protein in serum.

[0008] In some embodiments, the protein polymers described above include at least the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0009] In some embodiments, the protein polymer further includes at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0010] In some embodiments, the sum of the masses of sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens and sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer accounts for more than 40% of the total mass.

[0011] In some embodiments, the Serum albumin protein content is at least 38% of the total protein polymer content, and the Serotransferrin protein content is at least 2% of the total protein polymer content.

[0012] In some embodiments, the preparation of the above-mentioned protein polymer includes the following steps:

[0013] S1: Culture mesenchymal stem cells and create a stress environment using ultraviolet irradiation;

[0014] S2: Mesenchymal stem cells are lysed and purified to obtain protein polymers.

[0015] In some embodiments, the protein polymer satisfies at least one of the following properties:

[0016] 1) During molecular sieve size exclusion chromatography, under the conditions of a flow rate of 0.1 mL / min to 0.3 mL / min and PBS as the eluent, the elution volume of the first component peak is 12 mL to 13.2 mL, the elution volume of the second component peak is 15.2 mL to 17 mL, the elution volume of the third component peak is 17 mL to 20 mL, the elution volume of the fourth component peak is 29 mL to 31 mL, and the elution volume of the fifth component peak is 31 mL to 34 mL.

[0017] 2) During SDS-PAGE analysis, 4-20% pre-mixed gel was used for sample separation and analysis. The sample bands were mainly distributed between 11KD and 100KD, with the molecular weight decreasing from large to small. The first band was between 75KD and 100KD, and the second band was between 63KD and 75KD.

[0018] 3) For reversed-phase HPLC detection, the sample loading volume is 80μL~120μL, the column temperature is 20℃~30℃, the flow rate is 0.2mL / min~0.8mL / min, the detection wavelength is 260nm and / or 280nm, the mobile phase is PBS solution, and the peak elution time after sample separation is between 10min~40min, with characteristic peak 1 elution time being 13min~17min and characteristic peak 2 elution time being 20min~22min.

[0019] In some embodiments, the protein polymer satisfies the requirement of having a total of 5 characteristic peaks within the elution time of 10 to 40 min in reversed-phase chromatography, wherein the elution time of characteristic peak 1# is 13.3 min to 14.5 min, the elution time of characteristic peak 2# is 21.5 min to 22 min, the elution time of characteristic peak 3# is 22 min to 22.8 min, the elution time of characteristic peak 5# is 26.8 min to 27.8 min, and the elution time of characteristic peak 7# is 28.7 min to 30 min.

[0020] In some embodiments, the protein polymer satisfies the requirement of having ten characteristic peaks within the elution time of 10 to 40 min in reversed-phase chromatography. Specifically, the elution time of characteristic peak 1# is 13.3 min to 14.5 min, characteristic peak 2# is 21.5 min to 22 min, characteristic peak 3# is 22 min to 22.8 min, characteristic peak 4# is 23 min to 23.7 min, characteristic peak 5# is 26.8 min to 27.8 min, characteristic peak 6# is 27.8 min to 28.7 min, characteristic peak 7# is 28.7 min to 30 min, characteristic peak 8# is 30 min to 30.6 min, characteristic peak 9# is 30.6 min to 32 min, and characteristic peak 10# is 33.8 min to 34.8 min.

[0021] In some embodiments, the duration of ultraviolet irradiation is 1 hour to 30 hours; and / or, the intensity of ultraviolet irradiation is 10 μW / cm². 2 ~100μW / cm 2 ; and / or, the wavelength of ultraviolet light is 290nm to 340nm.

[0022] In a preferred example, the wavelength of the ultraviolet light is 290 nm to 325 nm.

[0023] In a specific and preferred example, the wavelength of the ultraviolet light is 300 nm to 320 nm.

[0024] In some implementations, the culture medium used for culturing is serum-free MSCs medium.

[0025] In some embodiments, the mesenchymal stem cells are selected from one of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placental-derived mesenchymal stem cells. In some embodiments, the administration route of the protein polymer is selected from one of intrathecal administration, intravenous administration, and combined intrathecal and intravenous administration. Attached Figure Description

[0026] Figure 1 is a photograph of the cell state after irradiation in Example 1.

[0027] Figure 2 is an SDS-PAGE electrophoresis image of the protein harvested in Example 1.

[0028] Figure 3 shows the elution curves for Example 2.

[0029] Figure 4 shows the elution curve of Example 3.

[0030] Figures 5 and 6 show the effects of different treatments on the anterior horn motor neurons of the spinal cord in SOD1 mutant mice.

[0031] Figure 7 shows the experimental results of the rotarod experiment on SOD1 mutant mice under different treatments.

[0032] Figures 8 and 9 show the effects of different treatments on the survival of SOD1 mutant mice.

[0033] Figures 10-12 show the ALS FRS-R and Norris scores of ALS patients in clinical trials.

[0034] Figure 13 shows the detection results of TDP-43 in the cerebrospinal fluid of patients with ALS. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the embodiments.

[0036] Example 1: Culture of human umbilical cord mesenchymal stem cells

[0037] Resuscitate one small P8 generation HUC-MSC (human umbilical cord mesenchymal stem cell) into a T25 culture flask and add 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium.

[0038] Irradiate cells with 300nm LED UV for 6 hours. Carefully remove supernatant from cells, wash twice with 1mL physiological saline, add 660μL of pure water and repeatedly pipette for 10min to lyse, filter through a 0.22μm filter membrane, and store at 4℃.

[0039] The ultraviolet irradiation conditions are as follows:

[0040] The cell state after irradiation is shown in Figure 1. The protein polymer stock solution was harvested at a concentration of 0.718 mg / mL, in a volume of 500 μL. The SDS-PAGE results of the harvested protein are shown in Figure 2.

[0041] Example 2: Molecular sieve purification and activity detection of protein polymers

[0042] 2-1 Molecular sieve purification of protein polymers:

[0043] Instrument: AKTA explorer;

[0044] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume approximately 30mL;

[0045] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water;

[0046] Ultraviolet absorption wavelength: 280nm, 260nm for reference;

[0047] Equilibrium chromatography column sequence:

[0048] First, rinse the chromatography column with 60 mL of purified water, then equilibrate the column with 60 mL of 1×PBS to zero the 280 nm UV absorbance.

[0049] Sample preparation: Repeat the method in Example 1 to harvest a total protein volume of 20 mL. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD to reduce the 20 mL sample to approximately 600 μL.

[0050] Experimental Procedure: After equilibrating the chromatography column, a 500 μl loading loop was used to load the sample at a flow rate of 0.4 mL / min. Elution with 1×PBS was performed at a flow rate of 0.2 mL / min until the peak was reached. Protein was collected starting from a UV absorbance of 4 mAU. The elution curve is shown in Figure 3. The elution volume for the first fraction (position 2) was 12 mL–13.2 mL; for the second fraction (position 9), it was 15.2 mL–17 mL; for the third fraction (position 12), it was 17 mL–20 mL; for the fourth fraction (position 16), it was 29 mL–31 mL; and for the fifth fraction (position 18), it was 31 mL–34 mL. The elution times for the five components were calculated to be 60-66 min, 76-85 min, 85-100 min, 145-155 min, and 155-170 min, respectively. Protein samples taken from positions 2, 9, 12, 16, and 18 in Figure 3 were lyophilized and stored for activity testing.

[0051] 2-2 Bioactivity assay of protein polymers

[0052] Day 1: Cell Plating: Dilute PC12 low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate at 6000 cells / well (96-well plate). Incubate overnight at 37°C with 5% CO2.

[0053] Day 2: Dilute the lyophilized sample and the unpurified sample (approximately 800 μg / mL) with DMEM + 5% FBS medium.

[0054] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0055] Hydrogen peroxide treatment: Discard 80 μL / well of cell culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and incubate at room temperature for 25 min.

[0056] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as a damage treatment control.

[0057] Add 50 μL / well of the diluted sample to the well after hydrogen peroxide treatment.

[0058] Discard the supernatant from the untreated wells and add 100 μL of complete culture medium per well as a cell growth control (PC).

[0059] Incubate at 37℃ for 2 days.

[0060] Day 5: Discard the culture supernatant, add 100 μL / well of complete culture medium, and set up blank control wells. Add 10 μL / well of CCK8 and incubate at 37℃ for 3.5 h. OD 450 Readings. Calculated after subtracting the culture medium blank.

[0061] The experimental results are shown in Table 1. Each elution fraction was repeated three times in parallel.

[0062] Table 1. Experimental data on bioactivity of different samples

[0063] As shown in Table 1, in the PC12 cell oxidative damage model, compared with NC, PC, and culture medium blanks, the unpurified samples demonstrated oxidative damage repair capabilities. Furthermore, the purified samples exhibited even stronger oxidative damage repair capabilities; for example, samples #2, #12, and #16 all showed strong oxidative damage repair abilities, superior to the unpurified samples. Sample #9 showed a slightly higher repair ability than the unpurified samples, while sample #18 showed a lower repair ability.

[0064] The purified sample #12 was analyzed by SDS-PAGE electrophoresis. SDS-PAGE showed that the sample bands were mainly distributed between 11KD and 100KD, with the first band between 75KD and 100KD and the second band between 63KD and 75KD, decreasing in molecular weight. Further analysis revealed that the purified protein polymer contained the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0065] The above two are the main proteins, accounting for more than 40% of the total mass of protein polymers. Other proteins include: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0066] Further analysis revealed that Serum albumin protein content was at least 38% of the total protein polymer content, and Serotransferrin protein content was at least 2% of the total protein polymer content.

[0067] Example 3: HPLC-SEC purification and protein polymer activity detection

[0068] Based on the difference in hydrophobicity, reversed-phase chromatography can be used to separate the protein polymers obtained by UV irradiation stimulation according to this invention. Under initial conditions, the concentration of organic components in the mobile phase is low, and the protein polymers have strong hydrophobic interactions with the stationary phase, resulting in almost complete adsorption by the stationary phase. When the concentration of organic components in the mobile phase reaches a specific level, the protein polymers are completely eluted from the stationary phase and no longer interact with it. Therefore, even minute changes in the organic components of the mobile phase can significantly affect the reversed-phase retention behavior of the protein polymers.

[0069] 3-1 HPLC-SEC purification

[0070] Sample information: The protein polymer prepared by the method in Example 1 was diluted to 1 mg / mL with PBS.

[0071] Mobile phase: PBS;

[0072] Detection conditions: injection volume 100 μL, column temperature 25℃, flow rate 0.4 ml / min, wavelength 280 nm, 260 nm; time: 48 min

[0073] The collection time ranges are approximately as follows: #1 13.3 min~14.5 min; #2 21.5 min~22 min; #3 22 min~22.8 min; #4 23 min~23.7 min; #5 26.8 min~27.8 min; #6 27.8 min~28.7 min; #7 28.7 min~30 min; #8 30 min~30.6 min; #9 30.6 min~32 (no obvious peak for A280) min; #10 33.8 min~34.8 min.

[0074] The elution curve is shown in Figure 4. Protein samples taken from positions 1-10 in Figure 4 were lyophilized and stored.

[0075] 3-2 Bioactivity assay of protein polymers

[0076] Day 1: Cell Plating: Dilute PC12 low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate at 6000 cells / well (96-well plate). Incubate overnight at 37℃ with 5% CO2.

[0077] Day 2: Dilute the lyophilized sample and the unpurified sample (approximately 800 μg / mL) with DMEM + 5% FBS medium.

[0078] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0079] Hydrogen peroxide treatment: Discard 80 μL / well of cell culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and incubate at room temperature for 25 min.

[0080] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as a damage treatment control.

[0081] Add 50 μL / well of the diluted sample to the well after hydrogen peroxide treatment.

[0082] Discard the supernatant from the untreated wells and add 100 μL of complete culture medium per well as a cell growth control (PC).

[0083] Incubate at 37℃ for 2 days.

[0084] Day 5: Discard the culture supernatant, add 100 μL / well of complete culture medium, and set up blank control wells. Add 10 μL / well of CCK8 and incubate at 37℃ for 3.5 h. OD 450 Readings. Calculated after subtracting the culture medium blank.

[0085] The experimental results are shown in Table 2.

[0086] Table 2. Bioactivity experimental data of different samples

[0087] Table 2 shows that, in the PC12 cell oxidative damage model, compared with NC, PC, and culture medium blanks, the unpurified samples possess oxidative damage repair capabilities. Furthermore, the purified samples exhibit even stronger oxidative damage repair capabilities; for example, samples 1#, 2#, 7#, 8#, and 10# all demonstrate strong oxidative damage repair abilities, surpassing those of the unpurified samples. Sample 5# shows a slightly lower repair ability than the unpurified sample.

[0088] Example 4: Application of the protein polymer of the present invention in the treatment of ALS (Amyotrophic Lateral Sclerosis).

[0089] The protein polymer obtained in Example 1 of this invention will be used to further study its application in the treatment of ALS.

[0090] 4-1 SOD1 mutant mouse experiment

[0091] The effects of the protein polymer of this invention on motor dysfunction in SOD1 mutant mice were studied using 100-day-old, half-male and half-female SOD1G93A mice (referred to as SOD1 mutant mice). SOD1 mutant mice were used as the model control group and experimental group, while 100-day-old, half-male and half-female C57BL / 6J mice were used as the normal control group, with 10 mice in each group.

[0092] The model control group received no medication, while the experimental group received protein polymers and riluzole. The protein polymers were administered via intrathecal, intravenous, or combined intrathecal and intravenous administration, while riluzole was administered via gavage. The dosing cycle was 14 days.

[0093] See Figures 5 and 6, SOD1 mutant mice (model control group, SOD1) G93A The number of motor neurons in the anterior horn of the spinal cord was significantly lower than that in the wild-type control (normal control group, WT). Treatment with 60 μg / kg protein polymer via intrathecal (it), intravenous (iv), and combined (i.t+iv) administration significantly increased the number of motor neurons, bringing them close to that of the wild-type. Riluzole treatment was slightly superior to intrathecal protein polymer administration, but inferior to the other two administration methods.

[0094] See Figure 7, using the rotarod test to reflect the motor ability of mice: with increasing age, the SOD1 mutant mice (SOD1...) G93A The time mice spent on the rotundus gradually decreased, indicating progressive loss of motor function. Treatment with different doses of protein polymers significantly reduced the decline in motor function, with the 20 μg / kg dose of protein polymer (SOD1) showing the most significant reduction. G93A The best results were observed with a protein polymer (20 μg / kg i.t. + iv). Riluzole treatment did not significantly improve motor function in mice.

[0095] See Figure 8. Compared with wild-type mice (WT), SOD1 mutant mice (SOD1) G93A The survival time of mice was significantly shortened. Treatment with different doses of protein polymers in combination significantly prolonged the survival time of mice, with the 20 μg / kg dose of protein polymer (SOD1) showing the most significant improvement. G93A The best results were observed with a protein polymer (20 μg / kg i.t. + iv). See Figure 9; riluzole did not prolong the survival of mice.

[0096] 4-2 Clinical Trials

[0097] Referring to Figure 10, a clinical trial was conducted using the protein polymer stock solution from Example 1 on 7 ALS patients in Hospital A and 2 ALS patients in Hospital B.

[0098] 4-2-1 Selection Criteria

[0099] 1. Patients diagnosed with amyotrophic lateral sclerosis (ALS) after medication;

[0100] 2. The score of each item of the Modified Amyotrophic Lateral Sclerosis Functional Scale (ALSFRS-R) must be ≥2 (of which the three items of dyspnea, orthopnea and respiratory failure must all be 4).

[0101] 3. Forced vital capacity (FVC%) ≥ 60%;

[0102] 4. The course of the disease is 2 years or less (calculated from the first appearance of any ALS symptoms);

[0103] 5. Age 45-70 (inclusive), gender not limited;

[0104] 6. Voluntarily participate in this clinical trial, provide informed consent, and sign the informed consent form.

[0105] 4-2-2 Exclusion Criteria

[0106] 1. Individuals diagnosed with familial ALS (based on family history);

[0107] 2. Individuals with significant cognitive impairment (MMSE scale: illiterate group ≤19 points, primary school group ≤22 points, junior high school and above group ≤26 points for more than 8 years of schooling);

[0108] 3. Significant difficulty swallowing;

[0109] 4. Severe renal insufficiency: creatinine clearance <30 mL / min (Cockcroft-Gault formula), or other known severe renal insufficiency;

[0110] 5. Severe liver function impairment: ALT, AST > 3 times the upper limit of normal, or other known liver diseases such as acute or chronic active hepatitis, cirrhosis, etc.;

[0111] 6. Screening period: Patients with acute myocardial infarction or who have undergone interventional treatment within the past 6 months, and patients with heart failure (NYHA class III-IV).

[0112] 7. Comorbid malignant tumors, hematological, digestive, or other serious systemic diseases;

[0113] 8. Allergic constitution;

[0114] 9. Pregnant and breastfeeding women;

[0115] 10. Participate in other clinical trials within 30 days prior to screening;

[0116] 11. Other situations where researchers deem the candidate unsuitable for enrollment.

[0117] 4-2-3 treatment plan

[0118] Patients were enrolled after signing informed consent and meeting the inclusion / exclusion criteria, and received protein polymer therapy in addition to routine treatment.

[0119] In this study, enrolled patients received standard treatment as the basis of care, along with lumbar puncture and intravenous therapy. Patients were randomly assigned after screening within one week. Treatment involved lumbar punctures twice weekly, or once daily intravenously when lumbar puncture was not required. The intravenous dose was 130 μg twice weekly (diluted in 100 mL of normal saline). Follow-up began after treatment and was conducted at day 11, week 6, and week 8 to collect information on safety and efficacy.

[0120] 4-2-4 Treatment Effect

[0121] Referring to Figure 10, the average increase in ALS R-R score by 1.44 points and the average increase in Norris score by 3.67 points before and after treatment in the 9 patients indicate that protein polymers can block the progression of ALS and show significant improvement.

[0122] Referring to Figures 11 and 12, after 2 weeks of treatment with protein polymers, both the ALSFRS-R and Norris scores improved; during the 4-week period after drug withdrawal, the ALSFRS-R gradually decreased; after 2 weeks of re-administration, the ALSFRS-R improved again. This indicates that protein polymers have a direct effect on the treatment of ALS.

[0123] TDP-43 exists in the amyloid form within patient cells such as neurons and glial cells, and is one of the main pathological marker proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal degenerative motor neuron disease. Referring to Figure 13, after 2 weeks of protein polymer administration, the concentration of TDP-43 in cerebrospinal fluid (CSF) decreased; during the 4-week drug withdrawal period, the concentration of TDP-43 in CSF increased somewhat; after 2 weeks of re-administration, the concentration of TDP-43 in CSF decreased again. This indicates that the protein polymer helps to induce benign changes in TDP-43 levels in CSF during drug administration.

[0124] The serum TDP-43 concentration in healthy individuals (2248 pg / mL) was significantly higher than that in ALS patients. Therefore, Hospital A tracked the changes in TDP-43 protein concentration in the cerebrospinal fluid and serum of four patients, as shown in Table 3. During the treatment cycle, patients received medication for 2 weeks, stopped medication for 4 weeks, and then received medication again for 2 weeks. It was observed that with the use of protein polymers, the TDP-43 concentration in the cerebrospinal fluid decreased, while the TDP-43 concentration in the serum increased, showing an overall benign change.

[0125] Table 3. Changes in TDP-43 protein concentration in cerebrospinal fluid and serum of patients.

[0126] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. Use of a protein polymer in the treatment of amyotrophic lateral sclerosis, characterised in that, The protein polymer is used for reducing the concentration of TDP-43 protein in cerebrospinal fluid and increasing the concentration of TDP-43 protein in serum.

2. Use of a protein polymer according to claim 1 in the treatment of frostbite, characterized in that, The protein polymer at least comprises the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

3. Use of a protein polymer according to claim 2 in the treatment of frostbite, characterized in that, The protein polymer further comprises at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

4. Use of a protein polymer according to claim 3 for the treatment of frostbite, characterized in that, The sum of the mass of the sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens in the protein polymer accounts for more than 40% of the total mass.

5. Use of a protein polymer according to any one of claims 1 to 4 for the treatment of frostbite, characterized in that, The preparation of the protein polymer comprises the following steps: S1: culturing mesenchymal stem cells and using ultraviolet irradiation to manufacture a stress environment; S2: lysing the mesenchymal stem cells and isolating and purifying to obtain the protein polymer.

6. Use of a protein polymer according to claim 5 for the treatment of frostbite, characterized in that, The protein polymer satisfies at least one of the following properties: 1) In the size exclusion chromatography, under the exclusion chromatography condition of a flow rate of 0.1 mL / min to 0.3 mL / min and an eluent of PBS, the elution volume of the first component is 12 mL to 13.2 mL, the elution volume of the second component is 15.2 mL to 17 mL, the elution volume of the third component is 17 mL to 20 mL, the elution volume of the fourth component is 29 mL to 31 mL, and the elution volume of the fifth component is 31 mL to 34 mL; 2) In the SDS-PAGE detection, using 4% to 20% precast gel for sample separation and detection, the sample bands are mainly distributed in 11 KD to 100 KD, and in order of decreasing molecular weight, the first band is located between 75 KD and 100 KD, and the second band is located between 63 KD and 75 KD; 3) In the reverse phase HPLC detection, under the condition of a sample volume of 80 μL to 120 μL, a column temperature of 20°C to 30°C, a flow rate of 0.2 mL / min to 0.8 mL / min, a detection wavelength of 260 nm and / or 280 nm, and a mobile phase of PBS solution, the peak time of the sample separation is between 10 min and 40 min, the peak time of characteristic peak 1 is 13 min to 17 min, and the peak time of characteristic peak 2 is 20 min to 22 min.

7. Use of a protein polymer according to claim 5 for the treatment of frostbite, characterized in that, The time of the ultraviolet irradiation is 1 h to 30 h. and / or the intensity of the ultraviolet irradiation is 10 μW / cm 2 ~ 100 μW / cm 2 ; And / or, the wavelength of the ultraviolet is 290 nm to 340 nm.

8. Use of a protein polymer according to claim 5 for the treatment of frostbite, characterized in that, The culture medium used in the culture is a serum-free MSCs culture medium.

9. Use of a protein polymer according to claim 5 for the treatment of frostbite, characterized in that, The mesenchymal stem cells are selected from one of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.

10. Use of a protein polymer according to any one of claims 1 to 4 in the treatment of frostbite, characterised in that, The administration mode of the protein polymer is selected from one of intrathecal administration, intravenous administration, and combined intrathecal and intravenous administration.

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