Use of elapidae postsynaptic neurotoxin in the treatment of over expression of inflammatory cytokines related diseases

Abstract

A method for treatment of the diseases related to overexpression of tumor necrosis factor- (TNF-) and/or interleukin-1 (IL-1) of a patient. The method comprises: administering a therapeutically effective dose of elapidae postsynaptic neurotoxin molecules (SEQ ID NOs. 1-21) and a pharmaceutically acceptable carrier. The diseases comprise rheumatoid arthritis, rheumatic arthritis, gouty arthritis, osteoarthritis, traumatic arthritis, ankylosing spondylitis, diabetes, diabetic peripheral neuropathy, diabetic retinopathy, systemic lupus erythematosus, neuropathic pain, cancer pains, myocarditis, pancreatic cancer, and liver cancer. The mature proteins or peptides of the elapidae postsynaptic neurotoxin molecules include any one of the amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 21, or have the homology of 70% or more to the amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 21 respectively.

Claims

1. A method for treating excessive expression of tumor necrosis factor-, (TNF-) and interleukin-1, (IL-1) in a mammal. Said method comprises administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of an Elapidae postsynaptic neurotoxin monomer molecule, (SEQ ID No.1-21) and a pharmaceutically acceptable carrier base, for use in inhibiting or reducing the concentration of tumor necrosis factor-, (TNF-) and interleukin-1, (IL-1) in blood and human body.

2. A method for treating excessive expression of tumor necrosis factor-, (TNF-) and interleukin-1, (IL-1) in a mammal. Said method comprises administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of an Elapidae postsynaptic neurotoxin monomer molecule, (SEQ ID No.1-21) and a pharmaceutically acceptable carrier base, for use in treating or preventing the disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1).

3. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to Rheumatoid arthritis.

4. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to rheumatic arthritis.

5. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to gouty arthritis.

6. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to traumatic arthritis.

7. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to osteoarthritis arthritis.

8. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to ankylosing spondylitis.

9. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to diabetes.

10. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to diabetic neuropathy.

11. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to diabetic retinopathy.

12. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to systemic lupus erythematosus.

13. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to neuropathic pain.

14. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to cancer pain.

15. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to myocarditis.

16. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to pancreatic cancer.

17. The disease associated with excessive expression of tumor necrosis factor-, (TNF-) and/or interleukin-1, (IL-1) of claim (2), wherein it refers to liver cancer.

18. The Elapidae postsynaptic neurotoxin of claim (2), wherein it is an Elapidae postsynaptic neurotoxin polypeptide having the amino acid sequence of SEQ ID No.1 to SEQ ID No.21, or an Elapidae neurotoxin polypeptide homologs having 70% or more homology with the Elapidae neurotoxin polypeptide of SEQ ID No.1 to SEQ ID No.21, and the biological function of the Elapidae postsynaptic neurotoxin polypeptide homologs is the same as or similar to that of the Elapidae neurotoxin polypeptide of the amino acid sequence ID No. 1 to SEQ ID No. 21.

19. The Elapidae postsynaptic neurotoxin polypeptides or Elapidae postsynaptic neurotoxin polypeptides homologs of claim (18), wherein it's further characterized in that they are isolated from natural snake venoms, or synthesized from chemical polypeptides, or obtained from prokaryotic or eukaryotic hosts using recombinant technology such as Bacteria, yeast, higher plants, insects and mammalian cells.

20. The method of claim (2) includes intravenous, intramuscular, subcutaneous, intra-articular, oral, sublingual, nasal, rectal, topical, intradermal, intraperitoneal, intrathecal, or transdermal administration, and the dose of the Elapidae postsynaptic neurotoxin includes from 1 g/Kg to 2 mg/kg each time.

Description

METHOD OF IMPLEMENTATION

Example A: The Method for Specific Isolation and Extraction of Postsynaptic Neurotoxin SEQ ID NO 1

[0021] Mature protein or polypeptide of the current invention refers to the Chinese patent application publication number: CN 110090296A as an example.

Example B: Method of Obtaining Bungarus multicinctus Postsynaptic Neurotoxin SEQ ID NO.4

[0022] The protein or polypeptide is produced by gene recombinant technology and is specified as follows:

[0023] 1. Cloning of Recombinant Expression Vector

[0024] A DNA sequence is synthesized according to the gene of Bangarus postsynaptic neurotoxin (SEQ ID NO. 4) provided on GenBank, PCR amplification is carried out on a target DNA sequence, a sequence encoding the intestinal kinase recognition site, and an NDE I enzyme digestion site is introduced at the 5 end of the upstream primer, and a stop codon and a BamHI enzyme digestion site are introduced at the 5 end of the downstream primer.

[0025] The gene containing the postsynaptic neurotoxin SEQ ID No. 4 is amplified by using a PCR method and cloned into a PBS-T vector, and the constructed recombinant sub-PBS-T-Bangarus postsynaptic neurotoxin SEQ ID No. 4 shall be under examination with analysis and identification.

[0026] 2. Protein Expression in E. coli.

[0027] The recombinant plasmid is transformed into the Escherichia coli expression vector pET15b, and the recombinant expression plasmid pET15b-postsynaptic neurotoxin SEQ ID No. 4 is constructed, and the correct recombinant transplanting to Escherichia coli BL21 (DE3)LysS and examined under analysis and identification. The monoclonal antibody is inoculated into a 5G LB culture medium, cultured overnight at 37 DEG C., the day is inoculated into a 50 ml LB culture medium according to a ratio of 1:100, and the culture is shaken at 37 DEG C. until the OD600 nm is equal to 0.4-0.6.

[0028] 3. Collection and Analysis of Expressed Products

[0029] The transformation is continued with 1 mmol/L of IPTG for 3 hours, the expression bacteria, which means the induced and transformed Escherichia coli BL21 (DE3), is centrifuged, the inclusion body is dissolved in the buffer solution, and the supernatant and the precipitate are subjected to SDS-PAGE electrophoresis detection after centrifugation and collection, and the target protein exists in the form of inclusion bodies.

[0030] 4. Affinity and Purification of Expressed Products

[0031] The inclusion body after ultrasonic breaking is dissolved in a buffer solution (6 mol/L of guanidine hydrochloride, 20 mmol/L of Tris-HCl, ph 8.0, 0.5 mol/L of Nacl, 5 mmol/L imidazole); the buffer solution is purified through a nickel-NTA column affinity chromatography, specifically, the buffer solution containing 20 mmol/L imidazole is washed to a baseline before loading, and finally, the buffer solution containing 300 mol/L imidazole is used for elution. The enterokinase is used to cut for obtaining the postsynaptic neurotoxin SEQ ID No. 4 protein.

[0032] 5. Renaturation of Expressed Product

[0033] The eluted protein is dialyzed with 6 mol/L guanidine hydrochloride, 0.1 mol/L Tris-HClHCl Ph8.0, 0.01 mol LEDTA, 0.1 mmol/L PMSF, 10 mmol/L DTT buffer solution, the concentration of DTT and guanidine hydrochloride in the buffer solution is gradually decreased, and then the buffer solution is dialyzed with 10-fold volume of 0.1 mol/L Tris-HCL Ph-8.0, 5 mol/L CuSo4, and 20% glycerol. An RP-HPLC method is used for detecting the renaturation result, and through the comparison of retention time with a standard sample, the renaturation substance is identified, and the renaturation product is refrigerated and stored.

[0034] 6. Amino Acid Sequence Determination

[0035] The peptide fragment coverage rate and the Edman degradation method are used for the analysis of the obtained postsynaptic neurotoxin, and the measured SEQ ID No. 4 sequence is compared with the amino acid sequence of the neurotoxin in the protein library, and after the confirmation of the consistency of the sequence, then the postsynaptic neurotoxin is used for the anti-inflammation test in the next step.

Example C: An Experiment of Anti-Inflammation Properties of Elapidae Postsynaptic Neurotoxin Monomer Molecule

[0036] 1. Animals and Grouping

[0037] 140 Wistar rats (200-240 g) are randomly divided into 14 groups, and each group is 10. The preparation method comprises the following steps: (group 1) only receiving sterile normal saline (0.95% of sodium chloride) (control group); (group 2) carrageenan inflammation modeling+oral normal saline 10 ml/kg (inflammation group); (Group 3) Carrageenan Inflammation modeling+oral Naja atra postsynaptic neurotoxin (SEQ ID NO 1) 200 g/kg prepared into a liquid state and continuous intragastric administration performed; (Group 4) Carrageenan Inflammation modeling+oral Naja atra postsynaptic neurotoxin (SEQ ID NO 1) 800 g/kg prepared into a liquid state and continuous intragastric administration performed. (Group 5-14) The postsynaptic neurotoxin of Bungarus multicinctus, Ophiophagus Hannah, Naja kaouthia, Dendroaspis polylepis, Bungarus fasclatus, through the same administration model, that means carrageenan inflammation modeling+oral administration of 200 g/kg and 800 g/kg two doses respectively and continuously intragastric administration, so that the rat is divided into 14 groups.

[0038] 2. Animal Inflammation Modeling

[0039] Except for the illumination group, 0.1 ml of sterile normal saline+carrageenan (CG, 1%) is injected into the rest 13 groups through pleural cavities for inflammation modeling. The preparation method comprises the following steps: 1 hour before molding, orally administration of normal saline or different types of postsynaptic neurotoxin with different doses respectively in each group, 6 hours after the carrageenan injection, collecting rat tail vein blood to detect the concentration of IL-1 beta and TNF-alpha.

[0040] 3 Measurement of Tumor Necrosis Factor Alpha (TNF-Alpha) and Interleukin 1 Beta (IL-1 Beta)

[0041] The serum of the collected rat tail vein blood is separated and collected by using a commercial ELISA assay kit, and biochemical evaluation is carried out on the concentration of the tumor necrosis factor alpha (TN F-alpha) and interleukin 1 beta (IL-1 beta) according to the instructions of experimental step of the kit's manufacturer.

[0042] 4. Experimental Results

TABLE-US-00002 TABLE 1 is a comparison of the blood average concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) among the rat control group, the inflammation group and six postsynaptic neurotoxin treatment groups. IL-1 TNF-a n = 10 (ng/L, x s) (ng/L, x s) Saline Control Group 35.20 4.78 42.70 5.93 Inflammation Group 52.40 6.74 61.30 7.86 SEQ ID No1(200 g/kg) 45.40 4.95* 53.70 6.32* SEQ ID No1(800 g/kg) 41.30 4.31*** 50.60 5.89** SEQ ID No4(200 g/kg) 46.70 5.12* 52.76 6.43* SEQ ID No4(800 g/kg) 39.80 5.01*** 48.80 5.41*** SEQ ID No9(200 g/kg) 44.50 4.97** 54.80 5.01* SEQ ID No9(800 g/kg) 41.70 4.23*** 51.20 5.76** SEQ ID No14(200 g/kg) 43.50 5.43** 53.01 6.87* SEQ ID No14(800 g/kg) 38.20 4.35*** 49.50 5.12** SEQ ID No17(200 g/kg) 46.02 4.53 *.sup. 52.20 6.35 * SEQ ID No17(800 g/kg) 43.80 3.98 ** .sup.50.10 6.02 ** SEQ ID No20(200 g/kg) 44.10 4.32 *.sup. 52.82 6.55 * SEQ ID No20(800 g/kg) 40.80 3.78 ** 47.81 5.72 ***

[0043] Experimental results show that total 12 postsynaptic neurotoxin treatment groups, in each treatment group, the average blood concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) of is lower than that of the inflammation group, and it shows a significant difference and dose-dependent effect between the treatment group and inflammation group. *representing P<0.05, **representing P<0.01, ***representing P<0.001.

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