Application of pharmaceutical composition in treating diabetic nephropathy

Abstract

The present invention relates to a pharmaceutical composition for treating diabetic nephropathy, wherein the composition comprises a screened cobra cardiotoxin having a specific amino acid sequence. The diabetic nephropathy (DKD) refers to chronic kidney disease caused by diabetes. The composition can reduce the urine albumin / creatinine ratio (UACR) of diabetic nephropathy and improve the estimated glomerular filtration rate (eGFR).

Description

[0001] Technical field: The present invention relates to the use of a composition in preparing a drug for treating diabetic nephropathy in patients. Background technology:

[0002] According to the "Guidelines for the Prevention and Treatment of Diabetic Kidney Disease in China (2021 Edition)", diabetic kidney disease (DKD) refers to chronic kidney disease caused by diabetes, mainly manifested by urine albumin / creatinine ratio (UACR) ≥ 30 mg / g and / or estimated glomerular filtration rate (eGFR) < 60 ml·min-1·(1.73 m 2 )- 1 The Guidelines recommend that patients with type 1 diabetes and type 1I diabetes with a course of more than 5 years should undergo urine albumin / creatinine ratio (UACR) testing and evaluation at the time of diagnosis to detect diabetic nephropathy (DKD) at an early stage, and should be screened at least once a year thereafter.

[0003] Clinically, diabetic nephropathy is divided into stages. The early stage of diabetic nephropathy is also called "persistent microalbuminuria stage". Urinary albumin (UAE) continues to rise to 20-200μg / min, resulting in microalbuminuria. However, if the disease progresses to the clinical diabetic nephropathy stage, persistent large amounts of albuminuria (UAE>200μg / min) or proteinuria greater than 500mg / 24 hours may occur. About 30% of patients may develop nephrotic syndrome. The patient has entered the clinical diabetic nephropathy stage. The disease often progresses progressively. If it is not actively controlled, it will enter end-stage renal failure, with obvious symptoms of uremia and the need for dialysis treatment. The diabetic population is huge, and a considerable proportion of patients will eventually develop diabetic nephropathy. In clinical practice, there is no effective treatment for diabetic nephropathy except symptomatic treatment. Although there have been studies and reports on using crude cobra venom to reduce 24-hour proteinuria in rats with diabetic nephropathy, the components of crude cobra venom are complex and varied. Known components include neurotoxins, cytotoxins, cardiotoxins, nerve growth factors, hemolysins (DLPs), CVA proteins, membrane active polypeptides, cobra venom factors, and other components, such as alkaline phosphamonoesterase, phosphodiesterase, acetylcholinesterase, L-amino acid oxidase, ribonucleases, proteases, etc. For crude cobra venom, mixed toxins will pose certain risks to clinical safety, and there is no clear real effective ingredient; at the same time, 24-hour proteinuria is not a real standard for the diagnosis and healing of diabetic nephropathy, and generally when diabetic patients have 24-hour proteinuria exceeding the normal range, the patient has entered the progressive stage of clinical diabetic nephropathy, and treatment and improvement of the condition become more difficult. Therefore, there have been no reports on the treatment and improvement of the two real diagnostic standards of diabetic nephropathy, urine albumin / creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR). Clinically, once early symptoms of diabetic nephropathy appear, i.e., persistent microalbuminuria, and the urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR) exceed normal values, treatment should be initiated to prevent the progression of the disease. However, there are currently no approved drugs that can truly prevent the progression of diabetic kidney, improve the urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR), or reduce microalbuminuria and / or other microproteinuria. Therefore, the development of a drug that can truly and effectively prevent the progression of diabetic nephropathy as soon as possible has become a clinical problem that needs to be urgently addressed. Summary of the invention:

[0004] The present invention relates to an application of a composition in preparing a drug for treating diabetic nephropathy, characterized in that the composition comprises a screened cobra cardiotoxin (Naja cardiotoxin) with a specific amino acid sequence, and is used for treating diabetic nephropathy in the early stage of patients with diabetic nephropathy once the urine albumin / creatinine ratio (UACR) is ≥30mg / g and (or) the estimated glomerular filtration rate (eGFR) is <60ml·min- 1 ·(1.73m 2 )- 1 The invention provides a pharmaceutical composition of a selected monomeric molecule of cobra cardiotoxin that can improve the urine albumin / creatinine ratio (UACR) and the estimated glomerular filtration rate (eGFR), and at the same time, the composition also has an effect on reducing urine microalbumin and some other urine microproteins such as immunoglobulin, β2 microglobulin, α1 microglobulin, and transferrin. Our research found that the cobra cardiotoxin with a specific amino acid sequence in the crude cobra venom has a significant therapeutic effect on improving the urine albumin / creatinine ratio, which is the gold standard for the diagnosis of early diabetic nephropathy, and also has a significant therapeutic effect on improving the estimated glomerular filtration rate (eGFR).

[0005] Streptozotocin (STZ)-induced diabetic nephropathy in rats can cause rats to develop typical manifestations of diabetic nephropathy, and the pathological changes in this animal model are similar to those of minimal change disease in human diabetic nephropathy[1].

[0006] The present invention establishes a diabetic nephritis and nephropathy model induced by streptozotocin (STZ) recognized by professionals in the field to observe the therapeutic efficacy of cobra cardiotoxin monomer molecules on diabetic nephropathy.

[0007] In some embodiments, we discovered for the first time that there are nearly 10 different cardiotoxin monomer molecules in each crude cobra venom, and two of them with specific amino acid sequences that have been screened through animal experiments can inhibit streptozotocin (STZ)-induced diabetic nephropathy in rats, specifically by reducing the urine albumin / creatinine ratio and improving the estimated glomerular filtration rate (eGFR). Their mechanism of action is achieved by activating the "cholinergic anti-inflammatory pathway".

[0008] In 2000, Lyudmila and Tracey KJ et al. first discovered that when the body is injured and an inflammatory response is generated to activate the immune system, its immune stimulation signal can be projected to the vagus nerve nucleus through the central nervous system, activating the efferent vagus nerve fibers, causing the peripheral nerve endings to release acetylcholine and bind to the alpha7nicotinic acetylcholinergic receptor (alpha7nicotinic acetylcholinergic receptor, a 7nAChR) with the alpha7 subunit on the immune cells. Then, the intracellular signal inhibits the release of inflammatory cytokines (interleukin-1, IL-1) and tumor necrosis factor-α (tumor necrosis factor-α, TNF-α) to regulate the inflammatory response. [2-8] This pathway that controls the inflammatory response through the efferent nerve-acetylcholine-nicotinic acetylcholine receptor is named the "cholinergic anti-inflammatory pathway" [2]. This is a regulatory mechanism that inhibits autoinflammatory response. Studies have shown that activating the "cholinergic anti-inflammatory pathway" can reduce inflammatory response and improve the prognosis of the disease. [9-11]

[0009] Cobra cardiotoxins all have the commonality of a three-finger protein structure in terms of functional structure, and the three-finger protein structure can selectively bind to various subunits of the acetylcholine receptor. This binding increases the concentration of free acetylcholine in the blood, thereby further activating the "cholinergic anti-inflammatory pathway." Therefore, cardiotoxins all have the commonality of a three-finger protein structure in terms of functional structure, which enables them to achieve consistent regulation of acetylcholine receptors, and acetylcholine receptors are involved in the regulation of immune inflammatory responses. [12-15] This also makes cobra cardiotoxins consistent in treating diabetic nephropathy in rats.

[0010] In some embodiments, the cobra cardiotoxin used is from Chinese cobra neurotoxin or Bengal cobra, and their mature peptide amino acid sequences have a high degree of homology. After optimization, the cardiotoxin (FASTA) sequences are as follows:

[0011] Chinese cobra cardiotoxin

[0012] SEQ ID No.1

[0013] lkcnklvpl fyktcpagkn lcykmfmvat pkvpvkrgci dvcpkssllv kyvccntdrc n

[0014] SEQ ID No.2

[0015] lkcnklipi asktctagkn lcykmfmmsd ltipvkrgci dvcpknsllv kyvccntdrc n

[0016] Bengal cobra cardiotoxin

[0017] SEQ ID No.3

[0018] lkcnklipia sktcpagknl cykmfmmsdl tipvkrgcid vcpknsllvk yvccntdrcn

[0019] SEQ ID No.4

[0020] lkcnklipla yktcpagknl cykmfmvsnk tvpvkrgcid acpknsllvk yvccntdrcn

[0021] Another advantage of the present invention is in production. Because the cobra cardiotoxin monomer molecule disclosed in the present invention has a clear amino acid sequence, it can be produced through genetic engineering, solving the practical problem of scarce snake venom resources. That is, if the cardiotoxin is obtained by separation and purification of natural snake venom, it is easier to achieve quality and purity control because of the clear amino acid sequence in the process, which lays a necessary foundation for the development of drugs of monomer components in snake venom.

[0022] The present invention is further described below in conjunction with specific embodiments, but the following embodiments are not intended to limit the present invention; at the same time, any equivalent replacements in the art made according to the disclosure of the present invention belong to the protection scope of the present invention.

[0023] Implementation Cases:

[0024] Case A: Obtaining the cobra cardiotoxin polypeptide SEQ ID No. 1

[0025] 1. Isolation and purification of crude venom of Chinese cobra

[0026] Dissolve 1g of crude Chinese cobra venom in 25ml of 0.025M ammonium acetate buffer at pH 6.0, centrifuge at low temperature, and take the supernatant; balance a TSK CM-650 (M) column with 0.025M ammonium acetate solution at pH 6.0; after loading, perform two-compartment step gradient elution with 0.1-0.5M and 0.7-1.0M ammonium acetate buffer at pH 5.9, UV detection parameters: 280nm; elution flow rate: 48ml / h; collect various toxin component peaks according to the recorded spectrum, and 11 protein peaks are eluted in the collected solution.

[0027] 2. Perform N-terminal amino acid sequencing on the eluted single peak protein

[0028] The method for determining the amino acid sequence comprises the following steps:

[0029] The eluted single peak protein was purified and desalted using a reverse phase high performance liquid chromatography (RP-HPLC) column (4.6×250mm, VYDACRP-C8, 5μm); the N-terminal amino acid sequence was determined using a protein sequencer; the protein with the N-terminal amino acid lkcnkl obtained by sequencing was analyzed by peptide coverage and Edman degradation method. Then, animal experiments were performed to select the cardiotoxin protein monomers with the best efficacy as the preferred candidate molecules, a total of 2.

[0030] Case B: Use of cobra cardiotoxin peptide SEQ ID No.1 to treat streptozotocin (STZ)-induced diabetic nephropathy in rats

[0031] 1. Experimental animals and groups

[0032] 40 Wistar rats weighing 280±10g were randomly divided into two groups, a normal control group of 10 and a model group of 30. Twenty rats that survived after successful modeling were randomly selected and randomly divided into a cobra cardiotoxin (SEQ ID No1) treatment group of 10, a model group of 10, and a normal control group of 10, totaling three groups, each with 10 rats.

[0033] 2. Modeling method

[0034] The rats in the modeling group ate and drank water normally. Each rat was first intraperitoneally injected with 0.5 ml of Freund's complete adjuvant (CFA). The next day, streptozotocin (STZ) solution was intraperitoneally injected. Before use, 0.1 mmol / L pH 4.5 citrate buffer was prepared into a concentration of 1%, and 55 mg / kg was intraperitoneally injected. One week later, tail vein blood was collected from the modeling group to test blood sugar. Random blood sugar was maintained above 16.7 mmol / L, and urine sugar 3+ to 4+ was considered a successful diabetes model. Then, after 3 months when the urine microalbumin (UALB) of the modeling group rats continued to increase and the albumin to urine creatinine ratio exceeded 30 mg / g, the three groups of rats began to receive different treatments. Ten rats in the normal control group and ten rats in the model group were gavaged with normal saline, and the treatment group was gavaged with 40 ug / kg solution of cardiotoxin (SEQ ID No1), once a day for 16 consecutive weeks.

[0035] 3. Observation indicators and detection methods

[0036] During the treatment, the three groups of rats were constantly collecting urine microalbumin and urine creatinine, and the ratio was always above the normal range. After the last treatment, the rats were placed in a metabolic cage to collect urine for 24 hours, and then blood was collected by decapitation, centrifuged at 3500r / min for 10min, and the supernatant was extracted into a cryopreservation tube and placed in a -80℃ refrigerator for later use. Enzyme-linked immunosorbent assay was used to detect urine microalbumin, and the operation was performed according to the instructions of the ELISA kit. Urine creatinine and blood creatinine were detected using a fully automatic biochemical analyzer.

[0037] 4. Experimental results (Table 1)

[0038] Calculation method:

[0039] Urine microalbumin to urine creatinine ratio (UACR): = urine albumin / urine creatinine

[0040] Estimated glomerular filtration rate (eGFR) = body weight (kg) × (140-age) / 72 × serum creatinine value (mg / L)

[0041] Unit: ml·min -1 ·(1.73m 2 ) -

[0042] Table 1, (SEQ ID No. 1 cardiotoxin was used in the treatment group, 10 rats in each group)

[0043]

[0044] * indicates P < 0.05 compared with the model group.

[0045] a) Urinary albumin (UALB): Compared with the control group, the UALB levels of the two groups of rats after modeling were significantly increased; compared with the model group, the UALB levels of the cobra cardiotoxin group were significantly decreased. * indicates that P < 0.05 between the cobra cardiotoxin group and the model group.

[0046] b) Serum creatinine: Compared with the control group, the serum creatinine of the two groups of rats after modeling was significantly increased; compared with the model group, the serum creatinine of the cobra cardiotoxin group was significantly decreased. * indicates that P < 0.05 compared with the model group.

[0047] c) Urine creatinine: Compared with the control group, the urine creatinine of the two groups of rats after modeling was significantly reduced; compared with the model group, the urine creatinine of the cobra cardiotoxin group was significantly increased. * indicates that P < 0.05 compared with the model group.

[0048] d) Urine microalbumin to urine creatinine ratio (UACR): Compared with the control group, the urine microalbumin to urine creatinine ratio (UACR) of the two groups of rats after modeling was significantly increased; compared with the model group, the urine microalbumin to urine creatinine ratio (UACR) of the cobra cardiotoxin group was significantly decreased. * indicates P < 0.05 compared with the model group.

[0049] e) Estimated glomerular filtration rate (eGFR): Compared with the control group, the estimated glomerular filtration rate (eGFR) of the two groups of rats after modeling was significantly reduced; compared with the model group, the estimated glomerular filtration rate (eGFR) of the cobra cardiotoxin peptide group was significantly increased. * indicates that P < 0.05 compared with the model group.

[0050] References:

[0051] 1. Li Shifen. Study on diabetic nephropathy model of SD rats induced by different methods of streptozotocin. Chinese Journal of Modern Medicine, 2010-14-007

[0052] 2. Tracey KJ. The inflammatory reflex. Nature, 2002, 420(6917): 853-859.

[0053] 3.Tracey, KJet al.Shock and tissue injury induced by recombinanthuman cachectin.Science 234,470–474(1986).

[0054] 4. Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality inmice. Science 285, 248–251 (1999).

[0055] 5.Tracey, KJ et al. Anti-cachectin / TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330, 662–664 (1987).

[0056] 6. Tracey, KJ & Cerami, A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu. Rev. Med. 45, 491–503 (1994).

[0057] 7. Wang Bojie et al., Clinical application of cholinergic anti-inflammatory pathway, Anesthesia and Monitoring Forum, 2005

[0058] 8. Lyudmila V. Borovikova et a1. Vagus nerve stimulation attenuates thesystemic inflammatory response to endotoxin. Nature 2000 405: 458–462

[0059] 9. Zheng Cheng. Study on the protective effect of cholinergic anti-inflammatory pathway in viral myocarditis in mice. Master's thesis of Wenzhou Medical University, 2014.

[0060] 10. Wang Bojie et al., Research progress on cholinergic anti-inflammatory pathway. International Journal of Anesthesiology and Resuscitation, 2006(04)

[0061] 11. Liu Chuiliang et al., Research progress on cholinergic anti-inflammatory pathway. International Journal of Anesthesiology and Resuscitation, 2007.01.018

[0062] 12. Zhou Guowu et al. α7 nicotinic acetylcholine receptor and inflammation regulation. Progress in Modern Biomedicine, 2009, 14.

[0063] 13. Liu Zhigang. α7 nicotinic acetylcholine receptor and the regulation of inflammation. China Medical Guide, 2014, 03

[0064] 14.Hong Wang et al;.Nicotinic acetylcholine receptor a7 subunit is an essential regulator of inflammation NATURE|VOL 421|23JANUARY 2003

[0065] 15.Stéphanie St-Pierre et al;.Nicotinic Acetylcholine ReceptorsModulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production andSurvival.PLOS ONE DOI:10.1371 / journal.pone.0150230February 29,2016

Claims

1. A pharmaceutical composition for use in preparing a drug for treating diabetic nephropathy, wherein the composition contains a therapeutically effective amount of cobra cardiotoxin having a specific amino acid sequence; the amino acid sequence of the cobra cardiotoxin is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The diabetic nephropathy includes albuminuria and renal function damage caused by diabetes.

3. The use according to claim 1, characterized in that The cobra cardiotoxin is separated and extracted from natural snake venom, or synthesized by chemical polypeptide, or produced from a prokaryotic or eukaryotic host using recombinant technology.

4. The use according to claim 1, characterized in that The cobra cardiotoxin with a specific amino acid sequence is combined with other effective ingredients for treating diabetic nephropathy to treat diabetic nephropathy.

5. The use according to claim 1, characterized in that: The composition can be administered orally, sublingually, nasally, rectally, intradermally or transdermally.

6. The use according to claim 1, characterized in that: The composition also contains a pharmaceutically acceptable carrier, which is selected from propylene glycol, hydroxypropyl-β-cyclodextrin, Tween 80, ethanol, hydroxypropyl methylcellulose (HPMC), methylcellulose, polyvinyl pyrrolidone, laurocapram, poloxamer, xanthan gum, carbomer, polyethylene glycol, glycerol, sorbitol, and borneol.

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