Use of pharmaceutical composition in treatment of diabetic nephropathy

Abstract

The present invention relates to a pharmaceutical composition for preparing a drug for treating diabetic nephropathy with a urinary albumin-to-urinary creatinine (urinary albumin / urinary creatinine) ratio exceeding a medically prescribed normal range and / or a renal filtration rate below a medically prescribed normal range. The pharmaceutical composition comprises a cobra postsynaptic neurotoxin. A patient with the diabetic nephropathy is characterized by a urinary albumin-to-urinary creatinine (urinary albumin / urinary creatinine) ratio exceeding a medically prescribed normal concentration range and / or a renal filtration rate below a medically prescribed normal range. The pharmaceutical composition can be used to ameliorate the aforementioned two major clinical symptoms in the patient with the diabetic nephropathy, and belongs to the field of biopharmaceuticals.

Description

Application of pharmaceutical composition in treating diabetic nephropathy Technical Field

[0001] The present invention relates to application of a composition in preparing a medicine for treating diabetic nephropathy, and belongs to the field of biopharmaceuticals. Background Art

[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, primarily manifested by a urine albumin / creatinine ratio (UACR) ≥ 30 mg / g and / or an estimated glomerular filtration rate (eGFR) < 60 ml·min-1·(1.73m2)-1, persisting for more than three months. The Guidelines recommend that patients with type 1 diabetes and type 1I diabetes with a duration of more than five years undergo urine albumin / creatinine ratio (UACR) testing and assessment at the time of diagnosis for early detection of diabetic kidney disease (DKD), and screening should be conducted at least annually thereafter.

[0003] Clinically, diabetic nephropathy is divided into stages. The early stage of diabetic nephropathy, also known as the "persistent microalbuminuria stage," is characterized by persistently elevated urinary albumin (UAE) levels of 20 to 200 μg / min, resulting in microalbuminuria. However, if the disease progresses to the clinical diabetic nephropathy stage, persistent macroalbuminuria (UAE>200 μg / min) or proteinuria greater than 500 mg / 24 hours may occur. About 30% of patients may develop nephrotic syndrome, marking the onset of clinical diabetic nephropathy. The disease is often progressive and, if not actively controlled, will lead to end-stage renal failure with significant uremia symptoms requiring dialysis. Therefore, early treatment of diabetic nephropathy is crucial to the prognosis of the disease.

[0004] Since most patients with diabetic nephropathy have no obvious symptoms in the early stages and the onset is insidious, and when obvious clinical manifestations appear, it has often entered the progressive stage, and it becomes very difficult to reverse the patient's pathological changes. Therefore, early diagnosis and treatment of diabetic nephropathy has become a top priority. The traditional single-indicator detection methods such as 24-hour proteinuria still need to be improved in accuracy and reliability. Although increased urinary albumin excretion is an early signal of worsening renal function in diabetic nephropathy, the traditional urine protein test is easily affected by factors such as urine concentration and sampling time, which will cause certain errors, namely false positives or false negatives. Therefore, a more reliable and accurate method is now used clinically, namely the ratio of urine microalbumin to urine creatinine to reflect urine albumin excretion, that is, urine creatinine is used for correction to eliminate the influence of urine concentration or sampling time on urine albumin detection.

[0005] Because trace albumin is almost entirely reabsorbed in the proximal convoluted tubule, while creatinine is not reabsorbed at all, and urinary creatinine has an excellent correlation with urine concentration and is not affected by renal basement membrane disease, the urine microalbumin to creatinine ratio (UACR) can more accurately reflect the early stage of kidney damage. In clinical tests of renal function in patients with diabetic nephropathy, the urine microalbumin to creatinine ratio (UACR) shows significant differences from the traditional 24-hour proteinuria test, so it is an independent diagnostic indicator different from the 24-hour urine protein test. (1‑5)

[0006] At present, this method has been used clinically for the detection of diabetic nephropathy, and is of great significance for the early diagnosis of diabetic nephropathy. The "Guidelines for the Prevention and Treatment of Diabetic Kidney Disease in China (2021 Edition)" has set the urine albumin / creatinine ratio (UACR) ≥ 30mg / g and / or estimated glomerular filtration rate (eGFR) <60ml·min‑1·(1.73m2)‑1, and lasting for more than 3 months as the gold indicator for diagnosing early diabetic nephropathy. However, there are almost no drugs in clinical practice that can truly improve the ratio of urine albumin to urine creatinine and / or renal filtration rate, so this is an unmet clinical need.

[0007] Although there have been reports on the use of cobra venom to reduce 24-hour proteinuria in diabetic nephropathy rats, the components of cobra venom are complex and varied, including neurotoxins, cytotoxins, nerve growth factor, hemolysin (DLP),

[0008] CVA protein, membrane active polypeptide, cobra venom factor and other components such as alkaline phosphatase, phosphodiesterase, acetyl

[0009] Cholinesterase, L-amino acid oxidase, ribonuclease, proteolytic enzyme, etc. For cobra crude venom, mixed toxins will pose certain hidden dangers to clinical safety, and the real effective ingredients are not clear; at the same time, 24-hour proteinuria is not the real standard for the diagnosis and recovery of diabetic nephropathy, and generally when a diabetic patient has 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 has become more difficult.

[0010] Clinically, once early symptoms of diabetic nephropathy appear, namely persistent microalbuminuria, and one of the two indicators, urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR), exceeds the normal value, 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 nephropathy, improve urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR), or reduce microalbuminuria and / or other microproteinuria. Therefore, the rapid development of a drug that can truly and effectively prevent the progression of diabetic nephropathy has become an urgent clinical issue that needs to be addressed. Summary of the Invention

[0011] To solve the above problems, the present invention provides a pharmaceutical composition for preventing the progression of diabetic nephropathy, reducing urine albumin / creatinine ratio (UACR) and / or increasing estimated glomerular filtration rate (eGFR), and a preparation and use method thereof.

[0012] The present invention relates to the use of a composition in preparing a medicament for treating diabetic nephropathy in a patient, reducing the urine albumin / creatinine ratio (UACR) and / or improving the estimated glomerular filtration rate (eGFR), characterized in that the composition contains cobra postsynaptic neurotoxin, and the patient has a history of diabetic nephropathy.

[0013] The present invention relates to a use of a composition for treating diabetic nephropathy in a patient, reducing the urine albumin / creatinine ratio (UACR) and / or increasing the estimated glomerular filtration rate (eGFR), characterized in that the composition comprises cobra postsynaptic neurotoxin, and the patient has a history of diabetic nephropathy.

[0014] The present invention relates to a composition for treating diabetic nephropathy in a patient, reducing the urine albumin / creatinine ratio (UACR) and / or increasing the estimated glomerular filtration rate (eGFR), characterized in that the composition contains cobra postsynaptic neurotoxin, and the patient has a history of diabetic nephropathy.

[0015] Streptozotocin (STZ) combined with unilateral nephrectomy and high-fat diet induced diabetic nephropathy in rats, which showed typical diabetic nephropathy symptoms. The pathological changes in this animal model were similar to those of minimal change disease in human diabetic nephropathy. (6)

[0016] The present invention establishes a diabetic nephropathy model induced by streptozotocin (STZ) combined with unilateral nephrectomy and high-fat diet, which is recognized by professionals in this field, to observe the therapeutic efficacy of cobra postsynaptic neurotoxin on the urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate of diabetic nephropathy.

[0017] The present invention relates to the use of a composition for preparing a method for treating the urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR) of diabetic nephropathy. The composition comprises a cobra postsynaptic neurotoxin and can provide timely treatment for early-stage kidney disease patients once the urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate (eGFR) are found to be below the normal range, thereby preventing or delaying the progression of kidney disease patients to the development of obvious clinical symptoms and proteinuria.

[0018] Cobra neurotoxins can be divided into presynaptic and postsynaptic neurotoxins (a-neurotoxins). In our experiments, we found that postsynaptic neurotoxins have the function of reducing urine albumin / creatinine ratio (UACR) and (or) increasing estimated glomerular filtration rate. The cobra neurotoxin preparations that have been marketed, such as cobotin, Nyloxin, and cobraxin, are all postsynaptic neurotoxins. Their molecules contain four pairs of disulfide bonds, which gather together to form a dense core. From this core, three rings extend like three fingers, so they are also figuratively called three-finger proteins. They all have the commonality of the three-finger protein structure in terms of function and structure, which also ensures that the cobra postsynaptic neurotoxins have the function of reducing urine albumin / creatinine ratio (UACR) and (or) increasing estimated glomerular filtration rate.

[0019] The therapeutic efficacy of toxins in the treatment of diabetic nephropathy based on urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate

[0020] consistency on.

[0021] Among cobra postsynaptic neurotoxins, those with the amino acid residues lechnqqs at the N-terminus and einccttdrcnn or einccttdrcn at the C-terminus, such as cobotin, have shown enhanced efficacy in inhibiting the urine albumin / creatinine ratio (UACR) and / or elevated estimated glomerular filtration rate in diabetic nephropathy. These neurotoxins, such as cobotin, share a common spatial structure and over 90% amino acid identity. These neurotoxins consist of a polypeptide chain of 61-62 amino acid residues, with significant homology in the composition and relative positions of the amino acid residues. Their functional structures all share a common three-finger protein structure, which contributes to the consistency of these cobra postsynaptic neurotoxins in their therapeutic efficacy in treating diabetic nephropathy with elevated urine albumin / creatinine ratio (UACR) and / or estimated glomerular filtration rate. The amino acid sequences (FASTA) of their mature proteins have the following characteristics:

[0022] SEQ ID No.1

[0023] lechnqqssq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngi einccttdrc nn

[0024] SEQ ID No. 2

[0025] lechnqqssq tpttktcsge tncykkwwsd hrgtiiergc gcpkvkpgvn lnccttdrcn n

[0026] SEQ ID No. 3

[0027] lechnqqsiq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngi einccttdrc nn

[0028] SEQ ID No.4

[0029] lechnqqssq apttktcsge tncykkwwsd hrgtiiergc gcpkvkpgvn lnccrtdrcn n

[0030] Cobra postsynaptic neurotoxin can be extracted from snake venom or obtained through recombinant technology.

[0031] In some embodiments, the cobra postsynaptic neurotoxin in the composition is any cobra postsynaptic neurotoxin.

[0032] In some embodiments, the amino acid sequence of the cobra postsynaptic neurotoxin in the composition is SEQ ID No. 1.

[0033] In some embodiments, the amino acid sequence of the cobra postsynaptic neurotoxin in the composition is SEQ ID No. 2.

[0034] In some embodiments, the amino acid sequence of the cobra postsynaptic neurotoxin in the composition is SEQ ID No. 3.

[0035] In some embodiments, the amino acid sequence of the cobra postsynaptic neurotoxin in the composition is SEQ ID No. 4.

[0036] In some embodiments, the composition comprises, in addition to cobra postsynaptic neurotoxin, at least one of cobra cardiotoxin, cobra phospholipase A2, or both cobra cardiotoxin and cobra phospholipase A2.

[0037] In some embodiments, the composition is administered once daily for 15-120 consecutive days.

[0038] In some embodiments, the composition is administered every 12 hours, twice daily, for 15-120 consecutive days.

[0039] In some embodiments, the dosage of cobra postsynaptic neurotoxin in the composition is 0.5-50 μg / kg administered once.

[0040] In some embodiments, the dosage of cobra postsynaptic neurotoxin administered in the composition is 10 μg / kg once.

[0041] In some embodiments, the dosage of cobra postsynaptic neurotoxin in the composition is 2 μg / kg.

[0042] Jin once.

[0043] In some embodiments, the dosage of cobra postsynaptic neurotoxin administered in the composition is 1 μg / kg once.

[0044] In some embodiments, the composition is in the form of a sublingual film.

[0045] In some embodiments, the composition is in an oral dosage form.

[0046] In some embodiments, the composition is in the form of an injection.

[0047] In some embodiments, the composition is in the form of a nasal spray.

[0048] In some embodiments, the composition is in a transdermal dosage form.

[0049] In some embodiments, the pharmaceutically acceptable excipient is selected from one or more of a protein stabilizer, a membrane excipient, a mucosal permeation promoting agent, a cosolvent, or a solvent.

[0050] In some embodiments, the protein stabilizer is mannitol.

[0051] In some embodiments, the film excipient is propylene glycol, polyethylene glycol, hydroxypropyl beta-cyclodextrin, Tween 80, hydroxypropyl methylcellulose (HPMC), methylcellulose, or xanthan gum.

[0052] In some embodiments, the mucosal permeation promoting agent is laurocapram, poloxamer, borneol, or dextroborneol.

[0053] In some embodiments, the co-solvent is propylene glycol.

[0054] In some embodiments, the diabetic nephropathy is a diabetic patient whose urine albumin / creatinine ratio (UACR) is elevated above the medically normal range and / or whose estimated glomerular filtration rate (eGFR) is lower than the medically normal range.

[0055] Another advantage of the present invention is its production. Because the postsynaptic cobra neurotoxin 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. Even if the neurotoxin is still obtained through the separation and purification of natural snake venom, the clear amino acid sequence in the process makes it easier to control the quality and purity, which lays the necessary foundation for the development of pharmaceuticals based on the monomer components in the snake venom.

[0056] The present invention will be further described below with reference to specific examples, but the following examples 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 shall fall within the scope of protection of the present invention. DETAILED DESCRIPTION

[0057] Example:

[0058] Example 1: Obtaining SEQ ID No. 1 and SEQ ID No. 2 of cobra postsynaptic neurotoxin

[0059] 1. Isolation and purification of crude venom of Chinese cobra

[0060] Dissolve 1 g of crude Chinese cobra venom in 25 ml of 0.025 M ammonium acetate buffer, pH 6.0, centrifuge at low temperature, and collect the supernatant; equilibrate a TSK CM-650(M) column with 0.025 M ammonium acetate solution, pH 6.0; load the column and perform dual gradient elution with 0.1-0.5 M and 0.7-1.0 M ammonium acetate buffer, pH 5.9, using UV detection parameters at 280 nm and an elution flow rate of 48 ml / h; collect peaks of various toxin components according to the recorded spectra, and elute 11 protein peaks in the collected solution.

[0061] 2. Amino acid sequencing of eluted single peak proteins

[0062] The amino acid sequence determination method is to purify and desalt the eluted single-peak protein using reverse-phase high-performance liquid chromatography (RP-HPLC); then, through animal experiments, the neurotoxin protein monomer with the best therapeutic effect is selected as the preferred candidate molecule. Finally, the sequence of the protein with the best therapeutic effect is analyzed using peptide coverage and Edman degradation method, and finally, protein SEQ ID NO 1-4 is obtained.

[0063] Example 2: Treating diabetic nephropathy rats with cobra postsynaptic neurotoxin SEQ ID No. 1 and SEQ ID No. 2

[0064] 1. Experimental Animals and Grouping

[0065] Forty Wistar rats weighing 280 ± 10 g were randomly divided into two groups: a normal control group of 10 and a modeling group of 30. Twenty surviving rats were randomly selected after successful modeling and divided into a postsynaptic cobra neurotoxin (SEQ ID No. 1) treatment group of 10, a model group of 10, and a normal control group of 10, for a total of 30 rats.

[0066] 2. Modeling method

[0067] Model rats were injected with 40 mg / kg of streptozotocin (STZ) via the jugular vein, while 10 normal control rats were injected with normal saline. The STZ-injected rats underwent right nephrectomy on day 10. Two weeks after recovery from surgery, the model rats were fed a high-fat diet, while the normal control rats were fed a basal diet.

[0068] The modeling rats were fed and drank normally. Four weeks later, tail vein blood was drawn for blood glucose testing. Random blood glucose levels remained above 16.7 mmol / L, and urine microalbumin (UALB) levels continued to rise. Three months after the albumin-to-urine creatinine ratio exceeded 30 mg / g, the three groups of rats began receiving different treatments. Ten rats in the control group and ten rats in the model group were gavaged with normal saline. The treatment groups were gavaged with a 20 μg / kg suspension of the neurotoxin (SEQ ID No. 1) in an oral film formulation. This was administered once daily for 16 consecutive weeks. The excipients in the oral film formulation include a protein stabilizer, a film excipient, a mucosal permeation enhancer, a solubilizer, and a solvent.

[0069] 3. Observation indicators and detection methods

[0070] Urine microalbumin and creatinine were collected from the three groups of rats throughout treatment. After the final treatment, the rats were placed in metabolic cages to collect urine for 24 hours. Blood was then collected by decapitation and centrifuged at 3500 rpm for 10 minutes. The supernatant was collected and placed in a cryovial and stored at -80°C. Urine microalbumin was detected using an enzyme-linked immunosorbent assay (ELISA) according to the kit's instructions. Urine and serum creatinine were measured using an automated biochemical analyzer.

[0071] 4. Calculation method and experimental results:

[0072] Urine microalbumin to urine creatinine ratio (UACR): = urine albumin / urine creatinine

[0073] Estimated glomerular filtration rate (eGFR) = body weight (kg) × (140 - age) / 72 × serum creatinine (mg / L)

[0074] Unit: ml min-1 (1.73m2)

[0075] Table 1, (SEQ ID No. 1 neurotoxin was used in the treatment group, 10 rats in each group)

[0076]

[0077]

[0078]

[0079] Use SEQ ID No. 2 to treat diabetic nephropathy rats after modeling according to the method of Example 2

[0080] Table 2, (SEQ ID No. 2 neurotoxin used in treatment groups, 10 rats in each group)

[0081]

[0082] Example 3

[0083] Treat diabetic nephropathy rats after modeling with SEQ ID No. 1 + cobra cardiotoxin (CDX) + cobra phospholipase A2 (PLA2) according to the method of Example 2 above

[0084] Table 3, (SEQ ID No. 1 + cobra cardiotoxin (CDX) + cobra phospholipase A2 (PLA2) for treatment groups, 10 rats per group)

[0085]

[0086] * indicates P < 0.05 compared with the postsynaptic cobra neurotoxin group and the model group

[0087] ** indicates P < 0.01 compared with the postsynaptic cobra neurotoxin group and the model group

[0088] 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 postsynaptic cobra neurotoxin group were significantly decreased.

[0089] Compared with the control group, the blood creatinine of the two groups of rats after modeling was significantly increased; compared with the model group, the blood creatinine of the postsynaptic cobra neurotoxin group was significantly decreased.

[0090] Urinary 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 postsynaptic cobra neurotoxin group was significantly increased.

[0091] The ratio of urine microalbumin to urine creatinine (UACR) of rats in the two groups after modeling was significantly increased compared with the control group; compared with the model group, the ratio of urine microalbumin to urine creatinine (UACR) of rats in the postsynaptic cobra neurotoxin group was significantly decreased.

[0092] The estimated glomerular filtration rate (eGFR) of the two groups of rats after modeling was significantly reduced compared with the control group; compared with the model group, the estimated glomerular filtration rate (eGFR) of the postsynaptic cobra neurotoxin peptide group was significantly increased.

[0093] References:

[0094] 1.Quick Reference on UACR&GFR, In Evaluating Patients with Diabetes for Kidney Disease.National institute of diabetes and digestive and kidney disease.

[0095] 2. Zhan Qiuyue et al., Diagnostic significance of urine microalbumin to urine creatinine ratio in early diabetic nephropathy. Diabetes New World, September 2023

[0096] 3. Meng Suling et al., Amyloid A, Cystatin C, and Urinary Microalbumin to Creatinine Ratio for Early Diabetic Nephrology

[0097] Diagnostic value analysis of the disease. Diabetes New World October 2023

[0098] 4.Elena A.Christofides et al;.Optimal Early Diagnosis and Monitoring of Diabetic Kidney Disease in Type 2Diabetes Mellitus:Addressing the Barriers to Albuminuria Testing.J Prim Care Community Health.2021 Jan-Dec.doi:10.1177 / 21501327211003683

[0099] 5. Yang Xuemei et al., Effects of Yishen Decoction Modified According to Symptoms on Renal Blood Flow and Urine Albumin-Cratinine Ratio in Patients with Chronic Glomerulonephritis. Medical and Health Science and Technology, Special Topic: Traditional Chinese Medicine, Classification Number: R277.5

[0100] 6. Li Shifen. Study on diabetic nephropathy model in SD rats induced by different methods of streptozotocin. Chinese Journal of Modern Medicine, 2010-14-007.

[0101] Type your sequence listing free description paragraph here.

Claims

1. A composition for treating diabetic nephropathy wherein the ratio of urine albumin to urine creatinine (urine albumin / urine creatinine) exceeds The composition is used in medicine for the treatment of renal insufficiency in the medically prescribed normal range and / or renal filtration rate lower than the medically prescribed normal range. The composition contains a therapeutically effective dose of cobra neurotoxin and pharmaceutically acceptable excipients.

2. The use according to claim (1), characterized in that The cobra neurotoxin in the pharmaceutical composition is a postsynaptic neurotoxin (a-neurotoxin), including short-chain and / or long-chain postsynaptic neurotoxins, preferably, they are cobra postsynaptic neurotoxins having the amino acid sequence of the mature protein shown in SEQ ID No.1-SEQ ID No.4; or respectively with SEQ ID No.1-SEQ ID No. The cobra postsynaptic neurotoxin in 4 has a mature protein with 90% or more homology, and the function of the mature protein is the same or similar to that of the cobra postsynaptic neurotoxin with the amino acid sequence shown in SEQ ID No.1 to SEQ ID No.

4.

3. The use according to claim (1), further characterized in that: The pharmaceutical composition also includes the combined use of cobra postsynaptic neurotoxin and other drugs for treating diabetic nephropathy to reduce the ratio of urine albumin to urine creatinine or increase renal filtration rate. The other drugs for treating diabetic nephropathy include cobra cardiotoxin and cobra phospholipase A2.

4. The use according to claim (1), characterized in that The pharmaceutically acceptable excipients in the pharmaceutical composition are selected from one or more of protein stabilizers, membrane excipients, mucosal permeation promoting agents, cosolvents or solvents.

5. The use according to claim (1), further characterized in that the protein stabilizer in the pharmaceutically acceptable excipients is mannitol, the membrane excipients are propylene glycol, polyethylene glycol, hydroxypropyl β-cyclodextrin, Tween-80, hydroxypropyl methylcellulose (HPMC), methylcellulose, xanthan gum, the mucosal permeation promoting agent is laurocapram, poloxamer, borneol, dextroborneol, and the cosolvent is propylene glycol.

6. The use according to claim (1), characterized in that The dosage form of the pharmaceutical composition is an oral dosage form, a sublingual dosage form, a nasal dosage form, a transdermal dosage form or an injection dosage form.

7. The use according to claim (1), characterized in that The dosage of the cobra postsynaptic neurotoxin in the pharmaceutical composition ranges from 0.5 μg / kg to 50 μg / kg per time, preferably 1-3 μg / kg per time; the composition is administered 1-3 times a day.

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