Application of poecilobdella manillensis in preparation of medicine for treating renal injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of poecilobdella manillensis in preparation of a medicine for treating renal injury. The invention provides a new application of poecilobdella manillensis in preparation of a medicine for treating renal injury, serum biochemical index detection proves that the poecilobdella manillensis can effectively reduce serum creatinine and blood urea nitrogen levels, increase serum albumin and total protein content, remarkably improve glomerular filtration function and protein metabolism function, and improve renal function. And the improvement degree of the traditional Chinese medicine composition is superior to that of a traditional Chinese medicine contrast. Although the curative effect is slightly poorer than that of a chemical drug group, the safety advantage is remarkable. The poecilobdella manillensis can comprehensively regulate renal function metabolism indexes, relieve kidney injury and avoid common side effects of chemical drugs, provides a new choice for clinical treatment of kidney injury, and is especially suitable for chronic kidney disease patients needing long-term treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Hirudo medicinalis in the preparation of drugs for treating kidney injury. Background Technology

[0002] Kidney injury is a shorthand for kidney damage. Mild cases are usually asymptomatic, while severe cases mainly present with symptoms such as hematuria, pain, fever, and even shock. The causes are mainly direct or indirect violent injuries (such as gunshot wounds, knife wounds, stab wounds, kidney surgery injuries, falls, car accidents, and other violent attacks) and pathological injuries (such as kidney stones, kidney tumors, and chronic nephritis).

[0003] Currently, treatment options for kidney injury mainly involve surgery or symptomatic treatment. Surgery is only used when the kidney damage is life-threatening. However, regardless of the surgical method used, it causes serious damage to the kidneys and the body, and in severe cases, kidney removal may be necessary. Therefore, symptomatic treatment is the primary approach, depending on the patient's kidney damage and symptoms. However, current treatment options are very limited, mainly relying on rest, anti-infection measures, hemostasis, pain relief, and nutritional support, ultimately depending on the patient's own recovery ability to cure the disease.

[0004] Hirudo medicinalis (Poecilobdella manillensis Lesson), a traditional Chinese medicine, has long been primarily used in the field of promoting blood circulation and removing blood stasis. Its pharmacological effects, such as anticoagulation, antithrombosis, and improvement of microcirculation, have been extensively studied and applied in the treatment of cardiovascular and cerebrovascular diseases. Current technology has confirmed that Hirudo medicinalis extract contains multiple active ingredients, including hirudin, hirudin analogues, and fibrinolytic enzymes. These components exert their therapeutic effects through mechanisms such as inhibiting thrombin activity, dissolving fibrin, and inhibiting platelet aggregation. Hirudo medicinalis has also been used as a directly orally administered traditional Chinese medicine to treat diseases such as amenorrhea due to blood stasis and hemiplegia due to stroke.

[0005] Patent CN115006431A discloses the application of Hirudo medicinalis in the preparation of a drug for treating vitiligo and a pharmaceutical formulation thereof. This patent provides a novel use of Hirudo medicinalis in the preparation of a drug for treating vitiligo, offering a new natural medicine for treating the refractory disease vitiligo. However, this patent does not cover the application of Hirudo medicinalis in the treatment of kidney damage, thus limiting its applicability.

[0006] Patent CN117362415A relates to the field of hirudin extraction and processing technology, specifically to emetic solution from *Hirudo medicinalis* and its application in large-scale hirudin extraction. This patent achieves a green and environmentally friendly method for mass production of hirudin from *Hirudo medicinalis* saliva by improving the breeding method of *Hirudo medicinalis*. However, this patent does not cover the application of hirudin in the treatment of kidney damage, thus limiting its applicability.

[0007] However, research on the application of Hirudo medicinalis in the field of kidney disease is currently lacking, especially regarding its efficacy in the treatment of kidney injury. Kidney injury is a common clinical condition with a complex pathogenesis involving multiple pathological processes such as inflammatory response, oxidative stress, and fibrosis, and existing treatment methods still have limitations. Summary of the Invention

[0008] To address the aforementioned shortcomings, this invention provides a method that utilizes leeches, a natural medicinal material, as a drug source, resulting in fewer toxic side effects and higher safety.

[0009] The technical solution of this invention is as follows: On the one hand, the present invention provides the use of Hirudo medicinalis in the preparation of medicaments for the prevention and / or treatment of kidney injury.

[0010] Specifically, the kidney injury can be chronic kidney injury or acute kidney injury.

[0011] Preferably, the kidney injury is chronic kidney injury.

[0012] Specifically, the leech is the main or sole active ingredient in the drug.

[0013] Specifically, the drugs include, but are not limited to: live Hirudo medicinalis, dried Hirudo medicinalis products, freeze-dried Hirudo medicinalis products, freeze-dried Hirudo medicinalis powder, Hirudo medicinalis saliva or extracts.

[0014] More specifically, the extract may be obtained by extracting one or more of the following: live Hirudo medicinalis, dried Hirudo medicinalis, freeze-dried Hirudo medicinalis, freeze-dried Hirudo medicinalis powder, or Hirudo medicinalis saliva.

[0015] Specifically, the drug is lyophilized leech powder.

[0016] More specifically, the lyophilized leech powder generally also includes a lyophilization protectant.

[0017] Preferably, the lyophilization protectant includes, but is not limited to, pH buffers, fillers, sugars, nonionic surfactants, ligands, etc. The pH buffer includes, but is not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, and acetic acid or its salts. The fillers include, but are not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugars can be disaccharides, such as sucrose or trehalose, any one or more. The nonionic surfactants include, but are not limited to, Tween, such as Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. Specifically, the lyophilization protectant may also include albumin, polyethylene glycol, etc.

[0018] Specifically, the medicine also includes one or more of the following: *Hedyotis diffusa*, *Edelweiss*, *Salvia miltiorrhiza*, *Spatholobus suberectus*, *Ligusticum chuanxiong*, *Scutellaria baicalensis*, *Forsythia suspensa*, or *Isatis indigotica*.

[0019] Preferably, the medicine also includes *Hedyotis diffusa* and *Edelweiss*.

[0020] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0021] Preferably, the pharmaceutically acceptable excipient is any one or more of the following: excipient, stabilizer, diluent, binder, preservative, lubricant, and antioxidant.

[0022] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0023] Specifically, the dosage forms of the drug include, but are not limited to, tablets, liquids, capsules, powders, suppositories, granules, oral liquids, pills, sprays, or liniments.

[0024] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0025] Specifically, the dosage of the drug, calculated based on the antithrombin activity of Hirudo medicinalis, is 0.1-100,000 units per day.

[0026] Specifically, the drug can also be used in combination with other drugs for the prevention and / or treatment of kidney damage.

[0027] More specifically, the combined use can be simultaneous or sequential.

[0028] The beneficial effects of this invention are as follows: (1) It can effectively reduce serum creatinine levels and significantly increase serum albumin and total protein content, indicating that it has a good kidney protective effect; in terms of blood urea nitrogen index, leech treatment also showed a significant reduction effect. These changes suggest that glomerular filtration function and protein metabolism function have been significantly improved.

[0029] (2) This invention uses leeches, a natural medicinal material, as the drug source, which has fewer toxic side effects and higher safety.

[0030] (3) The leech in this invention has significant therapeutic effects, can effectively treat kidney damage, improve kidney function, and provide new treatment options for patients with kidney damage.

[0031] (4)The source of Poecilobdella manillensis in the present invention is abundant, and the resources of Poecilobdella manillensis are widely distributed, which is conducive to large-scale production. Description of the Drawings

[0032] Figure 1 It is the pathological result diagram of the blank group.

[0033] Figure 2 It is the pathological result diagram of the model control group.

[0034] Figure 3 It is the pathological result diagram of the Poecilobdella manillensis group.

[0035] Figure 4 It is the pathological result diagram of the traditional Chinese medicine positive control group.

[0036] Figure 5 It is the pathological result diagram of the chemical medicine positive control group. Detailed Embodiments

[0037] The present invention will be further clearly and completely described through embodiments below. The following embodiments are only a part of the embodiments of the present invention, which are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are all conventional experiments unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0038] Example 1 1.1 Model establishment and drug administration (1)Animal model establishment SD rats, SPF grade, the weight range of the animals at the time of purchase: 180 - 220 g, and the weight of the animals in the same batch did not exceed 20% of the average weight. Breeding unit: Spf (Beijing) Biotechnology Co., Ltd., production license number: SCXK(Beijing)2019 - 0010. Collect serum by tail vein blood collection, and detect serum creatinine (CRE enzyme method), urea nitrogen (BUN diacetyl - monoxime method), albumin (ALB), and total protein content (TP) to ensure that the above indicators are normal.

[0039] During the experiment, the model was established by injection 3 times. Except for the blank group, the other groups (model control group, Poecilobdella manillensis group, traditional Chinese medicine positive control group, and chemical medicine positive control group) were injected with 0.1% doxorubicin hydrochloride solution (trade name: doxorubicin hydrochloride for injection; source: Shenzhen Wanle Pharmaceutical Co., Ltd.; 10 mg / vial; batch number: 2023E3) at a dose of 3 mg / kg by tail vein injection for each rat on the first day of model establishment. The blank control group was injected with an equal volume of sodium chloride injection, with 10 rats in each group.

[0040] On the 7th and 14th days after the first model establishment, the second and third tail vein injections of rats were carried out at a dose of 2 mg / kg, and the method and reagent source were the same as the first time.

[0041] Precautions: Doxorubicin should be prepared and used immediately, protected from light, and stored in an ice bath.

[0042] (2) Animal administration Following the third injection of 0.1% doxorubicin hydrochloride solution, the drug was administered orally twice daily. The blank control group and the model control group received sterile purified water; the leech group received 90 mg / kg of lyophilized leech powder; and the positive control group received 892.8 mg / kg of nephritis recovery tablets. -1 The positive control group for chemical drugs was given prednisolone acetate tablets at a dose of 1.24 mg / kg. -1 ; administer continuously for 28 days (4 weeks).

[0043] The preparation method of freeze-dried leech powder is as follows: (1) Freezing treatment: Take fresh leeches (Leech from Qiaocheng District, Bozhou, Anhui Province), rinse them quickly with sterile water 2-3 times, treat them with low temperature anesthesia (0-4℃ standing for 30 min), and then freeze them at -80℃ for 4 hours to obtain frozen leeches.

[0044] (2) Thawing and slicing: Take out the frozen leeches and thaw them at room temperature (or a specified thawing environment) for 0.5 hours; after they are completely thawed, use a precision slicing device to cut them into thin slices of uniform thickness of 0.1 cm.

[0045] (3) Vacuum freeze-drying: The sliced ​​leech slices are laid flat on the freeze-drying tray and placed in a vacuum freeze dryer. The temperature is set to -60℃ and the vacuum degree is 0Pa. The freeze-drying is continued for 24 hours to obtain freeze-dried leech slices.

[0046] (4) Crushing and sieving: The freeze-dried slices are transferred to a high-speed crusher for crushing. After crushing, they are sieved through a 40-mesh standard sieve to remove coarse particles and finally obtain freeze-dried leech powder with uniform particle size.

[0047] 1.2 Detection Indicators 1.2.1 Weight Measurement The body weight of rats was measured before administration (week 2) and after administration (week 6); Method: weighing by balance; Instrument: YP20001 balance.

[0048] 1.2.2 Determination of Biochemical Indicators The levels of creatinine (CRE enzymatic method), albumin (ALB), total protein (TP), and blood urea nitrogen (BUN, diacetyl-oxime method) in the serum of rats were measured before administration (week 2) and after administration (week 6); Instrument: OLYMPUS AU400.

[0049] 1.2.3 Kidney HE staining pathological examination After the above indicators were detected, kidney pathological sections were prepared from the rats. The specific steps are as follows: (1) Anesthetize rats (isoflurane or sodium pentobarbital) and perfuse their hearts (first flush the blood with physiological saline, then fix with 4% paraformaldehyde).

[0050] (2) Quickly remove both kidneys, remove the capsule, and cut along the coronal plane (preserving the cortex, medulla, and renal pelvis structures).

[0051] (3) Fix in 4% paraformaldehyde (24-48 hours, 4℃), then transfer to 70% ethanol for storage.

[0052] (4) Tissue dehydration and embedding, instrument: Leica ASP300S fully automatic dehydrator.

[0053] (5) Slicing: Leica RM2235 manual microtome, slice thickness: 3-4μm.

[0054] (6) Dyeing, dyeing machine: Leica ST5020 automatic dyeing machine.

[0055] 1.3 Results and Analysis Quantitative data are expressed as mean ± standard deviation.

[0056] 1.3.1 Weight The results are shown in Table 1: Table 1. Body weight and rate of change in body weight

[0057] Note: Significant difference in weight change rate relative to the control group; ***: P value < 0.001, indicating extremely significant difference; *: P value < 0.05, indicating significant difference.

[0058] 1.3.2 Determination of Biochemical Indicators The results are shown in Tables 2-5: Table 2. Serum albumin levels

[0059] Note: Significant difference relative to the control group; ***: P value < 0.001, indicating extremely significant difference; **: P value < 0.01, indicating highly significant difference.

[0060] Table 3 Serum creatinine levels

[0061] Note: Significant difference relative to the control group; ***: P value < 0.001 indicates extremely significant difference.

[0062] Table 4. Serum urea nitrogen content

[0063] Note: Significant difference relative to the control group; ***: P value < 0.001, indicating extremely significant difference; **: P value < 0.01, indicating highly significant difference.

[0064] Table 5 Total protein content in serum

[0065] Note: Significant difference relative to the control group; ***: P value < 0.001, indicating extremely significant difference; **: P value < 0.01, indicating highly significant difference.

[0066] The results in Tables 1-5 show that lyophilized leech powder significantly improved renal function during the treatment of kidney injury. Serum biochemical tests revealed that this treatment effectively reduced serum creatinine levels while significantly increasing serum albumin and total protein levels, indicating a good protective effect on the kidneys. Leech treatment also showed a significant reduction in blood urea nitrogen levels, suggesting a significant improvement in glomerular filtration function and protein metabolism.

[0067] Compared with the model group, the renal function indicators in the leech treatment group all significantly recovered to normal levels, and the degree of improvement was significantly better than that in the traditional Chinese medicine control group. Although the efficacy was slightly inferior to that of the chemical drug group, leech showed superior safety characteristics. This multifaceted improvement in renal function may be related to the multi-target mechanism of action of the active ingredients contained in leech, including anti-inflammatory, antioxidant, and tissue repair-promoting effects.

[0068] 1.3.3 Pathological Examination The pathological results of the blank group are as follows: Figure 1 As shown, the glomeruli in the kidney tissue are evenly distributed, and the number of cells and matrix in the glomeruli are uniform. The renal tubular epithelial cells are round and plump, and the brush borders are arranged neatly and regularly. There is no obvious proliferation of connective tissue interstitium between the urinary tubules. No obvious inflammatory changes were observed.

[0069] Pathological results of the model control group are as follows Figure 2 As shown, the kidney tissue is fibrotic, with mild proliferation of interstitial connective tissue and a large number of lymphocytes infiltrating (inflammatory); accompanied by renal tubular atrophy and irregular shape; many protein casts are visible, and eosinophilic material is visible in the tubular lumen; mild dilation of renal tubules is common; a small number of renal tubular epithelial cells show hydropic degeneration, cell swelling, and loose, pale cytoplasm; mild proliferation of matrix in the glomeruli, significant dilation and sclerosis of Bowman's capsule.

[0070] Pathological results of the *Hirudo medicinalis* group are as follows: Figure 3As shown, the kidney tissue showed mild fibrosis, mild proliferation of interstitial connective tissue, and a small amount of lymphocyte infiltration; many renal tubules were atrophied and irregular in shape; many protein casts were visible, and eosinophilic material was visible in the tubular lumen; mild dilation of renal tubules was occasionally seen; a small number of renal tubular epithelial cells showed hydropic degeneration, cell swelling, and loose, pale cytoplasm, without obvious sclerosis.

[0071] Pathological results of the positive control group of traditional Chinese medicine as follows Figure 4 As shown, the kidney tissue exhibits mild fibrosis, mild proliferation of interstitial connective tissue, and lymphocyte infiltration (inflammatory); numerous renal tubules are atrophied and irregular in shape; a small number of protein casts are present, and eosinophilic material is visible within the tubular lumen; mild dilation of renal tubules is occasionally observed; a small number of renal tubular epithelial cells show hydropic degeneration, cell swelling, and loose, pale cytoplasm; the glomerular matrix shows mild proliferation, but no obvious sclerosis is observed.

[0072] Pathological results of the positive control group for chemical drugs are as follows Figure 5 As shown, the glomeruli in the kidney tissue are evenly distributed, and the number of cells and matrix in the glomeruli are uniform; the tubular epithelial cells show hydropic degeneration, cell swelling, and loose, lightly stained cytoplasm; there is a small amount of interstitial proliferation; no obvious inflammatory changes were observed.

[0073] In summary, the trends of various indicators show that Hirudo medicinalis can comprehensively regulate kidney-related metabolic indicators and effectively reduce the degree of kidney damage. This natural medicine improves kidney function while avoiding the common side effects of chemical drugs, providing a new option for the clinical treatment of kidney injury, and is particularly suitable for patients with chronic kidney disease who require long-term treatment.

[0074] Comparative Example A comparative example was set up with reference to Example 1. The difference between the comparative example and Example 1 is shown in Table 6: Table 6

[0075] The method for detecting antithrombin is as follows: Accurately weigh 0.25 g of the lyophilized leech powder to be tested, add 15 mL of 0.9% sodium chloride solution, stir thoroughly, and extract for 30 min to obtain the active ingredient extract of leech. Filter (0.22 μm filter membrane) to obtain the leech active ingredient filtrate. Accurately measure 100 μL of the leech active ingredient filtrate and place it in a test tube. Add 200 μL of tris(hydroxymethyl)aminomethane hydrochloride buffer containing 0.5% bovine fibrinogen (based on coagulation), shake well, and place in a water bath (37℃±0.5℃) for 5 min. Add 5 μL of thrombin solution containing 40 units per mL dropwise (once per minute, gently shaken while adding) until coagulation. Record the volume of thrombin solution consumed and calculate the antithrombin activity of the lyophilized leech powder to be tested. The calculation formula is as follows: The antithrombin activity of Hirudo medicinalis (U) = C1×V1 / C2×V2.

[0076] In the formula, U is the thrombin activity unit per 1g of test sample (U / g), C1 is the mass concentration of thrombin solution (U / mL), C2 is the mass concentration of test sample solution (g / L), V1 is the volume of thrombin solution consumed (μL), and V2 is the amount of test sample solution added (μL).

[0077] The blood urea nitrogen content in rat serum was detected according to Example 1, and the results are shown in Table 7: Table 7

[0078] Note: Significant difference relative to the control group; ***: P value < 0.001, indicating extremely significant difference; **: P value < 0.01, indicating highly significant difference.

[0079] The above results indicate that the reduction in blood urea nitrogen in the leech group was significantly greater than that in the control group, demonstrating that it is superior to other leech products (such as broad-bodied golden thread leech and Japanese medicinal leech) and single anticoagulant components (antithrombin III and hirudin) in protecting renal function and reducing blood urea nitrogen. This suggests that the treatment of kidney injury by leeches does not rely on a single component, but rather on the synergistic effect of multiple components. In contrast, control groups 1-4 only contained some single components or similar components, lacking "multi-component synergy," and therefore their improvement in renal function was far inferior to that of leeches.

[0080] Application Example 1: Freeze-dried leech powder Freeze-dried leech powder is prepared and used in accordance with the Yunnan Provincial Standard for Traditional Chinese Medicine (YPBZ-0199-2013).

[0081] Application Example 2: Hirudo medicinalis oral solution Stimulate leeches with sour fruits to produce saliva. Collect the saliva, add 2-5 times the volume of purified water, centrifuge, discard the precipitate, add appropriate amounts of sucrose and methylparaben to the supernatant, then add more purified water to prepare a solution with an antithrombin activity of 20-30 units per ml. Filter through a 0.45-micron filter and bottle, 10 mL / bottle. Use 1-2 bottles each time.

[0082] Application Example 3: Compound Hirudo Tablets Prescription: 1 part freeze-dried leech powder, 30 parts *Hedyotis diffusa*, 30 parts *Edelweiss*.

[0083] Preparation: Extract the herbs *Hedyotis diffusa* and *Edelweiss* twice with water, collect the liquid, concentrate the liquid into a clear paste, spray dry it into a fine powder, add *Hirudo medicinalis* freeze-dried powder, an appropriate amount of pregelatinized starch, and magnesium stearate to the fine powder, compress into tablets, and the product is ready.

[0084] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. The use of Hirudo medicinalis in the preparation of drugs for the prevention and / or treatment of kidney injury.

2. The application according to claim 1, characterized in that, The kidney injury is either chronic or acute.

3. The application according to claim 2, characterized in that, The kidney injury mentioned is chronic kidney injury.

4. The application according to claim 1, characterized in that, The leech mentioned is the main or only active ingredient in the drug.

5. The application according to claim 4, characterized in that, The drug is live leech, dried leech, freeze-dried leech, freeze-dried leech powder, leech saliva, or extract; the extract is obtained by extracting one or more of live leech, dried leech, freeze-dried leech, freeze-dried leech powder, or leech saliva.

6. The application according to claim 5, characterized in that, The drug in question is lyophilized leech powder.

7. The application according to claim 6, characterized in that, The medicine also includes one or more of the following: *Hedyotis diffusa*, *Edelweiss*, *Salvia miltiorrhiza*, *Spatholobus suberectus*, *Ligusticum chuanxiong*, *Scutellaria baicalensis*, *Forsythia suspensa*, and *Isatis indigotica*.

8. The application according to any one of claims 1-7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients are any one or more of the following: excipients, stabilizers, diluents, binders, preservatives, lubricants, and antioxidants.

10. The application according to claim 9, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

11. The application according to claim 10, characterized in that, The dosage form of the drug is tablets, liquids, capsules, powders, suppositories, granules, pills, sprays, or liniments.

12. The application according to claim 11, characterized in that, The drug can be administered orally, intravenously, locally, intradermally, or subcutaneously.

13. The application according to claim 12, characterized in that, The dosage of the drug, calculated based on the antithrombin activity of Hirudo medicinalis, is 0.1-100,000 units per day.

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