Application of hexacosanol in preparation of medicine for relieving and / or treating chronic kidney disease

Abstract

The invention discloses an application of hexacosanol in preparation of a medicine for relieving and / or treating chronic kidney disease. Relates to the technical field of medicine products. It is found and proved for the first time that hexacosanol can effectively reduce serum creatinine (Scr) and urea nitrogen (BUN) levels of chronic kidney disease model animals and improve kidney pathological injuries, and shows a remarkable kidney protection effect. Besides, the invention provides a series of products containing hexacosanol, including pharmaceutical compositions for treating and / or relieving chronic kidney diseases and health products for assisting in improving and / or maintaining kidney health, and brand-new and safe natural active ingredient choices are provided for treatment and auxiliary management of chronic kidney diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical product technology, and more specifically to the use of hexacosanol in the preparation of medicaments for relieving and / or treating chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a clinical syndrome characterized by progressive damage to the structure and function of the kidneys, caused by various etiologies. Its progression is insidious, with few or no early symptoms, and many patients already exhibit significant decline in kidney function at the time of diagnosis. The final stage of CKD is end-stage renal disease (ESRD), requiring renal replacement therapy (such as dialysis or kidney transplantation), placing a heavy burden on patients, their families, and the social healthcare system. Currently, clinical management strategies for CKD primarily focus on controlling the underlying disease (such as diabetes and hypertension), slowing the decline in kidney function, and managing complications (such as anemia, mineral and bone metabolism abnormalities, and cardiovascular disease). Commonly used medications include angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor antagonists (ARBs), and sodium-glucose cotransporter 2 inhibitors (SGLT2i). However, existing therapies have certain limitations: First, the dosage of some drugs needs careful adjustment in patients with renal insufficiency, and adverse reactions may occur; second, patients often need to use multiple drugs in combination, increasing the risk of drug interactions and overall side effects; finally, existing treatments have limited effectiveness in reversing or significantly delaying key pathological processes such as renal fibrosis. Therefore, developing novel, highly effective, and safe therapeutic drugs or adjuvant interventions, especially active substances derived from natural products, has become an important direction in the current CKD research field.

[0003] Polyeicosanols are a class of long-chain fatty primary alcohols, widely found in lipids such as plant waxes and insect waxes in nature. Octacosanol, as a representative member of this family, has received considerable research attention for its physiological activities such as regulating blood lipids and combating fatigue. However, research and development on hexacosanol (n-hexacosanol, C26H54O), another important member of the polyeicosanol family, has lagged behind. Hexacosanol is abundant in nature, often existing in bound form in rice bran wax, sugarcane wax, sorghum husk wax, and various plants such as Artemisia annua. Notably, hexacosanol is the main component of polyeicosanols in the young leaves and seedlings of grains such as barley, wheat, and buckwheat, accounting for over 60%. Although some literature reports the extraction of hexacosanol from wheat seedlings and other plants, to date, there has been no systematic and in-depth research or reporting on its biological activities, particularly its potential efficacy in kidney protection, and its value has not been fully recognized and utilized.

[0004] Therefore, how to develop an active substance derived from natural products to effectively alleviate and treat chronic kidney disease is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the use of hexacosanol in the preparation of medicaments for alleviating and / or treating chronic kidney disease, thus successfully achieving the treatment of chronic kidney disease.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] The primary objective of this application is to provide the use of hexacosanol in the preparation of medicaments for alleviating and / or treating chronic kidney disease.

[0008] As a preferred technical solution, the drug has the following therapeutic effects:

[0009] (1) Used to lower serum creatinine levels in patients with chronic kidney disease;

[0010] (2) Used to reduce blood urea nitrogen levels in patients with chronic kidney disease;

[0011] (3) Reduce kidney damage and improve renal tubular and / or glomerular lesions;

[0012] (4) Reduce kidney inflammation;

[0013] (5) Reduce the degree of renal interstitial fibrosis.

[0014] Another object of this application is to provide: a pharmaceutical composition for relieving and / or treating chronic kidney disease, comprising a therapeutically effective amount of hexacosanol as an active ingredient; said therapeutically effective amount is 32 mg / kg / day body weight.

[0015] As a preferred technical solution, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0016] As a preferred technical solution, the pharmaceutical composition is formulated into any pharmaceutically acceptable dosage form.

[0017] As a more preferred technical solution, the dosage form is an oral preparation, an injection, a transdermal preparation, or a mucosal preparation;

[0018] As a more preferred technical solution, the oral preparation is a tablet, capsule, granule, powder, or oral liquid; the injection is an injection solution or lyophilized powder for injection.

[0019] As a preferred technical solution, the pharmaceutical composition further comprises at least one other active agent for treating chronic kidney disease and / or its complications.

[0020] As a preferred technical solution, the chronic kidney disease described in the above applications or in the above pharmaceutical compositions is chronic renal failure or chronic interstitial nephritis.

[0021] Another object of this application is to provide: the use of hexacosanol in the preparation of health products for the purpose of assisting in improving and / or maintaining kidney health.

[0022] As a preferred technical solution, the health product is a special medical purpose formula product or a nutritional supplement.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0024] Innovation: This invention reveals for the first time a new use of hexacosanol in improving kidney function, protecting the kidneys, and alleviating the progression of chronic kidney disease, thus expanding the application field of this natural ingredient.

[0025] Efficacy: Animal experiments have shown that hexacosanol can significantly reduce serum creatinine and blood urea nitrogen in animals with chronic kidney disease and alleviate pathological damage to kidney tissue, with a clear effect.

[0026] High safety profile: Hexacosanol is a naturally occurring ingredient with good biocompatibility and safety, and the risk of side effects with long-term use is low. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The following parameters were used to describe the appearance and shape of the kidneys in different groups: CK group represents the normal rat group; CKD group represents the chronic kidney disease model group; HEX+CKD group is the hexacosanol treatment group; and IN+CKD group is the cellulose (inulin) treatment group.

[0029] Figure 2 The following is a graph showing the kidney index of rats in different groups; where CK group represents normal rats; CKD represents chronic kidney disease model group; CKD+HEX is hexacosanol treatment group; CKD+IN is cellulose (inulin) treatment group; **** p<0.0001, **p<0.01, * p<0.05.

[0030] Figure 3The graph shows the changes in serum creatinine in rats of different groups; where CK group represents normal rats; CKD represents chronic kidney disease model group; CKD+HEX is the hexacosanol treatment group; CKD+IN is the cellulose (inulin) treatment group; **** p<0.0001, *** p<0.001, * p<0.05.

[0031] Figure 4 The graph shows the changes in serum urea nitrogen in rats of different groups; where CK group represents normal rats; CKD represents chronic kidney disease model group; CKD+HEX is hexacosanol treatment group; CKD+IN is cellulose (inulin) treatment group; **** p<0.0001.

[0032] Figure 5 Here are: HE staining images of kidney pathology in rats from different groups; where CK group represents normal rats; CKD represents chronic kidney disease model group; CKD+HEX is hexacosanol treatment group; and CKD+IN is cellulose (inulin) treatment group.

[0033] Figure 6 The images show Masson staining images of the kidneys of rats in different groups; where CK group represents the normal rat group; CKD represents the chronic kidney disease model group; CKD+HEX is the hexacosanol treatment group; and CKD+IN is the cellulose (inulin) treatment group. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] Determination of rat kidney morphology and kidney index

[0037] Forty male SD rats, SPF grade, 4 weeks old, and weighing between 18 and 20 g, were obtained from Hangzhou Medical College. The rats were acclimatized for one week under controlled environmental conditions (temperature 20-24℃; relative humidity 40-60%; 12-hour light / dark cycle). The rats were then randomly divided into four groups as follows:

[0038] Normal rat group (CK): 10 rats;

[0039] Chronic kidney disease model group (CKD): 10 animals;

[0040] Hexadecanool treatment group (HEX): 10 animals;

[0041] Cellulose (inulin) treatment group (IN): 10 animals.

[0042] Except for the CK group, the other three groups were administered adenine via gavage to establish a CKD model for two weeks. The dosage of 2.5% adenine was 250 mg / kg / day / mouse. After two weeks, the successful establishment of the model was verified. Then, intervention was carried out using hexacosanol at a dose of 200 mg / kg body weight / day and inulin at a dose of 5 g / kg body weight / day. After three weeks of intervention, renal function was measured using the instructions for use of the creatinine reagent kit and the blood urea nitrogen reagent kit (Nanjing Jiancheng). The weight of the rats was measured before sacrifice, and the kidneys of the rats were harvested after sacrifice. Figure 1 ) and measure kidney weight, calculate kidney index ( Figure 2 ).

[0043] Kidney index = kidney weight / rat body weight × 100%.

[0044] Results Analysis: The results are as follows Figure 1 As shown, the kidneys of rats in the CK group were reddish-brown in color and had a smooth and shiny surface; the kidneys of rats in the CKD model group showed compensatory enlargement, were whitish in color and had a granular texture on the surface; the kidney morphology of rats in the HEX group and IN group was improved, the color gradually became reddish, the swelling was relieved and the volume was reduced, with the improvement effect of the HEX group being better than that of the IN group.

[0045] like Figure 2 As shown, the kidney index of rats in the CKD model group was significantly higher than that in the CK group (P < 0.0001). The kidney index of rats in the HEX group and IN group was significantly lower than that in the CKD model group (P < 0.01, P < 0.05), indicating that the intervention measures have a certain positive effect on improving kidney morphology and reducing kidney damage.

[0046] Human equivalent dose (HED) = Animal dose × (Animal weight / Human weight)^0.33 ÷ Interspecies coefficient

[0047] Specific calculations:

[0048] Standard weight for rats: 200g (0.2kg)

[0049] Standard human body weight: 60kg

[0050] Interspecies coefficient from rat to human: 6.2

[0051] The equivalent dose for humans is approximately 200 × (0.2 / 60)^0.33 ÷ 6.2 ≈ 32 mg / kg / d. Therefore, when converting the effective dose in mice to the equivalent dose in humans, the equivalent dose is 32 mg / kg / d.

[0052] Example 2

[0053] Measurement of renal function in rats

[0054] (1) Determination of serum creatinine (Scr) content

[0055] Serum from rats in different groups in Example 1 was collected, and the Scr content was determined by the sarcosine oxidase method according to the kit instructions (Table 1).

[0056] Table 1. Scr reagent kit operation steps

[0057] Note: K is the dilution factor, calculated using the following formula:

[0058] K = (sample volume + enzyme solution A volume) / (sample volume + enzyme solution A volume + enzyme solution B volume) = 186 / 246.

[0059] The formula for calculating creatinine content is as follows:

[0060] Creatinine content (μmol / L) = (ΔA measured - ΔA blank) / (ΔA standard - ΔA blank) × C 标准 In the formula, C 标准 The standard solution concentration is 442 μmol / L.

[0061] (2) Determination of blood urea nitrogen (BUN) content

[0062] Serum from rats in different groups in Example 1 was collected, and the BUN content was determined by the diacetyl oxime colorimetric method, according to the kit instructions (Table 2).

[0063] Table 2. BUN Reagent Kit Operation Procedure

[0064] The formula for calculating blood urea nitrogen content is as follows:

[0065] Blood urea nitrogen level (mmol / L) = (A measurement - A blank) / (A standard - A blank) × C 标准 ×N

[0066] In the formula, C represents the standard solution concentration of 10 mmol / L (280.1 mg / L);

[0067] N is the dilution factor of the sample before testing.

[0068] Results Analysis: Based on Figure 3 , 4It was found that the levels of blood urea nitrogen and serum creatinine in the CKD model group were significantly higher than those in the CK group (P < 0.0001). After HEX and IN intervention, the levels of blood urea nitrogen and serum creatinine in the intervention group showed a decreasing trend, and the levels of serum creatinine and blood urea nitrogen in the HEX group decreased significantly (P < 0.001, P < 0.0001). This indicates that HEX intervention effectively improved renal function in CKD model rats and has a positive effect on the treatment of CKD.

[0069] Example 3

[0070] Observe the pathological morphology of rats

[0071] I. Kidney HE staining

[0072] (1) Embedded sections

[0073] This embodiment uses the conventional paraffin embedding and sectioning method to process the sample. The specific operation is as follows:

[0074] 1) After fixation, the tissue blocks are rinsed in a water tank with running water for 4 hours to complete the initial cleaning;

[0075] 2) Next, perform gradient ethanol dehydration, immersing the tissue pieces in 70% ethanol for 2 hours, 80% ethanol overnight, 90% ethanol for 2 hours, and anhydrous ethanol I and II for 1 hour each (considering that 100% alcohol will embrittle the tissue, the soaking time in anhydrous ethanol is shortened).

[0076] 3) After dehydration, the tissue blocks are placed in xylene solution for clearing treatment for about 30 minutes, and then transferred to a container containing a mixture of xylene and paraffin. They are then immersed in a 60°C oven for 2 hours to ensure that the tissue is completely immersed in paraffin.

[0077] 4) After the paraffin melts, embed the tissue block in the center of the metal frame, let it cool to form a wax block, trim the wax block and cut it into 5 μm thick slices using a microtome.

[0078] 5) Spread the thin slices fully in warm water, then transfer them to a glass slide to dry, and bake them in a 60°C oven for 2 hours.

[0079] (2) Dewaxing and preliminary dehydration of sections

[0080] After the sections are dried, they are sequentially placed in containers containing xylene I and xylene II, and immersed for 15 minutes each to dewax the sections. After dewaxing, the sections are placed in containers containing anhydrous ethanol I and anhydrous ethanol II, and immersed for 5 minutes each. Subsequently, for further dehydration, the sections are sequentially immersed in 95%, 85%, and 75% ethanol solutions, immersing for 2 minutes each time (residual liquid must be drained each time the sections are transferred).

[0081] (3) Staining

[0082] Staining operation was performed on the sections that had completed dewaxing and preliminary dehydration: First, the sections were immersed in hematoxylin staining solution for 5 min, then soaked in distilled water for 5 min, and then placed in 1% hydrochloric acid alcohol for 3 s. Then the sections were placed in a water bath, rinsed with running water for 20 min, soaked in distilled water for 2 min, and finally soaked in eosin staining solution for 3 min (the residual liquid should be drained each time the sections were transferred).

[0083] (4) Secondary dehydration, clearing and sealing

[0084] After staining, the sections need to be dehydrated again. Immerse the sections sequentially in 75%, 85%, and 95% ethanol solutions for 2 minutes each to remove water from the tissue. Then, immerse the sections sequentially in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, followed by xylene I and xylene II for 10 minutes each, to complete the clearing process. After clearing, drain the liquid from the sections, mount them by adding half a drop of neutral resin, cover with a coverslip, and allow to air dry at room temperature.

[0085] (5) Microscopic examination

[0086] After mounting, the staining effect of the specimen sections was observed using an optical microscope, and images were captured under the microscope. The experimental results are as follows: Figure 5 As shown.

[0087] Results analysis: such as Figure 5 As shown, in the CK group rats, the glomeruli and tubules of the kidneys were neatly arranged, with clear and normal basic structures, and no lesions were observed. In the CKD model group, severe glomerular lesions were observed, with compensatory tubular dilation and notching, a large amount of adenine brown crystals in the tubules, glomerular interstitial edema, enlarged vacuoles, and inflammatory cell infiltration. In the HEX and IN groups, the glomerular vacuoles were reduced to varying degrees, the lumen showed a narrowing trend, and the infiltration of inflammatory cells in the renal interstitium was reduced. The amount of adenine brown crystals was also reduced compared to the CKD model group. These results indicate that HEX and IN intervention can effectively slow down kidney damage, improve tubular and glomerular lesions, and reduce the inflammatory response in the kidneys.

[0088] II. Masson staining of the kidney

[0089] (1) Embedded sections

[0090] Same as HE staining procedure (1).

[0091] (2) Dewaxing and preliminary dehydration of sections

[0092] Same as HE staining procedure (2).

[0093] (3) Staining

[0094] After dewaxing and dehydrating the specimen sections, wipe them dry. Add Regaud hematoxylin staining solution to the sections, ensuring the staining solution evenly covers the tissue specimen. Observe the staining status of the cell nuclei after 6 minutes of staining. After staining, place the sections on a slide rack, rinse thoroughly with running water, and then immerse in 1% hydrochloric acid alcohol for 3 seconds. Remove and rinse in a water bath for 20 minutes, then immerse in distilled water for 2 minutes, and finally wipe dry with absorbent paper. Add Ponceau S and Acid Fuchsin staining solution to the sections, ensuring the tissue is completely covered with the staining solution. After 1 minute, rinse with 0.2% glacial acetic acid aqueous solution, wipe dry, add Ponceau S and Acid Fuchsin staining solution again, and rinse with 0.2% glacial acetic acid aqueous solution after 1 minute. Add 1% phosphomolybdic acid aqueous solution to the sections for differentiation for 5 minutes, then counterstain with aniline blue for 5 minutes, and finally rinse with 0.2% glacial acetic acid aqueous solution.

[0095] (4) Secondary dehydration, clearing and sealing

[0096] After staining, place the sections in 95% ethanol for 1 min. The clearing and mounting steps are the same as the HE staining steps (4).

[0097] (5) Microscopic examination

[0098] Same as HE staining step (5), experimental results are as follows Figure 6 As shown.

[0099] Results analysis: such as Figure 6 As shown, the renal tubular basement membrane and collagen fibers are stained blue, while the cytoplasm and erythrocytes are red. In the CK group, the kidney tissue structure of rats was intact, with no obvious blue areas between the renal interstitium and almost no fibrosis deposition. Compared to the CK group, the blue areas representing fibrosis in the CKD model group rat sections were significantly increased, indicating excessive deposition of extracellular matrix fibers in the kidneys and more severe renal interstitial fibrosis damage, reflecting the typical pathological characteristics of CKD renal interstitial fibrosis. Compared to the CKD model group, the blue areas in both the HEX and IN intervention groups were reduced, and the degree of renal interstitial fibrosis was reduced to varying degrees.

[0100] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of hexacosanol in the preparation of drugs for relieving and / or treating chronic kidney disease.

2. The application according to claim 1, characterized in that, The drug has the following therapeutic effects: (1) Used to lower serum creatinine levels in patients with chronic kidney disease; (2) Used to reduce blood urea nitrogen levels in patients with chronic kidney disease; (3) Reduce kidney damage and improve renal tubular and / or glomerular lesions; (4) Reduce kidney inflammation; (5) Reduce the degree of renal interstitial fibrosis.

3. A pharmaceutical composition for relieving and / or treating chronic kidney disease, characterized in that, It contains a therapeutically effective amount of hexacosanol as the active ingredient; the therapeutically effective amount is 32 mg / kg / day body weight.

4. The pharmaceutical composition according to claim 3, characterized in that, It also includes pharmaceutically acceptable carriers and / or excipients.

5. The pharmaceutical composition according to any one of claims 3-4, characterized in that, It is formulated into any pharmaceutically acceptable dosage form.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form is an oral preparation, an injection, a transdermal preparation, or a mucosal preparation; The oral preparation is a tablet, capsule, granule, powder, or oral liquid; the injectable preparation is an injection solution or lyophilized powder for injection.

7. The pharmaceutical composition according to any one of claims 3-6, characterized in that, It also contains at least one other active agent used to treat chronic kidney disease and / or its complications.

8. The chronic kidney disease described in the application of any one of claims 1-2 or in the pharmaceutical composition of any one of claims 3-7 is chronic renal failure or chronic interstitial nephritis.

9. The use of hexacosanol in the preparation of health products for the purpose of assisting in the improvement and / or maintenance of kidney health.

10. The application as described in claim 9, characterized in that, The health products mentioned are special medical purpose formula products or nutritional supplements.

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