Use of pde5 inhibitors for the preparation of a medicament for lowering uric acid and for renal protection

Abstract

The application belongs to the field of medicine, and specifically discloses application of a PDE5 inhibitor in preparation of a medicine for reducing uric acid and protecting kidneys. Research shows that, through concentration analogy, it is inferred that a PDE5 specific inhibitor is used for reducing uric acid and / or kidney protection, and therefore, the concentration of PDE5 inhibitors such as tadalafil is set to 0.001-0.1 mg / kg. It is found through animal experiments that tadalafil at the dose has a significant effect of reducing uric acid and a kidney protection function, and the dose applied to the human body (adults, body weight 60 kg) is 0.0075 mg-0.75 mg per day. The dose range is far lower than the recommended dosage of the medicine for treating ED, and is also far lower than the use dose of the existing public invention. The application increases the new indication range of the medicine for ED, and is expected to quickly enter clinical use.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of PDE5 inhibitors in the preparation of drugs for lowering uric acid and protecting the kidneys. Background Technology

[0002] Hyperuricemia (HUA) is a typical metabolic disease defined as a fasting serum uric acid level >420 μmol / L for men and >360 μmol / L for women under normal dietary conditions. The incidence of hyperuricemia is influenced by various factors, including genetics, diet, gender, lifestyle, age, medication, and economic development. The global rate of gout caused by hyperuricemia has increased over the past 20 years. Hyperuricemia is not only a major contributing factor to gout but is also closely related to hypertension, diabetes, cardiovascular disease, and uric acid nephropathy. Therefore, hyperuricemia has gradually become a serious public health disease threatening human life.

[0003] Hyperuricemia can be summarized by two main factors: excessive uric acid production and insufficient excretion. Drugs that reduce uric acid production primarily include allopurinol, febuxostat, and topiprostat, which lower serum uric acid concentrations by inhibiting xanthine oxidase activity, thus reducing uric acid synthesis. Drugs that promote uric acid excretion to alleviate hyperuricemia include probenecid, benzbromarone, rasinard, and dotinurad. However, all these drugs have certain side effects. Common side effects of allopurinol include gastrointestinal discomfort such as abdominal pain, diarrhea, nausea, and vomiting; in severe cases, it can cause kidney damage. Febuxostat may cause skin rashes, nausea, and abnormal liver function; most seriously, it increases the risk of cardiovascular events. Benzbromarone commonly causes gastrointestinal reactions such as diarrhea; its most concerning adverse reaction is hepatotoxicity, which may cause liver damage. Probenecid may cause gastrointestinal discomfort, dizziness, headache, and skin rashes; long-term use may affect kidney function. Therefore, developing uric acid-lowering drugs with fewer side effects and lower toxicity is particularly important. Summary of the Invention

[0004] It has been reported that tea has a uric acid-lowering effect, with its main active ingredient being epigallocatechin gallate (EGCG). This study demonstrated that EGCG (12.5 mg / kg, 25 mg / kg, 50 mg / kg) had a significant uric acid-lowering effect in a hyperuricemic mouse model and exhibited significant inhibitory activity against PDE5 in vitro. Tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine ketone derivatives (pyrazolopyrimidine ketone derivatives and their pharmaceutically acceptable salts, their preparation methods and applications, patent number ZL201010501383.2) are specific PDE5 inhibitors. EGCG's effect on PDE5... The half-maximal inhibitory concentration (IC50) of PDE5A is 24-400 times that of the aforementioned PDE5A-specific inhibitors. Therefore, it is estimated that the mouse dosage of the aforementioned PDE5A-specific inhibitor is 0.03-2 mg / kg. Since the metabolic rate of mice is 8-10 times that of humans, this translates to a human (adult, 60 kg) dosage of 0.225 mg-15 mg. The daily human dosage of sildenafil should not exceed 50 mg, and the daily human dosage of tadalafil and vardenafil should not exceed 20 mg. However, in the existing invention (phosphodiesterase type 5 inhibitors for lowering uric acid levels and their uses, application number: 202410405590.X), the dosage of tadalafil in mice is 5 mg / kg, and that of sildenafil is 10 mg / kg. The dosages of the aforementioned invention, when converted to human (adult, 60 kg) dosages, are 37.5 mg / kg and 75 mg / kg, respectively, far exceeding the single-dose human dosage of these drugs.

[0005] Based on previous uric acid-lowering mouse experiments with EGCG and in vitro PDE5 inhibitory activity, this invention, through concentration analogy, deduced the dosage of PDE5-specific inhibitors. Therefore, the concentration of PDE5 inhibitors such as tadalafil was set at 0.001-1 mg / kg. It was found that tadalafil at this dosage has a significant uric acid-lowering effect and renal protective function. In particular, at a dosage of 0.001-0.1 mg / kg, tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives all showed significant uric acid-lowering effects. The dosage for human use (adult, 60 kg) is 0.0075 mg-0.75 mg, which is far lower than the recommended dosage for treating erectile dysfunction (ED) and far lower than the dosages of existing publicly disclosed inventions. This invention expands the scope of indications for ED drugs and is expected to have significant clinical value.

[0006] This invention first provides the application of PDE5 inhibitors in the preparation of drugs for lowering uric acid and protecting the kidneys, wherein the dosage of the PDE5 inhibitor in the drug is 0.000125-0.0125 mg / kg.

[0007] Specifically, the drug is prepared as a single dose at a dosage of 0.0075 mg to 0.75 mg.

[0008] Preferably, the drug is prepared as a single dose with a standard dosage of 0.000125-0.0125 mg / kg, specifically with a standard dosage of 0.0075 mg / kg.

[0009] Specifically, the drug that lowers uric acid is used to treat patients with hyperuricemia, or the drug is used to prevent hyperuricemia; The PDE5 inhibitor is selected from one or more of tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives.

[0010] Preferably, the drug is used to lower uric acid and repair kidney pathological damage induced by hyperuricemia, and the drug is prepared such that the actual daily dose of the PDE5 inhibitor, which is the effective active ingredient in the drug, is 0.000125-0.0125 mg / kg body weight. Experiments have shown that the uric acid-lowering effect at this dose is even better than that at a higher dose of 0.125 mg / kg body weight.

[0011] Preferably, the drug is a sustained-release or controlled-release formulation; the daily release of the PDE5 inhibitor in the drug is 0.000125-0.0125 mg / kg of human body weight.

[0012] Preferably, the drug is a sustained-release or controlled-release drug; based on the weight of an adult weighing 60 kg, the daily release of the PDE5 inhibitor in the drug in an adult is 0.0075 mg-0.75 mg.

[0013] The present invention provides a pharmaceutical composition for treating or preventing hyperuricemia, wherein each unit of the pharmaceutical composition contains a dose of 0.36-0.52 mg of a PDE5 inhibitor and a pharmaceutically acceptable carrier.

[0014] Specifically, each unit of the drug contains 0.42-0.48 mg (e.g., 0.45 mg) of a unit dose of PDE5 inhibitor and a pharmaceutically acceptable carrier.

[0015] More specifically, the pharmaceutical composition is administered orally, preferably in tablet or capsule form; each tablet contains 0.36-0.52 mg of a PDE5 inhibitor, preferably 0.42-0.48 mg (e.g., 0.45 mg).

[0016] Specifically, the PDE5 inhibitor is selected from one or more of tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives.

[0017] Preferably, the pharmaceutical composition is used to lower uric acid and repair kidney pathological damage induced by hyperuricemia, and the pharmaceutical composition is prepared such that the actual daily dose of the PDE5 inhibitor, which is the effective active ingredient in the pharmaceutical composition, is 0.000125-0.0125 mg / kg human body weight.

[0018] Preferably, the pharmaceutical composition is a sustained-release or controlled-release drug, and the daily in vivo release of the PDE5 inhibitor in the pharmaceutical composition is 0.000125-0.0125 mg / kg of human body weight.

[0019] Preferably, the pharmaceutical composition further comprises a soybean globulin-tannic acid-zinc ion complex carrier, wherein the PDE5 inhibitor is encapsulated by the complex carrier to form drug-loaded composite microparticles.

[0020] Preferably, the preparation method of the compound drug is as follows: S1: Oxidized tannins modified soybean globulin Soy globulin was dispersed in distilled water, and the pH of the system was adjusted to 8.5-9.5. The mixture was incubated at 40-45℃ for 1-2 hours until completely dissolved. The resulting solution was boiled and then naturally cooled to room temperature to obtain a soy globulin solution. Tannic acid was dissolved in an aqueous solution with a pH of 8.5-9.5 and oxidized at 40-45℃ in an oxygen-rich environment for 0.5-1.5 hours to obtain an oxidized tannic acid solution. The oxidized tannic acid solution was mixed with the soy globulin solution and incubated at 40-45℃ for 1-3 hours. Then, hydrochloric acid was added to adjust the pH of the system to 7.5 to terminate the reaction. The solution was dialyzed for 20-28 hours in a closed environment using a dialysis bag with a molecular weight cutoff of 10000 Da to remove free tannic acid and obtain the oxidized tannic acid modified soy globulin solution. S2: Drug loading and self-assembly of composite microparticles A PDE5 inhibitor solution was prepared using dimethyl sulfoxide as a solvent. After sonication, the solution was incubated at 40-45°C under sealed, light-protected conditions with stirring to obtain a drug solution. The drug solution was then slowly added dropwise to a soybean globulin solution modified with oxidized tannins under continuous vortex conditions. The mixture was incubated at 40-45°C under sealed, light-protected conditions for 30-60 minutes, with intermittent sonication during incubation to ensure thorough binding of the drug to the carrier. Subsequently, zinc chloride solution was slowly added dropwise to the system, inducing flocculation and precipitation of the complex through zinc ion coordination, thus completing self-assembly. S3: Product post-processing The flocculent precipitate in the system was collected and freeze-dried to obtain a composite microparticle drug containing a PDE5 inhibitor encapsulated by a soybean globulin-tannic acid-zinc ion composite carrier.

[0021] The present invention also provides a medicine box containing the pharmaceutical composition, preferably comprising a package containing the pharmaceutical composition and instructions for use, wherein the instructions for use specify a dosage of 0.000125 mg-0.0125 mg / kg, more preferably 0.00125 mg-0.0125 mg / kg, more preferably 0.0025-0.0080 mg / kg, specifically such as 0.0075 mg / kg.

[0022] Preferably, the ultra-low dose PDE5 inhibitor significantly reduces drug toxicity and side effects and improves medication safety while achieving uric acid reduction and / or renal protection effects.

[0023] Preferably, the sustained-release or controlled-release drug adopts an ultra-low dose continuous release mode to achieve long-term uric acid reduction and kidney damage repair effects.

[0024] Preferably, the soy globulin is 7S soy globulin, 11S soy globulin, or a mixture thereof in any proportion.

[0025] Preferably, the PDE5 inhibitor is immobilized within the composite carrier through hydrophobic interactions, hydrogen bonding, and zinc ion coordination complexation.

[0026] Preferably, the hyperuricemia includes primary hyperuricemia and secondary hyperuricemia, and the renal protection is to repair the pathological damage of renal tubular dilation and glomerular atrophy caused by hyperuricemia.

[0027] <Technical Effects> Compared to existing technologies and conventional ED treatment regimens, this invention has several outstanding technical advantages, as follows: 1. Breaking through the limitations of existing drug dosages, achieving ultra-low dose and high-efficiency uric acid reduction. Existing publicly available PDE5 inhibitor uric acid-lowering technologies employ high-dose dosing regimens in mice, which, when converted to human doses, far exceed the safe drug threshold and are divorced from practical clinical applications. This invention creatively screens and validates an ultra-low-dose dosing range. At an extremely low daily dose of 0.000125-0.0125 mg / kg, it can significantly lower uric acid levels. The corresponding standard single-dose dosage for a 60kg human is only 0.0075-0.75 mg, far lower than the conventional ED treatment dose of PDE5 inhibitors and the dosages in existing uric acid-lowering patents.

[0028] 2. Expand the indications for PDE5 inhibitors and explore new medicinal value. Existing PDE5 inhibitors are only routinely used to treat erectile dysfunction in men. This invention demonstrates that ultra-low doses of tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives can effectively prevent and treat hyperuricemia, while also intervening in kidney lesions caused by hyperuricemia. This expands the new pharmaceutical indications for this class of drugs and has extremely high clinical translational potential and application value.

[0029] 3. It has both uric acid-lowering and kidney pathological damage-repairing effects. This invention's ultra-low dose PDE5 inhibitor can not only significantly reduce serum uric acid levels and improve the symptoms of hyperuricemia from the source, but also specifically repair typical kidney pathological damage induced by hyperuricemia. It can effectively improve organic kidney lesions such as renal tubular dilation and glomerular atrophy caused by hyperuricemia, achieving a dual therapeutic effect of "lowering uric acid and repairing kidney damage". It can be used for the prevention of hyperuricemia and the intervention treatment of combined kidney damage.

[0030] 4. Ultra-low dosage significantly improves medication safety and avoids drug toxicity and side effects. The dosage used in this invention is much lower than the clinical treatment dosage and the dosage reported in existing studies. While ensuring significant uric acid reduction and renal protection efficacy, it greatly reduces the risk of toxic side effects and adverse reactions caused by high-dose PDE5 inhibitors.

[0031] 5. Due to the extremely low therapeutic dose, sustained-release / controlled-release formulations can be used, offering good convenience. This invention can prepare ultra-low dose PDE5 inhibitors into sustained-release or controlled-release formulations, relying on a stable in vivo drug release mode to achieve long-lasting and stable uric acid-lowering and renal protection effects, avoiding instantaneous fluctuations in blood drug concentration, ensuring continuous drug delivery while reducing the frequency of administration, and significantly improving the comfort and convenience of clinical medication.

[0032] 6. Specialized composite carriers further enhance drug efficacy. This invention utilizes a soybean globulin-tannic acid-zinc ion biocompatible composite carrier to encapsulate PDE5 inhibitors. By relying on hydrophobic interactions, hydrogen bonds, and zinc ion coordination complexation, the drug is stably locked in place, effectively improving the water solubility and dispersibility of hydrophobic drugs and enhancing their in vivo stability. Simultaneously, the carrier enables slow drug release, reduces drug loss, and further amplifies the bioavailability of drugs at ultra-low doses. Compared to free drugs, the synergistic effect is more significant, the formulation structure is more stable, and the efficacy is more prolonged.

[0033] The drug of this invention can be used simultaneously for the prevention and treatment of both primary and secondary hyperuricemia, and is suitable for various hyperuricemia scenarios. Attached Figure Description

[0034] Figure 1Effect of EGCG on serum uric acid in potassium oxonate-induced hyperuricemia mice. Compared with the control group, ####p<0.0001; compared with the model group, ****p<0.0001.

[0035] Figure 2 Mouse kidney sections (H&E staining). A: Control group; B: Model group; C: Allopurinol; D: 12.5 mg / kg EGCG; E: 25 mg / kg EGCG; F: 50 mg / kg EGCG.

[0036] Figure 3 Effects of tadalafil (0.001, 0.01, 0.1, 1 mg / kg) on ​​serum uric acid in potassium oxonate-induced hyperuricemia mice. Compared with the control group, ####p < 0.0001; compared with the model group, ****p < 0.0001, **p < 0.01.

[0037] Figure 4 Mouse kidney sections (H&E staining). A: Control group; B: Model group; C: Allopurinol; D: 0.001 mg / kg tadalafil; E: 0.01 mg / kg tadalafil; F: 0.1 mg / kg tadalafil; G: 1 mg / kg tadalafil. Red arrows indicate glomeruli, and black arrows indicate renal tubules.

[0038] Figure 5 Effects of tadalafil (0.001, 0.01, 0.1 mg / kg) on ​​serum uric acid in mice with hypoxanthine combined with potassium oxonate-induced hyperuricemia. Compared with the control group, ####p<0.0001; compared with the model group, ****p<0.0001.

[0039] Figure 6 Mouse kidney sections (H&E staining). A: Control group; B: Model group; C: Allopurinol; D: 0.001 mg / kg tadalafil; E: 0.01 mg / kg tadalafil; F: 0.1 mg / kg tadalafil.

[0040] Figure 7 Effects of vardenafil, sildenafil, and the pyrazolopyrimidinone derivative WTCSO7 on serum uric acid in potassium oxonate-induced hyperuricemia mice. Compared with the control group, ####p<0.0001; compared with the model group, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0041] Figure 8 Images of suspensions of soy globulin-tannic acid (SG-TA) and soy globulin-tannic acid-zinc ions (SG-TA-Zn).

[0042] Figure 9 : Comparison of the drug loading capacity of tadalafil induced by different concentrations of tannic acid in the SG-TA-Zn ternary complex.

[0043] Figure 10 : Particle size distribution of SG-TA-Zn, SG-TA-Zn-DMSO and SG-TA-Zn@tadalafil complexes.

[0044] Figure 11 : Effects of SG-TA-Zn@tadalafil (0.001, 0.01, 0.1 mg / kg) on serum uric acid in hyperuricemic mice induced by potassium oxonate; where compared with control, #p < 0.0001; compared with model, ****p < 0.0001; compared with model, △△△△p < 0.0001.

[0045] Figure 12 : Mouse kidney sections (H&E staining); (a) is the blank control group; (b) is the hyperuricemia model group; (c) is the positive control group; (d) is SG-60TA-Zn@266 drug at 0.001 mg / kg; (e) is SG-60TA-Zn@266 drug at 0.01 mg / kg; (f) is SG-60TA-Zn@266 drug at 0.1 mg / kg. Detailed implementation methods

[0046] Example 1 1.1 Experimental animals Male, SPF-grade Kunming mice (18 ± 2 g), purchased from Beijing Speywood Biotechnology Co., Ltd., animal production license number: SCXK(Beijing)2024-0001.

[0047] 1.2 Experimental drugs EGCG.

[0048] 1.3 Experimental methods 1.3.1 Adaptive feeding of mice Before the experiment, the animals were placed in a controlled environment (25 ± 1 °C, relative humidity of 60 ± 10%), with a 12-hour light / dark cycle. Distilled water and feed were provided ad libitum, and the animals were allowed to adapt for 3 - 5 days.

[0049] 1.3.2 Grouping and treatment methods Oxylenate potassium modeling: After acclimatization for 3-5 days, experimental animals were randomly divided into groups of 8. Except for the control group, the other groups received intraperitoneal injection of oxylenate potassium, while the control group received intraperitoneal injection of 0.5% sodium carboxymethyl cellulose (0.1 mL / 10 g). Thirty minutes after the intraperitoneal injection, except for the control and model groups, the other groups received the drug via gavage. This treatment continued for 10 days. One hour after the last administration, blood and tissue samples were collected for further analysis.

[0050] Hypoxanthine combined with potassium oxonate modeling: Adaptive feeding and grouping were the same as for potassium oxonate modeling. Except for the control group, the other groups were first administered hypoxanthine by gavage, followed by intraperitoneal injection of potassium oxonate. The control group was intraperitoneally injected with 0.5% sodium carboxymethyl cellulose. 30 minutes after the intraperitoneal injection, except for the control group and the model group, the other groups were administered the drug by gavage. This treatment was continued for 10 days. One hour after the last administration, blood and tissue samples were collected for further analysis.

[0051] 1.3.3 Blood Sample Collection and Processing Ten days after each group of mice were administered the drug via gavage, blood samples were collected from the mice. After the group collection was completed, the blood samples were placed in a 37°C incubator and allowed to stand for 40 minutes. Then, they were centrifuged for 10 minutes at 4°C and 3000g using a benchtop high-speed centrifuge. The separated supernatant was transferred to an EP tube and frozen at -80°C.

[0052] 1.3.4 Sampling and Processing A certain amount of liver was weighed and rapidly cooled in liquid nitrogen, then transferred to a freezer at -80°C for storage. The left kidney was also rapidly cooled in liquid nitrogen and transferred to a freezer at -80°C for subsequent analysis. The right kidney was placed in 4% paraformaldehyde for slide preparation and analysis.

[0053] 1.3.5 Detection of serum uric acid in mice (1) Serum sample processing: Mix serum with perchloric acid (0.3M) and vortex. After 30 minutes in an ice-water bath, centrifuge for 10 minutes. Mix the supernatant with Na2HPO4 (0.2M) by vortexing and centrifuge for 10 minutes. Transfer the supernatant to a new centrifuge tube, filter it through a membrane, and use it as a liquid chromatography injection solution.

[0054] (2) Liquid chromatography conditions: Inertsil ODS-3 column was used, with 20mM potassium dihydrogen phosphate: methanol = 85:15 (V / V) as the mobile phase, column temperature 30℃, flow rate 1mL / min, injection volume 20μL, and detection wavelength 290nm.

[0055] 1.3.6 Staining of mouse kidney sections (HE staining) (1) Dehydration: Gradient dehydration method is adopted, using 75%, 85%, and 90% alcohol for overnight dehydration, and 95% and 100% alcohol for 1 hour dehydration.

[0056] (2) Transparent: Xylene treatment for 2 hours.

[0057] (3) Wax embedding: Drain the xylene and place it in a melted wax tank. After soaking, embed it.

[0058] (4) Sectioning: Use a microtome to cut sections (5μm), and use a glass slide to scoop them up and air dry.

[0059] (5) Dewaxing: Dewaxing is performed using xylene and graded ethanol.

[0060] (6) Staining: stain with hematoxylin for 4 minutes, differentiate with hydrochloric acid and ethanol, rinse with tap water to return to blue, stain with eosin, clear, and mount.

[0061] 1.4 Experimental Results A mouse model of hyperuricemia was established using potassium oxonate. The experimental results are as follows: Figure 1 As shown, EGCG at doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg significantly reduced serum uric acid in mice, with no significant difference among the three doses.

[0062] Based on kidney HE staining ( Figure 2 As can be seen, the potassium oxonate model group showed significant glomerular atrophy and renal tubular dilation. Although the allopurinol positive drug group could reduce serum uric acid in mice, its kidney damage was not improved. Instead, it aggravated glomerular atrophy and renal tubular dilation. After treatment with EGCG, not only was serum uric acid reduced, but glomerular atrophy and renal tubular dilation were also improved, showing a significant protective effect on the kidneys.

[0063] Example 2 2.1 Experimental Methods PDE inhibition assays were performed using a radiolabeled substrate method. For the enzymatic hydrolysis of radiolabeled substrates [3H]cAMP or [3H]cGMP to generate [3H]AMP or [3H]GMP, PDE reacted alone or with different concentrations of EGCG, tadalafil, sildenafil, vandenafil, or pyrazolopyrimidinone derivatives in the reaction mixture with [3H]cAMP or [3H]cGMP. The reaction was terminated by adding 200 µL of 0.2 M zinc sulfate while immersing the reaction tube in ice water. The reaction products [3H]AMP or [3H]GMP were precipitated by adding 200 µL of 0.2 N Ba(OH)₂, while unreacted [3H]cAMP or [3H]cGMP remained in the supernatant. The supernatant after centrifugation was transferred to a scintillation counter vial, and the radioactivity of the supernatant was measured using a liquid scintillation counter.

[0064] 2.2 Experimental results The IC of EGCG against PDE5 50 is 290 nM, showing a good inhibitory effect on PDE5. Compared with tadalafil, sildenafil, vardenafil, and pyrazolopyrimidinone derivatives, its IC 50 is 24 - 400 times that of the above-mentioned PDE5-specific inhibitors.

[0065] Table 1. In vitro inhibitory activity of PDE5A (IC 50 , nM)

[0066] Example 3 3.1 Experimental animals Male, SPF-grade Kunming mice (18 ± 2 g), purchased from Beijing Speyford Biotechnology Co., Ltd., animal production license number: SCXK(Beijing)2024 - 0001.

[0067] 3.2 Experimental drugs Tadalafil 3.3 Adaptive feeding of mice Same as Example 1 3.3.1 Grouping and treatment methods Potassium oxonate modeling: Same as Example 1 Hypoxanthine combined with potassium oxonate modeling: The adaptive feeding and grouping are the same as those of potassium oxonate modeling. Except for the control group, the other groups are first gavaged with hypoxanthine and then intraperitoneally injected with potassium oxonate. The control group is intraperitoneally injected with 0.5% sodium carboxymethylcellulose. 30 minutes after intraperitoneal injection, except for the control group and the model group, the other groups are gavaged with drugs and continuously treated for 10 days. 1 hour after the last drug administration, blood samples are collected and tissue samples are taken for further analysis.

[0068] 3.3.2 Blood sample collection and processing Same as Example 1.

[0069] 3.3.3 Tissue sampling and processing Same as Example 1.

[0070] 3.3.4 Detection of serum uric acid in mice Same as Example 1.

[0071] 3.3.5 Staining of mouse kidney sections (HE staining) Same as Example 1.

[0072] 3.4 Experimental results The experiment used tadalafil at doses of 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, and 1 mg / kg. All four doses had a significant effect on reducing uric acid, and the effects of tadalafil at 0.001 mg / kg, 0.01 mg / kg, and 0.1 mg / kg were better than those at 1 mg / kg. The effect of reducing uric acid was still relatively significant (p < 0.01), and there was no significant difference in uric acid levels between the treatment groups and the control group ( Figure 3 and Figure 4 ).

[0073] By establishing a mouse model of hyperuricemia accompanied by severe kidney injury, a model was established using hypoxanthine combined with potassium oxonate. The uric acid level in the model group increased to 5 times that of the control group. After treatment with tadalafil at 0.001 mg / kg, 0.01 mg / kg, and 0.1 mg / kg, the uric acid level decreased significantly (p < 0.0001). The uric acid level in the 0.1 mg / kg group reached no significant difference compared with the control group ( Figure 5 ).

[0074] As shown by observing the results of kidney sections Figure 6 . Compared with the control group, the model group showed severe renal tubular dilation and glomerular atrophy, resulting in relatively severe kidney injury. The positive control group, allopurinol group, also had obvious renal tubular dilation. Compared with the model group, the drug group significantly improved kidney injury, and the effect of kidney injury repair was more obvious at a concentration of 0.1 mg / kg compared with the model group.

[0075] Example 4 4.1 Experimental animals Male, SPF-grade Kunming mice (18 ± 2 g), purchased from Beijing Spepharm Biotechnology Co., Ltd., animal production license number: SCXK(Beijing)2024-0001.

[0076] 4.2 Experimental drugs Sildenafil, vardenafil, pyrazolopyrimidine derivative WTCS07 (5-(2-propoxy-5-(N-propoxycarbonylaminosulfonyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one).

[0077] 4.3 Adaptive feeding of mice Same as Example 1.

[0078] 4.3.1 Grouping and treatment methods Potassium oxonate modeling: Same as Example 1.

[0079] Hypoxanthine combined with potassium oxonate modeling: Same as Example 3.

[0080] 4.3.2 Blood sample collection and processing Same as Example 1.

[0081] 4.3.3 Sampling and Processing Same as Example 1.

[0082] 4.3.4 Detection of serum uric acid in mice Same as Example 1.

[0083] 4.4 Experimental Results The experiment evaluated the uric acid-lowering efficacy of vardenafil, sildenafil, and its derivative WTCS07. The results showed that the three compounds, at doses of 0.001 mg / kg, 0.01 mg / kg, and 0.1 mg / kg, [the following text appears to be unrelated and possibly a separate excerpt: "as shown in the experiment, vardenafil, sildenafil, and its derivative WTCS07, [the following text appears to be Figure 7 As shown, all three drugs had significant uric acid-lowering effects. Vardenafil showed no significant difference in uric acid levels compared to the control group at doses of 0.01 mg / kg and 0.1 mg / kg. Sildenafil showed uric acid-lowering effects at all three doses, but its uric acid level was still significantly higher than that of the control group. The derivative WTCS07 showed no significant difference in uric acid levels compared to the control group at all three doses.

[0084] Example 5 In this embodiment, a tannic acid-Zn-soybean globulin complex was constructed and used as a drug carrier to load tadalafil to obtain a drug complex. The tadalafil drug loading and the particle size distribution of the complex were then tested.

[0085] Soy globulin is widely available and biocompatible, making it an ideal natural drug-carrying matrix. However, when used alone, it suffers from problems such as loose structure, low drug loading efficiency, and poor stability. Tannic acid can modify the protein structure through covalent / non-covalent interactions, while zinc ions can promote complex assembly and enhance structural stability through coordination. This embodiment constructs a tannic acid-zinc ion-soy globulin ternary complex for the encapsulation of hydrophobic tadalafil. The experimental method is as follows: (1) Prepare experimental materials Soy protein isolate, tannic acid (TA), zinc ions (ZnCl2), tadalafil (hydrophobic), dialysis bag 10000Da.

[0086] (2) Extraction of soybean globulin 100g of soy protein isolate was weighed and thoroughly dispersed in 2L of distilled water. The pH of the system was adjusted to 9.0 using 1mol / L sodium hydroxide solution, and the mixture was incubated magnetically at 45℃ for 2 hours to ensure complete protein dissolution. After incubation, the mixture was centrifuged at 4℃ and 9000×g for 15 minutes, and the supernatant was collected. The remaining precipitate was extracted twice more using the same dissolution and centrifugation process, and the supernatants from the three extractions were combined. Sodium bisulfite was added to the combined supernatant to adjust its final concentration to 0.01mol / L, and then the pH of the system was adjusted to 6.4 using 2mol / L hydrochloric acid solution. The solution was incubated overnight at 4℃, and the next day it was centrifuged at 4℃ and 6500×g for 15 minutes, and the precipitate was collected. The precipitate was washed three times with distilled water and then freeze-dried to obtain soy 11S globulin.

[0087] Collect the supernatant from the 11S globulin separation process, add sodium chloride to adjust the final concentration to 0.25 mol / L, adjust the pH of the system to 5.0 with 2 mol / L hydrochloric acid solution, stir at room temperature for 1 h, and then centrifuge at 4 °C and 9500 × g for 15 min to remove insoluble intermediate components. Dilute the obtained supernatant twice with distilled water, adjust the pH again to 4.8 with 2 mol / L hydrochloric acid solution, centrifuge at 4 °C and 6500 × g for 15 min, collect the precipitate, wash it three times with distilled water, and freeze-dry it to obtain soybean 7S globulin. Mix the separated and purified soybean 7S globulin with 11S globulin to obtain soybean globulin (SG), and store it in a sealed sample bag at 4 °C.

[0088] (3) Modification of tannins on soybean globulin Soy globulin (SG) was dispersed in distilled water, and the pH of the system was adjusted to 9.0. The solution was incubated at 45°C for 1 hour to ensure complete dissolution. The solution was then heated to boiling and held at this temperature for 15 minutes, followed by natural cooling to 25°C for later use. Tannic acid (TA) was dissolved in an aqueous solution with a pH of 9.0 and oxidized at 45°C for 1 hour in an oxygen-rich environment to obtain an oxidized tannic acid solution. Different volumes of the oxidized tannic acid solution were mixed with the above SG solution, and the protein concentration in the mixture was uniformly adjusted to 10 g / L. The final tannic acid concentrations were set at 0 μmol / g TA, 10 μmol / g TA, 30 μmol / g TA, and 60 μmol / g TA, respectively. The mixed solution was incubated at 45°C for another 2 hours, and then 2 mol / L hydrochloric acid solution was added dropwise to adjust the pH to 7.5 to terminate the reaction.

[0089] A dialysis bag with a molecular weight cutoff of 10000 Da was used to dialyze the reaction solution for 24 hours at 4°C in a sealed environment to remove free tannic acid from the system. After dialysis, the solution was freeze-dried to obtain the soybean globulin-tannic acid (SG-TA) complex. Based on the difference in tannic acid concentration, the samples were named SG-0TA, SG-10TA, SG-30TA, and SG-60TA, respectively.

[0090] like Figure 8 The image shows physical images of soy globulin-tannic acid (SG-TA) and soy globulin-tannic acid-zinc ion (SG-TA-Zn) suspensions. When soy globulin (SG) and tannic acid (TA) are mixed, the solution appears pink. This is because soy 11S globulin is rich in amino groups, thiol groups, and hydrophobic cavities, which can combine with tannic acid through hydrogen bonds and hydrophobic interactions to form a charge-transfer complex. This complex is further oxidized in an aerobic environment to generate a quinone-protein conjugated system, and the synergistic effect of the two makes the system appear pink. Introducing zinc ions into the above mixture resulted in significant protein precipitation, confirming that zinc ions can induce the self-assembly of the soy globulin-tannic acid complex, ultimately forming aggregates and producing precipitation. Therefore, zinc ions can serve as an initiator for encapsulating drugs using the soy globulin-tannic acid complex.

[0091] (4) Encapsulation of Tadalafil by the carrier Tadalafil solutions were prepared using dimethyl sulfoxide (DMSO) as solvent. After sonication at 100 W for 2 min, the solutions were incubated at 45 °C in a sealed, dark environment with stirring for 30 min. Under rapid vortex conditions, 4 mL of tadalafil solutions of different concentrations were slowly added dropwise to 20 mL of SG-TA solution (pH 7.5). The mixture was incubated at 45 °C in a sealed, dark environment with stirring for 30 min, during which time it was intermittently sonicated at 100 W for 3 min. Subsequently, 15.7 mmol / L zinc chloride (ZnCl2) solution was slowly added dropwise to the system to induce complete flocculent precipitation of proteins. The precipitate was collected and freeze-dried to obtain tadalafil-loaded soybean globulin-tannic acid-zinc ion composite microparticles (SG-TA-tadalafil-Zn).

[0092] (5) Determine the drug loading and particle size of tadalafil. ① Drug loading determination Accurately weigh 10 mg of the composite microparticles and mix them thoroughly with 5 mL of DMSO to extract the tadalafil adhering to the surface of the microparticles. Subsequent operations are the same as the method for determining the total packaged amount. The tadalafil content on the surface of the composite microparticles is calculated using a standard curve, with the unit being μg / mL.

[0093] Depend on Figure 9It can be seen that when the tannic acid concentration is gradually increased from 0 μmol / g to 60 μmol / g, the drug loading capacity of the soy protein isolate-zinc ion complex for tadalafil shows regular changes: when the tannic acid concentration increases from 0 to 10 μmol / g, the drug loading capacity significantly decreases; when the concentration increases to 30 μmol / g, the drug loading capacity gradually recovers; after the concentration reaches 60 μmol / g, the drug loading capacity tends to be stable.

[0094] This change rule is jointly regulated by the multiple action mechanisms of tannic acid: low-concentration tannic acid will occupy the hydrophobic binding sites of the complex and simultaneously increase the surface hydrophilicity of the system, thereby inhibiting drug loading and causing a decrease in drug loading capacity; under medium- and high-concentration conditions, tannic acid and zinc ions form a metal-phenol complex network. Relying on its drug-locking effect and the moderate conformational relaxation effect of the binding protein molecules, it offsets the adverse effects brought by steric hindrance and promotes the recovery of drug loading capacity. When tannic acid is not introduced, the hydrophobic cavity inside the soy protein isolate has no spatial obstruction, which can ensure the efficient embedding of tadalafil, but the overall encapsulation stability is poor.

[0095] ② Particle size determination Take the freeze-dried SG-TA-Zn@tadalafil complex, resuspend it充分 in pure water,吸取 1 mL of the suspension into the sample detection cell, and use a nano particle size and zeta potential analyzer to measure the particle size and polydispersity index (PDI) of the complex. The detection parameters are set as follows: the real part of the sample refractive index is 1.46, the imaginary part is 0.00, and the refractive index of the dispersion medium is 1.33.

[0096] It can be seen from Figure 10 that the particle size of the blank SG-60TA-Zn ternary complex is about 3300 nm, and the polydispersity index PDI is about 0.08. The particle size distribution of the system is uniform and the stability is good. After adding DMSO solvent, the particle size of the carrier slightly increases, and the dispersibility still remains excellent; after loading tadalafil, the drug molecules fill the hydrophobic cavity inside the carrier, promoting the stretching of the carrier spatial structure, and the particle size further increases to 4300 nm, and the PDI slightly rises to 0.15. The above results confirm that the DMSO solvent has a weak impact on the carrier skeleton structure; tadalafil has been successfully loaded into the ternary carrier and causes a regular increase in the carrier particle size. This particle size change characteristic is consistent with the physicochemical change rule of the drug-loaded micro-nano particles.

[0097] Example 6 This example further tests the effect of the drug complex constructed in Example 5 on reducing uric acid and repairing kidney injury in hyperuricemia mice. The experimental method is as follows: (1)Experimental animals SPF-grade male Kunming mice with a body weight of 18±2 g were used in the experiment. They were purchased from Beijing Spearf Bio-Technology Co., Ltd., and the animal production license number is: SCXK(Beijing)2024-0001.

[0098] (2) Experimental drugs Self-made drug complex (SG-60TA-tadalafil-Zn), allopurinol (positive control drug) (3) Adaptive feeding of mice Before the experiment, the mice were housed in a controlled environment with the following conditions: temperature 25±1℃, relative humidity 60±10%, day-night light cycle of 12 h, and free access to food and water. The mice were acclimatized for 3-5 days.

[0099] (4) Grouping and processing methods After 7 days of acclimatization, mice were randomly divided into four groups of eight: a blank control group, a hyperuricemia model group, a drug intervention group, and an allopurinol positive control group.

[0100] The model was constructed according to Example 3. Thirty minutes after intraperitoneal injection, mice in the drug intervention group (excluding the blank control group and the hyperuricemia model group) were administered different doses of tadalafil and SG-60TA-tadalafil-Zn drug complex (based on pure tadalafil, doses were 0.001 mg / kg, 0.01 mg / kg, and 0.1 mg / kg, respectively) by gavage. The positive control group received an equal amount of allopurinol. This intervention was continued for 10 days. One hour after the last administration, blood and kidney tissue samples were collected from the mice for subsequent indicator detection and histological analysis.

[0101] (5) Blood sample collection and processing After 10 days of continuous administration, blood samples were collected from mice. The collected blood samples were placed in a 37°C incubator and allowed to stand for 40 minutes. Then, they were centrifuged for 10 minutes at 4°C and 3000 g using a benchtop high-speed centrifuge to separate the supernatant serum, which was then transferred to sterile centrifuge tubes and stored at -80°C for later testing.

[0102] (6) Sampling and processing Mouse kidney tissue was dissected and fixed in 4% paraformaldehyde fixative for subsequent tissue section preparation and HE staining analysis.

[0103] (7) Detection of serum uric acid in mice ① Serum sample processing Take the serum to be tested and mix it thoroughly with 0.3 M perchloric acid solution by vortexing. After standing in an ice-water bath for 30 min, centrifuge for 10 min and collect the supernatant. Mix the supernatant with 0.2 M disodium hydrogen phosphate solution by vortexing and centrifuge again for 10 min. After filtering the supernatant through a filter membrane, the test solution is obtained and used for high performance liquid chromatography (HPLC) injection detection.

[0104] ② Liquid chromatography detection conditions An Inertsil ODS-3 column was used with 20 mM potassium dihydrogen phosphate aqueous solution-methanol (v / v ratio 85:15) as the mobile phase, column temperature 30℃, flow rate 1 mL / min, injection volume 20 μL, and detection wavelength 290 nm.

[0105] (8) HE staining of mouse kidney sections ① Gradient dehydration: The fixed kidney tissue was dehydrated using a gradient of ethanol solutions: sequentially dehydrated overnight in 75%, 85%, and 90% ethanol solutions, followed by dehydration for 1 hour each in 95% and 100% ethanol solutions. ② Clearing: The dehydrated tissue was cleared in xylene solution for 2 hours. ③ Paraffin embedding: The xylene on the tissue surface was drained, and the tissue was fully immersed in molten paraffin, followed by paraffin embedding. ④ Sectioning: The embedded tissue blocks were sectioned using a microtome to a thickness of 5 μm. Intact sections were retrieved using glass slides and allowed to air dry. ⑤ Dewaxing and rehydration: The dried sections were dewaxed and rehydrated sequentially using xylene and gradient ethanol solutions. ⑥ Staining and mounting: The dewaxed sections were stained with hematoxylin for 4 minutes, differentiated with hydrochloric acid ethanol solution, rinsed with tap water to regain blue color, stained with eosin solution, cleared, and mounted with neutral resin to complete the section preparation for microscopic observation and analysis.

[0106] Depend on Figure 11 The results showed that the combined hypoxanthine and potassium oxonate modeling method was used to construct a mouse model of hyperuricemia with severe kidney injury. The serum uric acid level of the model group mice increased to 4 times that of the blank control group, indicating that the modeling was successful.

[0107] Intervention with different doses of tadalafil and the SG-60TA-tadalafil-Zn complex (0.001 mg / kg, 0.01 mg / kg, and 0.1 mg / kg based on pure tadalafil) significantly reduced serum uric acid levels in mice (p < 0.0001). Furthermore, the SG-60TA-tadalafil-Zn complex, at the same dose, further reduced serum uric acid levels in mice compared to free tadalafil. In other words, to achieve the same level of reduction in serum uric acid levels in mice, the dosage of SG-60TA-Zn complex required to further reduce tadalafil levels could be decreased, thereby further reducing the side effects of tadalafil.

[0108] Depend on Figure 12The results of H&E staining of kidney tissue sections showed that, compared with the blank control group, the kidneys of mice in the hyperuricemia model group exhibited obvious typical pathological changes such as renal tubular dilation and glomerular atrophy, indicating severe kidney tissue damage. The allopurinol positive control group still showed obvious pathological features of renal tubular dilation. Compared with the hyperuricemia model group, all drug complex intervention groups significantly improved the pathological damage of kidney tissue, with the SG-60TA-tadalafil-Zn complex at a dose of 0.1 mg / kg showing the most outstanding effect in repairing kidney damage.

[0109] The above experimental results confirm that the SG-TA-Zn drug delivery system constructed in this invention can significantly enhance the in vivo efficacy of tadalafil. Compared with free drugs, the soybean globulin-tannic acid-zinc ion drug delivery system can significantly enhance the kidney injury repair effect of tadalafil, basically restore the normal tissue structure of mouse kidneys, and significantly improve pathological symptoms such as renal tubular dilation and glomerular damage induced by hyperuricemia.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of PDE5 inhibitors in the preparation of drugs for lowering uric acid and protecting the kidneys, characterized in that, The dosage of the PDE5 inhibitor in the drug is 0.000125-0.0125 mg / kg.

2. The application as described in claim 1, characterized in that, The drug is prepared as a single dose at a dosage of 0.0075 mg to 0.75 mg.

3. The application as described in claim 2, characterized in that, The drug is prepared as a single dose according to the standard dosage of 0.000125-0.0125 mg / kg, specifically as a single dose of 0.0075 mg / kg.

4. The application as described in claim 3, characterized in that, The aforementioned uric acid-lowering drug is used to treat patients with hyperuricemia.

5. The application as described in claim 1, characterized in that, The drug is used to prevent hyperuricemia; The PDE5 inhibitor is selected from one or more of tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives.

6. The application as described in claim 1, characterized in that, The drug is used to lower uric acid and repair kidney pathological damage induced by hyperuricemia, and the drug is prepared such that the actual daily dose of the PDE5 inhibitor, which is the effective active ingredient in the drug, is 0.000125-0.0125 mg / kg of human body weight.

7. The application as described in claim 1, 5, or 6, characterized in that, The drug is a sustained-release or controlled-release formulation; the daily release of the PDE5 inhibitor in the drug is 0.000125-0.0125 mg / kg of human body weight.

8. The application as described in claim 7, characterized in that, The drug is a sustained-release or controlled-release formulation; based on an adult weighing 60 kg, the daily in vivo release of the PDE5 inhibitor in the drug is 0.0075 mg to 0.75 mg.

9. A pharmaceutical composition for treating or preventing hyperuricemia, characterized in that, Each unit of the drug contains 0.36-0.52 mg of a PDE5 inhibitor and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, Each unit of the drug contains 0.42-0.48 mg (e.g., 0.45 mg) of a unit dose of PDE5 inhibitor and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is administered orally, preferably in tablet or capsule form; each tablet contains 0.36-0.52 mg of PDE5 inhibitor, preferably 0.42-0.48 mg.

12. The pharmaceutical composition according to claim 9, characterized in that, The PDE5 inhibitor is selected from one or more of tadalafil, sildenafil, vandenafil, and pyrazolopyrimidine derivatives.

13. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is a sustained-release or controlled-release drug, and the daily in vivo release of the PDE5 inhibitor in the pharmaceutical composition is 0.000125-0.0125 mg / kg of human body weight.

14. The pharmaceutical composition according to claim 9 or 13, characterized in that, The pharmaceutical composition further comprises a soybean globulin-tannic acid-zinc ion complex carrier, wherein the PDE5 inhibitor is encapsulated by the complex carrier to form drug-loaded complex microparticles.

15. The pharmaceutical composition according to claim 14, characterized in that, The preparation method of the compound drug is as follows: S1: Oxidized tannins modified soybean globulin Soy globulin was dispersed in distilled water, and the pH of the system was adjusted to 8.5-9.

5. The mixture was incubated at 40-45℃ for 1-2 hours until completely dissolved. The resulting solution was boiled and then naturally cooled to room temperature to obtain a soy globulin solution. Tannic acid was dissolved in an aqueous solution with a pH of 8.5-9.5 and oxidized at 40-45℃ in an oxygen-rich environment for 0.5-1.5 hours to obtain an oxidized tannic acid solution. The oxidized tannic acid solution was mixed with the soy globulin solution and incubated at 40-45℃ for 1-3 hours. Then, hydrochloric acid was added to adjust the pH of the system to 7.5 to terminate the reaction. The solution was dialyzed for 20-28 hours in a closed environment using a dialysis bag with a molecular weight cutoff of 10000 Da to remove free tannic acid and obtain the oxidized tannic acid modified soy globulin solution. S2: Drug loading and self-assembly of composite microparticles A PDE5 inhibitor solution was prepared using dimethyl sulfoxide as a solvent. After sonication, the solution was incubated at 40-45°C under sealed, light-protected conditions with stirring to obtain a drug solution. The drug solution was then slowly added dropwise to a soybean globulin solution modified with oxidized tannins under continuous vortex conditions. The mixture was incubated at 40-45°C under sealed, light-protected conditions for 30-60 minutes, with intermittent sonication during incubation to ensure thorough binding of the drug to the carrier. Subsequently, zinc chloride solution was slowly added dropwise to the system, inducing flocculation and precipitation of the complex through zinc ion coordination, thus completing self-assembly. S3: Product post-processing The flocculent precipitate in the system was collected and freeze-dried to obtain a composite microparticle drug containing a PDE5 inhibitor encapsulated by a soybean globulin-tannic acid-zinc ion composite carrier.

16. A pillbox containing the pharmaceutical composition according to any one of claims 9 to 15, characterized in that, The package includes a pharmaceutical composition and instructions for use, wherein the instructions for use specify a dosage of 0.000125 mg to 0.0125 mg / kg, preferably 0.00125 mg to 0.0125 mg / kg, more preferably 0.0025 to 0.0080 mg / kg, specifically such as 0.0075 mg / kg.

Images