A pharmaceutical composition and its application

Abstract

This invention relates to the pharmaceutical field, specifically disclosing a pharmaceutical composition and its application. The composition comprises sibifluin, uridine, and adenosine, wherein the mass ratio of sibifluin:uridine:adenosine is 6.0-11.5:0.8-1.4:1.0. This pharmaceutical composition exhibits good safety and anti-inflammatory effects, demonstrates good efficacy in the treatment of phenylketonuria, and also shows good preventative and / or therapeutic effects against ovarian insufficiency.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and specifically to a pharmaceutical composition and its use. Background Technology

[0002] Phenylketonuria (PKU) is a common disorder caused by the metabolism of the aromatic amino acid phenylalanine. It is an autosomal recessive genetic disorder caused by a gene defect that results in a deficiency of phenylalanine hydroxylase (PAH) or its coenzyme tetrahydrobiopterin (BH4), leading to hyperphenylalaninemia (HPA). The accumulated phenylalanine and its intermediate metabolites damage the central nervous system, and if left untreated, it can lead to irreversible intellectual disability, mental and behavioral abnormalities, and other symptoms.

[0003] There is currently no cure for phenylketonuria (PKU). Management primarily involves medication combined with dietary control. Some commonly used medications include: Sapropterin Dihydrochloride, an oral synthetic form of tetrahydrobiopterin (BH4), which activates residual phenylalanine hydroxylase activity, helping to metabolize phenylalanine and lowering serum phenylalanine levels. It is suitable for PKU patients who respond to BH4 (approximately 10%-20% of patients). Suitability needs to be confirmed through genetic testing or a BH4 loading test. During treatment, regular monitoring of serum phenylalanine levels, liver function, and neurological symptoms is necessary. Adverse reactions such as headache and diarrhea may occur. Pegvaliase is a recombinant phenylalanine ammonia-lyase that converts phenylalanine into harmless metabolites, directly lowering blood phenylalanine levels. It is independent of phenylalanine hydroxylase activity and is primarily used in adult patients with PKU, especially those whose blood phenylalanine levels cannot be effectively managed through diet and sapropterin. Subcutaneous injection may cause injection site reactions and allergic symptoms; a skin allergy test is required before use, and blood phenylalanine levels and antibody production should be monitored during treatment. Sephience (Sepiapterin), as a prodrug of BH4, increases phenylalanine hydroxylase activity and lowers blood phenylalanine levels through a dual mechanism of action. Adverse reactions may include upper respiratory tract infection (19.8%), headache (15.3%), diarrhea (14.9%), abdominal pain (12.2%), and abnormal stool color (4.5%). Regular monitoring of blood phenylalanine concentration and nutritional status is necessary.

[0004] Therefore, it is necessary to develop a pharmaceutical composition for treating phenylketonuria with low adverse reactions. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a pharmaceutical composition and its application that has a good effect on improving the gastrointestinal adverse reactions of adenosine, in order to address the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution: In a first aspect, the present invention discloses a pharmaceutical composition.

[0007] In some embodiments, the pharmaceutical composition includes sibifluin, uridine, and adenosine.

[0008] In some embodiments, the mass ratio of sibiformin:uridine:adenosine is 6.0-11.5:0.8-1.4:1.0, in some embodiments it is 6.5-11.0:0.8-1.3:1.0, in some embodiments it is 7.0-10.0:0.8-1.3:1.0, in some embodiments it is 7.0-10.0:0.9-1.2:1.0, in some embodiments it is 7.8-9.4:0.9-1.2:1.0, and in some embodiments it is 8.7-10:0.9-1.2:1.0.

[0009] In some embodiments, the mass ratio of sibifluin to adenosine is 6.5:1.0, 7.0:1.0, 7.5:1.0, 8.0:1.0, 8.5:1.0, 8.7:1.0, 9.0:1.0, 9.5:1.0, 10.0:1.0, 10.5:1.0, or 11.0:1.0.

[0010] In some embodiments, the mass ratio of uridine to adenosine is 0.9:1.0, 1.0:1.0, 1.1:1.0, 1.2:1.0, or 1.3:1.0.

[0011] In some embodiments, the pharmaceutical composition contains 68.0-86.5% sibifluin, 5.0-16.0% uridine, and 6.5-14.0% adenosine, based on the total mass of sibifluin, uridine, and adenosine.

[0012] In some embodiments, the pharmaceutical composition contains 70.8-86.0% sibiforin based on the total mass of sibiforin, uridine, and adenosine; in some embodiments, it contains 71.8-85.5%; in some embodiments, it contains 73.8-85.0%; in some embodiments, it contains 75.8-84.5%; in some embodiments, it contains 77.8-84.0%; and in some embodiments, it contains 70.0%, 70.5%, 71.0%, 71.5%, 72.0%, 72.5%, 73.0%, and 7... 3.5%, 74.0%, 74.5%, 75.0%, 75.5%, 76.0%, 76.5%, 77.0%, 77.5%, 77.8%, 78.0%, 78.5%, 79.0%, 79.5%, 79.8%, 80.0%, 80.5%, 81.0%, 81.3%, 81.5%, 82.0%, 82.5%, 83.0%, 83.3%, 83.5%, 84.0%, 84.5%, 85.0%, 85.5%, or 86.0%.

[0013] In some embodiments, the pharmaceutical composition contains 6.0-15.5% uridine based on the total mass of sibifluin, uridine, and adenosine; in some embodiments, it contains 6.5-13.1%; in some embodiments, it contains 7.0-12.1%; in some embodiments, it contains 7.6-11.1%; and in some embodiments, it contains 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 7.6%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, or 15.5%.

[0014] In some embodiments, the adenosine content in the pharmaceutical composition, based on the total mass of sibifluin, uridine, and adenosine, is 7.0-13.5%, 7.5-13.1% in some embodiments, 8.0-12.1% in some embodiments, 8.3-11.1% in some embodiments, and 6.5%, 7.0%, 7.5%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.3%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, or 14.0%.

[0015] In a second aspect, the present invention discloses a pharmaceutical composition.

[0016] In some embodiments, the pharmaceutical composition includes sibifluin, uridine, and adenosine.

[0017] In some embodiments, the pharmaceutical composition contains 68.0-86.5% sibifluin, 5.0-16.0% uridine, and 6.5-14.0% adenosine, based on the total mass of sibifluin, uridine, and adenosine.

[0018] In some embodiments, the pharmaceutical composition contains 70.8-86.0% sibiforin based on the total mass of sibiforin, uridine, and adenosine; in some embodiments, it contains 71.8-85.5%; in some embodiments, it contains 73.8-85.0%; in some embodiments, it contains 75.8-84.5%; in some embodiments, it contains 77.8-84.0%; and in some embodiments, it contains 70.0%, 70.5%, 71.0%, 71.5%, 72.0%, 72.5%, 73.0%, and 7... 3.5%, 74.0%, 74.5%, 75.0%, 75.5%, 76.0%, 76.5%, 77.0%, 77.5%, 77.8%, 78.0%, 78.5%, 79.0%, 79.5%, 79.8%, 80.0%, 80.5%, 81.0%, 81.3%, 81.5%, 82.0%, 82.5%, 83.0%, 83.3%, 83.5%, 84.0%, 84.5%, 85.0%, 85.5%, or 86.0%.

[0019] In some embodiments, the pharmaceutical composition contains 6.0-15.5% uridine based on the total mass of sibifluin, uridine, and adenosine; in some embodiments, it contains 6.5-13.1%; in some embodiments, it contains 7.0-12.1%; in some embodiments, it contains 7.6-11.1%; and in some embodiments, it contains 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 7.6%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, or 15.5%.

[0020] In some embodiments, the adenosine content in the pharmaceutical composition, based on the total mass of sibifluin, uridine, and adenosine, is 7.0-13.5%, 7.5-13.1% in some embodiments, 8.0-12.1% in some embodiments, 8.3-11.1% in some embodiments, and 6.5%, 7.0%, 7.5%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.3%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, or 14.0%.

[0021] Thirdly, the present invention discloses a pharmaceutical composition.

[0022] In some embodiments, the pharmaceutical composition includes sibifluin, uridine, and adenosine.

[0023] In some embodiments, the mass ratio of sibiforin:uridine:adenosine is 7.0-10.0:0.9-1.2:1.0; based on the total mass of sibiforin, uridine, and adenosine, the content of sibiforin is 70.0-86.5%; and / or the content of uridine is 6.0-15.5%; and / or the content of adenosine is 7.0-13.5%.

[0024] In some embodiments, the content of sibiformin, based on the total mass of sibiformin, uridine, and adenosine, is 70.0-86.5%, in some embodiments 71.0-85.5%, and in some embodiments 70.0%, 70.5%, 71.0%, 71.5%, 72.0%, 72.5%, 73.0%, 73.5%, 74.0%, 74.5%, 75.0%, 75.5%, 76.0%, 76.5%, 77.0%, 77.5%, 77.8%, 78.0%, 78.5%, 79.0%, 79.5%, 79.8%, 80.0%, 80.5%, 81.0%, 81.3%, 81.5%, 82.0%, 82.5%, 83.0%, 83.3%, 83.5%, 84.0%, 84.5%, 85.0%, 85.5%, or 86.0%.

[0025] In some embodiments, the uridine content is 6.0-15.5% based on the total mass of sibiformin, uridine, and adenosine; in some embodiments, it is 6.5-13.0%; and in some embodiments, it is 6.0%, 6.5%, 7.0%, 7.5%, 7.6%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, or 13.0%.

[0026] In some embodiments, the adenosine content is 7.0-13.5% based on the total mass of sibiformin, uridine, and adenosine, and in some embodiments it is 8.0-11.8%, and in some embodiments it is 7.0%, 7.5%, 8.0%, 8.2%, 8.3%, 8.4%, 8.5%, 9.0%, 9.2%, 9.3%, 9.5%, 10.0%, 10.5%, 11.0%, 11.1%, 11.5%, 12.0%, 12.5%, 13.0%, or 13.5%.

[0027] In the pharmaceutical compositions described in the first to third aspects above, the pharmaceutical compositions further contain a pharmaceutically acceptable carrier.

[0028] In some embodiments, the pharmaceutically acceptable carrier is a diluent or lubricant or a combination thereof, and in some embodiments it is calcium hydrogen phosphate, sodium hydrogen phosphate, potassium bicarbonate, calcium bicarbonate, calcium carbonate, sodium carbonate, potassium carbonate, starch, glucose, magnesium stearate or a combination thereof.

[0029] The pharmaceutical composition of the present invention can be formulated into a suitable dosage form to facilitate drug administration. In some embodiments, the dosage form of the composition is a solid dosage form or a liquid dosage form, for example, it can be a capsule, granule or solution formulation.

[0030] In some embodiments, the mass ratio of the pharmaceutically acceptable carrier to sibifluin is 0.1-200.0, more preferably 0.5-200.0:100, more preferably 1.0-200.0:100; in some embodiments, it is 1.0:1, 5.0:1, 10.0:1, 15.0:1, 20.0:1, 25.0:1, 30.0:1, 35.0:1, 40.0:1, 45.0:1, 50.0:1, 55.0:1, 60.0:1, 65.0:1, 70.0:1, 75.0:1, 80. 0:1, 85.0:1, 90.0:1, 95.0:1, 100.0:1, 105.0:1, 110.0:1, 115.0:1, 120.0:1, 125.0:1, 130.0:1, 135.0:1, 140.0:1, 145.0:1, 150.0:1, 155.0:1, 160.0:1, 165.0:1, 170.0:1, 175.0:1, 180.0:1, 185.0:1, 190.0:1, 195.0:1, or 200.0:1.

[0031] In some embodiments, the pharmaceutically acceptable carrier is magnesium stearate; in some embodiments, the mass ratio of magnesium stearate to sibifluin is 0.1-5.0:100, in some embodiments it is 1-5:100, and in some embodiments it is 2:1, 3:1, or 4:1.

[0032] Fourthly, the present invention provides a pharmaceutical formulation.

[0033] In some embodiments, the pharmaceutical formulation includes the pharmaceutical composition described in the first to third aspects above and a pharmaceutically acceptable carrier.

[0034] In some embodiments, the pharmaceutically acceptable carrier is a diluent or lubricant or a combination thereof, and in some embodiments it is calcium hydrogen phosphate, sodium hydrogen phosphate, potassium bicarbonate, calcium bicarbonate, calcium carbonate, sodium carbonate, potassium carbonate, starch, glucose, magnesium stearate or a combination thereof.

[0035] In some embodiments, the formulation is a solid dosage form or a liquid dosage form.

[0036] In some embodiments, the mass ratio of the pharmaceutically acceptable carrier to sibifluin is 0.1-200.0, more preferably 0.5-200.0:100, more preferably 1.0-200.0:100; in some embodiments, it is 1.0:1, 5.0:1, 10.0:1, 15.0:1, 20.0:1, 25.0:1, 30.0:1, 35.0:1, 40.0:1, 45.0:1, 50.0:1, 55.0:1, 60.0:1, 65.0:1, 70.0:1, 75.0:1, 80.0:1, 85.0:1, 90.0:1, 95.0:1, 100.0:1, 105.0:1, 110.0: 1, 115.0:1, 120.0:1, 125.0:1, 130.0:1, 135.0:1, 140.0:1, 145.0:1, 150.0:1, 155.0:1, 160.0:1, 165.0:1, 170.0:1, 175.0:1, 180.0:1, 185.0:1, 190.0:1, 195.0:1, or 200.0:1; in some embodiments, the pharmaceutically acceptable carrier is magnesium stearate; in some embodiments, the mass ratio of magnesium stearate to sibifluin is 0.1-5.0:100, in some embodiments it is 1-5:100, in some embodiments it is 2:1, 3:1, or 4:1.

[0037] Fifthly, the present invention provides the use of the pharmaceutical compositions and pharmaceutical preparations provided in the first to third aspects of the foregoing invention, and the pharmaceutical formulations provided in the fourth aspect of the foregoing invention, in the preparation of medicaments for the prevention and / or treatment of phenylketonuria and / or nephritis and / or ovarian insufficiency and / or reduction of adenosine gastrointestinal side effects. In some embodiments, the ovarian insufficiency includes premature ovarian failure.

[0038] Sixthly, the present invention provides the use of sibifluin in the preparation of a medicament for the prevention or treatment of phenylketonuria and / or ovarian insufficiency. In some embodiments, sibifluin is used as the sole active ingredient in the preparation of a medicament for the prevention or treatment of phenylketonuria and / or ovarian insufficiency. In some embodiments, the ovarian insufficiency includes premature ovarian failure.

[0039] Phenylketonuria (PKU) is an autosomal recessive inherited amino acid metabolism disorder and one of the most common genetic diseases detected in newborn screening. Its core characteristic is a deficiency of a key enzyme that breaks down phenylalanine, leading to the accumulation of phenylalanine and its metabolites in the body. This causes irreversible damage to the nervous system, and without timely intervention, it can result in serious consequences such as intellectual disability and developmental delays. Early diagnosis and standardized treatment can significantly improve patient prognosis, reduce the harm caused by the disease, and help patients achieve a normal life.

[0040] The pharmaceutical compositions and formulations provided by this invention have good therapeutic effects on phenylketonuria, while exhibiting fewer gastrointestinal side effects. The pharmaceutical compositions and formulations provided by this invention also have good anti-inflammatory therapeutic effects.

[0041] The compositions described in this invention are compositions for the prevention and / or treatment of pyketonuria, and / or compositions for reducing gastrointestinal adverse reactions, and / or compositions for the prevention or treatment of nephritis, and / or compositions for the prevention and / or treatment of ovarian insufficiency.

[0042] The pharmaceutical preparations described in this invention are pharmaceutical preparations for the prevention and / or treatment of pyketonuria, and / or pharmaceutical preparations for reducing gastrointestinal adverse reactions, and / or pharmaceutical preparations for the prevention or treatment of nephritis, and / or pharmaceutical preparations for the prevention and / or treatment of ovarian insufficiency.

[0043] The structural formulas of adenosine, uridine, and hibifolin (also known as gossypol-8-O-β-D-glucuronide) described in this invention are as follows:

[0044] The term "treatment" as used in this invention generally refers to achieving a desired pharmacological and / or physiological effect. The effect may be preventative, but not necessarily preventative, in relation to the prevention or partial prevention of a disease, symptom, or condition (such as dermatitis). The effect may be therapeutic in relation to the partial or complete cure of a disease, condition, symptom, or adverse reaction attributable to that disease, condition, or condition. Specifically, the term "treatment" can include any treatment of a subject (especially a person) for a relevant symptom or disease, and may include any one or more of the following: (a) preventing the disease from occurring in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or condition. The term "pharmaceutically acceptable" refers to a substance that does not significantly affect the biological activity of the pharmaceutical composition of the present invention at that dosage. The term "pharmaceuticalally acceptable carrier" refers to a substance that does not significantly affect the biological activity of the pharmaceutical composition of the present invention at that dosage, particularly the biological activities listed in the experiments of the embodiments of this application. The pharmaceutically acceptable carrier may be selected from disintegrants such as sodium carboxymethyl starch, crospovidone, and sodium crospovidone carboxymethyl cellulose; fillers such as starch, lactose, microcrystalline cellulose, asiaticoside (an extract obtained by reflux extraction of Centella asiatica with 90% ethanol and subsequent drying), mannitol, and sucrose; binders such as PVP, hydroxypropyl methylcellulose (HPMC), and starch paste; lubricants / flow aids such as magnesium stearate and talc; and coating materials such as acrylic resin and gastric / enteric coating materials. These carriers may be one or more combinations thereof. Preferably, these carriers meet the quality standards for pharmaceutical excipients, for example, pharmaceutical excipients registered with the National Medical Products Administration. The pharmaceutical compositions and formulations of the present invention have biological activities including prevention and / or treatment of phenylketonuria, anti-inflammatory effects, and low gastrointestinal adverse reactions. In addition, preliminary exploratory experiments have shown that they have good preventive and / or therapeutic effects on ovarian insufficiency, especially good preventive and / or therapeutic effects on premature ovarian failure.

[0045] Premature ovarian insufficiency (POI) refers to a syndrome of decreased ovarian function that occurs in women before the age of 40. Clinical manifestations include infrequent, frequent, or absent menstruation for at least 4 months. Laboratory tests show two basal FSH (follicle-stimulating hormone) levels >25 U / L at intervals of more than 4 weeks, and fluctuating estrogen levels. The incidence of this disease is about 1% to 4%, and it is divided into primary POI, which is the absence of spontaneous menstruation, and secondary POI, which is the decline in function after the occurrence of normal menstruation. The previously referred to premature ovarian failure (POF) is the terminal stage of POI, specifically referring to a state of amenorrhea for more than 4 to 6 months before the age of 40, FSH >40 U / L, persistently low estrogen levels, and menopausal symptoms.

[0046] The contents of the active ingredients sibifluin, uridine, and adenosine contained in the pharmaceutical composition or pharmaceutical preparation of the present invention can be detected by conventional high performance liquid chromatography (HPLC) methods, and they have separation effects that meet the requirements of the pharmacopoeia, such as good resolution.

[0047] Specifically, the determination was performed according to high performance liquid chromatography (HPLC) (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). The detection method for sibiformin (HPLC Method A) is as follows: using octadecylsilane-bonded silica gel as the stationary phase (Agilent ZORBAX SB C18 column, 250 × 4.6 mm, 5 μm), accurately pipette 10 μl each of the reference solution and the test solution and inject them into the HPLC system. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed (0–20 min, 6% A → 17% A; 20–30 min, 17% A → 17% A; 30–50 min, 6% A → 17% A; 30–50 min, 17% A → 17% A). The chromatography was performed at a wavelength of 360 nm, a flow rate of 1.0 mL / min, and a column temperature of 30 °C. The chromatogram was recorded, and the content of Sibiforin was calculated by peak area using the external standard method with a correction factor of 1.09. The relative retention time of the analyte Sibiforin peak and the hyperoside reference standard peak was within 1.361 ± 5%. The reference standard solution was prepared by accurately weighing an appropriate amount of hyperoside reference standard and adding 70% ethanol to prepare a solution containing approximately 0.2 mg per mL. The test solution was prepared by accurately weighing an appropriate amount of sample (50 mg), placing it in a 50 mL volumetric flask, adding an appropriate amount of 70% ethanol, sonicating for 20 minutes, cooling, diluting to the mark with 70% ethanol, shaking well, filtering, and collecting the filtrate. The detection method for uridine and adenosine (HPLC method B) is as follows: A C18 column AQ-C18 (4.6 × 250 mm, 5 μm) is used with 0.1% formic acid solution (A) - acetonitrile (B) as the mobile phase. The detection wavelength is 260 nm, the flow rate is 1.0 mL / min, the column temperature is 30℃, and the signal acquisition time is 0 min to 12 min. Elution is performed (0~12 min, 2% B; 12~15 min, 2% B → 95% B). The sample is prepared by accurately weighing approximately 0.5 g of the sample, placing it in a 50 mL volumetric flask, adding 35 mL of 10% methanol, sonicating for 20 minutes, cooling, adding 10% methanol to the mark, and shaking well. The adenosine content is determined by external standard method, and the uridine content is calculated using the relative correction factor (correction factor is 1.32) with adenosine. The relative retention time of the uridine peak and the adenosine reference peak is within 0.808 ± 5%. The mass ratio in the pharmaceutical composition is calculated directly from the content data obtained by the detection method.

[0048] Beneficial effects: The pharmaceutical composition provided by this invention has a good therapeutic effect on phenylketonuria, while exhibiting fewer gastrointestinal side effects. Furthermore, the pharmaceutical composition provided by this invention also has a good therapeutic effect on nephritis and a good preventive and / or therapeutic effect on ovarian insufficiency. Attached Figure Description

[0049] Figure 1 Electron micrographs of cell states in the blank group, model group, and various embodiment groups in Test Example 1 of this invention.

[0050] Figure 2 Electron micrographs of the cell states in each comparative group of test example 1 of this invention. Detailed Implementation

[0051] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0052] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0053] Unless otherwise specified, the contents of Sibiformin (gossypol-8-O-β-D-glucuronide), uridine, and adenosine used in the following examples are above 98.0%.

[0054] Example 1 Preparation of pharmaceutical composition (S1) (the mass ratio of sibifluin:uridine:adenosine is 10:0.9:1) Weigh out 100 mg of adenosine, 90 mg of uridine, and 1000 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition (S1).

[0055] Example 2 Preparation of pharmaceutical composition (S2) (the mass ratio of sibifluin:uridine:adenosine is 7:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, and 700 mg of sibifluin according to the following weights, mix them, and prepare the drug composition (S2).

[0056] Example 3 Preparation of pharmaceutical composition (S3) (the mass ratio of sibifluin:uridine:adenosine is 8.7:1.2:1) Weigh out 100 mg of adenosine, 120 mg of uridine, and 870 mg of sibifluin according to the following weights, mix them, and prepare the drug composition (S3).

[0057] Example 4 Preparation of pharmaceutical composition (S4) (the mass ratio of sibifluin:uridine:adenosine is 10:1.2:1) Weigh out 100 mg of adenosine, 120 mg of uridine, and 1000 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition (S4).

[0058] Example 5 Preparation of pharmaceutical composition (S5) (the mass ratio of sibifluin:uridine:adenosine is 8.7:1.2:1) Weigh out 100 mg of adenosine, 120 mg of uridine, 870 mg of sibifluin, and 320 mg of quercetin-3'-O-glucoside as follows and mix them to prepare the drug composition (S5).

[0059] Example 6 Preparation of pharmaceutical composition (S6) (the mass ratio of sibifluin:uridine:adenosine is 10:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, and 1000 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition (S6).

[0060] Example 7 Preparation of pharmaceutical composition (S7) (the mass ratio of sibifluin:uridine:adenosine is 8.7:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, and 870 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition (S7).

[0061] Example 8 Preparation of pharmaceutical composition (S8) (the mass ratio of sibifluin:uridine:adenosine is 10:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, 1000 mg of sibifluin, 2000 mg of asiaticoside, and 10 mg of magnesium stearate according to the following weights. Mix them to prepare pharmaceutical composition S8.

[0062] Example 9 Preparation of pharmaceutical composition (S9) (the mass ratio of sibifluin:uridine:adenosine is 8.7:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, 870 mg of sibifluin, and 80 mg of calcium bicarbonate as follows, mix them, and prepare the pharmaceutical composition (S9).

[0063] Example 10 Preparation of pharmaceutical composition formulations (S1-10) (the mass ratio of sibifluin:uridine:adenosine is 10:0.9:1) Weigh out 100 mg of adenosine, 90 mg of uridine, 1000 mg of sibifluin, and 100 mg of starch as follows and mix them. Fill a portion of the mixture into capsules of 50 mg / capsule to prepare the drug composition capsule (S1-101). Pack the remaining portion into bags of 50 mg / bag to prepare the drug composition granules (S1-102).

[0064] Example 11 Preparation of pharmaceutical composition formulation (S1-11) (the mass ratio of sibifluin:uridine:adenosine is 10:0.9:1) Weigh out 100 mg of adenosine, 90 mg of uridine, 1000 mg of sibifluin, 100 mg of glucose, and 1 L of water as follows, mix them, and prepare a solution of the pharmaceutical composition (S1-11).

[0065] Example 12 Preparation of pharmaceutical composition formulation (S1-12) (the mass ratio of sibifluin:uridine:adenosine is 10:0.9:1) Weigh out 100 mg of adenosine, 90 mg of uridine, 1000 mg of sibifluin, and 47 mg of magnesium stearate as follows, mix them, and fill them into capsule shells (400 mg / capsule) to prepare capsules of the pharmaceutical composition (S1-12).

[0066] Example 13 Preparation of pharmaceutical composition formulation (S1-13) (the mass ratio of sibifluin:uridine:adenosine is 10:0.9:1) Weigh out 100 mg of adenosine, 90 mg of uridine, 1000 mg of sibifluin, 48 mg of magnesium stearate, and 25 mg of calcium bicarbonate as follows, mix them, and fill them into capsule shells (400 mg / capsule) to prepare capsules of the pharmaceutical composition (S1-13).

[0067] Example 14 Preparation of pharmaceutical composition (S14) (the mass ratio of sibifluin:uridine:adenosine is 10:1:1) Weigh out 100 mg of adenosine, 100 mg of uridine, 1000 mg of sibifluin, and 10 mg of magnesium stearate according to the following weights, mix them, and prepare the pharmaceutical composition S14.

[0068] Comparative Example 1: Preparation of the pharmaceutical composition (DS1) (the mass ratio of sibifluin: uridine: adenosine was 3:1:1). Weigh out 100 mg of adenosine, 100 mg of uridine, and 300 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition DS1.

[0069] Comparative Example 2: Preparation of the pharmaceutical composition (DS2) (the mass ratio of sibifluin:uridine:adenosine was 30:1:1). Weigh out 100 mg of adenosine, 100 mg of uridine, and 3000 mg of sibifluin according to the following weights, mix them, and prepare the pharmaceutical composition DS2.

[0070] Comparative Example 3: Preparation of the pharmaceutical composition (DS3) (the mass ratio of sibifluin:uridine:adenosine was 0:2:1). Weigh 100 mg of adenosine and 50 mg of uridine according to the following weights, mix them, and prepare the pharmaceutical composition DS3.

[0071] Test Example 1: Effects of different drug combinations on LPS-induced cell damage The efficacy of the drug composition was evaluated in an LPS-induced HK-2 cell injury model to assess its effect on nephritis.

[0072] 1. List of Abbreviations Table 1. List of Abbreviations

[0073] 2. Test substance The compositions prepared by the methods of Examples 1, 2, 3 and Comparative Examples 1, 2, and 3.

[0074] 3. Materials and Methods 3.1 Experimental Materials HK-2 cells.

[0075] 3.2 Experimental Reagents CCK8 kit, human IL-1β ELISA kit.

[0076] 3.3 Cell Culture Cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured at 37 °C and 5% CO2.

[0077] 4. Experimental Procedure 4.1 Model Establishment and Grouping Except for the normal control group of HK-2 cells which received no treatment, all other groups were induced to develop inflammatory cell models using 26 μg / mL LPS for 24 h, and then divided into model groups and drug treatment groups. The drug treatment groups were Example 1, Example 2, Example 3, and Comparative Example 1, Comparative Example 2, and Comparative Example 3. The control group and model group received no additional treatment and were cultured normally. Each drug treatment group was incubated with 1 μg / mL of the drug composition for 24 h, after which the cells were lysed, centrifuged, and the supernatant was collected for the determination of inflammatory factors.

[0078] 4.2 Drug Preparation Weigh the required amount of drug for each treatment group and add an appropriate amount of DMSO to prepare a 1 mg / mL stock solution. When adding the drug, dilute it with cell culture medium according to the drug dosage of each group.

[0079] 5. Sample Collection After drug administration, the cell culture supernatant and lower cell layer were collected by centrifugation. Cell lysis buffer was added to the lower cell layer, and the supernatant was collected by centrifugation and stored at -80°C for subsequent analysis.

[0080] 6. Data Statistical Analysis All data were analyzed using GraphPad Prism software to perform normality and homogeneity of variance tests. If the conditions were met, one-way ANOVA was used for inter-group comparisons; otherwise, Wilcoxon's test of arithmetic was used (compared to the blank control group). # P <0.05, ## P <0.01, ### P <0.001; compared with the model control group a P <0.05, aa P <0.01, aaa P <0.001).

[0081] 7. Results 7.1 Effects of drugs on IL-1β ELISA levels in an LPS cell injury model In this cell model, Examples 1, 2, and 3 all significantly reduced the IL-1β content in the cell model, while Comparative Examples 1 and 2 had no significant reducing effect. Specific results are shown in Table 2.

[0082] Table 2. IL-1β ELISA levels in each group of LPS cell injury models

[0083] Note: ### P <0.001, compared with the blank control group; aa P <0.01, aaa P <0.001, compared with the model control group.

[0084] 7.2 Microscopic observation of cell state Microscopic observation of cells in each group 24 h after drug administration revealed the following: Figure 1 , Figure 2 (Table 3) In the comparative group, no significant improvement was observed in cell shedding or other signs of disease by the naked eye, while the cell condition in the drug administration example group was significantly improved.

[0085] Table 3. Description of cell status in each group

[0086] Test Example 2: The therapeutic effects of different drug combinations on phenylketonuria 1. Test substance and grouping Control model group.

[0087] Dosing Example Group: Pharmaceutical compositions prepared using the methods of Examples 1, 2, 3, and 8; Comparative control group: Drug compositions prepared using the methods of Comparative Examples 1 and 2; Except for the control model group, the dosage of each administration example group and the administration comparison group was 70 mg / kg based on the total mass of sibifluin, uridine and adenosine, and the administration method was gavage.

[0088] 2. Experimental animals Pah enu2 Forty-two mice (phenylketonuria model mice, Jackson Laboratory, USA), half male and half female, weighing 20±2g, were used. They were acclimatized for 3 days at a room temperature of 23±2℃ with alternating light and dark lighting for 12 hours, and were provided with regular feed and access to drinking water. After 3 days of feeding, blood was collected from the tail vein to measure the phenylalanine content in the blood, which was used as the pre-administration phenylalanine concentration.

[0089] The model mice were randomly divided into 7 groups, with 6 mice in each group, half male and half female. The drug administration example groups were fed the drug compositions prepared according to methods 1, 2, 3, and 8, respectively, along with a conventional diet. The drug administration control example groups were fed the drug compositions prepared according to methods 1 and 2, respectively, along with a conventional diet. The control model group was fed a conventional diet. Each feeding consisted of 6g of feed, along with 5mL of drinking water, for 14 consecutive days. Blood was collected from the tail vein on day 14 after drug administration to detect the phenylalanine content in the serum, which was used as the post-drug administration phenylalanine concentration. The results are shown in Table 4 below.

[0090] Table 4. Effects of different drug combinations on Pah enu2 Effect of phenylalanine concentration in mouse blood

[0091] Note: ## P <0.01, compared with the control group.

[0092] Experimental results showed that the drug administration examples all significantly reduced the concentration of phenylalanine. Although the drug administration comparison groups also showed a reducing effect, their reducing effect was not as good as that of the drug administration examples.

[0093] Test Example 3: Observation of Adverse Reactions in Rats 1. Laboratory animals Fifty-four male SPF-grade SD rats, weighing 200-220g, were kept at a room temperature of 23±2℃ and under alternating light and dark lighting for 12 hours, while being fed regular feed and having access to their own drinking water.

[0094] 2. Test substance and grouping In this experiment, 54 SPF-grade female SD rats were randomly divided into 6 groups, with 9 rats in each group.

[0095] Dosing Example Groups: 3 groups, using the drug compositions prepared by the methods of Examples 1, 3, and 5, with a dosage of 210 mg / kg / day; Comparative groups: 2 groups, using the drug compositions prepared by methods of Comparative Examples 1 and 2, with a dosage of 210 mg / kg / d; Blank control group: Normal drinking water.

[0096] For the preparation of experimental drugs, use drinking water to make suspensions or solutions, and administer 2 mL each time.

[0097] 3. Test methods Male SD rats were acclimatized for 7 days and then randomly divided into four groups using a random number table: a blank control group, treatment example 1 group, treatment example 3 group, treatment example 5 group, treatment comparison group 1, and treatment comparison group 2. The rats were administered the medication by gavage once daily for 14 consecutive days. Their mental state, food and water intake, and body weight were closely monitored.

[0098] 4. Test Results 4.1 Gastrointestinal reactions In the blank control group, there were no significant changes in food intake and water intake. In the drug administration group, there were no significant changes in food intake and water intake, and no obvious diarrhea was observed. In the drug administration control groups, reduced food and water intake and some diarrhea were observed. Specifically, in drug administration control group 1 and drug administration control group 2, 4 and 1 rats, respectively, showed significantly reduced activity levels, and 6 and 1 rats, respectively, showed diarrhea.

[0099] 4.2 Changes in rat body weight Fourteen days after administration, the body weight of the blank control group increased significantly. Compared with the blank control group, there was no significant difference in body weight among the groups in each administration example. The body weight of rats in administration example 1 group decreased significantly, and three rats in administration example 2 group showed a significant decrease in body weight, which may be related to the decrease in food intake caused by gastrointestinal reactions. The specific changes in body weight are shown in Table 5.

[0100] Table 5. Changes in rat body weight

[0101] Note: ## P <0.01, compared with the blank control group.

[0102] The experimental results show that, under specific ratios of sibifluin, uridine, and adenosine, for example, a ratio of 7.0-10.0:0.9-1.2:1.0, this composition has a good therapeutic effect on phenylketonuria, reduces gastrointestinal reactions such as diarrhea caused by other component combinations, and also has good safety and anti-inflammatory effects, thus exerting a synergistic and detoxifying effect.

[0103] Test Example 4: An exploratory experiment on the role of ovarian insufficiency Experimental methods Animal model construction method: 10-week-old female C57 mice were injected intraperitoneally with 1,4-butanediol dimethylsulfonate (30 mg / kg) and cyclophosphamide (120 mg / kg) once a day for 7 consecutive days.

[0104] Grouping: Fifteen mice with ovarian insufficiency were randomly divided into three groups of five mice each: the model control group, drug group 1, and drug group 2.

[0105] Administration: Simultaneously with the establishment of the animal model, mice in each drug group were administered the drug via oral gavage once daily for 14 consecutive days. Drug group 1 received the drug composition prepared according to the method in Example 1 via oral gavage at a dose of 100 mg / kg; Drug group 2 received the drug composition prepared according to the method in Comparative Example 1 via oral gavage at a dose of 100 mg / kg; the model control group received physiological saline via oral gavage. The volume of the gavage fluid was 0.1 mL / 10 g based on the mouse's body weight. The control group mice received an equal volume of physiological saline via oral gavage daily. The drugs were prepared in physiological saline.

[0106] Treatment: After 14 days of drug administration, the mice were sacrificed and ovarian tissue was collected. One ovary was fixed with 4% paraformaldehyde, embedded in paraffin, and then serially sectioned (each section was 5 μm, and one section was collected every 5 sections). The 5 sections containing the largest facet of each ovary were stained with HE, and the primordial follicles, primary follicles, secondary follicles, corpus luteum, and atretic follicles were counted and statistically analyzed.

[0107] The results showed that, compared with the model control group, the number of follicles at all stages of the ovary of mice in drug group 1 was significantly increased, the number of corpora lutea was significantly increased, and the number of atretic follicles was decreased; compared with the model control group, the number of follicles at all stages of the ovary of mice in drug group 2 was significantly increased, the number of corpora lutea was slightly increased, and the number of atretic follicles was decreased.

[0108] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, It includes sibiformin, uridine, and adenosine, wherein the mass ratio of sibiformin:uridine:adenosine is 6.0-11.5:0.8-1.4:1.

0.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of sibifluin:uridine:adenosine is 6.5-11.0:0.8-1.3:1.

0.

3. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of sibifluin:uridine:adenosine is 7.0-10.0:0.8-1.3:1.

0.

4. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of sibifluin:uridine:adenosine is 7.0-10.0:0.9-1.2:1.

0.

5. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of sibifluin:uridine:adenosine is 7.8-9.4:0.9-1.2:1.

0.

6. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of sibifluin:uridine:adenosine is 8.7-10:0.9-1.2:1.

0.

7. The pharmaceutical composition according to any one of claims 1-6, characterized in that, It contains a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is present in a mass ratio of 0.1-200.0:100 to sibiflu.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is magnesium stearate, wherein the mass ratio of magnesium stearate to sibifluin is 0.1-5.0:

100.

10. The pharmaceutical composition according to claim 9, characterized in that, The mass ratio of magnesium stearate to Sibiforin is 1.0-5.0:

100.

11. A pharmaceutical preparation, characterized in that, The formulation comprises a pharmaceutical composition according to any one of claims 1-6 and a pharmaceutically acceptable carrier, wherein the formulation is a solid dosage form or a liquid dosage form.

12. The use of the pharmaceutical composition of any one of claims 1-10 or the pharmaceutical preparation of claim 11 in the preparation of a medicament for the prevention and / or treatment of phenylketonuria.

13. The use of the pharmaceutical composition according to any one of claims 1-10 or the pharmaceutical preparation according to claim 11 in the preparation of a medicament for the prevention and / or treatment of nephritis.

14. The use of the pharmaceutical composition according to any one of claims 1-10 or the pharmaceutical preparation according to claim 11 in the preparation of a medicament for the prevention and / or treatment of ovarian insufficiency.

15. The application according to claim 14, characterized in that, The aforementioned ovarian insufficiency includes premature ovarian failure.

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