Use of arginine and pharmaceutical composition containing arginine

Abstract

The present invention relates to the use of arginine, and a pharmaceutical composition containing arginine. First, provided in the present invention is an inflammation model successfully constructed by using zoledronic acid. The model can be used for screening a drug for inflammation-related diseases, thereby facilitating for research on inflammation-related mechanisms and pharmaceutical development. The construction method has good repeatability and stability. Further provided in the present invention is the use of arginine and a pharmaceutical composition thereof. Studies show that both arginine and the composition of arginine and zoledronic acid can effectively treat, prevent and / or relieve side reactions such as renal injury, and inflammation that occurs during the treatment by using zoledronic acid.

Description

Application of arginine and pharmaceutical composition containing arginine

[0001] This application claims priority from the following cases, the entire contents of which are incorporated herein by reference:

[0002] A Chinese patent application filed with the China Patent Office on November 30, 2023, with application number 202311631202.1 and titled “A pharmaceutical composition comprising zoledronic acid and capable of inhibiting its side effects and its use”;

[0003] Submitted to the China Patent Office on March 20, 2024, application number 202410317328.X, invention name "A method and application of bisphosphonate-induced mouse inflammation model"

[0004] A Chinese patent application was submitted to the China Patent Office on August 28, 2024, with application number 202411196488.X and invention name "Application of arginine in the preparation of drugs for preventing and treating renal damage caused by zoledronic acid". Technical Field

[0005] The present invention relates to the technical field of biomedicine, and in particular to the application of arginine and a pharmaceutical composition containing arginine. Background Art

[0006] Osteoporosis (OP) is a metabolic disease caused by a complex combination of endocrine, nutritional, immune, and genetic factors. It is a systemic bone disease characterized by decreased bone mass and damaged bone tissue microstructure, leading to increased bone brittleness and susceptibility to fractures. Osteoporosis is divided into two major categories: primary osteoporosis and secondary osteoporosis. Primary osteoporosis includes postmenopausal osteoporosis (type I), senile osteoporosis (type II), and idiopathic osteoporosis (including juvenile osteoporosis). Secondary osteoporosis refers to osteoporosis caused by any disease and / or drug that affects bone metabolism, as well as other clear causes.

[0007] Primary osteoporosis can be alleviated through drug therapy. Current anti-osteoporosis drugs are categorized by their mechanism of action as bone resorption inhibitors, bone formation promoters, dual-action drugs, drugs with other mechanisms, and traditional Chinese medicines. Bone resorption inhibitors include bisphosphonates, the RANKL monoclonal antibody (denosumab), selective estrogen receptor modulators (SERMs), and calcitonin. Bone formation promoters include parathyroid hormone (PTH) and PTH-related protein (PTHrP). Dual-action drugs include the sclerostin monoclonal antibody romosuzumab. Drugs with other mechanisms include active vitamin D and its analogs and vitamin K2.

[0008] The clinical use of bisphosphonates for the treatment of osteoporosis began in the 1990s and has evolved through three generations of drug development. Representative first-generation drugs include etidronate, clodronate, and tiludronate; second-generation products include pamidronate and alendronate; and third-generation products include ibandronate, risedronate, and zoledronate. All of these representative drugs are currently available in China.

[0009] Zoledronic acid is a third-generation bisphosphonate that inhibits osteoclast activity, induces osteoclast apoptosis, and inhibits bone resorption, reducing bone turnover. It significantly reduces fracture risk and increases bone mineral density in postmenopausal osteoporosis patients, making it the most effective bisphosphonate for preventing vertebral fractures. However, adverse reactions occur in approximately one-third of patients taking zoledronic acid. The most common adverse reactions are flu-like symptoms, including bone pain, fever, fatigue, chills, arthralgia and myalgia, arthritis, and subsequent joint swelling. These reactions typically occur within three days of administration and generally resolve within a few days. A small number of patients may also experience symptoms such as decreased plasma phosphate levels (approximately 20% of patients), nausea (approximately 5.8%), and vomiting (approximately 2.6%). Osteonecrosis of the jaw has been reported primarily in cancer patients receiving intravenous bisphosphonate therapy. Furthermore, several cases of zoledronic acid-induced renal injury have been reported in the literature.

[0010] The above analysis demonstrates that zoledronic acid, a bisphosphonate, has significant efficacy for osteoporosis, but also has relatively significant side effects. To address these side effects, research is focusing on two key areas: research and development of new osteoporosis treatments with improved efficacy and fewer side effects; and research on reducing zoledronic acid's side effects, including kidney damage, weight loss, and increased mortality.

[0011] Among them, there are many causes of kidney damage. In addition to zoledronic acid, many other drugs or causes can also cause kidney damage. For example, antibiotics and anticancer drugs (such as paclitaxel) are metabolized by the kidneys. If taken for a long time or in large doses, they will cause certain damage to the kidneys. Long-term high blood sugar can damage the blood vessels and cells of the kidneys, leading to a gradual decline in renal function. Sustained high blood pressure increases the pressure on the renal blood vessels, leading to damage to the kidney structure and function. In addition, trauma such as car accidents and falls from heights can also cause kidney trauma. Due to the different causes of kidney damage, subsequent treatment methods also have certain differences. Therefore, it is necessary to select the corresponding treatment drug based on the disease cause of kidney damage. For example, if kidney damage is caused by clinical hyperlipidemia, statins can be selected to lower lipids and play a protective role in the kidneys. If the kidney damage is caused by a nephrological disease, common drugs for treating basic kidney disease such as benazepril hydrochloride, captopril, and losartan potassium can be selected in the milder stage of the disease. Currently, the exact mechanism by which bisphosphonates cause renal damage is still unclear, and so far, no drug has been found that can effectively treat or prevent renal damage caused by zoledronic acid.

[0012] Summary of the Invention

[0013] In view of this, the present invention provides use of arginine in inhibiting the side effects of zoledronic acid and a pharmaceutical composition containing arginine.

[0014] First, the present invention found that intraperitoneal injection of bisphosphonates significantly reduced the survival rate and body weight of Kunming and BALB / c mice, while significantly increasing white blood cell counts, C-reactive protein levels, IL-6 levels, and TNF-α levels in these mice. Based on these observations, the severity of inflammation can be assessed, and a model for evaluating the degree of inflammation in animals has been established. This model can be used for drug screening, facilitating research on inflammation-related mechanisms and drug development.

[0015] Based on this inflammation model, the present invention has found that arginine can significantly reduce inflammatory factors (including IL-6 and TNF-α levels, etc.), and has a significant improvement effect on inflammation. Further research has found that arginine can effectively treat, prevent and / or alleviate the side effects related to the inflammatory mechanism caused by the use of zoledronic acid (such as kidney damage, weight loss, high mortality, abnormal coagulation function, etc.). At the same time, the present invention has found that a composition composed of zoledronic acid and arginine can effectively alleviate or prevent the occurrence of zoledronic acid side effects when treating osteoporosis.

[0016] The purpose of the first aspect of the present invention is to provide a method for constructing an animal inflammation model.

[0017] The second aspect of the present invention aims to provide a product.

[0018] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0019] The first aspect of the present invention provides a method for constructing an animal inflammation model, characterized by comprising the following steps:

[0020] The animals were administered bisphosphonate drugs.

[0021] Preferably, the animal comprises a mouse.

[0022] Preferably, the mouse comprises at least one of Kunming mice, BALB / c mice, SCID mice, C57BL / 6 mice, NOD mice, and ICR mice.

[0023] Preferably, the mice include Kunming mice and BALB / c mice.

[0024] Preferably, the inflammation model comprises an acute injury inflammation model.

[0025] Preferably, the bisphosphonate drug comprises one of etidronic acid or its salt, clodronic acid or its salt, pamidronic acid or its salt, alendronic acid or its salt, ibandronic acid or its salt, risedronic acid or its salt, and zoledronic acid or its salt.

[0026] Preferably, the bisphosphonate drug comprises one of risedronic acid or a salt thereof, and zoledronic acid or a salt thereof.

[0027] Preferably, the salt includes at least one of a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, and a salt with a basic or acidic amino acid. Preferred examples of metal salts include alkali metal salts such as sodium salts, potassium salts, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts, and the like; and aluminum salts. Preferred examples of salts with organic bases include salts with the following organic bases: trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Preferred examples of salts with basic amino acids include salts with the following basic amino acids: arginine, lysine, ornithine, and the like.

[0028] Preferably, the bisphosphonate drug comprises risedronic acid and / or zoledronic acid.

[0029] Preferably, the dosage of risedronic acid is 40-80 mg / kg.

[0030] Preferably, the dosage of zoledronic acid is 20-40 mg / kg.

[0031] Preferably, the administration method includes at least one of injection, gavage, and oral administration.

[0032] Preferably, the administration comprises injection.

[0033] Preferably, the injection includes at least one of intraperitoneal injection and intravenous injection.

[0034] Preferably, the injection comprises intraperitoneal injection.

[0035] Preferably, the administration is performed 1 to 3 times.

[0036] Preferably, the number of administrations includes 1 time.

[0037] Preferably, the construction method further comprises the step of raising the animal after administration.

[0038] Preferably, the number of days for raising animals is 1 to 7 days.

[0039] The second aspect of the present invention provides products for (a1)-(a5):

[0040] (a1) Study the mechanism of inflammation;

[0041] (a2) Screening drugs for treating inflammation;

[0042] (a3) Screening for drugs that prevent inflammation;

[0043] (a4) preparing a medicament for treating inflammation;

[0044] (a5) Preparing a drug for preventing inflammation.

[0045] The product includes the inflammation model obtained by the construction method.

[0046] The present invention also provides the use of the inflammation model or the product in at least one of the following aspects:

[0047] (a1) Study the mechanism of inflammation;

[0048] (a2) Screening drugs for treating inflammation;

[0049] (a3) Screening for drugs that prevent inflammation;

[0050] (a4) preparing a medicament for treating inflammation;

[0051] (a5) Preparing a drug for preventing inflammation.

[0052] The present invention also provides a method for screening drugs for treating, ameliorating and / or preventing inflammation-related diseases, characterized by comprising: administering a candidate drug to an animal inflammation model;

[0053] The following indicators were measured in the animal inflammation model group and the treatment group: white blood cell count, C-reactive protein level, IL-6 level, and TNF-α level;

[0054] If the index is significantly different between the candidate drug treatment group and the model group, the candidate drug is a drug for treating, improving and / or preventing inflammation-related diseases.

[0055] The animal inflammation model is the animal inflammation model described above or the animal inflammation model obtained by the method constructed above.

[0056] The present invention also provides the use of arginine in preparing medicine for preventing and treating kidney damage.

[0057] Kidney injury refers to the abnormal changes in the kidney's structure and function caused by various factors. In the present invention, kidney injury is mainly caused by drugs or pathology. Among them, drug-induced kidney injury is also called nephrotoxicity.

[0058] In some embodiments, the renal injury is zoledronic acid-induced renal injury (i.e., nephrotoxicity); wherein arginine prevents and treats zoledronic acid-induced renal injury, meaning that arginine antagonizes the renal injury caused by zoledronic acid. The prevention and treatment includes: ameliorating pathological renal damage and / or reducing the levels of blood biochemical indicators related to nephrotoxicity.

[0059] Among them, the pathological damage of the kidney includes at least one of the following: dark red urine, slight to mild inflammatory cell infiltration in the renal cortex / capsule / renal papilla, mild basophilic tubules in the cortex, mild to obvious dilation, slight to obvious degeneration / necrosis of the renal tubules, and moderate to obvious casts in the cortex / medullary.

[0060] The regulation of blood biochemical indicators related to nephrotoxicity includes: reducing urea and / or creatinine levels, and / or reducing inflammatory factor levels.

[0061] More specifically, the inflammatory factor level includes TNF-α.

[0062] In the above application provided by the present invention, the active ingredient of the drug is arginine, and the effective dosage of the arginine is 750 to 4500 mg / kg body weight.

[0063] In the above application of the present invention, the active ingredients of the drug include arginine and zoledronic acid, and the mass ratio of zoledronic acid to arginine is 1:150-600, specifically 1:150, 1:300, or 1:600.

[0064] In other embodiments, studies have shown that arginine has a significant preventive and therapeutic effect on diabetic renal injury. Therefore, the present invention also provides the use of arginine in the preparation of a medicament for preventing and treating diabetic renal injury. Such prevention and treatment includes: increasing podocyte viability, reducing levels of oxidative stress markers, promoting levels of anti-inflammatory factors in podocytes, and / or inhibiting levels of pro-inflammatory factors in podocytes.

[0065] The oxidative stress markers include ROS.

[0066] The podocyte pro-inflammatory factors include IL-6; the podocyte anti-inflammatory factors include IL-10.

[0067] The present invention provides a method for preparing a drug for preventing and treating kidney damage caused by diabetes, wherein the active ingredient of the drug is arginine. Furthermore, the concentration of the arginine is 0.5 to 1.5 mM, specifically 0.5 mM, 1 mM, or 1.5 mM.

[0068] In the aforementioned application, the arginine is L-arginine, D-arginine or DL-arginine. In a specific embodiment of the present invention, the arginine is L-arginine.

[0069] The present invention also provides a pharmaceutical composition for preventing renal damage caused by zoledronic acid, comprising arginine and zoledronic acid, wherein the mass ratio of zoledronic acid to arginine is 1:150 to 600, and specifically 1:150, 1:300, or 1:600.

[0070] The present invention also provides a method for treating, preventing, alleviating, and / or reducing renal damage caused by zoledronic acid, comprising administering arginine or a combination of arginine and zoledronic acid. Furthermore, in the composition, the mass ratio of zoledronic acid to arginine is 1:150 to 600, specifically 1:150, 1:300, or 1:600.

[0071] The present invention also provides a method for treating and / or preventing kidney damage caused by diabetes, comprising administering arginine at a concentration of 0.5 to 1.5 mM, specifically 0.5 mM, 1 mM, or 1.5 mM.

[0072] In the application and method of the present invention, arginine and zoledronic acid are administered simultaneously. In order to improve patient compliance, the present invention prepares arginine and zoledronic acid into a compound preparation, that is, the two are present in the form of a mixture in the same preparation.

[0073] In the present invention, the prevention and treatment include both prophylaxis and treatment. The simultaneous administration of arginine and zoledronic acid can be used to prevent the occurrence of renal damage caused by zoledronic acid. Administration of arginine after zoledronic acid can be used to treat renal damage caused by zoledronic acid and renal damage caused by diabetes. Administration of arginine after high glucose induction can treat renal damage caused by diabetes, and administration before high glucose induction can prevent renal damage caused by diabetes. The subjects of the prevention and treatment are humans or mammals, including species common in the art, including but not limited to cattle, horses, sheep, pigs, dogs, cats, rodents, and primates. The prevention and treatment effects are particularly pronounced in women or female mammals.

[0074] The present invention provides a pharmaceutical composition containing zoledronic acid and inhibiting its side reactions. The active ingredients of the pharmaceutical composition consist of zoledronic acid and the side reaction inhibitor; wherein the mass ratio of zoledronic acid to the side reaction inhibitor is 1:66-1000.

[0075] Furthermore, the mass ratio of zoledronic acid to the side effect inhibitor is 1:66.

[0076] Furthermore, the mass ratio of zoledronic acid to the side reaction inhibitor is 1:100.

[0077] Furthermore, the mass ratio of zoledronic acid to the side effect inhibitor is 1:150.

[0078] Furthermore, the mass ratio of zoledronic acid to the side effect inhibitor is 1:360.

[0079] Furthermore, the mass ratio of zoledronic acid to the side effect inhibitor is 1:720.

[0080] Furthermore, the side reaction inhibitor is arginine or a salt thereof.

[0081] Furthermore, the arginine is L-arginine, D-arginine or DL-arginine.

[0082] Furthermore, the pharmaceutical composition is in the form of powder, tablet, injection or pill.

[0083] Use of the above-mentioned pharmaceutical composition in preparing a drug for treating osteoporosis.

[0084] Studies have shown that administration of zoledronic acid can cause weight loss and high mortality in mice, and administration of a combination of arginine and zoledronic acid can significantly inhibit these two side effects. This shows that arginine can inhibit the side effects caused by zoledronic acid administration: weight loss and / or high mortality.

[0085] The use of the above-mentioned pharmaceutical composition in preparing medicine for treating inflammation or cold-like symptoms.

[0086] Furthermore, cold-like symptoms include fever, headache, nausea, bone pain, fatigue, chills, arthralgia, myalgia, etc.

[0087] The present invention has at least one of the following beneficial effects:

[0088] (1) The present invention proposes a method for constructing a mouse inflammation model, which simulates an inflammatory response by intraperitoneal injection of an overdose of bisphosphonate drugs. The severity of the inflammation can be determined by observing the survival and weight changes of Kunming mice and BALB / c mice. Intraperitoneal injection of bisphosphonate drugs can significantly reduce the survival rate and weight of Kunming mice and BALB / c mice, significantly increase the white blood cell count, C-reactive protein level, IL-6 level and TNF-α level of Kunming mice and BALB / c mice, and significantly increase the ALT and AST levels of SD rats. This method can be used to establish a new model for evaluating the degree of animal inflammation for drug screening, which is of great significance for inflammation-related research and drug development. Compared with the LPS-induced inflammation model, the method proposed in the present invention is simpler and easier to operate, and only requires intraperitoneal injection of bisphosphonate drugs. This administration method is closer to clinical medication and can better simulate the inflammatory response in the human body. In addition, the present invention can be used to evaluate the degree of animal inflammation and is suitable for research on inflammation-related mechanisms and drug screening, especially for the formulation improvement research of bisphosphonate drugs and the development of new drugs. Due to the simplicity and low side effects of this method, it also has certain application potential in the field of clinical treatment. Finally, the inflammation model proposed by the present invention can also be used to study inflammation-related mechanisms, providing new ideas and methods for the prevention and treatment of inflammatory diseases, and is of great significance for evaluating the anti-inflammatory effects of drugs and screening potential drugs. This invention is expected to provide a new experimental means for inflammation-related research, making it easier for experimenters to monitor the development of inflammation, improve research efficiency, reduce research costs, and is expected to play an important role in future drug development.

[0089] (2) The present invention used Beagle dogs as research subjects and found that zoledronic acid can cause weight loss and abnormal changes in the following indicators in Beagle dogs: increased absolute values ​​of blood cell count indicators WBC and Neut, and decreased PLT; increased coagulation indicator FIB, and prolonged APTT; increased blood biochemical indicator AST, and decreased P. The body weight and the above indicators in the test group given arginine and zoledronic acid did not change, or the changes were significantly lower than those in the zoledronic acid group. The results show that arginine can significantly inhibit the weight loss, inflammatory response, abnormal coagulation function, and abnormal changes in blood biochemical indicators caused by zoledronic acid.

[0090] (3) The present invention found that arginine can effectively reduce and improve the pathological damage of renal injury during the treatment with zoledronic acid; and reduce the levels of blood biochemical indicators related to nephrotoxicity, such as urea, creatinine and / or reduce the levels of inflammatory factors (such as TNF-α). This shows that arginine has a good therapeutic and preventive effect on renal injury caused by zoledronic acid. In addition, arginine can also reduce the oxidative stress and inflammatory response of podocytes induced by high glucose and improve the activity of podocytes, indicating that arginine also has a good therapeutic effect on renal injury caused by diabetes.

[0091] (4) The present invention uses arginine (L-arginine, D-arginine, or DL-arginine) to modify zoledronic acid (ZOL). The modified zoledronic acid formulation can effectively alleviate symptoms such as weight loss and inflammation during zoledronic acid treatment, eliminating the side effects associated with single zoledronic acid use. It is foreseeable that this formulation will have a broad market prospect in disease treatment and prevention, benefiting a large number of patients while achieving higher socioeconomic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 shows the effects of zoledronic acid and risedronic acid modeling on the survival rate and body weight of Kunming mice, where A represents the survival status of Kunming mice; B represents the weight change results of Kunming mice.

[0093] FIG2 shows the effects of zoledronic acid and risedronic acid on the survival rate and body weight of BALB / c mice, wherein A represents the survival of BALB / c mice; B represents the weight change results of BALB / c mice.

[0094] Figure 3 shows the effects of zoledronic acid and risedronic acid modeling on the number of white blood cells in mice, where A represents the number of white blood cells in Kunming mice after modeling; B represents the number of white blood cells in BALB / c mice after modeling; * indicates p<0.05; ** indicates p<0.01.

[0095] Figure 4 shows the effects of zoledronic acid and risedronic acid modeling on C-reactive protein levels in mice, where A represents the C-reactive protein level results of Kunming mice after modeling; B represents the C-reactive protein level results of BALB / c mice after modeling; * indicates p<0.05; ** indicates p<0.01.

[0096] Figure 5 shows the effects of zoledronic acid and risedronic acid modeling on IL-6 levels in mice, where A represents the IL-6 level in Kunming mice, and B represents the IL-6 level in BALB / c mice; * represents p < 0.05; ** represents p < 0.01.

[0097] Figure 6 shows the effects of zoledronic acid and risedronic acid on TNF-α levels in mice, where A represents the TNF-α level in Kunming mice, and B represents the TNF-α level in BALB / c mice; * represents p<0.05; ** represents p<0.01.

[0098] Figure 7 shows that arginine improves nephrotoxicity in Kunming mice treated with zoledronic acid; changes in body weight (A), survival (B), urea nitrogen (C), and serum creatinine (D) were recorded after intraperitoneal injection of the drug;

[0099] Figure 8 shows that arginine improves the renal toxicity of SD rats treated with zoledronic acid; changes in body weight (A, B), urea nitrogen (C), and serum creatinine (D) on day 8 were recorded after intravenous injection of the drug;

[0100] FIG9 shows that arginine inhibits the level of inflammatory factor TNF-α in KM mice induced by zoledronic acid;

[0101] FIG10 shows that arginine inhibits the level of inflammatory factor TNF-α in Balb / c mice induced by zoledronic acid;

[0102] FIG11 shows that arginine improves high glucose-induced podocyte injury;

[0103] FIG12 shows that arginine alleviates high glucose-induced oxidative stress in podocytes;

[0104] FIG13 shows that arginine alleviates the levels of inflammatory factors in podocytes induced by high glucose.

[0105] FIG14 shows the effects of the improved zoledronic acid formulation on the side effects caused by zoledronic acid (Kunming mice);

[0106] FIG15 shows the effects of different forms of arginine on the side effects of zoledronic acid in modified zoledronic acid formulations (Kunming mice);

[0107] FIG16 shows the effects of improved zoledronic acid formulations prepared with DL-arginine, D-arginine, and L-arginine on the side effects of zoledronic acid (Kunming mice);

[0108] FIG17 shows the effect of the improved zoledronic acid formulation prepared with DL-arginine on the side effects of zoledronic acid (Kunming mice). DETAILED DESCRIPTION

[0109] The present invention provides the application of arginine, the pharmaceutical composition containing arginine, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0110] Zoledronic acid used in the following examples was purchased from Maclean, product number Z834511; risedronic acid was purchased from Maclean, product number R822659.

[0111] In the present invention, the abbreviations, full Chinese names and detection methods of the blood cell count indicators are as follows: Note: This study only analyzed the above indicators measured by the fully automatic modular blood and body fluid analyzer, and other indicators measured by the instrument were not analyzed.

[0112] The abbreviations, Chinese names and detection methods of various coagulation function indicators are as follows:

[0113] The abbreviations, Chinese names and detection methods of various blood biochemistry indicators are as follows:

[0114] The abbreviations, full Chinese names and detection methods of the various indicators of urine analysis are as follows:

[0115] The present invention will be further described below in conjunction with the embodiments:

[0116] Example 1 A Kunming mouse inflammation model

[0117] Zoledronic acid (Zol) was dissolved in 1 mL of normal saline at a dose of 30 mg / kg per mouse and injected intraperitoneally into female 8-week-old Kunming mice. In this example, a total of 6 mice were treated, with the drug administered once on the first day.

[0118] Example 2 A Kunming mouse inflammation model

[0119] Risedronic acid (Ris) at a dose of 60 mg / kg per mouse was dissolved in 1 mL of normal saline and injected intraperitoneally into female 8-week-old Kunming mice. In this example, a total of 6 mice were treated, and the drug was administered once on the first day.

[0120] Example 3 A BALB / c mouse inflammation model

[0121] Zoledronic acid (Zol) at a dose of 30 mg / kg per mouse was dissolved in 1 mL of normal saline and injected intraperitoneally into female 8-week-old BALB / c mice. In this example, a total of 6 mice were treated, and the drug was administered once on the first day.

[0122] Example 4 A BALB / c mouse inflammation model

[0123] Risedronate (Ris) at a dose of 60 mg / kg per mouse was dissolved in 1 mL of normal saline and injected intraperitoneally into female 8-week-old BALB / c mice. Six mice were treated in this example, and the drug was administered once on the first day.

[0124] Example 5: A SD mouse inflammation model

[0125] Zoledronic acid was dissolved in 1 mL of normal saline at a dose of 5 mg / kg per SD rat and injected intraperitoneally into 8-week-old SD rats. Administration was continued once weekly for two consecutive weeks, for a total of two doses, i.e., on Days 1 and 8. The first dose was designated Day 1 (D1), and Day 15 represents the 15th day after the first dose. In this example, a total of 10 SD rats were treated, with 5 rats of each sex per group.

[0126] Comparative Example 1 Control treatment of Kunming mice

[0127] 1 mL of physiological saline was intraperitoneally injected into female 8-week-old Kunming mice. Six mice were treated in this control example and six mice were treated in this example. The drug was administered once on the first day.

[0128] Comparative Example 2 Control treatment of BALB / c mice

[0129] 1 mL of normal saline was intraperitoneally injected into female 8-week-old Kunming mice. A total of 6 mice were treated in this control example, and the drug was administered once on the first day.

[0130] Effect Example 1 Effects of Zoledronic Acid and Risedronic Acid Modeling on Survival Rate and Body Weight in Mice

[0131] On the first day of modeling, the modeling experimental treatments of Examples 1-4 and Comparative Examples 1-2 were performed. After the modeling treatments, the mice were raised normally, and the survival status and weight changes of the mice were recorded regularly every day.

[0132] The survival status of the Kunming mouse inflammation model constructed in Example 1 and Example 2 is shown in FIG1A , and the weight change results are shown in FIG1B .

[0133] As shown in Figure 1A, at 5 days after modeling, the survival rate of Kunming mice injected with zoledronic acid was 66.7%, and the survival rate of Kunming mice injected with risedronic acid was 66.7%. At 10 days after modeling, the survival rate of Kunming mice injected with zoledronic acid was 33.3%, and the survival rate of Kunming mice injected with risedronic acid was 16.7%. Compared with the control group, zoledronic acid and risedronic acid resulted in a reduced survival rate in Kunming mice. For acute injury and inflammation models, it is necessary to induce a severe inflammatory phenotype in the animals within a short period of time. The drug concentration used in modeling needs to be controlled within an appropriate range to avoid excessively high concentrations that lead to total animal death, while also avoiding low concentrations that result in a subtle phenotype. As shown in Figure 1A, the concentration used in this example is exactly the concentration at which the mice gradually die, the phenotype is clear, and the time is appropriate. Subsequent experiments will be conducted at this concentration. As shown in Figure 1B, the weight of Kunming mice injected with zoledronic acid (Zol) remains stable, while the weight of Kunming mice injected with risedronic acid continues to decline. Risedronic acid has a significant short-term irritation effect on Kunming mice.

[0134] The survival status of the BALB / c mouse inflammation model constructed in Example 3 and Example 4 is shown in FIG2A , and the weight change results are shown in FIG2B .

[0135] As shown in Figure 2A, on day 5 of modeling, the survival rate of BALB / c mice injected with zoledronic acid was 83.3%, while that of mice injected with risedronic acid was 100%. On day 10 of modeling, the survival rate of BALB / c mice injected with zoledronic acid was 33.3%, while that of mice injected with risedronic acid was 66.7%. Compared with the control group, zoledronic acid and risedronic acid decreased the survival rate of BALB / c mice.

[0136] As can be seen from Figure 2B, the body weight of BALB / c mice injected with zoledronic acid (Zol) and risedronic acid decreased, and both drugs had a short-term stimulating effect on BALB / c mice.

[0137] Effect Example 2 Effect of Zoledronic Acid and Risedronic Acid Modeling on the Number of White Blood Cells in Mice

[0138] 1. Experimental Methods

[0139] 1) Blood was collected from mice on the first and third days after the modeling treatment. The mice (Examples 1-4 and Comparative Examples 1-2) were fixed on a laboratory table to ensure that their bodies were in a stable state. Each mouse was individually numbered. A total of 24 mice were used for the four examples and two comparative examples. Blood was collected from the mouse orbits using a capillary blood collection tube to ensure that sufficient blood samples were collected. Gently wipe the area around the mouse's eyes to avoid blood contamination.

[0140] 2) Add 0.38 ml of leukocyte diluent (biosharp-BL1224A) to the test tube. Use a micropipette to accurately draw 20 μl of whole blood. Then wipe away any excess blood along the pipette tip with cotton. Check that the blood volume is sufficient.

[0141] 3) Insert a pipette into the bottom of the diluent in the small test tube and slowly drain the blood from the pipette. Then, draw up the white blood cell diluent to rinse the pipette three times. Immediately mix to create a white blood cell suspension. Allow to stand for a while until the red blood cells are completely destroyed, then mix again.

[0142] 4) Pipette 10 μl and flush it into the counting chamber along the gap between the coverslip and the counting chamber.

[0143] 5) After flushing, let the pool stand for 3 minutes. After the white blood cells have completely sunk, use a low-power microscope to count the number of white blood cells in the four large squares in the four corners. For the white blood cells on the line, use the principle of "record the top and not the bottom, count the left and not the right."

[0144] 6) Calculate the number of white blood cells according to the formula.

[0145] All reagents and consumables were commercially available conventional reagents and consumables, and the cell counting chamber was purchased from Reward Company.

[0146] 2. Experimental Results

[0147] After intraperitoneal injection of drugs, the number of white blood cells in Kunming mice was recorded, and the results are shown in Figure 3A. The white blood cell count of Kunming mice in the control group was 4×10 6 The number of white blood cells in Kunming mice increased after Zol and Ris injection, with the number of white blood cells in Zol-injected mice being 8.8×10 6 / mL, and 7.6×10 in Ris-injected mice 6 There was a significant difference in the 37.5% and 17.5% of the controls, respectively.

[0148] The number of leukocytes in BALB / c mice was recorded after intraperitoneal injection of drugs. The results are shown in Figure 3B. The number of leukocytes in BALB / c mice in the control group was 4×10 6 The number of white blood cells in BALB / c mice increased after Zol and Ris injection, with the number of white blood cells in Zol-injected mice increasing to 8.5×10 6 / mL, and 7.9×10 in Ris-injected mice 6 There was a significant difference in the 37.5% and 17.5% of the controls, respectively.

[0149] Effect Example 3 Effect of Zoledronic Acid and Risedronate Modeling on C-Reactive Protein Levels in Mice

[0150] 1. Experimental Methods

[0151] 1) Blood was collected from mice on the 1st and 3rd day after the modeling treatment. The mice (Examples 1-4 and Comparative Examples 1-2) were fixed on the experimental table to ensure that their bodies were in a stable state. Each mouse had an independent number. There were 24 mice in 4 embodiments and 2 comparative examples. Blood was collected from the mouse eye sockets using a capillary blood collection tube to ensure that enough blood samples were collected. Gently wipe the area around the mouse eyes to avoid blood contamination. Anticoagulation was performed using a hematocrit capillary tube (heparinized, inner diameter 1.1-1.2 mm). Heparinized hematocrit capillary tube (AS ONE / Azov -- 2-454-21).

[0152] 2) The collected blood samples were centrifuged (3000 rpm) for 10 min to separate the plasma for the detection of C-reactive protein.

[0153] 3) Add 100 μl of standard working solution to each standard reaction well for drawing a standard curve. Add 100 μl of plasma treated in step 2) to each detection well. Seal the plate and incubate at 37°C for 90 min.

[0154] 4) Discard the liquid, spin dry, and add 100 μl of biotinylated C-reactive protein antibody to each reaction well. Seal the plate and incubate at 37°C for 60 minutes. Wash and repeat four times.

[0155] 5) Add 100 μl of HRP-labeled streptavidin to each reaction well, seal the plate, and incubate at 37°C for 30 min. Wash and repeat four times.

[0156] 6) Add 90 μl of color developer (protect from light) to each reaction well, seal the plate, and incubate at 37°C in the dark for about 15 minutes.

[0157] 7) Add 50 μl of stop solution to each reaction well and immediately measure the OD value using a microplate reader at a wavelength of 450 nm.

[0158] 8) Use software to draw a standard curve with the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis.

[0159] The standard working solution, biotin-labeled C-reactive protein antibody, HRP-labeled streptavidin, colorimetric reagent, and stop solution were from the mouse C-reactive protein (CRP) ELISA kit (Sangon Biotechnology).

[0160] 2. Experimental Results

[0161] After intraperitoneal injection of the drugs, C-reactive protein (CRP) levels were recorded in the mice. The results for Kunming mice are shown in Figure 4A. The C-reactive protein concentration in the control group of Kunming mice was 0.79 ng / mL, compared with 0.83 ng / mL in Zol-injected mice and 0.82 ng / mL in Ris-injected mice. The results for BALB / c mice are shown in Figure 4B. The C-reactive protein concentration in the control group of BALB / c mice was 0.80 ng / mL, compared with 0.83 ng / mL in Zol-injected mice and 0.82 ng / mL in Ris-injected mice. Compared with the control group, C-reactive protein levels in mice injected with Zol and Ris were significantly elevated, indicating an acute inflammatory response in the mice following drug injection.

[0162] Effect Example 4 Effects of Zoledronic Acid and Risedronic Acid Modeling on Inflammatory Factor Levels in Mice

[0163] 1) Blood was collected from mice on the first and third days after the modeling treatment. The mice (Examples 1-4 and Comparative Examples 1-2) were fixed on a laboratory table to ensure that their bodies were in a stable state. Each mouse was individually numbered. A total of 24 mice were used for the four examples and two comparative examples. Blood was collected from the mouse orbits using a capillary blood collection tube to ensure that sufficient blood samples were collected. Gently wipe the area around the mouse's eyes to avoid blood contamination.

[0164] 2) The collected blood samples were centrifuged (3000 rpm) for 10 min, and plasma was collected for the detection of inflammatory factors.

[0165] 3) RNA was extracted and reverse transcribed into cDNA for RT-qPCR analysis using the following kits: Total RNA Extraction Kit (Promega LS1040), Reverse Transcription Kit (Bio-Guang Biotechnology BG0070), and SYBR (Bio-Guang Biotechnology BG0014). For detailed instructions, refer to the kit instructions.

[0166] 4) Record the levels of IL-6 and TNF-α in Kunming mice and BALB / c mice.

[0167] The primer sequences used are shown in Table 1.

[0168] Table 1 Primer sequence information

[0169] 2. Experimental Results

[0170] The IL-6 levels of mice after drug injection are shown in FIG5 , wherein A represents the IL-6 level of Kunming mice and B represents the IL-6 level of BALB / c mice.

[0171] The TNF-α levels of mice after drug injection are shown in FIG6 , wherein A represents the TNF-α level of Kunming mice and B represents the TNF-α level of BALB / c mice.

[0172] The results showed that Zol and Ris could significantly increase the IL-6 level of mice by about 2 to 4 times within 1 day, and significantly increase the TNF-α level of mice by about 2 to 7 times within 1 day.

[0173] In summary, Zol and Ris can cause obvious phenotypes in mice in the short term, leading to decreased survival rate and body weight, increased white blood cell count, increased C-reactive protein levels, and increased inflammatory factors IL-6 and TNF-α, indicating that mice produce acute inflammatory responses and can be used for mouse inflammation modeling.

[0174] Effect Example 5

[0175] Various formulations were prepared, with the mass ratios of the two components being ZOL:L-Arg = 1:150, ZOL:L-Arg = 1:300, and ZOL:L-Arg = 1:600. This was intended to reduce the side effects of zoledronic acid during clinical use. The administration method was the same as that of zoledronic acid in Example 5, with the following dosages:

[0176] The ZOL:L-Arg (1:150) group received the following doses: 5 mg / kg ZOL + 750 mg / kg L-Arg;

[0177] The ZOL:L-Arg (1:300) group received the following doses: 5 mg / kg ZOL + 1500 mg / kg L-Arg;

[0178] The dosage of ZOL:L-Arg (1:600) group was: 5 mg / kg ZOL + 3000 mg / kg L-Arg.

[0179] The above formulation was intravenously injected into SD rats, with five SD rats per sex per group. Changes in ALT and AST in the rats' blood biochemical profile were observed and recorded on Day 15 (15 days after the first dose). The results are shown in the table below. The blood biochemical parameters of the zoledronic acid group in Group 5 are those of the SD rat model established according to Example 5.

[0180] Table 2 Summary of blood biochemical changes Note: "*" indicates p ≤ 0.05 compared with the negative control group of the same sex during the same period; "↑" indicates an increase, and "↓" indicates a decrease. Groups 1 to 6 are the negative control group, the ZOL:L-Arg (1:150) group, the ZOL:L-Arg (1:300) group, the ZOL:L-Arg (1:600) group, the zoledronic acid (5 mg / kg) group, and the arginine (3000 mg / kg) group, respectively.

[0181] During the trial, compared with the negative control group of the same sex during the same period, treatment-related changes in blood biochemistry were observed in all treatment groups. Specifically, elevations in ALT and AST were observed only in the zoledronic acid group and the ZOL:L-Arg = 1:150, ZOL:L-Arg = 1:300, and ZOL:L-Arg = 1:600 ​​treatment groups, suggesting a correlation with zoledronic acid. For most parameters, the zoledronic acid group showed the greatest changes, while the treatment groups ZOL:L-Arg = 1:150, ZOL:L-Arg = 1:300, and ZOL:L-Arg = 1:600 ​​showed less changes. Arginine had a significant attenuating effect, with the ZOL:L-Arg = 1:150 group exhibiting a lower attenuating effect than the ZOL:L-Arg = 1:300 and ZOL:L-Arg = 1:600 ​​groups. The treatment groups ZOL:L-Arg = 1:300 and ZOL:L-Arg = 1:600 ​​exhibited similar attenuating effects.

[0182] Example 6

[0183] This study involved 12 Beagle dogs (6 per sex) randomly divided into groups 1 to 6, with 1 dog per sex per group. Group 1 was administered the negative control, sodium chloride injection. Groups 2 to 4 were administered the test product, zoledronic acid and its modified formulation, at doses of zoledronic acid:L-arginine (2:300 mg / kg, 2:600 mg / kg, and 2:1200 mg / kg, respectively). Group 5 was administered with test product component 1 (zoledronic acid, 2 mg / kg), and group 6 was administered with test product component 2 (L-arginine, 1200 mg / kg). These drugs were administered once weekly for one week, for a total of two doses. Changes in body weight, blood cell count, coagulation function, and biochemical parameters were observed and recorded. The results are shown in Tables 3 to 6.

[0184] 1. Weight

[0185] During the study, compared to the negative control group and before self-administration, both male and female animals in the Component 1 group (zoledronic acid 2 mg / kg) showed a gradual decrease in body weight from Day 4 to Day 14 (maximum change: males: -14.2%, females: -10.4%). Female animals in the test product group (2:300 mg / kg) showed a gradual decrease in body weight from Day 4 to Day 14 (maximum change: -7.5%). No abnormal changes in body weight related to drug administration were observed in the other groups. Since the body weight loss in the test product group (2:300 mg / kg) was less than that in the Component 1 group, and no similar changes were observed in the Component 2 group, it is believed that Component 2 can mitigate the weight loss caused by Component 1.

[0186] Table 3 Body weight changes of animals in each group (kg) Note: Compared with the negative control group and the pre-drug value, the bold value indicates a decrease.

[0187] 2. Blood cell count

[0188] During the experiment, compared with the negative control group and the pre-drug values, the absolute values ​​of WBC and Neut of male and female animals in the 2 mg / kg component 1 group increased, and PLT decreased 3 days after the first dose (D4); the absolute value of Neut increased on the day after the last dose (D9); the absolute and relative values ​​of Retic and PLT decreased on the day after the last dose (D9) and 1 week (D15); the extent of change in the above indicators were: WBC: 200.9%~216.8%, Neut: 180.9%~465.9%, absolute value of Retic: -44.1%~-81.7%, relative value of Retic: -32.7%~-77.3%, PLT: -23.4%~-83.1%. Absolute increases in WBC and Neut values ​​were observed in male animals in the 300 mg / kg test product group on the day following the first dose (D4) and in female animals on the day following the final dose (D9). The magnitude of these changes was 37.7% to 39.9% for WBC and 60.4% to 75.1% for Neut, respectively. No other drug-related abnormalities were observed in the other groups. The magnitude of these changes in the test product group was lower than in the component 1 group, or no similar changes were observed. Similar changes were also not observed in the component 2 group. Therefore, it is suggested that component 2 can alleviate the inflammatory response induced by component 1.

[0189] Table 4 Changes in some blood cell count indicators of animals in each group Note: Compared with the negative control group and the pre-drug value, bold indicates an increase or decrease.

[0190] 3. Coagulation function

[0191] During the experiment, compared with the negative control group and the pre-drug value, the APTT of the male and female animals in the 2 mg / kg component 1 group and the female and / or male animals in the 2:300 mg / kg, 2:600 mg / kg and 2:1200 mg / kg test product groups was prolonged on the next day (D9) and / or 1 week (D15) after the last dose. The prolongation time ranges of each group were: 6.9-10.3s, 9.5s, 3.6-5.2s, 4.2s; 2 mg / kg Increased FIB was observed in male and female animals in the Component 1 group and the 2:300 mg / kg, 2:600 mg / kg, and 2:1200 mg / kg test product groups on the day after the first dose (D2) and / or three days (D4), the day after the last dose (D9), and one week (D15), with the magnitude of change ranging from 41.6% to 267.5%, 8.1% to 224.4%, 10.6% to 181.9%, and 9.7% to 176.4%, respectively. The magnitude of change in these parameters in the test product groups was lower than in the Component 1 group, and a dose-response relationship was observed. Therefore, it is believed that Component 2 can effectively inhibit the FIB increase and APTT prolongation caused by Component 1, alleviating or improving coagulation abnormalities.

[0192] Table 5 Changes of APTT and FIB in animals in each group Note: Compared with the negative control group and the pre-drug value, red bold indicates prolonged or increased.

[0193] During the experiment, compared with the negative control group and the pre-drug value, the female animals in Group 2 of the 1200 mg / kg component showed an increase in FIB on D15. Since there were no drug-related abnormal changes in other indicators such as the animal's body weight, food intake, and blood cell count, and this was only seen in one sex, the increase in FIB was considered to have no important toxicological significance.

[0194] 4. Blood biochemical indicators

[0195] During the experiment, compared to the negative control group and pre-dose values, male and female animals in the 2 mg / kg component 1 group showed increased AST and decreased P levels three days after the first dose (D4), the day after the last dose (D9), and one week (D15). The magnitude of change was 244.8% to 821.7% for AST and -44.4% to -61.4% for P, respectively. Male animals in the 300 mg / kg test product group showed an increase in AST by 334.8% one week after the last dose (D15). In male and female animals treated with the 2:300 mg / kg, 2:600 mg / kg, and 2:1200 mg / kg test product groups, decreases in P were observed three days after the first dose (D4), the day after the last dose (D9), and one week after the final dose (D15). The magnitude of change ranged from 32.7% to 46.4%, 17.6% to 48.6%, and 38.2% to 52.3%, respectively. The magnitude of change in these parameters in the test product groups was lower than that in the component 1 group and was not observed in the component 2 group. Therefore, it is believed that component 2 can alleviate the increase in AST and decrease in P caused by component 1.

[0196] Table 6 Changes of AST and P in animals in each group

[0197] Example 7

[0198] (1) Kunming mouse animal experiment

[0199] First, we injected an overdose of zoledronic acid (30 mg / kg) intraperitoneally into Kunming mice to simulate the side effects of zoledronic acid, thereby establishing an animal model to evaluate the efficacy of improved formulations. We also prepared an improved formulation of zoledronic acid with a zoledronic acid:L-arginine ratio of 1:150, hoping to reduce the side effects of zoledronic acid during clinical use.

[0200] We then injected the modified zoledronic acid formulation into Kunming mice via intraperitoneal injection, observed and recorded the mice's survival and weight changes, and also tested urea, creatinine and other kidney toxicity-related indicators in the Kunming mice. The results are shown in Figure 7:

[0201] Kunming mice (8-week-old female, 25 ± 3 g) were randomly divided into three groups: NS, ZOL, and ZOL:L-Arg = 1:150, with five mice per group. The ZOL:L-Arg group received 30 mg / kg ZOL plus 4500 mg / kg L-Arg via intraperitoneal injection only on the first day; no further administration was performed. Changes in body weight (A), survival (B), blood urea nitrogen (C), and serum creatinine (D) were recorded after intraperitoneal injection. Data were analyzed using GraphPad Prism 8.

[0202] Compared with the zoledronic acid model group, the modified zoledronic acid formulation containing L-Arg effectively suppressed the side effects of zoledronic acid in the animal model, alleviated weight loss in Kunming mice, and effectively reduced mortality in the experimental animals. Furthermore, compared with the zoledronic acid model group, the L-Arg-modified zoledronic acid formulation significantly reduced blood urea nitrogen and blood creatinine levels.

[0203] The above results show that during the experiment, weight loss, death and blood biochemical (urea nitrogen and blood creatinine) changes in Kunming mice were related to intraperitoneal injection of zoledronic acid, while the use of the improved preparation can effectively alleviate the weight loss and death of mice, and blood biochemical indicators further prove that L-Arg treatment can effectively alleviate the renal toxicity caused by zoledronic acid.

[0204] (2) Sprague-Dawley rat animal experiment

[0205] First, we injected 5 mg / kg of zoledronic acid intravenously into SD rats to simulate the side effects of zoledronic acid, thereby establishing an animal model to evaluate the efficacy of formulation improvements. We also prepared multiple formulations with weight ratios of ZOL:L-Arg = 1:150, ZOL:L-Arg = 1:300, and ZOL:L-Arg = 1:600, hoping to reduce the side effects of zoledronic acid during clinical use.

[0206] SD rats were randomly divided into 6 groups (male and female): NS, ZOL, ZOL:L-Arg=1:150, ZOL:L-Arg=1:300, and ZOL:L-Arg=1:600, with 5 males and 5 females in each group, for a total of 10 rats.

[0207] The ZOL:L-Arg (1:150) group received the following doses: 5 mg / kg ZOL + 750 mg / kg L-Arg;

[0208] The ZOL:L-Arg (1:300) group received the following doses: 5 mg / kg ZOL + 1500 mg / kg L-Arg;

[0209] The dosage of the ZOL:L-Arg (1:600) group was: 5 mg / kg ZOL + 3000 mg / kg L-Arg.

[0210] We then intravenously administered the modified zoledronic acid formulation to SD rats, and observed and recorded changes in body weight, blood cell count, blood biochemistry, and urine color. Data were analyzed using GraphPad Prism 8. The results are shown in Tables 7–10 and Figure 8 : Body weight after intravenous injection is shown in Figures 8A and 8B , blood urea nitrogen on day 8 is shown in Figure 8C , and blood creatinine is shown in Figure 8D .

[0211] (1) Weight statistics

[0212] Table 7 Note: "*" indicates p ≤ 0.05 compared with the negative control group of the same sex during the same period; "↓" indicates a decrease. Groups 1 to 6 are the negative control group, the ZOL:L-Arg (1:150) group, the ZOL:L-Arg (1:300) group, the ZOL:L-Arg (1:600) group, the zoledronic acid (5 mg / kg) group, and the arginine (3000 mg / kg) group, respectively.

[0213] (2) Blood cell count statistics

[0214] Table 8 Note: "*" indicates p ≤ 0.05 compared with the negative control group of the same sex during the same period; "↓" indicates a decrease. Groups 1 to 6 are the negative control group, the ZOL:L-Arg (1:150) group, the ZOL:L-Arg (1:300) group, the ZOL:L-Arg (1:600) group, the zoledronic acid (5 mg / kg) group, and the arginine (3000 mg / kg) group, respectively.

[0215] (3) Blood biochemical statistics

[0216] Table 9 Note: "*" indicates p ≤ 0.05 compared with the negative control group of the same sex during the same period; "↑" indicates an increase, and "↓" indicates a decrease. Groups 1 to 6 are the negative control group, the ZOL:L-Arg (1:150) group, the ZOL:L-Arg (1:300) group, the ZOL:L-Arg (1:600) group, the zoledronic acid (5 mg / kg) group, and the arginine (3000 mg / kg) group, respectively.

[0217] During the trial, compared with the same-sex negative control group, animals in the low-, medium-, and high-dose arginine groups and the zoledronic acid group showed weight loss after the first dose. One week after the final dose, body weight remained lower than that of the same-sex negative control group, but weight gain was similar to that of the same-sex negative control group. The weight change trend in the component two group was similar to that of the same-sex negative control group. The weight loss was related to the intravenous administration of zoledronic acid, but the administration of arginine had a toxicity-reducing effect on zoledronic acid.

[0218] During the trial, compared with the negative control group of the same sex during the same period, drug-related changes in blood biochemistry (creatinine and urea) were observed in all treatment groups. A total of 8 animals (4 / 5♂, 4 / 5♀) in the zoledronic acid group died on Days 7, 10, and 13. Clinical observation from the time of drug administration until death revealed dark red urine. Microscopic examination revealed mild to mild inflammatory cell infiltration in the renal cortex, capsule, and papillae, mild basophilic tubules in the cortex, mild to marked dilatation and mild to marked degeneration / necrosis of the renal tubules, moderate to marked casts in the cortex and medulla, and altered blood biochemical parameters (increased UREA and Cre). Administration of arginine alleviated the pathological changes and related biochemical parameters of renal injury in rats.

[0219] (4) Urinalysis results

[0220] Table 10 Changes in urine color

[0221] During the trial, red urine was observed in animals in the zoledronic acid group after administration on day 8, which is consistent with clinical observations of dark red urine (including the discovery of dead animals). Since red urine was only observed in the zoledronic acid group and was related to zoledronic acid, and was not observed in the ZOL:L-Arg=1:150, ZOL:L-Arg=1:300, and ZOL:L-Arg=1:600 ​​groups, it is believed that arginine has a significant attenuating effect.

[0222] (III) Elisa detection of TNF-α levels

[0223] (1) Experimental groups:

[0224] Control: normal saline (Balb / c, half male and half female, totaling 10 rats; KM, half male and half female, totaling 10 rats);

[0225] Model: 0.75 mg / ml zoledronic acid (Balb / c, half male and half female, totaling 10, KM, half male and half female, totaling 10);

[0226] Intervention group: Zoledronic acid: L-arginine = 1:150 (Balb / c half male and half female, totaling 10, KM half male and half female, totaling 10)

[0227] (2) Experimental steps:

[0228] Mice were collected and injected with drugs on the first day. On the fourth day, all blood was collected, centrifuged, and the plasma was subjected to ELISA. The detection indicator: TNF-α.

[0229] (3) Elisa test method:

[0230] 1. Reagent preparation: Prepare various test reagents, diluted standards and samples to be tested.

[0231] 2. Determine the enzyme label strips: Calculate the enzyme label strips required for the sample and standard to be tested, take the enzyme label strips out of the aluminum foil bag, put the remaining enzyme label strips back into the aluminum foil bag, seal the bag, and store at low temperature.

[0232] 3. Soak the plate: Add 1× wash solution (350 μL / well) to the plate. Let it sit for 30 seconds, then discard the liquid in the wells and pat the plate dry. The amount of liquid significantly affects the test results. Ensure that no wash solution remains after the final pat.

[0233] 4. Sample loading and incubation: Add each gradient standard and diluted sample to be tested at 100 μL / well, ensuring that the sample is loaded within 15 minutes, and incubate at room temperature for 2 hours.

[0234] 5. Wash the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μL / well) and wash the plate 5 times, and pat the ELISA plate dry.

[0235] 6. Detection antibody incubation: Add detection antibody prepared to working concentration to the ELISA plate at 100 μL / well and incubate at room temperature for 2 hours.

[0236] 7. Wash the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μL / well) and wash the plate 5 times, and pat the ELISA plate dry.

[0237] 8. Enzyme conjugate incubation: Add the enzyme conjugate prepared to the working concentration into the ELISA plate, 100 μL / well, and incubate at room temperature for 20 minutes.

[0238] 9. Wash the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μL / well) and wash the plate 5 times, and pat the ELISA plate dry.

[0239] 10. Color development: Return the substrate solution to room temperature 10 minutes before use. Add the substrate solution to the ELISA plate at 100 μL / well and incubate at room temperature in the dark for 15 minutes.

[0240] 11. Stop: Add 50 μL / well stop solution to the ELISA plate. The color will turn from blue to yellow. Gently shake the plate until the color is even.

[0241] 12. Reading: Read the absorbance at 450nm / 630nm within 10 minutes. Calculate the TNF-α concentration. See Figures 9-10 for the results.

[0242] (4) Result analysis

[0243] The results showed that arginine could significantly inhibit the increase in the level of inflammatory factor TNF-α caused by zoledronic acid, and improve the renal damage caused by the inflammatory response induced by zoledronic acid, especially in female mice.

[0244] (IV) Verification of the efficacy of arginine on diabetic kidney damage—MPC-5 cell experiment

[0245] 1. Construction of a cell model: Mouse renal podocyte MPC-5 cells were treated with high glucose (40 mM) to simulate a diabetic nephropathy cell model. High glucose treatment resulted in: (1) decreased cell viability; (2) decreased expression of podocin, a protein involved in the normal development and maintenance of the glomerular filtration barrier; and (3) significant increases in oxidative stress and inflammatory markers. This indicates that a cell model reflecting diabetic kidney damage has been successfully established and used to evaluate the effects of arginine on kidney damage.

[0246] 2. Verification of the efficacy of arginine on diabetic kidney damage

[0247] (1) Arginine improves high glucose-induced podocyte injury

[0248] First, cells were treated with different concentrations of arginine (0, 0.1, 0.5, 1, 2, 5, 10, 15, and 20 mM) for 24 hours. CCK-8 analysis revealed that low, medium, and high doses of arginine (0.5, 1, and 1.5 mM) were safe for cell survival. MPC-5 cells were then cultured in 12-well plates, and cell viability was assessed 24 hours after the different treatments. The dosages are shown in Table 11, and the results are shown in Figure 11.

[0249] Table 11

[0250] After 24 hours of high-glucose treatment, podocyte viability decreased significantly compared to the control group. Cell viability in the low-, medium-, and high-dose arginine groups was significantly increased compared to the broth group, with similar effects to those seen in the glucose-lowering drug metformin-treated group. This suggests that arginine treatment can improve cell viability in diabetic nephropathy models. A dose-dependent increase in podocin protein expression was also observed in the arginine-treated groups, with the increase in podocin protein levels in the high-dose arginine group being even more pronounced than in the metformin-treated group. This suggests that arginine treatment can promote the normal development of the glomerular filtration barrier and maintain its function.

[0251] (2) Arginine alleviates high glucose-induced oxidative stress in podocytes

[0252] By using the DCFH-DA probe for reactive oxygen species (ROS) fluorescence staining, it was observed that after high glucose treatment, the intracellular ROS level increased significantly, and arginine could reverse the increase of ROS in podocytes induced by high glucose, and its induction effect in the high-dose group was better than that in the metformin-treated group, indicating that arginine can alleviate high glucose-induced podocyte oxidative stress and improve renal damage caused by high glucose-induced oxidative stress, as shown in Figure 6.

[0253] (3) Arginine reduces the level of inflammatory factors in podocytes induced by high glucose

[0254] Oxidative stress often triggers inflammation, leading to the release of inflammatory cytokines. By measuring inflammatory cytokines, it was observed that high glucose upregulates the expression of the pro-inflammatory cytokine IL-6 and downregulates the expression of the anti-inflammatory cytokine IL-10. Arginine significantly inhibits IL-6 expression and increases IL-10 levels, with the induction effect being greater in the high-dose group than in the metformin-treated group (see Figure 7).

[0255] 3. Experimental Results and Analysis

[0256] Analysis of experimental results showed that arginine treatment can improve podocyte viability, reduce the levels of oxidative stress markers, and alleviate inflammatory response (reduce the level of pro-inflammatory factor IL-6 and increase the level of anti-inflammatory factor IL-10), further reflecting the potential of arginine to treat diabetic nephropathy by protecting podocytes from high glucose-induced oxidative stress and damage.

[0257] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0258] Example 8

[0259] 1. Constructing an animal model

[0260] Overdose of zoledronic acid was injected intraperitoneally into mice (e.g., Kunming mice) to simulate the side effects of zoledronic acid. The specific side effects of the mice after drug absorption were: (1) weight loss; (2) death in severe cases. Also observed were: lethargy and matted fur. This indicates that an animal model for zoledronic acid side effects has been successfully established and used to evaluate the effects of the improved zoledronic acid formulation on zoledronic acid side effects.

[0261] 2. Construction of an improved formulation of zoledronic acid

[0262] Different improved zoledronic acid preparations are formed by mixing zoledronic acid and L-arginine, DL-arginine or D-arginine in different mass ratios, and the mass ratios include but are not limited to the following ratios: ZOL:L-Arg=1:66, ZOL:L-Arg=1:100, ZOL:L-Arg=1:150, ZOL:L-Arg=1:180, ZOL:L-Arg=1:360, ZOL:DL-Arg=1:150, ZOL:DL-Arg=1:180, ZOL:DL-Arg=1:360, ZOL:D-Arg=1:100, ZOL:D-Arg=1:180, ZOL:D-Arg=1:360, ZOL:D-Arg=1:720.

[0263] Example 8

[0264] 1. Efficacy verification of the improved zoledronic acid formulation

[0265] Various modified zoledronic acid formulations were intraperitoneally injected into Kunming mice (8-week-old females, 25±3g). The mice were randomly divided into five groups (7 mice per group): NS, ZOL, ZOL:L-Arg = 1:66, ZOL:L-Arg = 1:100, and ZOL:L-Arg = 1:150. The survival and weight changes of the mice were observed and recorded. The results are shown in Figure 1. Figure 14 shows the survival data in A, B is the secondary graph of Figure A, and shows the time to median lethality. C shows the weight change, and D is the secondary graph of Figure C, showing the weight loss of each group on day 6. In Figure C, the ZOL:L-Arg = 1:66 group was terminated on day 5, indicating that only one Kunming mouse remained in this group. The ZOL group was terminated on day 9, indicating that only one Kunming mouse remained in this group. The ZOL:L-Arg = 1:100 group was terminated on day 12, indicating that only one Kunming mouse remained in this group, which is no longer statistically significant.

[0266] As shown in Figure 14, the improved zoledronic acid formulation with a ratio of ZOL:L-Arg = 1:100 significantly suppressed the side effects of the original formulation in animal models, effectively reducing the mortality rate of experimental animals and alleviating weight loss in the Kunming mice.

[0267] 2. Comparison of the effects of arginine and its analogs

[0268] (1) Preparation of modifiers: L-histidine, N-toluenesulfonyl-L-arginine, L-arginine-L-pyroglutamic acid, D-arginine hydrochloride, L-arginine ethyl acetate dihydrochloride, L-arginine methyl ester dihydrochloride, N-nitro-L-arginine, L-lysine hydrochloride and metformin hydrochloride were selected as analogs of L-arginine hydrochloride and mixed with zoledronic acid at a mass ratio of 1:100 to form a variety of preparation formulas.

[0269] (2) We then injected various modified zoledronic acid preparations intraperitoneally into Kunming mice, which were randomly divided into 12 groups (8-week-old females, 25±3g): NS, ZOL, ZOL:L-Arg=1:100, ZOL:4ZOL+L-His=1:100, ZOL:Tos-Arg-OH=1:100, ZOL:L-Arg-L-pyr=1:100, ZOL:D-Arg=1:100, ZOL:L-Arg ethyl-vinegar dihydrochloride=1:100, ZOL:L-Arg methyl-vinegar dihydrochloride=1:100, ZOL:N'-Nitro-L-Arg=1:100, ZOL:L-Lys=1:100, and ZOL:Dimethylbiguanide hydrochloride=1:100, with 5 mice in each group. The survival and weight changes of Kunming mice were observed and recorded. The results are shown in Figure 15 . In Figure 15 , A shows the survival status, and B is a secondary graph of Figure A, indicating the time of first death. C shows the weight change, and D is a secondary graph of Figure C, indicating the weight loss of each group on day 6. The mice in the ZOL:L-Arg ethyl-vinegar dihydrochloride = 1:100, ZOL:L-Arg methyl-vinegar dihydrochloride = 1:100, and ZOL:Dimethylbiguanide hydrochloride = 1:100 died immediately after drug injection and are therefore not shown in the figure.

[0270] As shown in Figure 15, compared to the standard zoledronic acid (ZOL) group, the L-Arg and D-Arg modified zoledronic acid formulation significantly suppressed the side effects of zoledronic acid alone in the animal model, alleviated weight loss in the Kunming mice tested, and effectively reduced the mortality rate of the experimental animals. The other arginine analogs tested did not achieve these results.

[0271] 3. The role of DL-arginine, D-arginine and L-arginine in improved preparations

[0272] (1) Preparation of the modifying agent: DL-arginine and zoledronic acid were mixed at different mass ratios of 1:150 and 1:180; D-arginine, L-arginine and zoledronic acid were mixed at a mass ratio of 1:180 to form different modified zoledronic acid preparations: ZOL:DL-Arg=1:150, ZOL:DL-Arg=1:180, ZOL:D-Arg=1:180, ZOL:L-Arg=1:180;

[0273] (2) Kunming mice were randomly divided into 6 groups (8-week-old female, 25±3 g): NS, ZOL, ZOL:DL-Arg = 1:150, ZOL:DL-Arg = 1:180, ZOL:D-Arg = 1:180, ZOL:L-Arg = 1:180; NS and ZOL groups each had 5 mice; ZOL:DL-Arg = 1:150, ZOL:DL-Arg = 1:180, ZOL:D-Arg = 1:180, ZOL:L-Arg = 1:180 groups had 10 mice. After intraperitoneal injection of drugs, the survival and weight changes of Kunming mice were recorded. The results are shown in Figure 16. Figure 16 (A) shows the survival of Kunming mice, and Figure 16 (B) is the secondary graph of Figure A, which shows the time to median lethality. C is the change in body weight, and D is the secondary graph of C, which shows the weight loss of each group on the sixth day. Note: The disappearance of the curve in Figure C means that only one Kunming mouse is left in this group, which is no longer statistically significant.

[0274] As shown in Figure 16, the improved zoledronic acid formulations ZOL:DL-Arg = 1:150, ZOL:DL-Arg = 1:180, ZOL:D-Arg = 1:180, and ZOL:L-Arg = 1:180 can all significantly inhibit the side effects of a single zoledronic acid formulation in an animal model, alleviate weight loss in the test Kunming mice, and effectively reduce the mortality rate of experimental animals.

[0275] 4. Study on the addition ratio of DL-arginine in the improved preparation

[0276] (1) preparing the improver: mixing DL-arginine and zoledronic acid at a mass ratio of 1:360 (the mass of zoledronic acid in Example 1 is reduced by half) to form a zoledronic acid improved formulation: ZOL:DL-Arg=1:360;

[0277] (2) Kunming mice were randomly divided into three groups (8-week-old females, 25 ± 3 g): NS, ZOL, and ZOL:DL-Arg = 1:360, with 5 mice in each group. After intraperitoneal injection of the drug, the survival and weight changes of the Kunming mice were recorded. The results are shown in Figure 17. Figure 17 (A) shows the weight change, and Figure 17 (B) is a secondary graph of Figure A, showing the weight loss of each group on the sixth day.

[0278] As shown in FIG17 , the improved zoledronic acid formulation ZOL:DL-Arg=1:360 can significantly inhibit the side effects of a single zoledronic acid formulation in an animal model and alleviate the weight loss of the test Kunming mice.

[0279] 5. Study on the ratio of DL-arginine added to the improved preparation (SD-rat)

[0280] (1) Preparation of the improver: DL-arginine and zoledronic acid were mixed at a mass ratio of 1:360 and 1:720 to form a zoledronic acid improved preparation: ZOL:DL-Arg = 1:360; ZOL:DL-Arg = 1:720.

[0281] (2) SD rats were randomly divided into five groups (9-week-old female, 170 ± 20 g): NS, 1 / 2ZOL, 1 / 2ZOL:DL-Arg = 1:360, 1 / 4ZOL, and 1 / 4ZOL:DL-Arg = 1:720, with 3 rats in each group. The body weight changes of the SD rats were recorded after intraperitoneal injection of the drug. The results are shown in Table 12.

[0282] Table 12 shows the effects of the modified zoledronic acid formulation prepared with DL-arginine on the side effects of zoledronic acid (SD rats)

[0283] As shown in Table 12, the improved zoledronic acid formulations ZOL:DL-Arg=1:360 and ZOL:DL-Arg=1:720 can significantly inhibit the side effects of a single zoledronic acid formulation in an animal model and alleviate the weight loss of the test Kunming mice (p<0.05).

[0284] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing an animal inflammation model, characterized in that: The following steps are involved: The animals were administered bisphosphonate drugs.

2. The construction method according to claim 1, characterized in that: The animals include mice; Preferably, the mouse includes at least one of Kunming mouse, BALB / c mouse, SCID mouse, C57BL / 6 mouse, NOD mouse, and ICR mouse; preferably, the mouse includes Kunming mouse and BALB / c mouse.

3. The construction method according to claim 2, characterized in that: The bisphosphonate drug includes at least one of etidronic acid and its salt, clodronic acid or its salt, pamidronic acid or its salt, alendronic acid or its salt, ibandronic acid or its salt, risedronic acid or its salt, and zoledronic acid or its salt; Preferably, the bisphosphonate drug comprises at least one of risedronic acid or a salt thereof, and zoledronic acid or a salt thereof.

4. The construction method according to claim 3, characterized in that: The salt includes at least one of a metal salt, an ammonium salt, a salt formed with an organic base, and a salt formed with a basic amino acid; Preferably, the metal salt comprises at least one of sodium salt, potassium salt, calcium salt, magnesium salt, barium salt and aluminum salt; Preferably, the salt formed with the organic base includes at least one of trimethylamine salt, triethylamine salt, methylpyridine salt, 2,6-dimethylpyridine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, cyclohexylamine salt, dicyclohexylamine salt, and N,N'-dibenzylethylenediamine salt; Preferably, the salt formed with the basic amino acid includes at least one of arginine salt, lysine salt and ornithine salt.

5. The construction method according to any one of claim 4, characterized in that: The bisphosphonate drugs include risedronic acid and / or zoledronic acid.

6. The construction method according to claim 5, characterized in that: The dosage of risedronic acid is 40 to 80 mg / kg.

7. The construction method according to claim 6, characterized in that: The dosage of zoledronic acid is 20-40 mg / kg.

8. The construction method according to claim 7, characterized in that: The administration method includes at least one of injection, intragastric administration, and oral administration; Preferably, the administration method comprises injection; Preferably, the injection includes at least one of intraperitoneal injection and intravenous injection; Preferably, the injection comprises intraperitoneal injection; Preferably, the administration frequency includes 1 to 3 times; Preferably, the number of administrations includes 1 time.

9. The construction method according to any one of claims 1 to 8, characterized in that: The construction method also includes the step of raising the animal after administration; Preferably, the number of days for raising animals is 1 to 7 days.

10. The animal inflammation model prepared by the construction method according to any one of claims 1 to 9.

11. Products for (a1) to (a5): (a1) Study the mechanism of inflammation; (a2) Screening drugs for treating inflammation; (a3) Screening for drugs that prevent inflammation; (a4) preparing a medicament for treating inflammation; (a5) preparing drugs for preventing inflammation; The product includes an animal inflammation model obtained by the construction method according to any one of claims 1 to 9.

12. Use of the inflammation model according to claim 10 or the product according to claim 11 in at least one of the following aspects: (a1) Study the mechanism of inflammation; (a2) Screening drugs for treating inflammation; (a3) Screening for drugs that prevent inflammation; (a4) preparing a medicament for treating inflammation; (a5) Preparing a drug for preventing inflammation.

13. A method for screening drugs for treating, improving and / or preventing inflammation-related diseases, characterized in that: These include: administering candidate drugs to animal inflammation models; The following indicators were measured in the animal inflammation model group and the treatment group: white blood cell count, C-reactive protein level, IL-6 level, and TNF-α level; If the index is significantly different between the candidate drug treatment group and the model group, the candidate drug is a drug for treating, improving and / or preventing inflammation-related diseases; The animal inflammation model is the animal inflammation model according to claim 10 or the animal inflammation model obtained by the construction method according to any one of claims 1 to 9.

14. Use of arginine in the preparation of a drug for preventing, treating or alleviating the side effects caused by zoledronic acid; The side reactions include: Zoledronic acid use causes at least one of the following: Kidney damage, liver damage, inflammatory response, weight loss, increased mortality, or cold-like symptoms.

15. The use according to claim 14, characterized in that: The side effect is kidney damage, and the prevention and treatment includes: improving the pathological damage of the kidney and / or regulating the levels of blood biochemical indicators related to kidney toxicity.

16. The use according to claim 15, characterized in that The pathological damage of the kidney includes at least one of the following: dark red urine, slight to mild inflammatory cell infiltration in the renal cortex / capsule / renal papilla, mild basophilic tubules in the cortex, mild to obvious dilation, slight to obvious degeneration / necrosis of the renal tubules, and moderate to obvious casts in the cortex / medullary area; The regulation of blood biochemical indicators related to nephrotoxicity includes: reducing urea and / or creatinine levels, and / or reducing inflammatory factor levels.

17. The use according to claim 16, characterized in that The inflammatory factors include TNF-α.

18. The use according to any one of claims 14 to 17, characterized in that: The active ingredient of the medicine is arginine, or a combination of arginine and zoledronic acid.

19. The use according to claim 18, characterized in that In the composition, the mass ratio of zoledronic acid to arginine is 1:150-600.

20. Use of arginine in the preparation of drugs for preventing and treating kidney damage caused by diabetes; The control includes: Improve podocyte activity, reduce the level of oxidative stress markers, and promote The levels of anti-inflammatory factors in podocytes and / or the levels of pro-inflammatory factors in podocytes were inhibited.

21. The use according to claim 20, characterized in that The oxidative stress markers include ROS; the podocyte pro-inflammatory factors include IL-6; and the podocyte anti-inflammatory factors include IL-10.

22. The use according to any one of claims 14 to 21, characterized in that: The arginine is L-arginine, D-arginine or DL-arginine.

23. A method for treating, preventing, alleviating and / or reducing renal damage, characterized in that: It includes: administering arginine, or a combination of arginine and zoledronic acid; The renal damage includes renal damage caused by zoledronic acid administration and / or renal damage caused by diabetes.

24. The method according to claim 23, characterized in that The arginine is L-arginine, D-arginine or DL-arginine; The administration concentration of the arginine is 0.5-1.5 mM.

25. A pharmaceutical composition comprising zoledronic acid and capable of inhibiting its side effects, characterized in that: The active ingredients of the pharmaceutical composition consist of zoledronic acid and a side effect inhibitor; the mass ratio of the zoledronic acid to the side effect inhibitor is 1:66-1000.

26. The pharmaceutical composition according to claim 25, characterized in that The mass ratio of zoledronic acid to the side reaction inhibitor is 1:66, 1:100, 1:150, 1:300, 1:360 or 1:

600.

27. The pharmaceutical composition according to claim 26, characterized in that The side reaction inhibitor is arginine or a salt thereof.

28. The pharmaceutical composition according to claim 25, characterized in that The pharmaceutical composition comprises arginine and zoledronic acid; the mass ratio of zoledronic acid to arginine is 1:150-600.

29. The pharmaceutical composition according to claim 27 or 28, characterized in that The arginine is L-arginine, D-arginine or DL-arginine.

30. The pharmaceutical composition according to any one of claims 25 to 29, characterized in that The pharmaceutical composition is in the form of powder, tablet, injection or pill.

31. Use of the pharmaceutical composition according to any one of claims 25 to 30 in the preparation of a medicament for treating osteoporosis.

32. Use of the pharmaceutical composition according to any one of claims 25 to 30 in the preparation of a medicament for treating inflammation or cold-like symptoms.

Images