A hyperuricemia non-human primate model and a method for constructing the same

Abstract

The application discloses a hyperuricemia non-human primate model and a construction method thereof, and belongs to the technical field of experimental animals. The construction method comprises the following steps: using a non-human primate, i.e., a cynomolgus monkey, as a model animal, orally administering potassium oxonate and intravenously injecting myo-inositol, so that the blood uric acid content of the cynomolgus monkey is significantly increased within 0.5-8 hours, and the process can be inhibited by a uric acid-lowering drug allopurinol, and based on this, a hyperuricemia model of a non-human primate can be formed. Compared with a model constructed by using rodents, the hyperuricemia non-human primate model constructed by the application can better simulate the related symptoms and physiological processes of human hyperuricemia, and provides a preclinical effect evaluation tool for the research and development of nutrients and drugs in related fields.

Description

Technical Field

[0001] This invention belongs to the field of experimental model construction, specifically relating to a non-human primate model of hyperuricemia and its construction method. Background Technology

[0002] Hyperuricemia is defined as elevated serum uric acid levels. Generally, a serum uric acid level greater than 6 mg / dL in women or greater than 7 mg / dL in men is sufficient for a diagnosis. According to worldwide population surveys, the prevalence of hyperuricemia in adults is approximately 0.68-3.9%, and the incidence is increasing globally. Numerous studies have shown a significant correlation between hyperuricemia and hypertension, obesity, diabetes, cardiovascular disease, and kidney disease, thus having a substantial impact on overall health.

[0003] Dietary purines account for about one-third of the daily serum uric acid production in the human body, with the remainder synthesized endogenously. Foods rich in purines, such as red meat and seafood, alcohol (especially beer rich in yeast purines), and sugary drinks, can also increase serum uric acid concentration and the risk of gout. Serum uric acid levels will also rise when purine degradation is accelerated or excretion is reduced (such as in renal insufficiency) during periods of high cell turnover (such as hemolysis, rhabdomyolysis, and tumor lysis). Currently, there are three main treatment strategies for hyperuricemia: (1) Inhibiting uric acid production: reducing uric acid production by competitively inhibiting xanthine oxidase, with representative drugs being allopurinol and febuxostat; (2) Promoting uric acid excretion: increasing uric acid excretion by blocking the reabsorption of urate by the renal tubules, with representative drugs being probenecid and benzbromarone; (3) Promoting uric acid metabolism: reducing uric acid levels by ingesting uricase to convert uric acid into allantoin, with representative drugs being Pegloticase. However, all of the aforementioned uric acid-lowering drugs have varying degrees of side effects, mainly including gastrointestinal discomfort, skin rash, and liver toxicity. In 2018, the FDA issued a warning about the potential liver and kidney toxicity and increased all-cause mortality associated with febuxostat and other drugs. Therefore, developing more effective and safer management mechanisms for hyperuricemia is crucial in clinical practice. However, currently used animal models are not suitable for preclinical studies of hyperuricemia.

[0004] Currently, animal models of hyperuricemia are mainly constructed using rodents and birds, with the biggest difference between the two being the expression of uricase. Rodents, including rats and mice, express uricase, which converts serum uric acid into allantoin, a more soluble form, which is then excreted in the urine. Therefore, methods for establishing rodent models mainly include: 1) long-term administration of the uricase inhibitor potassium oxonate; 2) high-purine diet modeling; 3) modeling reduced excretion due to kidney injury; and 4) uricase gene knockout modeling. Birds, on the other hand, do not express uricase, and serum uric acid is excreted through the cloaca in feces; a high-purine diet can cause elevated serum uric acid levels. However, both drug-based and purine-based models suffer from stability issues. Furthermore, physiologically, their uric acid metabolism pathways differ significantly from those in humans, and the mechanisms underlying hyperuricemia also differ. Neither model can accurately simulate the pathogenesis of hyperuricemia in humans, and they also differ in the metabolic compensation responses induced by hyperuricemia. For example, CN 116942675 A discloses a method for establishing a mouse gout model induced by long-term hyperuricemia. The method involves maintaining a high-temperature, high-humidity environment and administering potassium oxonate to mice daily, but without exogenous purine supplementation. Even with uricase inhibition, the serum uric acid levels in rodents take 2–4 weeks to rise, far exceeding the uric acid elevation seen in humans. Therefore, a more human-like animal model is needed for the development of preclinical nutritional interventions or drugs for hyperuricemia.

[0005] In summary, the genetic differences between rodents and birds and humans cannot effectively simulate the development and progression of hyperuricemia in humans. Furthermore, the use of conventional blood collection methods for pharmacokinetic studies in small animals requires a significant number of animals, which is detrimental to animal welfare. Therefore, establishing a non-human primate model that is similar to human lesions, has a complete disease spectrum, is stable and reliable, and can better simulate the pathogenesis of hyperuricemia in humans has become a promising research direction. Non-human primates, due to their nearly identical cardiopulmonary anatomy and physiology, are a good model animal for simulating human uric acid metabolism and studying related metabolic diseases. Therefore, this invention aims to establish a non-human cynomolgus monkey model of hyperuricemia. Summary of the Invention

[0006] To overcome the problems existing in the prior art, the purpose of this invention is to provide a non-human primate model of hyperuricemia and its construction method. Compared with models constructed using rodents and birds as model animals, the non-human primate model of hyperuricemia provided by this invention has the advantage of better simulating the physiological process of human hyperuricemia, and is of great significance for the research and development of nutrients and drugs in related fields.

[0007] The objective of this invention is achieved by at least one of the following technical solutions.

[0008] The purpose of this invention is to address the problem that existing hyperuricemia models differ significantly from human physiological processes, and to provide a method for constructing a non-human primate model of hyperuricemia.

[0009] This invention provides a method for constructing a non-human primate model of hyperuricemia. The method involves administering purine alone or in combination with purine and potassium oxonate to non-human primates to induce symptoms of hyperuricemia.

[0010] Furthermore, in the construction method, the non-human primate is a cynomolgus monkey.

[0011] Furthermore, the age range of the crab-eating macaques is 5-25 years.

[0012] Furthermore, the crab-eating macaques are kept in individual cages.

[0013] Furthermore, in the construction method, the type of purine is inosine, i.e., inosine nucleoside.

[0014] Furthermore, in the construction method, the single dose of the purine is 100-200 mg / kg.

[0015] Furthermore, the single-dose dose of the potassium oxonate is 100-500 mg / kg.

[0016] Furthermore, the method of combined administration of purine and potassium oxonate is to administer purine and potassium oxonate once, or to administer potassium oxonate once daily followed by a single administration of purine.

[0017] Furthermore, in the construction method, the administration of the purine includes, but is not limited to, intravenous injection, oral administration, intramuscular injection, and subcutaneous injection, and the administration frequency is once or multiple times.

[0018] Furthermore, in the construction method, the potassium oxonate is administered via methods including but not limited to intravenous injection, oral administration, intramuscular injection, and subcutaneous injection, and is administered once or multiple times.

[0019] The present invention also provides a method for constructing a non-human primate model of hyperuricemia. The non-human primate model of hyperuricemia constructed by the method shows that the blood uric acid content of the model animals increases significantly within 0.5-8 hours after injection of inosine, reflecting a disordered state of purine metabolism in the body.

[0020] The non-human primate model of hyperuricemia constructed in this invention has the following advantages and beneficial effects compared with the prior art:

[0021] (1) The non-human primate model of hyperuricemia constructed in this invention is closer to humans in terms of physiology, pathology, and anatomical structure than the models constructed using rodents and birds as model animals. It is more suitable for preclinical research on treatment methods, drugs and nutrients related to hyperuricemia in humans.

[0022] (2) The method of constructing the model in this invention is simple and the model results are stable. It fills the gap in non-human primate models of hyperuricemia and can be well promoted and applied to the research of hyperuricemia.

[0023] (3) The combined administration of the present invention can increase the peak uric acid level and inhibit uricase, which can better simulate the phenomenon of increased uric acid level after meals in humans. The present invention combines exogenous purine supplementation with potassium oxonate administration, which is closer to the state of increased uric acid in humans. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the construction process of the non-human primate model of hyperuricemia in Example 1 of the present invention.

[0025] Figure 2 The kinetic curves of serum uric acid metabolism in cynomolgus monkeys after modeling with inosine at 100 mg / kg and 200 mg / kg in Example 1 are shown.

[0026] Figure 3 This is a statistical graph showing the area under the blood uric acid metabolism kinetics curves of the control group and the experimental group of cynomolgus monkeys in Example 1.

[0027] Figure 4 This is a flowchart illustrating the construction process of the non-human primate model of hyperuricemia in Embodiment 2 of the present invention.

[0028] Figure 5 The kinetic curves of serum uric acid metabolism in cynomolgus monkeys were obtained by using inosine at 200 mg / kg in Example 2 and repeated three times.

[0029] Figure 6 This is a statistical graph showing the area under the blood uric acid metabolism kinetics curves of the control group and the experimental group of cynomolgus monkeys in Example 2.

[0030] Figure 7 This is a flowchart illustrating the construction process of the non-human primate model of hyperuricemia in Example 3 of the present invention.

[0031] Figure 8 The kinetic curves of serum uric acid metabolism in cynomolgus monkeys after modeling with inosine 200 mg / kg + potassium oxonate 100 / 250 / 500 mg / kg in Example 3 are shown.

[0032] Figure 9 This is a statistical graph of the area under the kinetic curve of uric acid metabolism in mice in Example 3.

[0033] Figure 10 This is a flowchart illustrating the construction process of the non-human primate model of hyperuricemia in Example 4 of the present invention.

[0034] Figure 11 The kinetic curves of serum uric acid metabolism in cynomolgus monkeys after intervention with potassium oxonate 250 mg / kg for a single treatment or for 7 consecutive days and modeling with inosine 200 mg / kg are shown in Example 4.

[0035] Figure 12 This is a statistical graph showing the area under the blood uric acid metabolism kinetics curves of the control group and the experimental group of cynomolgus monkeys in Example 4.

[0036] Figure 13 This is a flowchart illustrating the construction process of a non-human primate model of hyperuricemia using the positive drug febuxostat in Example 5 of this invention.

[0037] Figure 14 The image shows the kinetic curve of serum uric acid metabolism in cynomolgus monkeys modeled with inosine 200 mg / kg using febuxostat in Example 5.

[0038] Figure 15 This is a statistical graph showing the area under the blood uric acid metabolism kinetics curves of the control group and the experimental group of cynomolgus monkeys in Example 5. Detailed Implementation

[0039] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0040] Example 1

[0041] A method for constructing a non-human primate model of hyperuricemia includes the following steps:

[0042] This experiment used three adult cynomolgus macaques aged 7-8 years. After 7 days of acclimatization feeding, baseline uric acid levels were measured on day 0. Inosine 100 mg / kg was administered intravenously on day 7, and inosine 200 mg / kg was administered intravenously on day 14 to establish the cynomolgus model. During the experiment, the macaques were housed individually with normal feed. Before each intravenous inosine injection, the macaques were fasted overnight, and their weight was measured between 7:30 and 8:00 AM the following morning. After weighing, the macaques were restrained with their left inner arm exposed upwards. Hair was removed using an animal shaver, and the vein was wiped with an alcohol swab to ensure venous distension. Inosine was prepared as a solution with physiological saline and administered intravenously at a volume of 10 mL / kg, injected parallel to the needle. Hemostasis was achieved by applying pressure with a dry cotton ball after injection. Blood samples were collected from the cynomolgus macaques via limb veins at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. 2 mL of whole blood was collected using a separating gel coagulation tube, centrifuged at 3000 r / min and 4℃ for 15 min, and the serum was collected to determine the uric acid content.

[0043] Experimental results

[0044] Figure 1-3 The graph shows the changes in serum uric acid levels within 8 hours in non-human primate models of hyperuricemia established using inosine at concentrations of 100 mg / kg and 200 mg / kg. Figure 2 and Figure 3 It can be seen that both intravenous injection concentrations of inosine can cause a significant increase in serum uric acid levels in cynomolgus monkeys, leading to acute hyperuricemia. Therefore, it can be concluded that the non-human primate model of hyperuricemia has been successfully established.

[0045] Example 2

[0046] A method for constructing a non-human primate model of hyperuricemia includes the following steps:

[0047] Experimental results:

[0048] This experiment used three adult cynomolgus macaques aged 7-8 years. After 7 days of acclimatization, inosine (200 mg / kg) was administered intravenously on days 0, 7, and 14 to establish a model of hyperuricemia in cynomolgus macaques, verifying the stability of the model. During the experiment, the macaques were housed individually with normal feed. Before each intravenous inosine injection, the macaques were fasted overnight and weighed between 7:30 and 8:00 AM the following morning. After weighing, the macaques were restrained with their left inner arm exposed upwards. Hair was removed using an animal shaver, and the vein was wiped with an alcohol swab to ensure venous distension. Inosine was prepared as a solution with physiological saline and administered intravenously at a volume of 10 mL / kg, injected parallel to the needle. Hemostasis was achieved by applying pressure with a dry cotton ball after injection. Blood samples were collected from the limb veins of the macaques at 0h, 0.5h, 1h, 2h, 4h, and 8h. 2 mL of whole blood was collected using a separating gel coagulation tube, centrifuged at 3000 r / min and 4℃ for 15 min, and the serum was collected to determine the uric acid content.

[0049] Experimental results

[0050] Figure 4-6 This study investigated the stability of hyperuricemia in cynomolgus monkeys using repeated modeling with inosine at 200 mg / kg. The results showed that a single injection of 200 mg / kg inosine stably increased serum uric acid in cynomolgus monkeys, and the uric acid level decreased to baseline after washing. This indicates that the animals can still be used for research on uric acid-lowering interventions after washing, improving animal utilization and reducing the need for animal interventions.

[0051] Example 3

[0052] A method for constructing a non-human primate model of hyperuricemia includes the following steps:

[0053] This experiment used three adult cynomolgus macaques aged 7-8 years. After 7 days of acclimatization, the following treatments were administered: day 0, intravenous injection of inosine 200 mg / kg; day 7, intravenous injection of inosine 200 mg / kg plus oral administration of potassium oxonate 100 mg / kg; day 14, intravenous injection of inosine 200 mg / kg plus oral administration of potassium oxonate 250 mg / kg; and day 21, intravenous injection of inosine 200 mg / kg plus oral administration of potassium oxonate 500 mg / kg. These measures were used to verify the significance of hyperuricemia modeling in cynomolgus macaques. During the experiment, the macaques were housed individually with normal feed. Before each intravenous injection of inosine, the macaques were fasted overnight, and their weight was measured between 7:30 and 8:00 AM the following morning. Subsequently, potassium oxonate was administered orally, with a single dose given 1 hour before the intravenous injection. After restraining the macaques, the inner side of their left arm was exposed upwards. Hair was removed using an animal shaving tool, and the arm was wiped with an alcohol swab to make the veins distended. Inosine was prepared into a solution using physiological saline and administered intravenously at a volume of 10 mL / kg. After injection, hemostasis was achieved by applying pressure with a dry cotton ball. Blood samples were collected from the cynomolgus monkeys via limb veins at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. 2 mL of whole blood was collected using a separating gel coagulation tube and centrifuged at 3000 rpm for 15 min at 4°C. Serum was then collected to determine uric acid levels.

[0054] Experimental results:

[0055] Figure 7-9 This study presents the results of repeated modeling using inosine 200 mg / kg and inosine 200 mg / kg plus potassium oxonate 100 / 250 / 500 mg / kg to increase peak serum uric acid levels in a cynomolgus monkey model of hyperuricemia. The results show that a single injection of 200 mg / kg inosine plus potassium oxonate 100 / 250 / 500 mg / kg significantly increased peak uric acid and the area under the curve (AUC) compared to inosine alone. Furthermore, the potassium oxonate dosage was positively correlated with serum uric acid levels, further demonstrating the successful establishment of the cynomolgus monkey hyperuricemia model.

[0056] Example 4

[0057] A method for constructing a non-human primate model of hyperuricemia includes the following steps:

[0058] This experiment used three adult cynomolgus macaques aged 7-8 years. After 7 days of acclimatization, on day 0, they were given intravenous inosine 200 mg / kg and oral potassium oxonate 250 mg / kg to establish the cynomolgus model. After 6 days of single-cell rearing on normal feed, they were washed off. Starting on day 8, they were given daily oral potassium oxonate 250 mg / kg to establish the model. On day 14, after oral potassium oxonate, they were given intravenous inosine 200 mg / kg to verify the effect of repeated potassium oxonate administration on the degree of uric acid elevation in cynomolgus macaques in response to high-purine stimulation. During the experiment, they were kept in single-cell rearing on normal feed. Before intravenous inosine injection to establish the model, the cynomolgus macaques were fasted overnight and weighed between 7:30 and 8:00 the next morning. Subsequently, potassium oxonate was administered orally, with a single dose given 1 hour before the intravenous injection. After restraining the cynomolgus macaques, the inner side of their left arm was exposed upwards. The hair was removed using an animal shaving tool, and the arm was wiped with an alcohol swab to make the vein distended. Inosine was prepared into a solution using physiological saline and administered intravenously at a volume of 10 mL / kg. After injection, hemostasis was achieved by applying pressure with a dry cotton ball. Blood samples were collected from the cynomolgus monkeys via limb veins at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. 2 mL of whole blood was collected using a separating gel coagulation tube and centrifuged at 3000 rpm for 15 min at 4°C. Serum was then collected to determine uric acid levels.

[0059] Experimental results:

[0060] Figure 10-12 This study aimed to differentiate between repeated modeling using potassium oxonate 250 mg / kg for 7 days plus a single dose of inosine 200 mg / kg and modeling using a single dose of potassium oxonate 250 mg / kg plus inosine 200 mg / kg. Results showed that repeated administration of potassium oxonate could accumulate to a certain level in cynomolgus monkeys and reduce uricase activity without affecting basal uric acid levels. This led to increased sensitivity of cynomolgus monkeys to exogenous inosine intake and a more significant increase in serum uric acid levels, thus establishing a more severe model of hyperuricemia in cynomolgus monkeys.

[0061] Example 5

[0062] A method for constructing a non-human primate model of hyperuricemia includes the following steps:

[0063] This experiment used three adult cynomolgus macaques aged 7-8 years. After 7 days of acclimatization, inosine 200 mg / kg was administered intravenously on day 0 and again on day 7. Febuxostat (0.5 mg / kg) was used as a positive control to verify the model's sensitivity to uric acid-lowering drugs. During the experiment, the macaques were housed individually on normal feed. Before intravenous inosine administration, the macaques were fasted overnight, and their weight was measured between 7:30 and 8:00 AM the following morning. Subsequently, febuxostat was administered orally, with a single dose given 1 hour before the intravenous injection. After restraining the macaques, the inner side of their left arm was exposed upwards. Hair was removed using an animal shaving tool, and the arm was wiped with an alcohol swab to ensure vein distension. Inosine was prepared into a solution using physiological saline and administered intravenously at a volume of 10 mL / kg. After injection, hemostasis was achieved by applying pressure with a dry cotton ball. Blood samples were collected from the cynomolgus monkeys via limb veins at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. 2 mL of whole blood was collected using a separating gel coagulation tube and centrifuged at 3000 rpm for 15 min at 4°C. Serum was then collected to determine uric acid levels.

[0064] Experimental results:

[0065] Figure 13-15 The study investigated changes in serum uric acid levels in cynomolgus monkeys after modeling with inosine 200 mg / kg and intervention with febuxostat. Results showed that febuxostat significantly reduced peak serum uric acid levels in cynomolgus monkeys under high-purine stimulation, demonstrating the applicability of this positive control drug in the model and further validating its potential use for screening uric acid-lowering nutritional interventions or drugs.

[0066] The non-human primate model of hyperuricemia constructed in this invention is closer to humans in terms of physiology, pathology, and anatomical structure compared to rodent models, making it more suitable for the development of treatment methods, drugs, and nutrients related to human hyperuricemia. The modeling method is simple, and the results are stable, filling the gap in the field of non-human cynomolgus monkey models for hyperuricemia and enabling its widespread application in research on hyperuricemia.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a hyperuricemic non-human primate model, characterized by, A cynomolgus monkey is used as a model animal, and before administration of inosine, a single or continuous multi-day oral administration of potassium oxonate at a dose of 100-500 mg / kg, and a single intravenous injection of inosine at a dose of 100-200 mg / kg are performed; the blood uric acid content of the cynomolgus monkey is significantly increased within 0.5-8 h after administration of inosine, thereby constructing a hyperuricemia model.

2. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The cynomolgus monkey is 5-25 years old.

3. The method of claim 1 or 2, wherein the method is performed by administering to the non-human primate a urate-lowering agent. The cynomolgus monkey is single-caged.

4. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The administration dose of inosine is 200 mg / kg.

5. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The single administration dose of potassium oxonate is 250 mg / kg when potassium oxonate is administered in combination.

6. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The administration mode is that potassium oxonate is orally administered for 7 consecutive days, and then inosine is intravenously injected.

7. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The administration mode of potassium oxonate is oral administration.

8. The method of claim 1, wherein the non-human primate model of hyperuricemia is constructed by administering to the non-human primate a urate-lowering agent. The method further comprises a step of verifying the model using a uric acid-lowering drug after the model is constructed, and the uric acid-lowering drug comprises febuxostat.

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