Induction and construction method of a cynomolgus monkey hyperuricemia model and application thereof

Abstract

The present application relates to the technical field of hyperuricemia modeling, and particularly relates to a method for inducing and constructing a cynomolgus monkey hyperuricemia model and application thereof, comprising the following steps: S1: selecting male cynomolgus monkeys as model animals; S2: administering an inducing agent to the experimental animals, wherein the inducing agent is composed of hydrochlorothiazide, potassium oxonate and adenine. The inducing agent is added to ordinary monkey feed for normal feeding of the experimental animals, the intake dose of hydrochlorothiazide is 0.1 g / kg / day, the intake dose of potassium oxonate is 0.2 g / kg / day, the intake dose of adenine is 0.1 g / kg / day, and 10% fructose water is used to replace ordinary drinking water at the same time. The present application can induce a stable hyperuricemia state in the cynomolgus monkey body by means of dietary induction, combined use of hydrochlorothiazide, potassium oxonate and adenine, compared with a traditional model induced by a single intraperitoneal injection of a drug, the present application better simulates the pathogenic cause of human hyperuricemia, and is easier to operate.

Description

Technical Field

[0001] This invention relates to the field of hyperuricemia modeling technology, specifically to a method for inducing and constructing a hyperuricemia model in cynomolgus monkeys and its application. Background Technology

[0002] According to the "Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout in China (2019)," regardless of gender, a serum uric acid level exceeding 420 μmol / L measured on multiple separate days is considered hyperuricemia. Hyperuricemia is a continuous and slow physiological and pathological process; therefore, its treatment will also be an extremely long and sustained process. Currently, as of 2021, there are approximately 120 million people with hyperuricemia in my country, demonstrating that hyperuricemia has become a significant health problem in the country.

[0003] There are many contributing factors to hyperuricemia, including obesity, alcohol consumption, and excessive intake of high-purine foods. Based on uric acid excretion rate (UUE) and fractional uric acid excretion (FEUA), hyperuricemia can currently be classified into types such as poor renal excretion, excessive renal burden, mixed type, and others. The root causes of hyperuricemia can be summarized into two categories: excessive uric acid production and insufficient excretion. This can also be related to abnormal uric acid metabolism, involving adenine and guanine. In the body, both adenine and guanine are eventually converted to xanthine under the action of deaminase and oxidase, and then converted to uric acid under the action of xanthine oxidase. When there is excessive uric acid production, abnormal uric acid metabolism, or insufficient excretion, uric acid can accumulate in the body. It is generally accepted that a serum uric acid concentration higher than 420 μmol / L in men and higher than 360 μmol / L in women is considered a high level of uric acid.

[0004] High levels of uric acid in the blood can cause a range of diseases, including gout. Gout is a recurrent acute inflammatory reaction caused by the interaction of monosodium urate (MSU) crystals with the tissue microenvironment. When uric acid levels are too high, it precipitates in the joints, forming tophi (gouty nodules), which then trigger an inflammatory response, leading to gout. Therefore, gout is also an important indicator for diagnosing hyperuricemia, a disease caused by hyperuricemia. Studies have also shown that hyperuricemia is a significant risk factor for diabetes. Kidney damage and nephropathy are another major threat to health caused by hyperuricemia, and some studies suggest that hyperuricemia may be a marker of declining kidney function.

[0005] Establishing animal models of hyperuricemia is a powerful tool for studying the pathogenesis of hyperuricemia, the damage mechanisms of high uric acid to the body, and the development of uric acid-lowering drugs. Currently, the most common animals used to construct hyperuricemia models are mice and poultry such as chickens and quails. Mice contain uricase, which hydrolyzes uric acid produced in the mouse body, ultimately generating urea which is excreted. Therefore, researchers often use the uricase inhibitor potassium oxonate to inhibit uric acid degradation, thereby establishing a mouse model of hyperuricemia. Poultry such as chickens and quails, like humans, lack uricase, and the uric acid they produce is excreted through a mixture of urine and feces. Therefore, researchers establish poultry models of hyperuricemia by feeding chickens or quails high-purine foods or uric acid.

[0006] Existing Chinese patent document CN201710037421.5 discloses a method for constructing an animal model of acute hyperuricemia, including administering inosine to rhesus monkeys to obtain an animal model of acute hyperuricemia. This invention uses inosine as an inducer of elevated serum uric acid levels, administering it to rhesus monkeys to establish an animal model of hyperuricemia for evaluating the efficacy of uric acid-lowering drugs. The main drawbacks are: short duration of hyperuricemia and instability of the hyperuricemia model. As the patent presents, after intraperitoneal injection of inosine at a concentration of 50 mg / ml and a dose of 100 mg / kg into rhesus monkeys, serum uric acid levels reached their peak 0.5 hours later, and returned to near normal levels after 2 hours. However, currently, few uric acid-lowering drugs can significantly reduce serum uric acid levels within 2 hours, so this model is not suitable for screening uric acid-lowering drugs.

[0007] Chinese patent document CN202010869342.2 discloses a method for constructing a mouse model of acute hyperuricemia, which includes the following steps: feeding experimental mice with a diet containing ribbonfish daily for 7-8 days; and administering hypoxanthine and potassium oxonate daily for the last two days of the modeling experiment. The main drawbacks are: the model is constructed using mice, which, although common and easily manipulated experimental animals, differ significantly from humans in anatomical structure, physiological function, and metabolic processes. This modeling method can induce kidney damage in the model mice. Generally, only persistent hyperuricemia causes kidney damage; therefore, the kidney damage observed in this model is not caused by acute hyperuricemia but by the modeling method itself. Furthermore, because the model mice have kidney damage, they are not suitable as a model for screening uric acid-lowering drugs, as many uric acid-lowering drugs work by promoting renal excretion of uric acid. Summary of the Invention

[0008] The purpose of this invention is to provide a method for inducing and constructing a hyperuricemia model in cynomolgus monkeys and its application, to better simulate the pathogenesis of hyperuricemia in humans, to establish a platform for screening anti-hyperuricemia drugs, and to establish a stable hyperuricemia model in cynomolgus monkeys through dietary induction. The model of this invention exhibits relatively stable serum uric acid levels and prolonged hyperuricemia, which can better reflect the pathological process of hyperuricemia in humans, providing a powerful tool for studying the damage and mechanisms of hyperuricemia in humans and for screening anti-hyperuricemia drugs.

[0009] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0010] A method for inducing and constructing a hyperuricemia model in cynomolgus monkeys, comprising the following steps:

[0011] S1: Male cynomolgus monkeys were selected as the model animals;

[0012] S2: Administer an inducer to the experimental animals, the inducer being composed of hydrochlorothiazide, potassium oxonate, and adenine.

[0013] Furthermore, the inducer was added to the normal monkey feed for the experimental animals, with the intake dose of hydrochlorothiazide being 0.1 g / kg / day, the intake dose of potassium oxonate being 0.2 g / kg / day, and the intake dose of adenine being 0.1 g / kg / day. At the same time, 10% fructose water was used instead of ordinary drinking water.

[0014] Furthermore, the modeling process lasted for 30 days, and the concentration of uric acid in the blood and urine of the cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured on days 10, 20 and 30 of the modeling process.

[0015] Furthermore, before modeling, the concentration of uric acid in the blood and urine of the experimental cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured and recorded. This data was then compared with the uric acid concentration in the blood and urine of the modeled cynomolgus monkeys to determine whether the hyperuricemia cynomolgus monkey model had been successfully established.

[0016] On the other hand, this invention proposes the application of the above method in the screening of anti-hyperuric acid drugs.

[0017] The beneficial effects of this invention are:

[0018] The most commonly used model of hyperuricemia is the mouse hyperuricemia model, which is mainly induced by injection of the uricase inhibitor potassium oxonate. Therefore, this model cannot accurately reflect the pathological process of hyperuricemia in humans. Furthermore, the anatomical structure, physiological function, and metabolic processes of mice differ significantly from those of humans; therefore, research results obtained using this model may not be applicable to humans. Moreover, this modeling method easily leads to kidney damage in mice, making it unsuitable for screening uric acid-lowering drugs. This is because most uric acid-lowering drugs exert their effects by promoting uric acid excretion through the kidneys, and most uric acid-lowering drugs are themselves excreted through the kidneys.

[0019] Currently, some researchers have established a hyperuricemia model in rhesus monkeys using intraperitoneal injection of inosine. However, intraperitoneal injection is not very convenient for rhesus monkeys, which are primates. Furthermore, the duration of hyperuricemia is relatively short, with levels returning to normal within 2 hours of inosine injection. Since few uric acid-lowering drugs can significantly reduce serum uric acid levels within 2 hours, this model is not suitable for screening uric acid-lowering drugs.

[0020] Compared to methods using mice or poultry to construct hyperuricemia models, this invention, through dietary induction in cynomolgus monkeys, establishes a non-human primate hyperuricemia model that better simulates the pathogenesis of human hyperuricemia and can be used to study the pathogenesis of hyperuricemia. This invention, through dietary induction combined with the use of hydrochlorothiazide, potassium oxonate, and adenine, can induce a stable hyperuricemic state in cynomolgus monkeys. Compared to traditional models induced by a single intraperitoneal injection of drugs, this better simulates the causes of human hyperuricemia and is easier to operate. Furthermore, the cynomolgus monkey model of hyperuricemia constructed using this invention exhibits relatively stable serum uric acid levels over a long period. Therefore, it can be used to study the damage and mechanisms of prolonged hyperuricemia on the body and can serve as a screening platform for anti-hyperuricemia drugs. The adenine and fructose used in this modeling method can provide precursors for uric acid production. Potassium oxonate can inhibit a small amount of uricase activity in cynomolgus monkeys, while hydrochlorothiazide can inhibit uric acid excretion from the kidneys through competitive inhibition with uric acid. Therefore, these four substances can play a synergistic role in increasing blood uric acid in cynomolgus monkeys by providing precursors for uric acid production, inhibiting uric acid degradation, and inhibiting uric acid excretion, respectively.

[0021] The hyperuricemia model constructed using this invention, while significantly increasing uric acid levels, did not cause significant damage to the liver and kidney function of cynomolgus monkeys; indicators such as ALT, AST, UREA, and CREA showed no significant changes. This indicates that the model, while inducing hyperuricemia, causes minimal interference with the animals' physiological state, allowing for a more focused study of the effects of hyperuricemia itself on the body, rather than other non-specific damage caused by the model induction method. This provides a more accurate and reliable method for screening and evaluating anti-hyperuricemia drugs. Especially for studies requiring observation of long-term drug effects, this model has irreplaceable value. It is not only suitable for drug screening but can also be used to explore the pathophysiological mechanisms of hyperuricemia and its complications (such as gout and kidney disease). Through a long-term stable hyperuricemia model, the effects of uric acid on various systems of the body can be observed more meticulously and comprehensively, providing a powerful research platform for revealing the pathogenesis and pathological processes of hyperuricemia.

[0022] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of blood uric acid concentration at different modeling time points in this invention;

[0025] Figure 2 This is a schematic diagram of uric acid concentration in urine at different modeling time points according to the present invention;

[0026] Figure 3 This is a schematic diagram of 24-hour urinary uric acid excretion at different modeling time points according to the present invention;

[0027] Figure 4 This diagram illustrates that the present invention does not cause significant damage to the liver function of cynomolgus monkeys during the modeling process.

[0028] Figure 5 This diagram illustrates that the invention did not cause significant damage to the kidney function of cynomolgus monkeys during the modeling process. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] The method for inducing and constructing a hyperuricemia model in cynomolgus monkeys as described in this embodiment includes the following steps:

[0032] S1: Male cynomolgus monkeys were selected as the model animals;

[0033] S2: Administer an inducer to the experimental animals, the inducer being composed of hydrochlorothiazide, potassium oxonate, and adenine.

[0034] In this embodiment, the inducer was added to the normal monkey feed for the experimental animals. The intake dose of hydrochlorothiazide was 0.1 g / kg / day, the intake dose of potassium oxonate was 0.2 g / kg / day, and the intake dose of adenine was 0.1 g / kg / day. At the same time, 10% fructose water was used instead of ordinary drinking water.

[0035] In this embodiment, the modeling process lasted for 30 days, and the concentration of uric acid in the blood and urine of the cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured on the 10th, 20th and 30th days of modeling.

[0036] In this embodiment, the concentration of uric acid in the blood and urine of the experimental cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured and recorded before modeling. This data was then compared with the uric acid concentration in the blood and urine of the modeled cynomolgus monkeys to determine whether the hyperuricemia cynomolgus monkey model had been successfully established. The verification steps included:

[0037] 1. Compare the blood uric acid levels of cynomolgus monkeys before and after modeling.

[0038] 2. Compare the uric acid levels in the urine of cynomolgus monkeys before and after modeling.

[0039] 3. Compare the total uric acid levels in the urine of cynomolgus monkeys 24 hours before and after modeling.

[0040] In this embodiment, potassium oxonate used in the modeling process can inhibit the activity of uricase in cynomolgus monkeys, preventing uric acid degradation. Hydrochlorothiazide is a diuretic that promotes urination, reduces blood volume, and increases the reabsorption of urate by the renal tubules. Adenine and fructose are precursors or substrates for uric acid synthesis.

[0041] Example 2

[0042] Experimental reagents

[0043] Uric acid test kit, Merck Sigma-Aldrich, catalog number: MAK077-1KT; hydrochlorothiazide, MedChemExpress, catalog number: HY-B0252; potassium oxonate, aladdin, catalog number: P137112-100g; adenine, ThermoFisher, catalog number: A14906.

[0044] laboratory animals

[0045] Male crab-eating macaques, weighing 6-8 kg and aged 3.5 to 4.5 years, were purchased from Yunnan Yingmao Biotechnology Co., Ltd.

[0046] Experimental instruments

[0047] PHERAstar FSX multi-functional microplate reader, BMG LABTECH; high-speed low-temperature centrifuge, KATE Laboratory Instruments Co., Ltd., model: TGL16M; fully automated biochemical analyzer 7170, Hitachi.

[0048] Experimental methods

[0049] Before modeling, the baseline uric acid concentration in the blood and urine of the cynomolgus monkeys was measured to compare with the uric acid concentration in the blood and urine of the model group of cynomolgus monkeys in the future, so as to determine whether the hyperuricemia cynomolgus monkey model has been successfully constructed.

[0050] Four cynomolgus macaques were fed a model diet consisting of regular monkey food supplemented with hydrochlorothiazide, potassium oxonate, and adenine. The intake doses of hydrochlorothiazide, potassium oxonate, and adenine were 0.1 g / kg / day, 0.2 g / kg / day, and 0.1 g / kg / day, respectively. A 10% fructose solution was provided in place of regular drinking water.

[0051] The model was established for 30 days. Blood and urine uric acid concentrations and 24-hour urinary uric acid excretion in cynomolgus monkeys were measured on days 10, 20, and 30. The data were compared with the corresponding values ​​on day 0, and paired t-tests were used for statistical analysis. **p<0.01, ***p<0.001.

[0052] Experimental results

[0053] (1) Serum uric acid levels in hyperuricemic cynomolgus monkeys at different time points after modeling. Experimental results are shown in […]. Figure 1 During the continuous induction of hyperuricemia in cynomolgus monkeys, blood samples were collected from the monkeys at different time points to measure the uric acid concentration. For example... Figure 1As shown, before modeling, the baseline uric acid level in the cynomolgus monkeys' blood was approximately 52 μM / L. On day 10 of continuous modeling, the blood uric acid concentration in the cynomolgus monkeys rose to approximately 95.3 μM / L. On day 20 of modeling, the blood uric acid level in the model monkeys reached a peak of approximately 160 μM / L under the current induction conditions, about three times the normal value, and remained stable thereafter. On day 30 of modeling, the blood uric acid concentration remained at a high level.

[0054] Depend on Figure 1 The results showed that on the 10th day of modeling, the blood uric acid level of the cynomolgus monkeys increased significantly. On the 20th day of modeling, the blood uric acid level reached its peak and could continue to be maintained. Therefore, the model of hyperuricemia in cynomolgus monkeys was successfully established.

[0055] (2) Uric acid levels in the urine of hyperuricemic cynomolgus monkeys at different time points after modeling. Experimental results are shown in […]. Figure 2 During the continuous induction of hyperuricemia in cynomolgus monkeys, urine samples were collected at different time points to measure the uric acid concentration. For example... Figure 2 As shown, before modeling, the baseline uric acid concentration in cynomolgus monkey urine was approximately 38 μM / L. On day 10 of continuous modeling, the uric acid concentration in cynomolgus monkey urine increased significantly, reaching approximately 65 μM / L. On day 20 of modeling, the urinary uric acid concentration in the model monkeys reached a peak of approximately 120 μM / L under the current induction conditions, about three times the normal value, and remained stable thereafter. On day 30 of modeling, the urinary uric acid concentration remained at a high level.

[0056] (3) 24-hour urinary uric acid excretion in hyperuricemic cynomolgus monkeys at different time points after modeling. Experimental results are shown in […]. Figure 3 During the continuous induction of hyperuricemia in cynomolgus monkeys, 24-hour urine samples were collected from the monkeys at different time points to measure the total uric acid content. Figure 3 As shown, before modeling, cynomolgus monkeys excreted approximately 7 μM of uric acid daily through urine. On day 10 of continuous modeling, the 24-hour urinary uric acid excretion of cynomolgus monkeys significantly increased, reaching approximately 13 μM. On day 20 of modeling, the 24-hour urinary uric acid excretion of the model monkeys reached its peak at approximately 24 μM, more than three times the normal uric acid excretion, and remained stable thereafter. On day 30 of modeling, the 24-hour uric acid excretion of the model monkeys remained at a high level of over 20 μM.

[0057] like Figure 4 As shown, compared with before modeling, there were no significant changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in cynomolgus monkeys at any time point after modeling, indicating that this method of inducing hyperuricemia in cynomolgus monkeys did not significantly damage liver function. Figure 5As shown, compared with before modeling, there were no significant changes in blood urea nitrogen (UREA) and serum creatinine (CREA) in cynomolgus monkeys at any time point after modeling, indicating that this method of inducing hyperuricemia in cynomolgus monkeys did not cause significant damage to their renal function.

[0058] In inducing a stable hyperuricemia model in cynomolgus monkeys, several attempts were made using methods similar to those used in mice for hyperuricemia or gout, but all failed. For example, administering adenine alone to cynomolgus monkeys at doses of 0.1 g / kg / day or even higher resulted in no significant increase in serum uric acid levels after about a month of induction. Furthermore, excessive adenine dosage in the monkey feed (exceeding 2 mg / g) led to a significant decrease in appetite. Additionally, an attempt was made to combine 0.1 g / kg / day of adenine with 10% fructose in drinking water to provide more precursors for uric acid production. However, because fructose is sweet, cynomolgus monkeys readily consume water containing fructose, leading to increased water intake, urine output, and the excretion of large amounts of uric acid. Subsequent improvements to the modeling methods led to the development of the aforementioned method that can induce stable hyperuricemia in cynomolgus monkeys. Compared to mouse or poultry hyperuricemia models, both mice and poultry differ significantly from humans in anatomy and metabolic processes. Therefore, using these models to study the damage mechanisms of hyperuricemia may not be applicable to humans, and antiuric acid drugs screened using these models may be ineffective in humans. In contrast to existing primate hyperuricemia models, which are acute models with short durations (serum uric acid levels return to normal within 2 hours after intraperitoneal drug injection), the cynomolgus monkey hyperuricemia model induced by diet in this invention has a longer duration and more stable high-concentration serum uric acid levels. Therefore, it can be used to study the damage and mechanisms of sustained hyperuricemia, and also for screening anti-hyperuric acid drugs.

[0059] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for inducing and constructing a hyperuricemia model in cynomolgus monkeys, characterized in that, Includes the following steps: S1: Male cynomolgus monkeys were selected as the model animals; S2: Administer an inducer to the experimental animals. The inducer consists of hydrochlorothiazide, potassium oxonate, and adenine. The inducer was added to the normal monkey feed for the experimental animals. The intake dose of hydrochlorothiazide was 0.1 g / kg / day, the intake dose of potassium oxonate was 0.2 g / kg / day, and the intake dose of adenine was 0.1 g / kg / day. At the same time, 10% fructose water was used instead of ordinary drinking water. The modeling process lasted for 30 days. On days 10, 20 and 30 of the modeling process, the concentration of uric acid in the blood and urine of the cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured. Before modeling, the concentration of uric acid in the blood and urine of the experimental cynomolgus monkeys and the amount of uric acid excreted in urine over 24 hours were measured and recorded. This data was then used to compare with the concentration of uric acid in the blood and urine of the modeled cynomolgus monkeys to determine whether the hyperuricemia cynomolgus monkey model had been successfully established.

2. The application of the method described in claim 1 in the screening of anti-hyperuric acid drugs.

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