Application of nucleic acid molecule specifically targeting liver in preparation of medicine for treating obesity and complications thereof

Abstract

The invention discloses application of a specific liver targeting nucleic acid molecule in preparation of a medicine for treating obesity and complications thereof, and belongs to the technical field of biomedicine. The nucleic acid molecule is GalNAc-PRKAR1ASIRNA, the GalNAc-PRKAR1ASIRNA is obtained after the 3'tail end of PRKAR1ASIRNA is subjected to GalNAc coupling modification, and the base sequence of the PRKAR1ASIRNA is shown in the figure 1 in the specification. In-vivo experiments prove that the GalNAc-PRKAR1ASIRNA has a positive treatment effect on obesity and complications thereof, specifically, the GalNAc-PRKAR1ASIRNA obviously improves obesity induced by high fat diet, reduces the content of serum triglyceride, total cholesterol and blood sugar, reduces the content of liver fat, improves the athletic ability of mice, and has a remarkable treatment effect on obesity and complications thereof, so that the GalNAc-PRKAR1ASIRNA has a remarkable treatment effect on obesity and complications thereof, and the GalNAc-PRKAR1ASIRNA has a remarkable treatment effect on obesity and complications thereof. And candidate drugs are provided for treatment of obesity and complications thereof. And the GalNAc-PRKAR1ASIRNA specifically targets the liver tissue, so that compared with the existing obesity and complication treatment medicines, the GalNAc-PRKAR1ASIRNA has the advantages of smaller toxic and side effects and obvious curative effect, and makes obvious progress compared with the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the application of a nucleic acid small molecule GalNAc-PRKAR1A siRNA in the preparation of a drug for treating obesity and its complications. Background Art

[0002] Obesity and its complications are major challenges faced by global public health. Obesity often causes a series of complications, including type II diabetes, non-alcoholic fatty liver disease, cardiovascular diseases, hyperlipidemia, hypertension, metabolic syndrome, and certain types of cancers, thus seriously affecting human health.

[0003] The treatment means and strategies for obesity also show diversification. However, the current treatment measures have their respective application limitations and disadvantages, which limit their clinical effects to a certain extent. Among them, drug treatment remains an important way to treat obesity and its complications, and has achieved relatively significant clinical effects in the long-term treatment of obesity. Currently, the approved drugs on the market include orlistat, phentermine, topiramate, artesunate, etc. However, the current drug treatment means still face many challenges: (1) The treatment effects vary greatly among different individuals, making it difficult to achieve personalization and general high efficiency; (2) Many drugs have serious side effects and damages to the nervous system and cardiovascular system, and the safety of use is not high enough; (3) The action mechanisms of some drugs are not very clear, resulting in potential off-target toxicity; (4) The clearance rate of some drugs is low, and it is easy to cause long-term drug accumulation in tissues and organs, which damages health, etc. Therefore, there is an urgent need to develop new drugs for treating obesity to improve the effectiveness and safety of the current treatment.

[0004] Hepatocytes specifically express asialoglycoprotein receptors (ASGPRs), which can specifically recognize protein substrates with N-acetylgalactosamine groups (GalNAc), take them into endosomes through endocytosis, and release free substrate molecules. GalNAc-siRNA technology couples artificially synthesized small interfering RNA (siRNA) with N-acetylgalactosamine groups (GalNAc). Through the above-mentioned action, the molecule enters the hepatocytes and releases free siRNA, exerting the effect of silencing the expression of the target gene. At present, the application of GalNAc-siRNA technology in the field of oligonucleotide disease treatment has made a breakthrough progress, and more and more drugs have been approved for market or are in the clinical trial stage, such as Givlaari and fitusiran. Compared with other gene therapy drugs, GalNAc-siRNA drugs often show a series of advantages such as high efficacy, high specificity, low toxicity, and low off-target effects, and therefore have broad application prospects. Especially in the field of siRNA for the treatment of liver diseases, GalNAc targeting technology has almost dominated.

[0005] The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway is one of the intracellular signal transduction pathways mediated by G protein-coupled receptors (GPCRs), and is involved in the regulation of various intracellular physiological processes. The PKA holoenzyme protein consists of two regulatory subunits (PRKAR1A and PRKAR1B) and two catalytic subunits (PRKACA and PRKACB), and each subunit has different subtypes.

[0006] Our previous patent application, "An application of Prkar1a as a target for the treatment of obesity and its complications", explains that specific targeting of liver Prkar1a has the effect of treating obesity. In this patent, we used GalNAc-siRNA targeting liver PRKAR1A to specifically silence mouse liver Prkar1a. The results showed that obese mice significantly lost weight and their nutritional metabolism levels were significantly improved, suggesting that our GalNAc-siRNA technology has potential for application in the treatment of obesity and its complications. Summary of the invention

[0007] In order to solve the defects and shortcomings of current treatment methods, the primary purpose of the present invention is to provide a small nucleic acid molecule for use in preparing a drug for treating obesity and its complications. The small nucleic acid molecule is a GalNAc-modified siRNA, namely GalNAc-PRKAR1A siRNA.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] The present invention provides the use of GalNAc-siRNA targeting the Prkar1a gene (PRKAR1A-encoding gene), namely GalNAc-PRKAR1ASIRNA, in the preparation of a medicament for treating obesity and its complications.

[0010] On the one hand, the present invention provides the use of a nucleic acid small molecule GalNAc-PRKAR1ASIRNA in the preparation of a medicament for treating obesity and its complications, and the use is for non-diagnostic and non-therapeutic purposes.

[0011] Preferably, the nucleic acid small molecule is GalNAc-PRKAR1ASIRNA; the GalNAc-PRKAR1ASIRNA is obtained by conjugating and modifying the 3′ end of PRKAR1ASIRNA with GalNAc, and the base sequence of the PRKAR1ASIRNA is as shown in SEQ ID NO.1.

[0012] Preferably, the PRKAR1ASIRNA specifically silences Prkar1a in the liver (human GeneID: 5573; mouse Gene ID: 19084) after being modified with GalNAc.

[0013] Preferably, the obesity and its complications include at least one of the following: obesity, obesity-related hyperlipidemia, obesity-related hypertension, obesity-related atherosclerosis, and obesity-related fatty liver.

[0014] Preferably, the medicament includes the active ingredient nucleic acid small molecule GalNAc-PRKAR1ASIRNA and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0015] Preferably, the excipient includes any one or more of starch, sucrose, trehalose, mannitol, and sorbitol.

[0016] Preferably, the carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres, and hydrogels.

[0017] Preferably, the content of GalNAc-PRKAR1ASIRNA in the medicament is 1% - 99%.

[0018] Preferably, the medicament includes any one of tablets, capsules, granules, dripping pills, emulsions, liquids, patches, solutions, ointments, and injections.

[0019] Preferably, the nucleic acid small molecule GalNAc-PRKAR1ASIRNA can achieve at least one of the following effects: reducing the body weight and liver weight of obese individuals, and reducing blood lipid, blood glucose, and liver lipid levels.

[0020] The present invention has found through research that GalNAc-PRKAR1A siRNA can achieve specific knockdown of Prkar1a in the liver; GalNAc-PRKAR1A siRNA significantly reduces nutritional obesity, liver weight, and adipose tissue weight; GalNAc-PRKAR1A siRNA significantly reduces blood lipid and liver lipid levels and decreases liver lipid accumulation. The above phenotypes have been corroborated in a liver Prkar1a knockdown mouse model constructed by other means in the patent "An Application of Using Prkar1a as a Therapeutic Target for Obesity and Its Complications".

[0021] Based on the above research results, the present invention provides an application of GalNAc-PRKAR1A siRNA-mediated liver-specific knockdown of Prkar1a in the preparation of drugs for obesity and its complications.

[0022] The present invention specifically includes the following content:

[0023] The application of GalNAc-PRKAR1A siRNA in the treatment of obesity.

[0024] The application of GalNAc-PRKAR1A siRNA in the treatment of hyperlipidemia.

[0025] The application of GalNAc-PRKAR1A siRNA in the treatment of hypertension.

[0026] The application of GalNAc-PRKAR1A siRNA in the treatment of atherosclerosis.

[0027] The application of GalNAc-PRKAR1A siRNA in the treatment of fatty liver-related diseases.

[0028] The above-mentioned applications include other modifications with a structure similar to GalNAc for targeting liver Prkar1a to treat obesity and related metabolic diseases.

[0029] The application of various siRNA sequences targeting Prkar1a and different modification pattern types are all within the scope of protection of this patent, not limited to those used in the examples.

[0030] The present invention has verified that GalNAc-PRKAR1A siRNA targeting liver Prkar1a effectively improves nutritional obesity, reduces body lipid accumulation, and maintains the body's glucose and lipid metabolism homeostasis. This study provides new ideas and means for the treatment of obesity.

[0031] Details of the Prkar1a gene in the present invention can be found in the National Center for Biotechnology Information database (NCBI Genbank, https: / / www.ncbi.nlm.nih.gov) (human gene ID: 5573; mouse gene ID: 19084). Deletion, expression inhibition or inactivation of the Prkar1a gene or its encoded protein can be achieved by conventional means of molecular cell biology, such as gene editing mediated by viral vectors, gene silencing, expression of dominant negative mutants, administration of PKA activity agonists, etc.

[0032] The aforementioned application of the present invention is for non-diagnostic and non-therapeutic purposes.

[0033] Advantages of the present invention:

[0034] The research of the present invention found that GalNAc-siRNA-induced knockdown of Prkar1a in mouse liver significantly improved high-fat diet-induced obesity ( Figure 3 ), reduced adipose tissue weight ( Figure 3 ), improved nutritional fatty liver, and improved hyperlipidemia and hyperglycemia ( Figure 4 , 5 ). The present invention is a powerful supplement and extension of our previous patent "An Application of Prkar1a as a Therapeutic Target for Obesity and Its Complications", further enhancing the clinical application potential of targeting liver Prkar1a.

[0035] Currently marketed weight loss drugs have the disadvantages of multiple dosing, poor weight loss effect, and large side effects. This study observed that GalNAc-PRKAR1ASIRNA could effectively control the body weight of mice for a long time after a single administration ( Figure 3 ), and the amplitude of its silencing Prkar1a expression was conveniently controllable. These characteristics effectively improved the druggability of targeting liver Prkar1a.

[0036] In summary, the present invention first applied GalNAc-PRKAR1ASIRNA to target liver Prkar1a for improving obesity and its complications. GalNAc-PRKAR1ASIRNA not only alleviated high-fat diet-induced obesity, reduced liver fat content and adipose tissue weight, but also had the effect of resisting obesity-induced hyperglycemia. These results demonstrated the potential of GalNAc-PRKAR1ASIRNA to be developed into a new type of obesity treatment drug and its application prospect in clinical treatment.

[0037] The present invention provides a new strategy for the treatment of obesity and its complications, and is expected to be applied to people who have the need for weight control and improvement of body nutritional energy metabolism clinically, so as to achieve the purpose of improving human health level. Description of the Drawings

[0038] Figure 1 Structural diagram of the trimeric GalNAc-PRKAR1A siRNA modification used in the examples.

[0039] Figure 2 To knockdown Prkar1a in the liver of GalNAc-PRKAR1A siRNA-treated mice, C57 / BL6 male mice were sampled and analyzed 7 days after treatment with GalNAc-siControl or GalNAc-PRKAR1A siRNA. The two groups of mice were abbreviated as siCtr and siPrk, respectively; A: qPCR was used to detect the knockdown efficiency of Prkar1a in the kidney; B: qPCR was used to detect the knockdown efficiency of Prkar1a in the liver; C: Western blot was used to detect the knockdown efficiency of Prkar1a in the liver.

[0040] Figure 3 To improve nutritional obesity by GalNAc-PRKAR1A siRNA treatment, C57 / BL6 male mice were fed a high-fat diet for 16 weeks and then divided into two groups, which were injected with GalNAc-siControl and GalNAc-PRKAR1A siRNA, respectively; A: Growth curve; B: Weight of inguinal white adipose tissue; C: Weight of epididymal white adipose tissue; D: Rotarod fatigue tester, continuous distance of mice.

[0041] Figure 4 To improve nutritional fatty liver by GalNAc-PRKAR1A siRNA treatment, samples were taken 21 days after drug administration in mice; A: Liver weight; B: Liver triglyceride content.

[0042] Figure 5 To improve blood glucose and blood lipids by GalNAc-PRKAR1A siRNA treatment, serum samples were collected 21 days after drug administration in mice; A: Serum glucose level; B: Serum triglyceride (TG) content; C: Serum total cholesterol (TC) content.

[0043] The above results are expressed as mean ± standard error of the mean (SEM); p: coefficient of significance of difference; p > 0.05 indicates no significant difference (ns), 0.01 < p < 0.05 indicates significant difference (*), 0.001 < p < 0.01 indicates extremely significant difference (**), and 0.0001 < p < 0.001 indicates extremely extremely significant difference (***) (Student's t-test). Detailed implementation manners

[0044] The following describes the detailed implementation manners of the present invention in detail. The following examples are only for illustrating the present invention and do not limit the scope of the present invention.

[0045] The present invention shows through the following experiments that GalNAc-PRKAR1A siRNA induces Prkar1a knockdown in the livers of mice and significantly improves nutritional obesity, reduces adipose tissue weight, improves hepatic lipid deposition, and improves blood lipid and blood glucose levels, thereby alleviating obesity and its complications. The following experimental methods, unless otherwise specified, are all conventional technical methods in the art and are carried out according to the technical conditions described in professional literature or the instructions of related products; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0046] Statistical analysis was performed using GraphPad Prism 9 software. The results are expressed as mean ± standard error of the mean (SEM), and the differences between groups were analyzed by Student's t-test. p > 0.05 indicates no significant difference (ns), 0.01 < p < 0.05 indicates significant difference (*), 0.001 < p < 0.01 indicates extremely significant difference (**), 0.0001 < p < 0.001 indicates extremely extremely significant difference (***), and so on.

[0047] In the present invention, the Chinese name of GalNAC is N-acetylgalactosamine.

[0048] Example 1 Knockdown of Prkar1a gene in the livers of mice by GalNAc-PRKAR1A siRNA

[0049] Experimental method:

[0050] (1) Different specific siRNAs were designed against the murine Prkar1a sequence, and the Prkar1a expression silencing efficiency was detected and compared in an in vitro cultured murine cell line to obtain highly efficient silencing siRNA; then modified siPrkar1a ( Figure 1 ) was synthesized for in vivo experiments.

[0051] (2) 8-week-old wild-type C57 / BL6 mice were divided into 2 groups and injected with GalNAc-siControl and GalNAc-PRKAR1A siRNA respectively. After 1 week, samples were taken to confirm the Prkar1a knockdown efficiency in the liver ( Figure 2 ).

[0052] Experimental results:

[0053] Figure 2 The results of AqPCR showed that GalNAc-PRKAR1A siRNA did not affect the Prkar1a expression level in the kidneys; Figure 2 The results of BqPCR detection showed that GalNAc-PRKAR1A siRNA successfully knocked down the mRNA level of Prkar1a in the livers of mice; Figure 2The results of Western blot detection showed that GalNAc-PRKAR1A siRNA successfully knocked down the protein level of PRKAR1A in the liver of mice. The above results indicated that the method used in the present invention could successfully knock down Prkar1a in the liver without affecting the expression of Prkar1a in other tissues such as the kidney.

[0054] Example 2 Experimental method for GalNAc-PRKAR1A siRNA targeting liver Prkar1a to improve nutritional obesity and its complications:

[0055] (1) After male C57 / BL6 mice were fed a high-fat diet for 16 weeks, they were divided into 2 groups and injected with GalNAc-siControl and GalNAc-PRKAR1A siRNA respectively. The body weights of the mice were continuously observed and other indexes were detected ( Figures 3 - 5 ).

[0056] Experimental results:

[0057] (1) Figure 3 A showed that under the condition of high-fat diet feeding, the body weights of normal control mice were always maintained at a high level, while the body weights of mice after GalNAc-PRKAR1A siRNA treatment began to gradually decrease. This indicated that knocking down Prkar1a in the liver of mice induced by GalNAc-PRKAR1A siRNA could effectively treat nutritional obesity. In addition, around 20 days, the body weights of mice in the GalNAc-PRKAR1A siRNA treatment group gradually rebounded, indicating that the process of Prkar1a knockdown induced by GalNAc-PRKAR1A siRNA was reversible and would not cause adverse symptoms such as cachexia.

[0058] (2) Figure 3 B and 3C showed that under the condition of high-fat diet feeding, the weights of inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of mice in the GalNAc-PRKAR1A siRNA treatment group were significantly decreased (the results of sampling and analysis at 21 days). Figure 3 D showed that GalNAc-PRKAR1A siRNA treatment significantly improved the exercise ability of mice.

[0059] (3) Figure 4 A showed that under the condition of high-fat diet feeding, the liver weight of mice in the GalNAc-PRKAR1A siRNA treatment group had a decreasing trend compared with that of normal control mice; Figure 4 B showed that the determination results of triglyceride (TG) in liver samples showed that the TG content in the liver of mice in the GalNAc-PRKAR1A siRNA treatment group also had a decreasing trend compared with that of normal control mice.

[0060] (4) Figure 5The lipid measurement results of serum samples of A and B showed that under the condition of high-fat diet feeding, the contents of triglyceride (TG) and total cholesterol (TC) in the serum of mice in the GalNAc-PRKAR1A siRNA treatment group were significantly decreased. These results indicate that the knockdown of Prkar1a in the liver of mice induced by GalNAc-PRKAR1A siRNA significantly improves obesity-related hyperlipidemia.

[0061] (5) Figure 5 Figure C shows that there is a tendency for the blood glucose level of mice in the GalNAc-PRKAR1A siRNA treatment group to decrease. These results indicate that the knockdown of Prkar1a in the liver of mice induced by GalNAc-PRKAR1A siRNA significantly improves obesity-related hyperglycemia.

[0062] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately improve the experimental parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention.

Claims

1. Use of a nucleic acid molecule specifically targeting the liver in the preparation of a drug for treating obesity and its complications, characterized in that: The nucleic acid molecule is GalNAc-PRKAR1A SIRNA; the GalNAc-PRKAR1A SIRNA is obtained by modifying the 3′ end of PRKAR1A SIRNA through GalNAc coupling.

2. The use according to claim 1, characterized in that The PRKAR1A SIRNA base sequence is shown in Figure 1.

3. The use according to claim 1, characterized in that: The PRKAR1A SIRNA is modified with GalNAc to specifically silence liver Prkar1a (human Gene ID: 5573; mouse Gene ID: 19084).

4. The use according to any one of claims 1-2, characterized in that: The obesity and its complications include at least one of the following: obesity, obesity-related hyperlipidemia, obesity-related hypertension, obesity-related atherosclerosis, and obesity-related fatty liver.

5. The use according to any one of claims 1 to 3, characterized in that: The drug uses the GalNAc-PRKAR1A SIRNA as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

6. The use according to claim 4, characterized in that: The excipients include any one or more of starch, sucrose, trehalose, mannitol and sorbitol.

7. The use according to claim 4, characterized in that: The carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres and hydrogels.

8. The use according to claim 4, characterized in that The content of the GalNAc-PRKAR1A SIRNA in the medicine is 1%-99%.

9. The use according to any one of claims 1 to 7, characterized in that: The medicine includes any one of tablets, capsules, granules, pills, emulsions, liquids, patches, solutions, ointments and injections.

10. The use according to any one of claims 1 to 8, characterized in that: The nucleic acid small molecule GalNAc-PRKAR1A SIRNA can achieve at least one of the following effects: reducing the weight of obese individuals, and lowering blood lipids, blood sugar and liver fat levels.

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