Central targeting system for treating type 2 diabetes mellitus based on fibroblast growth factor

Abstract

The invention relates to the technical field of targeted drug delivery, in particular to a central targeting system for treating type 2 diabetes mellitus based on fibroblast growth factors. The method comprises the following steps: firstly, designing a genetic loop capable of expressing an FGF variant, and delivering the genetic loop into a body through intravenous injection or subcutaneous injection; the genetic loop entering the body expresses the target drug by using the liver as a biological reaction generator. In order to enable the polypeptide to have the capability of penetrating through a blood brain barrier to target a central nervous system, an optimized central targeting peptide is expressed in a genetic loop at the same time, and a drug can be efficiently delivered to the central nervous system in a targeted mode. The invention discloses a central targeting system for treating type 2 diabetes mellitus based on fibroblast growth factors. The central targeting system can effectively convey FGF (fibroblast growth factors) into the brain to improve insulin resistance of diabetes mellitus. According to the present invention, the long-term treatment effect is produced, the adverse reaction is avoided, the problem of low central administration compliance is solved, the advantage of low production cost is provided, and the new idea and the central system targeting administration mode are provided for diabetes treatment.

Description

Technical Field

[0001] The present invention relates to a central targeting system for treating type 2 diabetes based on fibroblast growth factor, and particularly to the construction of a method for central targeting of protein polypeptide drugs, belonging to the technical field of targeted drug delivery. Background Art

[0002] Due to sedentary lifestyles and overnutrition, the prevalence of diabetes has reached epidemic proportions globally. In particular, type 2 diabetes (T2D) has become a major health and economic burden worldwide. Although there are various drugs available for the treatment of type 2 diabetes, no hypoglycemic drug can cure diabetes, and diabetic patients need to take medication for life. In addition, it is currently not possible to achieve sustained remission of T2D: most current T2D treatments require at least once-daily dosing, and even the longest-acting semaglutide, tirzepatide, etc. on the market also require once-weekly injection. Therefore, there is an urgent need to develop long-term effective treatment methods.

[0003] The fibroblast growth factor (FGF) family has a total of 22 members. Except for FGF11, they all act in a secreted manner, regulating a large number of basic biological processes in embryonic development, tissue homeostasis / repair, and metabolism. Pharmacological studies in diabetic and obese animal models have shown that several members of the FGF family, including endocrine FGF19 / FGF21 and autocrine / paracrine FGF1, FGF4, have great potential in the treatment of obesity and type 2 diabetes. These FGF members all have potent antihyperglycemic effects when administered peripherally, and more strikingly, single intracerebroventricular (i.c.v.) administration of FGF1 and FGF4 can cause sustained diabetes remission in T2D rodents, which is in sharp contrast to the transient effects seen when these FGFs are administered peripherally.

[0004] Despite their effective hypoglycemic effects, the risk of tumorigenesis caused by FGF1 / FGF4 administration has dampened the enthusiasm for developing FGF1 / FGF4-based diabetes drugs. However, their mitogenic and antidiabetic activities can be separated: cell proliferation activity is mainly induced by FGFR3 and FGFR4, while metabolic activity is mainly mediated by FGFR1. Currently, some engineered variants have been developed to reduce their mitogenic properties to inhibit tumorigenesis, while retaining their metabolic activity through mutations. However, whether these variants maintain metabolic activity has not been tested. In addition, in terms of treatment, central injection also has the problem of low patient compliance. Currently, methods for intranasal delivery as an alternative to central injection are being developed, but this method has not been adopted in approved prescription drugs, and it is not clear whether intranasal delivery is sufficient to provide the same long-term benefits as central injection.

[0005] In summary, to address the key issues in the treatment of type 2 diabetes with fibroblast growth factor, this project developed a central-targeted drug delivery system that can deliver engineered FGF variants to the central nervous system through routes other than direct intracranial application (subcutaneous injection, intravenous injection). This not only preserves their metabolic activity but also avoids the risk of tumorigenesis. At the same time, it solves the problem of low patient compliance, providing new ideas for the development of central-targeted drugs and having great innovative significance for the treatment of type 2 diabetes. Summary of the Invention

[0006] The main objective of the present invention is to address the above-mentioned problems. Through genetic engineering means, engineered FGF variants are delivered to the central nervous system by routes other than direct intracranial application (subcutaneous injection, intravenous injection) to improve T2D.

[0007] The objective of the present invention can be achieved by adopting the following technical solutions:

[0008] A central-targeted system for treating type 2 diabetes based on fibroblast growth factor, whose coding sequence is SEQ ID NO.1 - SEQ ID NO.2.

[0009] A polynucleotide, whose coding sequence is fibroblast growth factor of SEQ ID NO.3 - SEQ ID NO.4.

[0010] A polynucleotide, whose sequence is SEQ ID NO.5, can encode a central-targeted peptide.

[0011] A recombinant plasmid, which includes the polynucleotide with the sequence of SEQ ID NO.1 - SEQ ID NO.2.

[0012] A host cell, which contains the said recombinant plasmid.

[0013] A host cell, which is derived from an Escherichia coli.

[0014] A host cell, in which the codons of the said recombinant plasmid have been optimized for expression in the said host cell.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This patent provides a central-targeted system for treating type 2 diabetes based on fibroblast growth factor. It successfully modifies protein polypeptide drugs through genetic engineering means and conducts functional verification. It is confirmed that the modified drug can effectively cross the blood-brain barrier and specifically reach the central nervous system without affecting its biological activity, exerting a long-lasting hypoglycemic effect without causing adverse reactions.

[0017] Compared with the commonly used hypoglycemic drugs in current clinical practice, its main advantages are as follows: 1) Long-lasting efficacy: Most clinical treatments for T2D require at least once-daily administration. Even the longest-acting semaglutide, tirzepatide, etc. on the market need to be injected once a week, while the drug of the present invention can maintain the hypoglycemic effect for at least 11 weeks; 2) Few side effects: Many clinical T2D drugs have adverse reactions such as hypoglycemia, weight gain, or bone loss. However, the engineered FGF variant modified in the present invention not only removes its own mitogenic activity (side effect), but also has the unique advantage that high-dose administration does not cause hypoglycemia; 3) Convenient administration: The present invention optimizes by adopting the method of LNP encapsulation. It can be administered subcutaneously, targeting the drug delivery system to the liver for in vivo self-assembly to express the engineered FGF variant with central targeting effect. Compared with intravenous injection, the administration is more convenient, and the patient compliance is higher. At the same time, LNP encapsulation can reduce the in vivo clearance rate of the drug and increase the bioavailability of the drug; 4) Low production cost: The price of the domestic semaglutide hypoglycemic drug commonly used in China is about 1100 yuan / 1.5 mL. The latest drug, tirzepatide, has not been listed in China yet, and the current selling price of a box is about 12757 yuan, and patients need to purchase it abroad by themselves. However, the drug of the present invention has a long dosing interval and low production cost, greatly reducing the cost for patients. Brief Description of the Drawings

[0018] Figure 1 Construction of a Central Targeting System for Treating Type 2 Diabetes Based on Fibroblast Growth Factor

[0019] Figure 2 Schematic Diagram of Targeted Central Delivery of Engineered FGF Variant by Intravenous Injection

[0020] Figure 3 Schematic Diagram of Targeted Central Delivery of Engineered FGF Variant by Subcutaneous Injection

[0021] Figure 4 Drug Administration Effect of Treating Type 2 Diabetes Based on Fibroblast Growth Factor Detailed Description of the Specific Embodiment

[0022] The present invention will be described in detail below through specific embodiments. The described embodiments are only a part of the embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Similarly, any similar small protein or polypeptide drug can also be evaluated for its biological function using corresponding tests. Based on the embodiments of the present invention, any person of ordinary skill in the art within the scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and all belong to the protection scope of the present invention.

[0023] Example 1: The present invention provides a technical solution: a construction method of a central targeting system for treating type 2 diabetes based on fibroblast growth factor, comprising the following steps: completed using the BLOCK-iT Pol II miRRNAi Designer of Invitrogen. The designer provides the sequences of two DNA oligos, which are annealed and hybridized and cloned into the pcDNA6.2-GW / EmGFP-miR linearized vector. The plasmid for expressing the genetic circuit was synthesized and constructed by GenScript Biotech (Nanjing, China). The plasmid was transformed into Escherichia coli DH5α competent cells (Tsingke, TSC01, Beijing, China), cultured in LB medium (containing 50 μg / mL spectinomycin) in a shaker at 37 °C for 14 hours, and purified using the EndoFree Maxi Plasmid Extraction Kit V2 (Tiangen, DP120, Beijing, China) according to the manufacturer's instructions. The purified plasmid was sequenced to ensure that the inserted sequence was correct. The full length of the successfully sequenced sequence is SEQ ID NO.1 and SEQ ID NO.2, named pcDNA6.2-(d)9R-RVG-FGF1 ΔHBS and pcDNA6.2-(d)9R-RVG-FGF4 (attached Figure 1 ).

[0024] Example 2: Administration effect of fibroblast growth factor in treating type 2 diabetes

[0025] A. Type 2 diabetic mice were intravenously injected once with a control empty vector or 10 mg / kg of the expression plasmid. The principle of this method is that first, a genetic circuit capable of expressing an FGF variant is designed and delivered into the body by intravenous injection. The genetic circuit that enters the body uses the liver as a biological reaction generator to express the drug protein. Since a central targeting peptide is simultaneously expressed in the genetic circuit, the expressed FGF variant has the ability to cross the blood-brain barrier and target the central nervous system, thereby exerting its effect (taking pcDNA6.2-(d)9R-RVG-FGF1 ΔHBS as an example, attached Figure 2 ).

[0026] B. Type 2 diabetic mice were subcutaneously injected once with a control empty vector or an expression plasmid encapsulated in 50 μg of LNP. The principle of this method is to utilize the liver-targeting function of LNP to efficiently deliver the genetic circuit capable of expressing the FGF variant into the liver via subcutaneous administration, and use the liver as a biological reaction generator to express the engineered drug protein. Since a central targeting peptide is simultaneously expressed in the genetic circuit, the expressed FGF variant has the ability to cross the blood-brain barrier and target the central nervous system, thus exerting its effect (taking pcDNA6.2-(d)9R-RVG-FGF1 ΔHBS as an example, attached Figure 3 ).

[0027] C. A blood glucose meter (Abbott Laboratories, USA) was used to measure blood glucose in tail blood to evaluate the therapeutic effect of diabetes. ITT was performed on mice fasted for 6 hours, and GTT was performed on mice fasted for 12 hours. Blood glucose values were immediately measured 15, 30, 60, and 120 minutes before and after intraperitoneal injection of human crystalline insulin (1 U / kg) or glucose (1 g / kg). Other metabolic indicators were measured using commercially available kits. The current experimental results show that the engineered FGF variant can maintain stable blood glucose reduction for at least 11 weeks (taking pcDNA6.2-(d)9R-RVG-FGF1 ΔHBS as an example, attached Figure 4 ).

Claims

1. A central targeting system for treating type 2 diabetes based on fibroblast growth factor, characterized in that: It can be administered by subcutaneous injection and intravenous injection, self-assemble and release FGF in the liver, and specifically target the central nervous system to improve type 2 diabetes in a long-term manner.

2. The central targeting system according to claim 1, characterized in that, This is a polynucleotide with a sequence of SEQ ID NO.1 - SEQ ID NO.

2.

3. A polynucleotide according to claim 2, characterized in that, The fusion protein (d)9R-RVG-FGF1^ with the coding sequence of SEQ ID NO.3 HBS .

4. A polynucleotide according to claim 2, characterized in that, Its coding sequence is the fusion protein (d)9R-RVG-FGF4 of SEQ ID NO.

4.

5. A polynucleotide according to claim 2, characterized in that, Its sequence is SEQ ID NO.

5.

6. A polynucleotide according to claim 2, characterized in that, Its sequence is SEQ ID NO.

6.

7. A polynucleotide according to claim 2, characterized in that, Its sequence is SEQ ID NO.7.

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