Application and therapeutic drugs of niclosamide in the treatment of congenital hyperinsulinemic hypoglycemia

Abstract

The present invention belongs to the field of biomedicine and relates to the use of niclosamide in the treatment of congenital hyperinsulinemic hypoglycemia and a therapeutic agent. The niclosamide of the present invention can inhibit insulin secretion by inhibiting mitochondrial function. The improved niclosamide formulation of the present invention can achieve the purpose of treating congenital hyperinsulinemic hypoglycemia after oral administration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of niclosamide in treating congenital hyperinsulinemic hypoglycemia and a therapeutic drug. Background Art

[0002] Neonatal hypoglycemia is a serious condition that threatens the health and even life of newborns. Recurrent episodes of severe hypoglycemia are not only life-threatening but can also cause permanent and irreversible brain damage. The primary cause of hypoglycemia is congenital hyperinsulinemic hypoglycemia (CHI). The etiology is complex (over 11 causative genes have been identified), involving multiple components of the insulin secretion regulatory pathway. This manifests as excessive or inappropriate insulin secretion, leading to hypoglycemia. Although the mechanisms of excessive insulin secretion vary among different subtypes of hypoglycemia, excessive insulin secretion consumes significant amounts of ATP. Current research suggests that several major subtypes of CHI involve abnormally active mitochondrial function in pancreatic β-cells. For example, CHI caused by loss-of-function mutations in ATP-dependent potassium channels in pancreatic β-cells. Studies of surgically resected pancreatic islets have revealed increased mitochondrial numbers and elevated basal oxygen consumption in these subtypes of CHI. Therefore, it is hypothesized that inhibiting mitochondrial function, and thus insulin secretion, could be used to treat CHI, ultimately increasing blood glucose.

[0003] Niclosamide is an anthelmintic and a salicylamide derivative. Its antiparasitic mechanism is to inhibit mitochondrial oxidative phosphorylation within parasite cells, leading to reduced ATP production. It is a mitochondrial uncoupler. This study found that niclosamide inhibits basal insulin secretion and amino acid-stimulated insulin secretion in pancreatic islets of CHI mice. Furthermore, intraperitoneal injection of niclosamide can increase blood glucose levels in CHI mice. However, conventional oral or intraperitoneal administration of niclosamide, after dissolving it, has limited effects on blood glucose due to formulation deficiencies, far lower than that achieved by intraperitoneal injection. However, improved formulations have significantly enhanced the blood glucose improvement in these mice, demonstrating that oral administration of niclosamide can be effective in treating CHI. Summary of the Invention

[0004] To address the above issues, the present invention provides a use of niclosamide in the treatment of congenital hyperinsulinemic hypoglycemia and a medicament for treating congenital hyperinsulinemic hypoglycemia. The niclosamide of the present invention inhibits insulin secretion by inhibiting mitochondrial function; an improved niclosamide formulation can achieve the purpose of treating congenital hyperinsulinemic hypoglycemia after oral administration.

[0005] The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

[0006] One aspect of the present invention provides a use of niclosamide in treating congenital hyperinsulinemic hypoglycemia.

[0007] Preferably, the niclosamide is an inhibitor of insulin function. Specifically, the niclosamide can inhibit insulin secretion by inhibiting mitochondrial function.

[0008] Preferably, the concentration of niclosamide is 40-500 nM.

[0009] Another aspect of the present invention provides a drug for treating congenital hyperinsulinemic hypoglycemia, wherein the drug comprises a lyophilized powder of a niclosamide-hydroxypropyl-β-cyclodextrin mixture.

[0010] Preferably, the niclosamide-hydroxypropyl-β-cyclodextrin is prepared according to the following method: hydroxypropyl-β-cyclodextrin and niclosamide are placed in a container at a mass ratio of 3:1, and an appropriate amount of water is added for suspension. After magnetic stirring at room temperature for 8 hours, freeze-drying is performed to obtain a niclosamide-hydroxypropyl-β-cyclodextrin mixture dry powder.

[0011] Preferably, the concentration of the niclosamide-hydroxypropyl-β-cyclodextrin mixture dry powder in the drug is 2 mg / mL.

[0012] Preferably, the concentration of niclosamide in the drug is 0.5 mg / mL.

[0013] Preferably, the medicine further comprises sodium carboxymethylcellulose.

[0014] Preferably, the concentration of the sodium carboxymethyl cellulose is 0.5%.

[0015] Preferably, the drug is administered orally.

[0016] By means of the above technical solution, the present invention has at least the following advantages:

[0017] (1) The present invention has found through research that niclosamide, an anthelmintic, has a significant effect of inhibiting insulin secretion, thereby providing application prospects in the treatment of hypoglycemia caused by congenital hyperinsulinemia.

[0018] (2) The improved niclosamide preparation of the present invention can achieve the purpose of treating congenital hyperinsulinemic hypoglycemia after oral administration.

[0019] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Graph showing the inhibitory effects of different concentrations of niclosamide on glucose-stimulated insulin secretion;

[0021] Figure 2 This is a diagram showing the effect of niclosamide on inhibiting insulin secretion in SUR1 knockout (SUR1-KO) mice;

[0022] Figure 3 This figure shows the effect of intraperitoneal injection of niclosamide on blood glucose in wild-type mice;

[0023] Figure 4 This figure shows the effect of intraperitoneal injection of niclosamide on blood glucose in SUR1-KO mice;

[0024] Figure 5 This is a graph showing the effect of oral administration (or gavage) of niclosamide on blood glucose in wild-type mice;

[0025] Figure 6 This figure shows the effect of oral administration (oral gavage) of niclosamide on blood glucose in SUR1-KO mice;

[0026] Figure 7 This figure shows the effect of oral administration (oral gavage) of niclosamide on blood glucose in SUR1-KO mice;

[0027] Figure 8 is the HPLC chromatogram of niclosulphite standard solution;

[0028] Figure 9 is the linear regression equation of niclosamide;

[0029] Figure 10 This is the test result of niclosamide-hydroxypropyl-β-cyclodextrin;

[0030] Figure 11 This figure shows the effect of oral (oral) administration of the modified formulation of niclosamide on the blood glucose of SUR1-KO mice. DETAILED DESCRIPTION

[0031] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1 In vitro islet perfusion detection of the effect of niclosamide on insulin secretion

[0033] 1. Islet Isolation

[0034] 1) After anesthesia, the mouse was fixed on a surgical board. The abdominal cavity was opened and the bile duct entering the duodenum was sealed with hemostats. Then, a 2 mg / ml collagenase solution was injected into the bile duct using a 5 ml syringe and a 31.5 gauge needle until the pancreas was fully inflated.

[0035] 2) Dissect the pancreas, remove fat and non-pancreatic tissue, and transfer the pancreas to a 50 ml centrifuge tube. Add 3 ml of 2 mg / ml collagenase solution and shake in a 37°C water bath for 4-5 minutes.

[0036] 3) Immediately after shaking, add Hanks buffer to 50 ml in a 50 ml centrifuge tube at 2000 rpm. After centrifugation, remove the supernatant and add 5 ml of Histopaque-1119 solution to the remaining tissue pellet and shake to mix.

[0037] 4) Slowly add 5 ml of Histopaque-1077 solution, and then slowly add 5 ml of Hanks buffer. The centrifuge tube speed is 2000 rpm.

[0038] 5) Remove the islet tissue from the layered liquid, pick out the purified islets under a dissecting microscope, wash them, and then culture them.

[0039] 2. Islet culture:

[0040] 1) Preparation of RPMI1640 culture medium: 10 mM glucose, 2 mM glutamine, 10% fetal bovine serum, 2 g / L sodium bicarbonate, 100 units / ml penicillin, 10 µg / ml streptomycin. Adjust the pH to 7.2.

[0041] 2) Culture the isolated and purified islets in RPMI1640 medium in an incubator at 37°C, 5% CO2, and 95% humidity for 1-2 days.

[0042] 3. Pancreatic islet perfusion test:

[0043] 1) Prepare buffer: 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, and 2.5 mM CaCl2. Adjust the pH to 7.4 and add 0.25% bovine serum albumin.

[0044] 2) Prepare stimulation solution containing 25 mM glucose.

[0045] 3) Manually pick 120-150 islets of equal size and place them in the islet chamber. Depending on the experimental requirements, place them in a water bath.

[0046] 4) Place the prepared reaction solutions in a water bath (37°C) and insert the corresponding injection tubes. The perfusion program is shown in Table 1 below:

[0047] Table 1 Perfusion procedures

[0048]

[0049] Note: A is buffer, B is stimulation solution, and C is 30 mM potassium chloride. Curves 1 to 6 are ramp perfusions, in which A and B are gradually mixed, with A decreasing from 100% to 0 and B increasing from 0 to 100%.

[0050] The secreted insulin was collected in a 96-deep-well plate by Collector Fraction Waters at a rate of 1 mL / min and stored in a -20°C refrigerator until the hormone was measured.

[0051] 4. Insulin secretion value detection:

[0052] 1) Transfer 10 μL / well of the solution collected from the islet perfusion experiment in a 96-deep-well plate to a 384-well plate;

[0053] 2) Add the antibody according to the HTRF insulin assay kit instructions, mix thoroughly by vortexing, and incubate at room temperature for 2 hours. Read the readings using the BMG Clariostar microplate reader HTRF program and calculate the hormone secretion value based on the standard curve. The effect of the stimulus or drug on insulin secretion is confirmed by measuring insulin secretion.

[0054] 5. Niclosamide inhibits pancreatic islet function in wild-type mice: Figure 1 The glucose-stimulated insulin secretion assay in perfused wild-type mouse islets, shown here, shows that under a glucose gradient of 0.5 mM / min, insulin secretion in the DMSO solvent control group rapidly rises after exceeding a glucose threshold of 5.5 mM and enters a plateau phase. Addition of 40 nM, 200 nM, and 500 nM niclosamide, respectively, reveals a significant inhibitory effect on glucose-stimulated insulin secretion. 40 nM increases the glucose threshold to 7.0 mM, and 200 nM increases it to 9.0 mM. Furthermore, maximal insulin secretion is also inhibited in a dose-dependent manner. Increasing the niclosamide dose to 500 nM completely inhibits glucose-stimulated insulin secretion, while maintaining no significant effect on 30 mM KCl stimulation. This demonstrates that the islets still have the capacity to secrete insulin, but exhibit a significant dose-dependent inhibitory effect on glucose-stimulated insulin secretion.

[0055] 6. Niclosamide inhibits insulin secretion in ATP-dependent potassium channel knockout mice:

[0056] 1) Development of SUR1 Knockout Mice: Using CRISPR / Cas9 gene knockout technology, crRNA binds to tracrRNA (trans-activating RNA) through base pairing, forming double-stranded RNA. This tracrRNA:crRNA binary complex guides the Cas9 protein to cleave double-stranded DNA at a specific site within the crRNA guide sequence. At sites complementary to the crRNA guide sequence, the HNH nuclease domain of the Cas9 protein cleaves the complementary strand, while the Cas9 RuvC-like domain cleaves the non-complementary strand, achieving knockout of the target gene and generating a knockout mouse model.

[0057] 2) Niclosamide inhibits pancreatic islet function in SUR1 knockout (SUR1-KO) mice: Figure 2 Shown are insulin secretion stimulated by amino acid ramping (0.4 mM / min) and glucose ramping (0.83 mM / min). Baseline insulin secretion in SUR1-KO mice was significantly elevated, and 2.5 µM niclosamide significantly inhibited basal insulin secretion. SUR1-KO islets are sensitive to amino acid-stimulated insulin secretion but insensitive to glucose. Niclosamide did not alter this characteristic, but significantly inhibited amino acid-stimulated insulin secretion. Islet secretion in response to KCl was similar, indicating that islets retain secretory capacity despite niclosamide inhibition.

[0058] Example 2 Study on the Effect of Conventional Niclosamide Preparations on Blood Glucose in Living Animals

[0059] 1. Conventional preparation of niclosamide: Dissolve 25 mg of niclosamide in 5.5 ml of DMSO. Slowly add 1 ml of castor oil, mix thoroughly, and then slowly add 33.5 ml of 0.5% sodium carboxymethylcellulose. Administer a dose of 10 mg / kg body weight. A solvent control group was prepared identically to the niclosamide solvent except that the solvent was omitted.

[0060] 2. Effects of intraperitoneal injection of niclosamide on blood glucose in wild-type mice: Figure 3 As shown, male wild-type mice were fasted overnight, and blood glucose in tail blood was measured every 30 minutes after intraperitoneal injection of niclosamide (10 mg / kg body weight). The results showed that the blood glucose level in the niclosamide-treated group was significantly higher than that in the solvent control group at 30 and 60 minutes.

[0061] 3. Effects of intraperitoneal injection of niclosamide on blood glucose in SUR1-KO mice: Figure 4As shown, male SUR1-KO mice were fasted overnight and then intraperitoneally injected with niclosamide (10 mg / kg body weight). Blood glucose was measured every 30 minutes after the injection. The results showed that blood glucose levels in the niclosamide group were significantly higher than those in the vehicle control group at 30, 60, 90, and 120 minutes. This indicates that niclosamide significantly increases blood glucose in SUR1-KO mice.

[0062] 4. Effects of oral (oral) administration of niclosamide on blood glucose in wild-type mice: Figure 5 Results showed that wild-type mice fasted overnight were given niclosamide (10 mg / kg body weight) by gavage and blood glucose levels were measured every 30 minutes after intraperitoneal injection. Results showed that blood glucose levels were significantly higher than those in the vehicle control group only at 30 minutes. Compared to intraperitoneal administration, the effect was significantly weakened.

[0063] 5. Effects of oral (or intragastric) administration of niclosamide on blood glucose in SUR1-KO mice: Figure 6 The results show that SUR1-KO mice were fasted overnight and then intraperitoneally injected with niclosamide (10 mg / kg body weight). Blood glucose levels in tail blood were measured every 30 minutes. The results showed that there was no significant difference between the blood glucose levels in the control group and the solvent control group at each time point. Compared with intraperitoneal administration, the traditional formulation of niclosamide did not increase the blood glucose level in SUR1-KO mice. In order to further verify whether the traditional formulation of niclosamide has an effect on the blood glucose level in SUR1-KO mice, Figure 7 As shown in Figure 2, the experimental protocol was adjusted to increase the dose of niclosamide to 20 mg / kg body weight after fasting SUR1-KO mice for 4 hours, but the results showed that the blood glucose after oral gavage was still no different from that of the solvent control.

[0064] Based on the above data, we can conclude that niclosamide can treat congenital hyperinsulinemic hypoglycemia by inhibiting insulin secretion at the pancreatic islet level. After niclosamide is dissolved in traditional reagents, the effect of intraperitoneal administration is significant, but the effect basically disappears after oral administration. This shows that traditional preparation methods cannot achieve the purpose of treating hypoglycemia after oral administration, and improvements in the preparation formula are needed.

[0065] Example 3 Preparation of New Dosage Forms of Niclosamide

[0066] 1. HPLC detection of niclosamide standard:

[0067] 1) Chromatographic conditions:

[0068] An XDB-C18 column (4.6 × 250 mm, 5 μm) was used; the mobile phase consisted of methanol: 0.02 mol / L ammonium acetate buffer (85:15, v / v); the flow rate was 0.8 mL / min; the UV detector wavelength was 340 nm; the column temperature was 30°C; and the injection volume was 20 μL. Under these conditions, the niclosamide sample was analyzed and determined, with the corresponding peak position at 4.375 min (e.g., Figure 8 shown).

[0069] 2) Linear relationship:

[0070] Accurately weigh 5.0 mg of niclosamide standard into a 10 mL volumetric flask. Dissolve the solution in a certain volume of mobile phase. Once completely dissolved, dilute to 10 mL with mobile phase to prepare a 0.5 mg / mL niclosamide standard solution. Dilute with mobile phase to prepare standard solutions with concentrations of 0.2, 0.5, 1.0, 2.0, and 5.0 μg / mL, respectively.

[0071] The standard curve was drawn with the peak area of ​​niclosamide as the ordinate and the concentration as the abscissa, and the regression equation was obtained: y = 78.029x -1.8719 (R² = 0.9999) (e.g. Figure 9 shown).

[0072] 2. Niclosamide preparation scheme:

[0073] A novel preparation is prepared by combining niclosamide with a cyclodextrin derivative, wherein the cyclodextrin derivative used is hydroxypropyl-β-cyclodextrin.

[0074] 1) Preparation: Place hydroxypropyl-β-cyclodextrin and niclosamide in a container at a mass ratio of 3:1. Add an appropriate amount of water to suspend the mixture. Stir magnetically at room temperature for 8 hours, then freeze-dry to obtain a niclosamide-hydroxypropyl-β-cyclodextrin mixture powder.

[0075] 2) Determination of niclosamide content in freeze-dried niclosamide-hydroxypropyl-β-cyclodextrin niclosamide sample:

[0076] Dissolve 5.9 mg of niclosamide-hydroxypropyl-β-cyclodextrin in 50 mL of double-distilled water, stir thoroughly, and filter through a syringe filter to obtain a clear mobile phase. This mobile phase was then diluted 20-fold using an XDB-C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of methanol:0.02 mol / L ammonium acetate buffer (85:15, v / v). The flow rate was 0.8 mL / min, the UV detector wavelength was 340 nm, the column temperature was 30°C, and the injection volume was 20 μL.

[0077] 3) The experimental results are as follows: Niclosamide-hydroxypropyl-β-cyclodextrin test results are as follows Figure 10 As shown in the figure, the retention time is 4.10 min, the peak area is 102.9244, and the calculated content is 2.323 μg / mL.

[0078] 3. Effect of niclosamide-hydroxypropyl-β-cyclodextrin freeze-dried powder on blood glucose in mice

[0079] 1) Experimental Materials:

[0080] 36 male SUR1-KO mice

[0081] 2) Experimental compounds:

[0082] (1) Dissolve niclosamide-hydroxypropyl-β-cyclodextrin lyophilized powder in 0.5% sodium carboxymethylcellulose to achieve a drug concentration of 0.5 mg / mL: Weigh 40 mg of niclosamide-hydroxypropyl-β-cyclodextrin lyophilized powder and dissolve it in 20 mL of 0.5% sodium carboxymethylcellulose to prepare a solution with a lyophilized powder concentration of 2 mg / mL. The final niclosamide concentration is 0.5 mg / mL.

[0083] (2) Solvent control: Hydroxypropyl-β-cyclodextrin + 0.5% sodium carboxymethylcellulose: Weigh 30 mg of hydroxypropyl-β-cyclodextrin powder and dissolve it in 20 mL of 0.5% sodium carboxymethylcellulose.

[0084] 3) Experimental steps:

[0085] (1) Grouping: Experimental group: niclosamide-hydroxypropyl-β-cyclodextrin (niclosamide concentration: 0.5 mg / mL);

[0086] Solvent control group: hydroxypropyl-β-cyclodextrin.

[0087] (2) Fasting: Take away the mouse's food, replace the bedding, and continue fasting without stopping the water supply.

[0088] (3) After fasting for 2 hours, the rats were gavaged orally at 20 μL / g body weight. Blood glucose was measured before gavage, 1 hour, 2 hours, and 3 hours. The gavage was repeated at 3 hours, and the blood glucose level after repeated gavage was measured.

[0089] 4) Result analysis:

[0090] like Figure 11As shown in the figure, after oral administration (niclosamide dose of 20 mg / kg body weight) of niclosamide-hydroxypropyl-β-cyclodextrin, the blood glucose level in the drug group continued to rise, and was significantly higher than that in the solvent control group 2 hours after administration. This blood glucose-raising effect lasted until 4 hours after administration. Following the second oral administration, the blood glucose-raising effect of niclosamide-hydroxypropyl-β-cyclodextrin continued until the end of the experiment 8 hours after fasting, which was similar to the Figure 7 The comparison showed that the new preparation significantly enhanced the blood glucose-raising effect of niclosamide in SUR1-KO mice.

[0091] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of niclosamide or its preparation in the preparation of a medicament for treating congenital hyperinsulinemic hypoglycemia; The preparation is a freeze-dried powder of a niclosamide-hydroxypropyl-β-cyclodextrin mixture.

2. The use according to claim 1, characterized in that The niclosamide is an inhibitor that inhibits the secretion function of insulin.

3. The use according to claim 1, characterized in that The concentration of niclosamide is 100-400 nM.

4. The use according to claim 1, characterized in that The niclosamide-hydroxypropyl-β-cyclodextrin mixture freeze-dried powder is prepared according to the following method: hydroxypropyl-β-cyclodextrin and niclosamide are placed in a container at a mass ratio of 3:1, and an appropriate amount of water is added to suspend the mixture. After magnetic stirring at room temperature for 8 hours, the mixture is freeze-dried to obtain a niclosamide-hydroxypropyl-β-cyclodextrin mixture dry powder.

5. The use according to claim 1, characterized in that The concentration of the niclosamide-hydroxypropyl-β-cyclodextrin mixture dry powder in the drug is 2 mg / mL.

6. The use according to claim 5, characterized in that The final concentration of niclosamide in the drug is 0.5 mg / mL.

7. The use according to claim 1, characterized in that The medicine also contains sodium carboxymethylcellulose.

8. The use according to claim 7, characterized in that The concentration of the sodium carboxymethyl cellulose is 0.5%.

9. The use according to claim 1, characterized in that The drug is administered orally.

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