Compound miglittol liposome and application thereof in preparation of hypoglycemic drugs

Abstract

The invention relates to a compound miglittol liposome and application thereof in preparation of a hypoglycemic drug, and belongs to the technical field of pharmaceutical preparations, the compound miglittol liposome comprises miglittol, scopoletin, distearoyl phosphatidyl ethanolamine-polyethylene glycol, egg yolk phospholipid, cholesterol and sodium cyclamate, and the preparation method comprises the following steps: mixing the miglittol, the scopoletin, the distearoyl phosphatidyl ethanolamine-polyethylene glycol, the egg yolk phospholipid, the cholesterol and the sodium cyclamate; a film dispersion method is adopted for preparation. The scopoletin and the miglitol are entrapped in the optimized liposome, so that the problems of low drug loading capacity, poor stability, leakage and the like of the traditional liposome are solved, and the stability and the transmission efficiency of the drug are remarkably improved. Compared with single administration, the compound miglittol lipidosome can effectively reduce blood sugar fluctuation, and the combined use of the two medicines shows a remarkable synergistic blood sugar reducing effect, so that the compound miglittol lipidosome has a better clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a compound miglitol liposome and its use in the preparation of hypoglycemic drugs. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Diabetes, as a global chronic metabolic disease, has been showing a continuous upward trend in its incidence rate in recent years. Diabetes not only leads to long-term abnormal blood glucose levels but also triggers a series of serious complications, such as cardiovascular diseases, neuropathy, retinopathy, and nephropathy, etc. These complications seriously affect the quality of life of patients and even endanger their lives.

[0004] Currently, there are a wide variety of drugs used in clinical treatment of diabetes. Miglitol, as a kind of α-glucosidase inhibitor, occupies a certain position in the treatment of diabetes. It mainly reduces postprandial blood glucose by inhibiting α-glucosidase in the small intestine and slowing down the digestion and absorption of carbohydrates. However, there are many limitations in using miglitol alone. On the one hand, after long-term medication, its hypoglycemic effect is often difficult to reach an ideal state and cannot effectively control the continuous and stable blood glucose. On the other hand, this drug is prone to cause gastrointestinal adverse reactions, such as abdominal distension, abdominal pain, diarrhea, etc., which makes the tolerance of some patients to the drug poor and the compliance reduced, thus affecting the treatment effect.

[0005] Scopoletin is a natural coumarin compound. In recent years, studies have found that it has certain potential in blood glucose regulation. Chu Zixuan disclosed in "Research Progress on the Pharmacological Activity and Pharmacokinetics of Scopoletin" that scopoletin can reduce blood glucose. It can inhibit α-glucosidase and α-amylase in streptozotocin (STZ)-induced diabetic mice, thereby reducing postprandial hyperglycemia. The half-maximal inhibitory concentration (IC50) of scopoletin for α-glucosidase and α-amylase are 85.12 and 37.36 μmol / L respectively. (Chu Zixuan, Lu Min, Xiong Shan. Research Progress on the Pharmacological Activity and Pharmacokinetics of Scopoletin [J]. Chemical Research, 2019, 30(04): 434-440.) However, the current research on its mechanism of action is not yet perfect, and its hypoglycemic effect is also relatively limited when used alone, making it difficult to be widely used as a single drug in the treatment of diabetes.

[0006] As a new type of drug carrier, liposomes have unique structural and performance advantages. It is mainly composed of lipid materials such as phospholipids to form a bilayer membrane, which can encapsulate drugs. Liposomes have good biocompatibility and are not likely to cause immune rejection reactions after entering the human body; their targeting ability can enable drugs to accumulate in specific tissues or organs, improving the drug efficacy while reducing the toxic and side effects on other normal tissues; moreover, liposomes also have the characteristics of slow release, which can prolong the action time of drugs in the body, reduce the number of drug administrations, and improve the convenience and compliance of patients' medication. Based on these advantages of liposomes, co-encapsulating miglitol and scopoletin in liposomes is expected to overcome the deficiencies of the two drugs when used alone and develop a more efficient and safe hypoglycemic drug. Summary of the Invention

[0007] Overcoming the deficiencies of the prior art, the present invention provides a compound miglitol liposome with high stability and good hypoglycemic effect. The components in the compound miglitol liposome are calculated by weight as follows: miglitol 5 - 10 parts, scopoletin (CAS: 92 - 61 - 5) 0.2 - 0.7 parts, distearoyl phosphatidylethanolamine - polyethylene glycol (DSPE - PEG, CAS: 474922 - 77 - 5) 5 - 12 parts, egg yolk phospholipid 2 - 6 parts, cholesterol 1 - 3 parts, sodium cyclamate 0.2 - 0.8 parts, and it is prepared by the thin - film dispersion method.

[0008] The preparation method of the compound miglitol liposome is as follows:

[0009] (1) Dissolve distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, and cholesterol in an organic solvent, heat and rotary evaporate in a water bath at 40°C - 50°C until 1 / 4 - 1 / 2 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0010] (2) Drop an aqueous solution of sodium cyclamate into the lipid film in step (1), add a buffer solution containing miglitol for hydration, and perform high - pressure homogenization to obtain the compound miglitol liposome.

[0011] Furthermore, the organic solvent is selected from at least one of chloroform, methanol, and ethanol; the buffer solution is selected from at least one of acetic acid - sodium acetate buffer solution, citric acid - sodium citrate buffer solution, and potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer solution; the pH of the buffer solution is 4.0 - 5.5; the dropping rate of the aqueous solution of sodium cyclamate is 0.05 ml / s - 0.25 ml / s; the parameters of high - pressure homogenization are a pressure of 60 - 100 MPa, a cycle number of 4 - 6 times, a flow rate of 30 - 60 ml / min, and a temperature of 30 - 40°C.

[0012] To meet the clinical treatment needs, facilitate drug application and storage, and enable convenient drug administration, the compound miglitol liposome can be formulated with pharmaceutically acceptable excipients. For example, when formulated into an injection, it can be used for emergency cases or patients who cannot take oral medications; when formulated into an oral preparation, such as tablets, capsules, etc., it is convenient for patients to take by themselves, improving the convenience of medication.

[0013] The present invention also provides the use of the compound miglitol liposome in the preparation of hypoglycemic drugs.

[0014] Compared with the prior art, the technical effect of the present invention lies in:

[0015] To overcome the deficiencies of the two drugs when used alone, the present invention encapsulates scopoletin and miglitol in liposomes. However, it is found that when two substances with different water solubilities are encapsulated in liposomes, there are defects such as low drug loading, poor stability, and easy leakage, which will affect their efficacy, increase side effects and the burden on patients, and may lead to higher production costs. Therefore, improving the drug loading and stability of liposomes is the key to enhancing their clinical application value. The present invention selects liposome materials preferably and optimizes the preparation process, improving the stability of the drug-loaded liposomes and the efficiency of drug delivery.

[0016] Compared with the single administration of miglitol, the compound miglitol liposome of the present invention has the advantage of reducing blood glucose fluctuations. The combined use of scopoletin and miglitol also shows an obvious synergistic effect, and the hypoglycemic effect is superior to that of single administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Blood glucose levels of rats at 0:00, 4:00, 8:00, 12:00, 16:00, 20:00, and 24:00 during drug administration.

[0018] Figure 2 : Glycated hemoglobin content in rats of each group after drug administration.

[0019] Figure 3 : Drug loading of miglitol in the compound miglitol liposome.

[0020] Figure 4 : Drug loading of scopoletin in the compound miglitol liposome. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] In order to make the objectives and technical solutions of the present invention clearer, the following, in conjunction with embodiments, further describes the present invention. However, the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. Those skilled in the art should understand that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0022] Example 1

[0023] Formulation:

[0024] Dosage (g) Miglitol 8 Scopoletin 0.4 Distearoyl phosphatidylethanolamine - polyethylene glycol 8 Egg yolk phospholipid 4 Cholesterol 2 Sodium cyclamate 0.5

[0025] Preparation method:

[0026] (1) Add 120 ml of chloroform to distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phosphatide, and cholesterol, heat and rotary evaporate in a 45°C water bath until 1 / 3 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0027] (2) Dropwise add 10 ml of sodium cyclamate aqueous solution to the lipid film in step (1) at a dropping rate of 0.15 ml / s, add 60 ml of citric acid - sodium citrate buffer solution with pH = 4.8 ± 0.2 containing miglitol for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35°C) to obtain compound miglitol liposomes.

[0028] Example 2

[0029] Formulation:

[0030] Dosage (g) Miglitol 5 Scopoletin 0.2 Distearoyl phosphatidylethanolamine - polyethylene glycol 5 Egg yolk phospholipid 2 Cholesterol 1 Sodium cyclamate 0.2

[0031] Preparation method:

[0032] (1) Add 150 ml of methanol to distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phosphatide, and cholesterol, heat and rotary evaporate in a 40°C water bath until 1 / 4 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0033] (2) Dropwise add 15 ml of sodium cyclamate aqueous solution to the lipid film in step (1) at a dropping rate of 0.05 ml / s, add 80 ml of acetic acid - sodium acetate buffer solution with pH = 4.2 ± 0.2 containing miglitol for hydration, and perform high-pressure homogenization (pressure 60 MPa, number of cycles 6 times, flow rate 30 ml / min, temperature 30°C) to obtain compound miglitol liposomes.

[0034] Example 3

[0035] Formulation:

[0036] Dosage (g) Miglitol 10 Scopoletin 0.7 Distearoyl phosphatidylethanolamine - polyethylene glycol 12 Egg yolk phospholipid 6 Cholesterol 3 Sodium cyclamate 0.8

[0037] Preparation method:

[0038] (1) Add 200 ml of ethanol to distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phosphatide, and cholesterol, heat and rotary evaporate in a 50°C water bath until 1 / 2 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0039] (2) Add 20 ml of sodium cyclamate aqueous solution dropwise to the lipid film in step (1) at a dropping rate of 0.25 ml / s. Then add 100 ml of potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer solution with pH = 5.3 ± 0.2 containing miglitol for hydration, and perform high-pressure homogenization (pressure 100 MPa, number of cycles 4 times, flow rate 60 ml / min, temperature 40 °C) to obtain the compound miglitol liposome.

[0040] Comparative Example 1

[0041] Formulation:

[0042] Dosage (g) Miglitol 8 Scopoletin 0.4 Distearoyl phosphatidylethanolamine - polyethylene glycol 8 Egg yolk phospholipid 4 Cholesterol 2

[0043] Preparation method:

[0044] (1) Mix 120 ml of chloroform with distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, and cholesterol, heat and rotate-evaporate in a 45 °C water bath until 1 / 3 of the solution remains. Then add scopoletin and continue to rotate-evaporate until a lipid film is formed;

[0045] (2) Add 60 ml of citric acid - sodium citrate buffer solution with pH = 4.8 ± 0.2 containing miglitol to the lipid film in step (1) for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35 °C) to obtain the compound miglitol liposome.

[0046] Comparative Example 2

[0047] Formulation:

[0048] Dosage (g) Miglitol 8 Scopoletin 0.4 Distearoyl phosphatidylethanolamine - polyethylene glycol 8 Egg yolk phospholipid 4 Cholesterol 2 Sodium cyclamate 0.5

[0049] Preparation method:

[0050] (1) Mix 120 ml of chloroform with distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, and cholesterol, heat and rotate-evaporate in a 45 °C water bath until 1 / 3 of the solution remains. Then add scopoletin and continue to rotate-evaporate until a lipid film is formed;

[0051] (2) Add 60 ml of citric acid - sodium citrate buffer solution with pH = 4.8 ± 0.2 containing sodium cyclamate and miglitol to the lipid film in step (1) for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35 °C) to obtain the compound miglitol liposome.

[0052] Comparative Example 3

[0053] Formulation:

[0054]

[0055]

[0056] Preparation method:

[0057] (1) Mix distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, cholesterol with 120 ml of chloroform, heat and rotary evaporate in a 45°C water bath until 1 / 3 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0058] (2) Pour 10 ml of sodium cyclamate aqueous solution into the lipid film obtained in step (1), add 60 ml of citric acid - sodium citrate buffer solution with pH = 4.8 ± 0.2 containing miglitol for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35°C) to obtain compound miglitol liposomes.

[0059] Comparative Example 4

[0060] Formulation:

[0061] Dosage (g) Miglitol 8 Distearoyl phosphatidylethanolamine - polyethylene glycol 8 Egg yolk phospholipid 4 Cholesterol 2 Sodium cyclamate 0.5

[0062] Preparation method:

[0063] (1) Mix distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, cholesterol with 120 ml of chloroform, heat and rotary evaporate in a 45°C water bath until a lipid film is formed;

[0064] (2) Dropwise add 10 ml of sodium cyclamate aqueous solution to the lipid film obtained in step (1) at a dropping rate of 0.15 ml / s, add 60 ml of citric acid - sodium citrate buffer solution with pH = 4.8 ± 0.2 containing miglitol for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35°C) to obtain compound miglitol liposomes.

[0065] Comparative Example 5

[0066] Formulation:

[0067]

[0068]

[0069] Preparation method:

[0070] (1) Mix distearoyl phosphatidylethanolamine - polyethylene glycol, egg yolk phospholipid, cholesterol with 120 ml of chloroform, heat and rotary evaporate in a 45°C water bath until 1 / 3 of the solution remains, add scopoletin, and continue rotary evaporation until a lipid film is formed;

[0071] (2) Add 10 ml of sodium cyclamate aqueous solution dropwise to the lipid film in step (1) at a dropping rate of 0.15 ml / s, add 60 ml of citric acid-sodium citrate buffer solution with pH = 4.8 ± 0.2 for hydration, and perform high-pressure homogenization (pressure 80 MPa, number of cycles 5 times, flow rate 45 ml / min, temperature 35 °C) to obtain compound miglitol liposomes.

[0072] Comparative Example 6

[0073] Miglitol tablets (National Medicine Approval No. H20045403)

[0074] Pharmacodynamic experiment

[0075] The inventors carried out relevant pharmacodynamic experimental studies to prove the efficacy of the compound miglitol liposomes of the present invention in treating diabetes. The following experimental studies were all carried out on the basis of proving the drug safety through acute toxicity test and long-term toxicity test, and the administration doses in the experimental studies were all within the safe dose range. The experimental data of the present invention were statistically analyzed using SPSS 22.0 software, and the measurement data were expressed as , one-way ANOVA was used for comparison among multiple groups, and independent sample T-test was used for analysis between two groups. P < 0.05 was considered statistically significant.

[0076] Experimental animals: SD rats, SPF grade, 180 - 220 g, experimental animal license number: SYXK (Lu) 20180008.

[0077] Model establishment: Before the experiment, the rats were adaptively fed for 1 week under standard conditions, fed with high-calorie feed for 5 weeks, fasted for 12 h, the fasting blood glucose was measured from the tail vein, and the body weight was weighed. Intraperitoneally inject 1% streptozotocin (STZ) at 60 mg / kg. STZ was dissolved in 0.1 mol / L citric acid-sodium citrate buffer solution with pH 4.2, and the drug amount each time was used up within 10 min. After 10 days, blood was taken from the tail to measure the whole blood glucose. Those with blood glucose level > 10.0 mmol / L were considered successful in model establishment, and those with blood glucose level > 15.0 mmol / L were considered stable. The successfully modeled rats were divided into a model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 4 group, Comparative Example 5 group, and Comparative Example 6 group, with 10 rats in each group. The rats in the blank group were intraperitoneally injected with an equal volume of citric acid-sodium citrate buffer solution and raised normally.

[0078] 2.3 Administration

[0079] Rats in each administration group were intragastrically administered with the corresponding drugs (rats in the groups of Examples 1 to 3 were administered with the drugs of Examples 1 to 3, calculated as miglitol, 27 mg / kg; rats in the group of Comparative Example 4 were administered with the drug of Comparative Example 4, calculated as miglitol, 27 mg / kg; rats in the group of Comparative Example 5 were administered with the drug of Comparative Example 5, calculated as scopoletin, 1.35 mg / kg; rats in the group of Comparative Example 6 were administered with the miglitol tablets of Comparative Example 6, calculated as miglitol, 27 mg / kg). Rats in the blank group and the model group were intragastrically administered with an equal amount of normal saline once a day for 7 consecutive days.

[0080] Observation indicators

[0081] Blood glucose levels of rats during drug administration

[0082] The blood glucose levels of rats during drug administration were measured by the glucose oxidase method at 0:00, 4:00, 8:00, 12:00, 16:00, 20:00, and 24:00 throughout the day, and a blood glucose-time curve was made. Figure 1 The blood glucose levels of rats at 0:00, 4:00, 8:00, 12:00, 16:00, 20:00, and 24:00 during drug administration are shown. The blood glucose fluctuations of rats in the groups of Examples 1 to 3 of the present invention are small and relatively stable.

[0083] Glycated hemoglobin in rats after drug administration

[0084] The content of glycated hemoglobin in rats after drug administration was measured using a kit. Figure 2 The content of glycated hemoglobin in rats after drug administration in each group is shown. There was a significant difference in the content of glycated hemoglobin between the rats in the groups of Examples 1 to 3 and the rats in the group of Comparative Example 4, P < 0.01.

[0085] Quality evaluation of compound miglitol liposomes

[0086] Appearance properties

[0087] Before and after the accelerated test, the appearance of compound miglitol liposomes was observed with the naked eye to check whether it was uniform, whether there was precipitation, delamination, or color change. If precipitation, delamination, or color change occurred, it might indicate problems with the stability of the liposomes, such as drug leakage, lipid oxidation, etc. The conditions for the accelerated test were a temperature of 40 ± 2°C and a relative humidity of 75% ± 5%.

[0088] Table 1 Appearance properties of compound miglitol liposomes before and after the accelerated test

[0089]

[0090]

[0091] Table 1 presents the appearance properties of compound miglitol liposomes on the 0th day and the 180th day of the accelerated test under different examples and comparative examples. By comparing the examples and comparative examples, it can be clearly seen that the compound miglitol liposomes in the examples have advantages in terms of the stability of appearance properties. Precipitation occurred in Comparative Examples 1, 2, and 3 on the 180th day of the accelerated test, indicating that certain physical changes occurred to the compound miglitol liposomes in the comparative examples during the accelerated test. It is possible that partial damage occurred to the structure of the liposomes, and the drug or other components precipitated out of the system.

[0092] Drug loading amount

[0093] The drug loading amount determines the drug dose that the liposomes can carry, which is of great significance for determining the dosing dose and therapeutic effect. An appropriate drug loading amount can reduce the dosing volume and frequency while ensuring the therapeutic effect. Measuring the amount of drug contained in the liposomes before and after the accelerated test can indirectly reflect the structural stability of the liposomes, understand whether the drug degrades under accelerated conditions, and provide data support for formulating the quality standard of the drug.

[0094] Figure 3 is the drug loading amount of miglitol in the compound miglitol liposomes. Figure 4 is the drug loading amount of scopoletin in the compound miglitol liposomes, showing that the compound miglitol liposomes in Examples 1 to 3 of the present invention encapsulate more effective components of miglitol and scopoletin, and have higher drug delivery efficiency. By comparing the drug loading amount results before and after the accelerated test, it can be known that the compound miglitol liposomes in Examples 1 to 3 of the present invention have high stability, meaning that the effective components can better maintain their original chemical structure and biological activity during storage and use and play their roles.

Claims

1. A compound miglitol liposome, characterized in that: The components of the compound miglitol liposome are calculated in parts by weight as follows: 5 to 10 parts of miglitol, 0.2 to 0.7 parts of scopoletin, 5 to 12 parts of distearoyl phosphatidylethanolamine-polyethylene glycol, 2 to 6 parts of egg yolk phospholipids, 1 to 3 parts of cholesterol, and 0.2 to 0.8 parts of sodium cyclohexylaminosulfonate. The compound miglitol liposome is prepared by a thin film dispersion method.

2. The compound miglitol liposome according to claim 1, characterized in that The components in the compound miglitol liposome are calculated by weight as follows: 8 parts of miglitol, 0.4 parts of scopoletin, 8 parts of phospholipid-polyethylene glycol, 4 parts of egg yolk phospholipids, 2 parts of cholesterol, and 0.5 parts of sodium cyclamate.

3. The compound miglitol liposome according to claim 1, characterized in that: The thin film dispersion method is: (1) dissolving phospholipid-polyethylene glycol, egg yolk phospholipids and cholesterol in an organic solvent, heating in a water bath at 40°C to 50°C and performing rotary evaporation until 1 / 4 to 1 / 2 of the solution remains, adding scopoletin, and continuing rotary evaporation until a lipid film is formed; (2) adding an aqueous solution of sodium cyclohexylaminosulfonate to the lipid film of step (1), adding a buffer solution containing miglitol to hydrate the film, and homogenizing the film under high pressure to obtain composite miglitol liposomes.

4. The compound miglitol liposome according to claim 3, characterized in that: The organic solvent is selected from at least one of chloroform, methanol and ethanol; preferably, the organic solvent is chloroform.

5. The compound miglitol liposome according to claim 3, characterized in that: The buffer solution is selected from at least one of acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution, and potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution; preferably, the buffer solution is citric acid-sodium citrate buffer solution.

6. The compound miglitol liposome according to claim 3, characterized in that: The pH of the buffer solution is 4.0-5.5; preferably, the pH of the buffer solution is 4.8±0.

2.

7. The compound miglitol liposome according to claim 3, characterized in that: The speed of adding the sodium cyclohexylaminosulfonate aqueous solution is 0.05 ml / s to 0.25 ml / s; preferably, the speed of adding the sodium cyclohexylaminosulfonate aqueous solution is 0.15 ml / s.

8. The compound miglitol liposome according to claim 3, characterized in that: The parameters of the high-pressure homogenization are pressure 60-100 MPa, cycle number 4-6 times, flow rate 30-60 ml / min, and temperature 30-40°C; preferably, the parameters of the high-pressure homogenization are pressure 80 MPa, cycle number 5 times, flow rate 45 ml / min, and temperature 35°C.

9. The compound miglitol liposome according to claim 1, characterized in that: The compound miglitol liposome is prepared into a preparation with pharmaceutically acceptable excipients.

10. Use of the compound miglitol liposome according to claim 1 in the preparation of hypoglycemic drugs.

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