CN101278913B - Novel polyethylene glycol-phospholipid entrapped prostaglandin E1 micelle preparation - Google Patents

Abstract

The present invention provides a prostaglandin E1 micelle preparation with high encapsulation efficiency and low initial release for intravenous injection, which contains therapeutically effective doses of prostaglandin E1, PEG derivatized phospholipids, hydrogenated phospholipids, and pharmaceutically Acceptable adjuvants, wherein the PEG derivatized phospholipids are formed by combining polyethylene glycol molecules with nitrogenous bases on phospholipid molecules through covalent bonds, and the total phospholipids of the sum of PEG derivatized phospholipids and hydrogenated phospholipids are mixed with prostate The molar ratio of the element E1 is in the range of 80:1-500:1. The nanoparticle drug delivery system of the present invention is a freeze-dried preparation. After reconstitution by adding a physiologically acceptable solvent, it forms nano drug-loaded micelles with high encapsulation efficiency and low initial release. After intravenous injection, It can significantly improve the bioavailability of prostaglandin E1, and has a stronger and longer-lasting pharmacological effect on blood vessels with microcirculation disorders.

Description

A PEG derivatized phospholipid-encapsulated micellar preparation of prostaglandin E1

technical field

The invention relates to a prostaglandin E1 (PGE1) micelle preparation for intravenous injection.

Background technique

The relevant background technology of the prostaglandin E1 (PGE1) nanoparticle drug delivery system has been discussed in detail in the applicant's previous patent (ZL 200410057147), involving the pharmacological properties of prostaglandin E1 and liposomes, microemulsions, polymers, etc. The drug-loading properties of ethylene glycol (PEG) derivatized phospholipid micelles are based on the many advantages of PEG derivatized phospholipid micelles as drug carriers, especially in the prostaglandin E1 nanomicelle particle drug delivery system prepared by encapsulating prostaglandin E1 Exhibited excellent pharmaceutical properties and stability, the patent (ZL 200410057147) describes a prostaglandin E1 micelles for intravenous injection, which is a kinetically stable system, has good stability, and has a target in vivo Increase the distribution of drugs in the lesion, improve the efficacy of drugs and reduce side effects.

Through further experimental research, the results show that the lipid ratio and phospholipid ratio of the traditional Chinese medicine in the prescription are closely related to the encapsulation efficiency and release rate of drug-loaded micelles. The encapsulation rate and release rate of drug-loaded micelles are the basis for micellar drugs to exert their biological characteristics , so on the basis of further research on the relationship between the lipid ratio and phospholipid ratio of the prescription traditional Chinese medicine and the encapsulation efficiency and release degree of drug-loaded micelles, we further optimized and limited the lipid ratio and phospholipid ratio of the prescription Chinese medicine, and prepared a compound with high encapsulation efficiency and low initial release of prostaglandin E1 micelles.

Contents of the invention

The purpose of the present invention is to provide a kind of excellent prostaglandin E1 micelle preparation, it is a kind of kinetically stable system, has good stability, and has targeting effect in vivo, increases the distribution of medicine in lesion, improves the medicine curative effect and reduce side effects.

The present invention is based on the patent (ZL 200410057147) to further optimize and limit the lipid ratio and phospholipid ratio of prescription traditional Chinese medicine, and provide a prostaglandin E1 micelle preparation with better encapsulation rate and release degree.

The present invention provides a prostaglandin E1 micelle preparation for intravenous injection, which contains a therapeutically effective amount of prostaglandin E1, PEG derivatized phospholipids, hydrogenated phospholipids, and pharmaceutically acceptable adjuvants, wherein the PEG derivatized phospholipids are Polyethylene glycol molecules are formed by combining covalent bonds with nitrogenous bases on phospholipid molecules, and the molar ratio of total phospholipids to prostaglandin E1 in the sum of PEG derivatized phospholipids and hydrogenated phospholipids is 80:1-500:1 within range.

In a preferred embodiment of the invention, the PEG derivatized phospholipid is polyethylene glycol 2000 derivatized distearoylphosphatidylethanolamine.

In a preferred embodiment of the present invention, the molar ratio of hydrogenated phospholipids to total phospholipids ranges from 50% to 90%.

In a preferred embodiment of the present invention, the molar ratio of total phospholipids to prostaglandin E1 is in the range of 160:1-240:1.

In the present invention, the pharmaceutically acceptable adjuvants include antioxidants, pH regulators, and osmotic pressure regulators.

In a preferred embodiment of the present invention, the micellar preparation is in a freeze-dried form, and is reconstituted by adding a physiologically acceptable solvent immediately before use. After the micellar preparation is reconstituted, a solution containing 0.5 μg/ml-10 μg/ml prostaglandin E1 and 0.01 mM-50 mM total phospholipids is formed, which is used for intravenous injection.

In a preferred embodiment of the present invention, after the micelle preparation is reconstituted, the pH of the drug-loaded micelle solution is in the range of 4.5-5.5, preferably in the range of 4.8-5.2.

In order to better understand the content of the present invention, some technical terms are explained below.

"Micelle" means that when the concentration of amphiphilic molecules in aqueous solution exceeds the critical micelle concentration (CMC), they can spontaneously polymerize to form micelles. Unlike liposomes, micelles do not have a bilayer, and their structure is that the hydrophobic part Inside, a hydrophobic core is formed, and the hydrophilic part forms a hydrophilic surface outward. The micelle particle size is small (less than 200nm), and the average particle size is about 20-30nm, so it is not only a thermodynamically stable system, but also a kinetically stable system. In addition, the micelle particles are not easy to aggregate and stratify, and the loading capacity is high. When the concentration is high, a higher amount of drug can be carried.

"Phospholipid", the molecular structure of phospholipid is similar to that of fat, the difference is that there are only two fatty acids attached to the glycerol molecule, while the third hydroxyl of phospholipid is combined with phosphoric acid to form a lipid, and there is a nitrogenous base combined with phosphoric acid . This structure of phospholipids makes it an amphiphilic molecule. One end of its phosphoric acid and nitrogenous base is polar, easy to absorb with water, forming the hydrophilic head of phospholipid molecule, and its fatty acid end is non-polar, not absorbing with water, forming the head of phospholipid molecule Hydrophobic tail, the phospholipids involved in the present invention are often used in conjunction with several phospholipids including PEG derivatized phospholipids.

"Therapeutically effective amount" refers to the amount of prostaglandin E1 that produces a therapeutic effect. According to the present invention, the unit dose of prostaglandin E1 is 1-1000 ug, preferably 5-100 ug, and the optimal unit dose is 10 ug, and the dose will be adjusted according to the needs of each special individual.

"Release" refers to the rate and degree of drug release from micellar preparations under specified conditions. It is generally believed that only when the drug remains in the micelles can it delay its metabolism in the blood circulation and continuously accumulate in the lesion. , in order to better exert the drug effect, so the slower the release of the micellar preparation in the simulated body fluid, the more conducive to its drug effect.

"Encapsulation efficiency" refers to the molecular ratio of the drug encapsulated by the micelles to the total amount of drug in the prostaglandin E1 micelle preparation. Only when the drug is encapsulated in micelles can the biological properties of micellar drugs be exerted. Therefore, in theory, the higher the encapsulation rate, the more conducive to the development of the biological properties of micellar drugs.

"Drug-to-lipid ratio" refers to the molar ratio of total phospholipids to drugs (prostaglandin E1) in the prescription.

Drug-to-lipid ratio = total moles of phospholipids/moles of prostaglandin E1

"Phospholipid ratio" refers to the ratio between different phospholipid components in the total phospholipids in the prescription. In the present invention, in addition to containing PEG derivatized phospholipids, the total phospholipids also contain hydrogenated phosphatidylcholine (HSPC). The drug encapsulation efficiency and release rate of the bundle have a significant impact.

The prostaglandin E1 micelle preparation of the present invention adopts PEG derivatized phospholipid derivatized phospholipid as a carrier, and at the same time, in order to control the release of the drug from the micelle preparation, it is used in conjunction with other phospholipid materials, and through certain pharmacy means, the A therapeutic amount of prostaglandin E1 is wrapped in the formed nano-micelle, and a certain amount of antioxidant, osmotic pressure regulator, and pH regulator are added as required.

In the present invention, the PEG derivatized phospholipid is PEG derivatized phospholipid 2000 derivatized distearoylphosphatidylethanolamine.

The prostaglandin E1 micelle preparation of the present invention adopts PEG derivatized phospholipid derivatized phospholipids as a carrier, and simultaneously adds hydrogenated phosphatidylcholine (HSPC) to the prescription, and utilizes the strong hydrophobicity of hydrogenated phosphatidylcholine, which can Modulation of drug encapsulation and release in micelles.

The prostaglandin E1 micelle preparation of the present invention has an encapsulation rate of over 80%, and a better drug encapsulation rate should be over 90%; in simulated body fluids, the release rate within 0.5 hours does not exceed 50%.

The prostaglandin E1 micelle preparation of the present invention, its encapsulation efficiency is relevant with prescription Chinese medicine lipid ratio, phospholipid ratio, along with the raising of phospholipid dosage in prescription, the encapsulation efficiency of medicine improves, in order to guarantee the drug encapsulation of micellar preparation The sealing rate is more than 80%, and the ratio of traditional Chinese medicine to fat in the prescription should be in the range of 80:1-500:1, and the preferred range is 160:1-240:1.

In the prostaglandin E1 micelle preparation of the present invention, the drug release rate of the micelle is related to the lipid ratio of the prescription traditional Chinese medicine and phospholipids. The slower the release rate of the drug, the longer the retention time of the drug in the micelle, which is more conducive to its drug effect. play. Under simulated body fluid conditions, the release rate of the prostaglandin E1 micelles preparation in the present invention does not exceed 50% in 0.5 hours.

Determination of release in simulated body fluids: Take an appropriate amount of prostaglandin E1 micelle preparation for injection, add physiological saline to prepare a 50 μg/mL solution, take 2 mL and place it in a dialysis bag (molecular weight cut-off is 14,000), seal both ends , take Krebs-Henseleit buffer (pH7.4) 40mL as the dissolution medium, place it in a shaker, 37°C, 100 rpm, take samples at the specified time, measure the content according to the HPLC method, and measure the prostaglandin in the dialysis bag The concentration of E1 was used to calculate the release rate of prostaglandin E1 micelles at the specified time points.

The release rate of micellar drugs is related to the ratio of drug to lipid. In order to ensure that the drug release of micellar preparations is slow enough, the molecular ratio of total phospholipids and drugs in the prescription should be in the range of 80:1 to 500:1, preferably 160:1. 1～240:1.

The release rate of micellar drugs is related to the ratio of phospholipids. When the ratio of drug to lipid is kept the same, as the proportion of HSPC in total phospholipids increases, the release rate of drugs decreases. In order to ensure the release rate of drugs from micelles, this In the prostaglandin E1 micelle preparation described in the invention, the molar ratio of HSPC to the total phospholipids is 50%-90%, preferably 70%-90%.

The micelle preparation of prostaglandin E1 involved in the present invention is in a freeze-dried form. After reconstitution by adding a physiologically acceptable solvent before use, drug-loaded micelles are formed. The solvent used can be water for injection, normal saline, glucose injection, etc. liquid etc.

After the micellar preparation of prostaglandin E1 involved in the present invention is redissolved, the pH value of the medicinal solution is closely related to the encapsulation rate, release rate and stability of the drug. There are carboxyl groups in the prostaglandin E1 molecule, and the improvement of pH can increase The salt-forming ratio of the drug (increased hydrophilicity of the drug) leads to a decrease in the encapsulation rate of the drug. Therefore, in the case of the same other factors in the prescription, reducing the pH value of the drug is conducive to the improvement of the encapsulation rate and the control of the drug. release speed. In addition, according to the stability study results, if the pH value of the medicine is too low, it is not conducive to the storage of the preparation. According to the needs, the drug delivery system of the present invention can add a pH regulator (various buffer systems such as citric acid-sodium citrate, acetic acid- Sodium acetate, phosphate, etc.), adjust the pH range to 4.5-5.5, and the optimum pH range is 4.8-5.2.

After the micelle preparation of prostaglandin E1 involved in the present invention is reconstituted, the concentration of prostaglandin E1 is 0.5 μg/ml to 10 μg/ml, the particle size range of the particles is below 200 nm, and the average volume particle size is less than 100 nm. It is used for intravenous Injection route of administration.

Specifically, the micellar drug delivery system described in this invention is made by the following preparation method: dissolve prostaglandin E1, phospholipids, and fat-soluble additives in an organic solvent, place them in an eggplant-shaped bottle, and use a rotary evaporator to . Evaporate the organic solvent to form a thin and uniform phospholipid film on the surface of the eggplant-shaped bottle, dissolve the water-soluble additive (water-soluble antioxidant, osmotic pressure regulator, pH value regulator) in water, and add the aqueous solution to the eggplant-shaped bottle. In the bottle, oscillate for hydration, ultrasonically disperse, pass through a membrane for sizing, and finally pass through a 0.22um microporous membrane to filter and sterilize, and freeze-dry according to needs.

Description of drawings

Accompanying drawing 1 is Wistar rat iv administration prostaglandin E1 micelle (test product T) and the mean whole blood drug concentration-time curve (logC-t) comparison of different time behind prostaglandin E1 for injection (reference product R) .

Accompanying drawing 2 is the content comparison of medicine in each tissue after 5 minutes after Wistard rat iv administration test article and reference article.

Accompanying drawing 3 is Wistardd rat iv administration test product and reference product 20 minutes after the drug content comparison in each tissue.

The following examples are mainly used to further illustrate the present invention, but not to limit the scope of the present invention.

Detailed ways

Embodiment 1: the determination of prescription and its release rate of different drug lipid ratio and phospholipid ratio prostaglandin E1 micelle preparation

See Table 1 for prescription

        Table 1 Prescription Form of Prostaglandin E1 Micellar Preparations with Different Drug-to-Lipid Ratio and Phospholipid Ratio

drug fat ratio 1:160 1:240 1:240 1:240 PEG 2000-DSPE: HSPC 1:4 1:2 1:4 1:3 Encapsulation rate% Greater than 90% Greater than 90% Greater than 90% Greater than 90% 0.5 hour release % 20.86 7.15 20.73 23.49 pH value after reconstitution with water 4.97 4.92 4.85 4.97

Preparation process: Prostaglandin E1, polyethylene glycol 2000 derivatized distearoylphosphatidylethanolamine (PEG 2000-DSPE), hydrogenated phospholipid (HSPC) were weighed according to the above prescription ratio, dissolved in an appropriate amount of chloroform, and placed in eggplant-shaped In the bottle, use a rotary evaporator to evaporate the organic solvent to form a thin and uniform phospholipid film on the surface of the eggplant-shaped bottle; dissolve water-soluble additives (VC, VE, EDTA) in water, and adjust the pH value with 5.0% sodium hydroxide To the specified range, the aqueous solution is added into an eggplant-shaped bottle, hydrated by shaking at 60°C, dispersed by ultrasonic, (pay attention to nitrogen protection during the preparation process), and freeze-dried in separate packages.

After freeze-drying, the appearance of the sample is white loose lumps. After reconstitution with water, drug-loaded micelles are formed, and the pH value of the drug solution is 4.8-5.2. The encapsulation efficiency is greater than 90%, and the average particle size is about 50nm. Hourly release is less than 50%, available for intravenous injection.

The research of the release degree of embodiment 2 prostaglandin E1 micelle preparation, encapsulation efficiency and drug-lipid ratio, phospholipid ratio

PEG-derivatized phospholipid 2000-DSPE and HSPC are the main materials for forming micelles, and their ratio determines the drug-loading properties and drug-release properties of the micelles. The drug-loaded micelles should have a high encapsulation efficiency and slow drug release as much as possible. Speed can ensure its excellent drug pharmacokinetic properties. Drug-to-lipid ratio and phospholipid ratio have a decisive impact on the encapsulation efficiency and release rate of drug-loaded micelles.

        Table 2 Comparative study on encapsulation efficiency of prostaglandin E1 micelles with different drug-lipid ratios

prescription number 1 2 3 4 5 drug fat ratio 1:10 1:30 1:40 1:80 1:160 PEG 2000-DSPE: HSPC 2:1 2:1 2:1 1:4 1:4 Encapsulation rate% 56.78 69.05 85.85 Greater than 90% Greater than 90%

The results show that the proportion of total phospholipids in the prescription increases, and the encapsulation efficiency of the medicine increases. When the lipid ratio of the Chinese medicine in the prescription exceeds 80:1, and the proportion of HSPC in the total phospholipids exceeds 50%, the encapsulation efficiency of the medicine is greater than 90%.

Table 3 Comparison of the release rate of prostaglandin E1 micelles with different drug-to-lipid ratios and phospholipid ratios

Comparison of Ratio of Prostaglandin E1 Micellar Release

The results show that the drug release degree of micelles is related to the lipid ratio and phospholipid ratio of the prescription. The higher the proportion of total phospholipids in the prescription, the slower the drug release. When the lipid ratio exceeds 80:1 and the proportion of HSPC in total phospholipids exceeds 50%, the release degree of micelles is less than 50% in 0.5 hours.

Preparation of Example 3 Prostaglandin E1 Micellar Preparation and Its Stability Research

See Table 3 for the prescription:

         Table 3 Prostaglandin E1 Micellar Preparation Prescription

Concentration (uM) Weight (ug) Prostaglandin E1 112.8 200 PEG2000-DSPE 6000 84000 HSPC 18000 69000 VE 423 1000 VC 11354 10000 EDTA-Ca 538 1000 citric acid 10417 10000 5.0%NaOH Adjust the pH to 4.5±0.2 water 5.0ml

Preparation process: Weigh prostaglandin E1, polyethylene glycol 2000 derivatized distearoylphosphatidylethanolamine (PEG 2000-DSPE) and hydrogenated phospholipid (HSPC) according to the above prescription ratio, dissolve them in an appropriate amount of chloroform, and place them in 100ml eggplant In the eggplant-shaped bottle, use a rotary evaporator to evaporate the organic solvent to form a thin and uniform phospholipid film on the surface of the eggplant-shaped bottle; dissolve water-soluble additives (VC, VE, EDTA) in water, and adjust the pH with 5.0% sodium hydroxide value to the specified range, the aqueous solution was added to an eggplant-shaped bottle, hydrated by shaking at 60°C, ultrasonically dispersed, (pay attention to nitrogen protection during the preparation process), and freeze-dried in separate packages.

After freeze-drying, the appearance of the sample was a white loose block. After reconstitution with water, the measured average particle size was 52.4nm.

The freeze-dried samples were placed at 2-8°C, and samples were taken in October, March, June, September, and December to observe the changes in various indicators of the samples. The results are shown in Table 4.

Take this product, add water to make a solution containing 2 μg per 1ml, and measure the pH value according to the law (Appendix VI H of Part Two of the Chinese Pharmacopoeia in 2005).

Take this product for particle size, add physiological saline to make a solution containing 11 μg of prostaglandin E per 1 ml, let it stand for 10 minutes, and use it as the test solution, and use the dynamic laser scattering particle size measurement method to measure the average particle size and particle size range of micelles .

Table 4 Results of Stability Study

Embodiment 4 prostaglandin E1 micelle pharmacokinetic research in vivo

Single-dose administration of prostaglandin E1 micelles for injection (test product T) and common domestic preparations of prostaglandin E1 for injection (reference product R) by liquid chromatography-mass spectrometry (LC/MS) Pharmacokinetics study, to investigate the similarities and differences in drug metabolism and tissue distribution between the test product and the reference product. Wistar rats were injected intravenously with the test product and the reference product respectively, blood was collected from the orbit at different times after administration, the blood drug concentrations of the test product and the reference product were measured, and the pharmacokinetic parameters were calculated. The main pharmacokinetic parameters of Wistar rats iv administration prostaglandin E1 test article and reference article are shown in Table 5. The AUC of the two preparations is different, the half-life of the elimination phase is basically the same, the bioavailability of the test product is higher than that of the reference product, and the drug-time curve is shown in Figure 1.

In addition, Wistar rats were killed 5min and 20min after iv administration, and the tissue distribution of each major organ was measured, and the similarities and differences between the test product and the reference product were compared. The distribution of the test product is basically equivalent, and the tissue content of the test product in the heart, liver, spleen, muscle and fat is higher than that of the reference product 20min after the administration, and the tissue content diagram is shown in accompanying drawing 2 and accompanying drawing 3.

       Table 5 Comparison of Pharmacokinetic Parameters

Pharmacokinetic parameters Units Test product Reference product

A ng/ml 2717 2216

A l/min 3.34 3.59

B ng/ml 7.92 2.67

B l/min 0.15 0.14

V _(d) L/kg 0.07 0.09

T _1/2α min 0.21 0.19

T _1/2β min 4.39 4.76

K ₂₁ l/min 0.17 0.15

K ₁₀ l/min 3.16 3.49

K ₁₂ l/min 0.18 0.10

AUC (ng/ml)*min 863.9 635.2

CL _(s) L/kg/min 0.23 0.31

Embodiment 5 prostaglandin E1 micelle is to the influence of animal microcirculation blood vessel

Objective: To observe the pharmacological effect of prostaglandin E1 micelles for injection on microcirculation under normal physiological conditions, and compare with prostaglandin E1 lipid microsphere injection and prostaglandin E1 for injection.

METHODS: The golden hamster cheek pouch arteriole third-fourth order observation model was prepared, and different doses (0.5 μg, 1.0 μg, 2.0 μg/animal·0.4ml) of prostaglandin E1 micelles for injection and prostaglandin E1 for injection were injected intravenously , Monitor and analyze changes in blood pressure, tube diameter, flow rate and spontaneous movement before injection and at 0, 5, 10, 15, 20, 25, and 30 minutes after injection.

Results: Prostaglandin E1 micelles for injection not only have strong vasodilation and antihypertensive effects, but also activate self-discipline movement, maintain caliber and micro-blood perfusion, and at the same dose, the effect is stronger than that of prostaglandin E1 lipid Microsphere injection and prostaglandin E1 for injection. The reason may be related to the dosage form and action characteristics of micelles. At the same time, this effect has a certain dose dependence, but it can play a better role at a low dose.

Claims (7)

1. used for intravenous injection PGE1 micellar preparation, it contains PGE1, PEG derivatization phospholipid, hydrogenated phospholipid and the pharmaceutically acceptable adjuvant for the treatment of effective dose, wherein said PEG derivatization phospholipid is a Macrogol 2000 derivatization DSPE, the total phospholipids of PEG derivatization phospholipid and hydrogenated phospholipid sum and the mol ratio of PGE1 are in 80: 1～500: 1 scope, and the molar ratio scope that hydrogenated phospholipid accounts for total phospholipids is 50%～90%.

2. micellar preparation according to claim 1, wherein the mol ratio of total phospholipids and PGE1 is in 160: 1～240: 1 scope.

3. micellar preparation according to claim 1, wherein said pharmaceutically acceptable adjuvant comprises antioxidant, pH value regulator and osmotic pressure regulator.

4. micellar preparation according to claim 1, wherein said micellar preparation is a lyophilized form, faces with the physiologically acceptable solvent of preceding adding to redissolve.

5. micellar preparation according to claim 1, after wherein the micellar preparation described in the claim 1 being redissolved, formation contains the solution of the 0.5 μ g/ml～PGE1 of 10 μ g/ml and the total phospholipids of 0.01mM～50mM, is used for the intravenous administration approach.

6. micellar preparation according to claim 1, after wherein micellar preparation redissolved, the pH value that forms polypeptide drug-loaded micelle solution was in the 4.5-5.5 scope.

7. micellar preparation according to claim 1, after wherein micellar preparation redissolved, the pH value that forms polypeptide drug-loaded micelle solution was in the 4.8-5.2 scope.

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