CN103690512B - A kind of deoxypodophyllotoxin polymer micelle lyophilized formulations - Google Patents

Abstract

The invention provides a lyophilized preparation of deoxypodophyllotoxin polymer micelles, wherein the polymer micelles are composed of amphiphilic block polymers, preferably polyethylene glycol monomethyl ether-poly(D, L) propane The lactide block copolymer, the preparation method of the preparation comprises dissolving deoxypodophyllotoxin and the block polymer in an organic solvent, driving the solvent to form a gel, and hydrating the gel to obtain micelles , and further made into a freeze-dried preparation; the freeze-dried preparation can be used to treat tumors, and the encapsulation rate of drug loading is above 90%. Stable for 4 to 24 hours.

Description

A kind of deoxypodophyllotoxin polymer micelles freeze-dried preparation

technical field

The invention relates to a freeze-dried pharmaceutical composition and a preparation method thereof, in particular to a composition loaded with deoxypodophyllotoxin micelles, a preparation method and a freeze-dried preparation thereof.

Background technique

Deoxypodophyllotoxin is a compound extracted and purified from lignin plants, and its structural formula is as follows:

Experiments in the 1990s have proved that deoxypodophyllotoxin has an in vitro inhibitory effect on P-388 leukemia, human lung cancer A-549 and colon cancer HT-29 cell lines. Since deoxypodophyllotoxin is insoluble in water, it cannot be used to prepare intravenous injection preparations, which limits its application in the preparation industry and clinical practice. In order to improve its water solubility, various methods have been adopted in the prior art.

Chinese patent CN101693112A discloses an inclusion compound of deoxypodophyllotoxin and β-cyclodextrin; Chinese patent CN102380104A discloses an inclusion compound of deoxypodophyllotoxin and hydroxypropyl-β-cyclodextrin improved preparation method. The above patents solve the problem of water solubility of deoxypodophyllotoxin, but the nephrotoxicity of β-cyclodextrin compounds and the hidden danger of causing pancreatic tumors have been confirmed by animal experiments. It is meaningful to look for excellent solubilizing materials with less toxic and side effects.

Polymeric micelles are composed of multiple amphiphilic copolymers, which spontaneously form micelles in water, with their hydrophobic ends facing inwards and hydrophilic ends facing outwards, showing a typical core-shell structure. Its hydrophobic core can solubilize hydrophobic drugs (such as paclitaxel, doxorubicin, etc.). Block polymers have greater softness and, because the polymer composition is easy to determine, form polymer micelles more reproducibly than random copolymer micelles. Biodegradable polymers such as block copolymers of lactide and polyethylene glycol monomethyl ether have excellent biocompatibility, drug permeability and biodegradability, and have attracted the attention of researchers. One of the most studied amphiphilic block copolymers for drug release systems is diblock polymers. The hydrophilic block of the amphiphilic block copolymer is mainly polyethylene glycol monomethyl ether, which has good hydrophilicity, low interfacial free energy in contact with water, and has an extended structure in water, so it has a high Chain mobility can provide a certain spatial stabilization effect. The high hydrophilicity of the surface of the drug carrier can reduce the leakage of the drug, and can also reduce the phagocytosis of the drug by the vascular endothelial network system, which not only improves the safety of the drug, but also prolongs the blood circulation time of the drug. The hydrophobic block usually uses polyester, polyoxypropylene, polystyrene or polyamino acid, which form amphiphilic copolymers with polyethylene glycol monomethyl ether, and can form various micelles.

The purpose of polymer micelles is to be used for injection and should be specially prepared to meet the requirements for injection. The inventors have found that polyethylene glycol monomethyl ether-poly (D, L) lactide block copolymer (mPEG-PDLLA) is suitable for injection, obtained after special preparation, and discloses paclitaxel and polysaccharides based on mPEG-PDLLA. For the patent of cetaxel, see ZL03105348.3, ZL200610145383.7, 201010001047.1, 201010114289.1 for details.

In order to improve the druggability of deoxypodophyllotoxin, we tried to study other ways to solubilize deoxypodophyllotoxin, however, the solubility of deoxypodophyllotoxin could not be solved by adding co-solvents such as ethanol, isopropanol and glycerol; further research Attempts to solubilize via micelles have been attempted with other block polymers such as poloxamers or surfactants such as Tween 80, respectively. but precipitated immediately.

In further research, we were surprised to find that deoxypodophyllotoxin has greater solubility and better stability in the polymer micelles of mPEG-PDLLA, while other drug-loading systems cannot achieve the same purpose.

Contents of the invention

In order to improve the solubility of deoxypodophyllotoxin in water, facilitate the preparation of intravenous administration, and avoid the side effects caused by the use of cyclodextrin, we use mPEG-PDLLA as a drug carrier to prepare deoxypodophyllotoxin Compared with the prior art, the micelle has the advantages of high encapsulation efficiency, good stability, small amount of carrier and easy preparation of freeze-dried preparations.

The invention provides a drug composition of deoxypodophyllotoxin polymer micelles freeze-dried preparation, the composition contains the drug active ingredient deoxypodophyllotoxin and the drug carrier material polyethylene glycol monomethyl ether-polyester block Copolymer; wherein the mass ratio of deoxypodophyllotoxin to polyethylene glycol monomethyl ether-polyester block copolymer is 1:3 to 1:10.

Wherein the polyethylene glycol monomethyl ether-polyester block copolymer is an amphiphilic copolymer, and its hydrophobic part is polyester, selected from: polyglycolide, polylactide, glycolide-acrylic acid A lactide copolymer whose hydrophilic portion is polyethylene glycol monomethyl ether. A preferred copolymer is: polyethylene glycol monomethyl ether-poly(D, L) lactide block copolymer.

The molecular weight of polyethylene glycol monomethyl ether in the polyethylene glycol monomethyl ether-poly(D, L) lactide block copolymer described in the present invention is 2000, and polyethylene glycol monomethyl ether and poly(D , L) The mass ratio of lactide is 3:7-9:1, preferably 3:7-6:4.

Polyethylene glycol monomethyl ether-poly (D, L) lactide block copolymer described in the present invention is also referred to as: mPEG-PDLLA, the block copolymer can be according to document (Zhang, X., Jackson, Prepared by the method reported in J.K., Burt, H.M., 1996. Development of amphiphilic diblockcopolymersasmicellacarriersoftaxol. Int. J. Pharm. 132, 195-206.).

According to needs, pharmaceutically acceptable pharmaceutical adjuvants can also be added to the polymer micelle composition of the present invention, such as co-solvents, pH regulators, lyoprotectants or lyophilized proppants with a scaffolding effect.

For this reason, the preferred formula of the present invention consists of the following:

Deoxypodophyllotoxin 1-2 parts by weight

mPEG-PDLLA20004/63-7 parts by weight

5-10 parts by weight of pharmaceutical excipients.

The present invention also provides the preparation method of the polymer micelle composition of the present invention, and the steps are as follows:

(1) dissolving mPEG-PDLLA and deoxypodophyllotoxin in an organic solvent;

(2) Concentrating and driving the organic solvent into a gel;

(3) adding water or an aqueous solution of pharmaceutical excipients to the concentrated gel, heating to a certain temperature for hydration if necessary, to obtain a micellar solution loaded with deoxypodophyllotoxin;

(4) The above micellar solution is freeze-dried to obtain freeze-dried powder.

Wherein, in step (1), the organic solvent used is selected from acetonitrile, methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, and chloroform. The mass ratio of polyethylene glycol monomethyl ether to poly(D, L) lactide in the mPEG-PDLLA used is preferably 3:7-6:4, and the molecular weight of polyethylene glycol monomethyl ether is preferably 2000. The mass ratio of deoxypodophyllotoxin and mPEG-PDLLA used is 1:3 to 1:10. The dosage of the organic solvent is 1-25ml of organic solvent per 150mg of deoxypodophyllotoxin.

In step (2), the concentrated organic solvent can be concentrated under normal pressure or under reduced pressure. During concentration, the temperature ranges from room temperature to the boiling point of the organic solvent used.

In step (3), the heating hydration temperature is 20-60° C., and the hydration time is 5-60 minutes. Micellar hydration can add water to the gel obtained in the previous step, shake or stir at an appropriate temperature to obtain hydrated micelles; a rotary evaporator can also be used for hydration.

Preferably, the organic solvent used in step (1) is methanol.

In step (2), the method for concentrating the organic solvent is carried out by concentrating under reduced pressure, which can be concentrated using a rotary evaporator or similar technical means.

In step (3), the heating hydration temperature is 40°C. Pharmaceutical excipients include excipients with freeze-drying protection and scaffolding effects, such as mannitol, trehalose, sorbitol, glucose, sucrose, lactose, polyethylene glycol 2000, polyethylene glycol 4000, glycine, dextran and mPEG- PDLLA, preferably mPEG-PDLLA. For every 150 mg of deoxypodophyllotoxin-made micelles, 0.75 g to 3 g of excipients can be used. Preferably 1.5 g.

The preferred preparation method of the present invention, the steps are as follows: dissolve deoxypodophyllotoxin and mPEG-PDLLA20004/6 in methanol, evaporate until the methanol is exhausted, add the residue to the aqueous solution of mPEG-PDLLA20004/6 for hydration, filter the After filtering, the filtrate was subpackaged and freeze-dried to obtain a white loose lumpy powder.

After screening, the present invention finds that the micelles prepared by combining deoxypodophyllotoxin and mPEG-PDLLA have good water solubility, stability and resolubility, and can be prepared into freeze-dried pharmaceutical preparations for injection, especially using mPEG-PDLLA That is, as a micelle copolymer and as a freeze-dried proppant to aid dissolution, the quality of the drug is greatly improved, and the production process is simplified.

Description of drawings

Figure 1 Atomic force microscope image of deoxypodophyllotoxin polymer micelles

detailed description

The present invention will be further described through specific examples below, but it should be pointed out that the following examples cannot constitute any limitation to the present invention.

The mPEG-PDLLA described in the following example is recorded as mPEG-PDLLAXY/Z according to the molecular weight of mPEG and the mass ratio of mPEG:PDLLA, where X represents the molecular weight of mPEG, and Y/Z represents the mass ratio of mPEG and PDLLA, such as mPEG-PDLLA20004 /6 means that the molecular weight of mPEG is 2000, and the mass ratio of mPEG and PDLLA is 4/6.

Example 1 Preparation and compounding of deoxypodophyllotoxin solution with ethanol as cosolvent

Put 150 mg of deoxypodophyllotoxin and 10 mL of ethanol in an eggplant-shaped bottle, and heat to dissolve completely. When the above solution was added to 500 mL of 0.9% sodium chloride solution, crystallization could be seen immediately.

Example 2 Preparation and redissolution of deoxypodophyllotoxin polymer micelles with polysorbate 80 as solubilizer

Put 150 mg of deoxypodophyllotoxin and 801500 mg of polysorbate in an eggplant-shaped bottle, add 20 ml of ethanol to dissolve completely, and remove the ethanol under reduced pressure. An oily product was obtained.

Take 1000 mg of this product, add it to 500 mL of 0.9% sodium chloride solution, shake it, and it can be seen that a large amount of crystals are precipitated and turbid.

Example 3 Preparation and reconstitution of deoxypodophyllotoxin polymer micelles using poloxamer 188 as solubilizer

Put 150 mg of deoxypodophyllotoxin and 1,884,000 mg of poloxamer in an eggplant-shaped bottle, add 20 ml of ethanol to dissolve completely, and remove the ethanol under reduced pressure. The product was obtained as a white block.

Take 2000 mg of this product, add it to 500 mL of 0.9% sodium chloride solution, shake it, and it can be seen that a large number of crystals are precipitated and turbid.

All the above are comparative examples, which cannot reach the purpose of the present invention.

The present invention adopts mPEG-PDLLA as carrier material, specific examples are as follows:

Example 4 Preparation of deoxypodophyllotoxin polymer micelles for injection without protective agent

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20004/6450mg in an eggplant-shaped bottle, add 12ml of methanol to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until a transparent gel is attached to the bottom of the bottle. Add 25ml of water for injection, heat in a water bath at 50°C, and rotate the film to hydrate for 20 minutes to obtain opalescent micelles. Filter with a 0.22 μm filter membrane, and divide the filtrate into vials of 5 ml per bottle, and freeze-dry to obtain a white loose block powder.

Example 5 Preparation and physical properties of deoxypodophyllotoxin polymer micelles for injection using mPEG-PDLLA20004/6 as protective agent

1. Preparation of deoxypodophyllotoxin micelles

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20004/6450mg in an eggplant-shaped bottle, add 12ml of methanol to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until the methanol is completely driven out, and a transparent film is attached to the bottom of the bottle. Add 25ml of 30mg/mlm PEG-PDLLA20004/6 aqueous solution, heat in a water bath at 40°C, and spin the film to hydrate for 20 minutes to obtain opalescent micelles. Filter with a 0.22 μm filter membrane, and divide the filtrate into vials of 5 ml per bottle, and freeze-dry to obtain a white loose block powder.

2. Morphological observation of drug-loaded micelles

The lyophilized preparation was redissolved in water to a concentration of 1 mg/ml of deoxypodophyllotoxin, and diluted to 10 μg/ml. Take 1 drop of the solution and put it on a cover glass, and observe it under an atomic force microscope. The results are shown in Figure 1. It can be seen that the micelles are spherical or quasi-spherical. However, the deoxypodophyllotoxin polymer micelles in Example 4 without a protective agent were difficult to reconstitute, required heating, and were unstable after reconstitution.

3. Determination of content and encapsulation efficiency

The lyophilized preparation was redissolved in physiological saline until the concentration of deoxypodophyllotoxin was 6 mg/ml. Take 0.1 ml of the reconstituted solution and place it in a 10 ml measuring bottle, make it to volume with methanol, and measure the absorbance A ₁ at a wavelength of 294 nm with an ultraviolet-visible spectrophotometer. Polymers have no absorption at this wavelength. Accurately weigh an appropriate amount of deoxypodophyllotoxin reference substance, dissolve it with methanol and quantitatively dilute it to a control solution containing about 60 μg/ml of deoxypodophyllotoxin, and measure the absorbance A ₀ at 294 nm. The content of deoxypodophyllotoxin in the complex solution was obtained from the concentrations of A ₁ , A ₀ and the reference solution.

Another 2ml of the complex solution was taken in a centrifuge tube, and ultracentrifuged at 10000rpm for 5 minutes, and 0.1ml of the supernatant was placed in a 10ml measuring bottle, and the volume was fixed with methanol, and the absorbance A2 was measured at a wavelength of 294nm with a UV _- visible spectrophotometer, including The calculation formula of encapsulation rate is as follows: encapsulation rate%=A ₂ /A ₁ ×100%.

The measured deoxypodophyllotoxin content was 5.86mg/ml, and the encapsulation efficiency was 97.27%.

4. Investigation of reconstitution time and particle size stability

Take 1 bottle of lyophilized preparation, add 5ml of normal saline to redissolve, and measure the time required for lyophilized powder to redissolve into light blue clear micellar solution is about 1min. The particle size was measured by dynamic light scattering method, the scattering angle was 90°, and the data was collected by spherical model. The reconstituted micellar solution was placed in a 25°C water bath, and the particle size change within 24 hours was monitored to investigate the kinetic stability of the micelles. The measured particle size is 49.8nm, and it can be stable for about 24h.

Example 6 Preparation and physical properties of deoxypodophyllotoxin polymer micelles for injection using lactose as protective agent

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20005/5750mg in an eggplant-shaped bottle, add 20ml of methanol to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until the methanol is completely driven out, and it is attached to the bottom of the bottle in the form of a transparent film. Add 25ml of 30mg/ml lactose aqueous solution, heat in a water bath at 40°C, rotate the film for 20 minutes to hydrate, and obtain opalescent micelles. Filter with a 0.22 μm filter membrane, pack in vials with 5ml per bottle, and freeze-dry to obtain a white loose block powder. Add 5ml of normal saline to redissolve, and form opalescent micelles. The content of deoxypodophyllotoxin is determined to be 5.32mg/ml by UV spectrophotometry, and the reconstitution time is about 3min. Can be stable for about 16h.

Example 7 Preparation and physical properties of deoxypodophyllotoxin polymer micelles for injection with polyethylene glycol 2000 as protective agent

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20006/41000mg in an eggplant-shaped bottle, add 15ml of ethanol to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until the ethanol is completely driven out, and it is attached to the bottom of the bottle in the form of a transparent film. Add 25ml of a 30mg/ml polyethylene glycol 2000 aqueous solution, heat in a water bath at 40°C, and spin the film to hydrate for 15 minutes to obtain opalescent micelles. Filter with a 0.22 μm filter membrane, pack in vials with 5ml per bottle, and freeze-dry to obtain a white loose block powder. Add 5ml of normal saline to redissolve, and form opalescent micelles. The content of deoxypodophyllotoxin is determined to be 5.57mg/ml by UV spectrophotometry, and the redissolution time is about 4min. Can be stable for about 8h.

Example 8 Preparation and physical properties of deoxypodophyllotoxin polymer micelles for injection with dextran D40 as protective agent

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20006/41350mg in an eggplant-shaped bottle, add 20ml of isopropanol to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until the isopropanol is completely removed, and it is attached to the bottle in the form of a transparent film end. Add 25ml of 50mg/ml dextran D40 aqueous solution, heat in a water bath at 40°C, and hydrate the film by rotating for 35 minutes to obtain opalescent micelles. Filter with a 0.22 μm filter membrane, pack in vials with 5ml per bottle, and freeze-dry to obtain a white loose block powder. Add 5ml of normal saline to redissolve, and form opalescent micelles. The content of deoxypodophyllotoxin is determined to be 5.58mg/ml by UV spectrophotometry, and the redissolution time is about 3min. Can be stable for about 4h.

Example 9 Preparation and physical properties of deoxypodophyllotoxin polymer micelles for injection using poloxamer 188 as protective agent

Put 150mg of deoxypodophyllotoxin and mPEG-PDLLA20005/5450mg in an eggplant-shaped bottle, add 12ml of acetonitrile to dissolve completely, heat in a water bath at 40°C and evaporate under reduced pressure until the acetonitrile is completely driven out, and it is attached to the bottom of the bottle in the form of a transparent film. Add 25ml of 30mg/ml poloxamer 188 aqueous solution, heat in a water bath at 40°C, spin the film for 30 minutes to hydrate, and obtain opalescent micelles. Filter with a 0.22 μm filter membrane, pack in vials with 5ml per bottle, and freeze-dry to obtain a white loose block powder. Add 5ml of normal saline and vibrate and redissolve, forming opalescent micelles. The content of deoxypodophyllotoxin was determined to be 5.86mg/ml by UV spectrophotometry. The reconstitution time is about 2min. The encapsulation efficiency is 97.27%, and the particle size is 49.8nm. , can be stable for about 8h.

Claims (5)

1. A polymer micelle composition of deoxypodophyllotoxin, characterized in that, the formula is as follows: Deoxypodophyllotoxin 1-2 parts by weight mPEG-PDLLA20004/63-7 parts by weight as micellar copolymer mPEG-PDLLA20004/65-10 parts by weight as lyophilized proppant Wherein, in the mPEG-PDLLA20004/6, the mass ratio of polyethylene glycol monomethyl ether to poly(D, L) lactide is 4:6, and the molecular weight of polyethylene glycol monomethyl ether is 2000. 2. the preparation method of polymer micelle composition described in claim 1 is characterized in that, step is as follows: (1) mPEG-PDLLA20004/6 and deoxypodophyllotoxin are dissolved in organic solvent; (2) organic The solvent is concentrated and driven out to a gel state; (3) adding an aqueous solution of mPEG-PDLLA20004/6 as a lyophilized proppant to the concentrated gel, heating to a certain temperature for hydration, and obtaining deoxypodophyllotoxin-loaded (4) lyophilization of the polymer micelle solution to obtain a lyophilized powder. 3. the described preparation method of claim 2 is characterized in that, the organic solvent described in step (1) is selected from: acetonitrile, methyl alcohol, ethanol, Virahol, THF, dioxane, methylene dichloride; Step (3) Heating to a certain temperature for hydration, the hydration temperature is 20-60°C. 4. The preparation method according to claim 2, characterized in that the steps are as follows: dissolving deoxypodophyllotoxin and mPEG-PDLLA20004/6 in methanol, evaporating until the methanol is exhausted, and adding the residue to the proppant as freeze-dried proppant Hydrate mPEG-PDLLA20004/6 in an aqueous solution, filter with a filter membrane, divide the filtrate, and freeze-dry to obtain a white loose block powder. 5. The preparation method according to claim 4, characterized in that, the steps are as follows: put 150 mg of deoxypodophyllotoxin and mPEG-PDLLA20004/6450 mg in an eggplant-shaped bottle, add 12 ml of methanol to dissolve completely, and heat in a water bath at 40 ° C to reduce Evaporate under high pressure until the methanol is completely driven out, and it is attached to the bottom of the bottle in the form of a transparent film. Add 25ml of 30mg/ml mPEG-PDLLA20004/6 aqueous solution as a lyophilized proppant, heat in a water bath at 40°C, and rotate the film for 20 minutes to obtain milk The optical micelles were filtered with a 0.22 μm filter membrane, and the filtrate was divided into 5 ml vials and freeze-dried to obtain a white loose block powder.