CN1682704A

CN1682704A - Levodopa nano preparation and its preparing method

Info

Publication number: CN1682704A
Application number: CNA2004100305595A
Authority: CN
Inventors: 周文斌; 刘晓英
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2004-04-14
Filing date: 2004-04-14
Publication date: 2005-10-19
Anticipated expiration: 2024-04-14
Also published as: CN100333721C

Abstract

The present invention relates to medical preparation, and a kind of nanometer preparation with levodopa as effective component and polymer as carrier for treating Parkinson's disease and Parkinson's syndrome. The nanometer levodopa preparation is prepared through synthesizing hydrophilic block copolymer PEO-b-PAA in one step, and subsequent loading nanometer levodopa particle. Animal test shows that compared with available levodopa preparation, the preparation of the present invention has obviously lowered peak dopamine concentration in blood and 500-600 times higher peak dopamine concentration in brain, with the lasting time being as long as 28 days.

Description

Levodopa nano preparation and preparation method thereof

The technical field is as follows:

the invention belongs to a medical preparation, and relates to a nano preparation for treating Parkinson's disease and Parkinson's syndrome by using levodopa as an active ingredient and using a high-molecular polymer as a carrier.

Background art:

parkinson's Disease (PD) is a chronic nervous system degenerative disease characterized by tremor, myotonia, hypokinesia, and abnormal posture, which is a common condition in the elderly. The pathogenesis of the traditional Chinese medicine is brain substantia nigra cell degeneration, and DA is seriously lost due to the fact that Dopamine (DA) cannot be synthesized in compact plaques; levodopa (LD) is still the gold standard therapeutic drug for Parkinson's disease so far, but has more obvious side effects, so that the application of levodopa in clinic is limited. Dopamine is produced by decarboxylation in the periphery due to most of LD. Dopamine does not easily penetrate the blood brain barrier, so that LD entering the central nervous system is not 1 percent of the dosage. Clinical application of MEDOPA can reduce adverse reaction by using peripheral decarboxylase inhibitor (carbidopa). However, with the slow progress of the Parkinson disease, the dosage of the MEDOPA is gradually increased, and the incidence rate of adverse reactions is increased. If the decarboxylation of LD at the periphery can be avoided, the blood brain barrier permeability of LD can be improved, and the long-acting slow-release preparation of LD can be prepared, the long-term application of large dose of LD can be effectively avoided, and the adverse reaction caused by LD can be reduced or delayed.

The medicine carrying nanometer particle is the product of combining nanometer technology and modern medicine and pharmacology and is one new medicine preparation for controlled and delayed release. The nano-particles comprise nano-particles (nanoparticles) and nano-capsules (nanocap sules), the nano-particles are solid colloidal particles with the diameter of 10-500 nm, and active parts (medicines, bioactive materials and the like) are positioned in the particles through dissolution and encapsulation effects or positioned on the surfaces of the particles through adsorption and adhesion effects.

Since Birrenbach reports drug-loaded nanoparticles for the first time in 1976, the nanoparticles have been widely used in the medical field as drug sustained-release and targeting carriers, but the use of sustained-release nanoparticles for treating Parkinson's disease has not been reported at home and abroad.

The materials used for preparing drug-loaded nanoparticles at present are mainly divided into two categories, namely natural polymers and synthetic polymers. The liposome is used as a drug carrier in clinic, but the liposome has poor stability, difficult control of drug release and low entrapment rate, and limits the application range of the liposome.

Recently, the amphiphilic block copolymer prepared nano micelle is greatly regarded as a delivery and release carrier system of fat-soluble drugs and genes. The shell-core nano micelle is tried to be used for entrapping adriamycin, and the entrapment rate is more than 60 percent for the first time. But the synthesis and preparation of the polymer are limited due to the hydrophobicity of the hydrophobic section of the polymer, the requirements on a solvent and a drug structure are very high, the large-scale production is not facilitated, and the industrialization of nano drug loading is also not facilitated.

The invention content is as follows:

the invention aims to solve the problems of poor blood brain barrier permeability, large side effect and short half-life period of the existing LD treatment for the Parkinson disease by utilizing the sustained-release and passive targeting technology of the L-dopa (LD) loaded nanoparticles for the first time. The levodopa nanoparticles have proper particle size and particle shape, more than 30% of drug-loading amount and more than 80% of encapsulation efficiency by adopting a proper preparation and purification method, the circulation time in a human body is prolonged, and the levodopa nanoparticles are nontoxic.

The technical scheme of the invention is as follows:

firstly, adopting a one-step method (ARPT method) to synthesize an amphiphilic block copolymer PEO-b-PAA by using PEO-Br as a macroinitiator according to the following route:

CH₃(OCH₂CH₂)_nOCOCH(Br)CH₃

a) preparation of macroinitiator PEO-Br

Weighing 10g of PEO (Mn 5000), vacuum drying for 5 hours, dissolving in 150ml of toluene, adding 0.42ml of triethylamine (0.003mol), and performing ice-water bath at 0 ℃; CH is dripped within half an hour₃CH (Br) COBr0.003ml, and stirred at room temperature for reaction overnight; filtering, precipitating, extracting, drying and reacting overnight to obtain white solid powder macroinitiator PEO-Br.

b) Synthesis of amphiphilic block polymer PEO-b-PtBA

2.064g of macroinitiator and CuBr58.9mg are taken to be put into a reactor, nitrogen is filled, 8ml of redistilled tBA monomer is added, liquid nitrogen is put into the reactor for freezing, the reactor is vacuumized, nitrogen is filled, water is dissolved, and the steps are repeated for three times; adding catalyst PMDETA0.42ml, reacting in oil bath at 80 ℃ for half an hour, and quickly freezing by liquid nitrogen to stop the reaction; selecting 80 ℃ and reacting for 0.5 hour as reaction conditions, a diblock polymer with the PAA end length of about 25 repeating units can be obtained. Experiments have demonstrated that PEO-b-PAA of this length has the best self-assembly properties.

c) Purification of the product

Washing with deionized water, filling a chromatographic column with 200-300 mesh neutral aluminum trioxide as a stationary phase for four times of chromatography, distilling a product, and drying in vacuum to obtain white solid powder PEO-b-PtBA.

d) Hydrolysis

Weighing PEO-b-PtBA1.2g, dissolving with 15ml dichloromethane, bubbling nitrogen for half an hour, injecting 1.4ml of trimethyl iodosilane, reacting in an oil bath at 26 ℃ for 3 hours, after the reaction is finished, decompressing, draining, dissolving with 2-3ml of THF, dropping sodium metabisulfite solution with pH of 1 until yellow color is eliminated, and stirring for about one hour; after 48 hours of dialysis, the mixture was freeze-dried to obtain PEO-b-PAA solid.

Then preparing the loaded LD nano particles:

ultrasonic dissolving 10mgPEO-b-PAA in 6ml deionized water continuously at low temperature (0 ℃), dissolving 32.5mgLD in 10ml deionized water, dripping into PEO-b-PAA solution at low temperature, dissolving to 10ml, and obtaining the Levodopa (LD) -carrying nano particle (1 ‰) preparation with LD: AA being 1.0 by molecular assembly technology.

The levodopa nano preparation prepared by the method is characterized in that: with PEO₁₁₃-b-PAA₂₅Is a carrier, and the molecular structural formula is as follows:

the structural formula of levodopa enclosed in the core is as follows:

the advantages and positive effects of the invention are fully expressed as follows:

1) the biocompatibility is good. Because the two blocks of the polymer are hydrophilic, the water solubility is good. Furthermore, PEO and PAA are highly biocompatible, have been authenticated by ADSL and are FDA approved for use in humans.

2) Preparing a polymer micelle solution with a fixed structure and a concentration of up to 20% by adopting a one-step method; the solid powder of the polymer micelle can be easily obtained from the solution, and a foundation is laid for large-scale industrial production of the loaded LD nano particles.

3) 48 Kunming mice (20 +/-2 g) are randomly divided into two groups, each group comprises 24 mice, LD aqueous solution and LD-loaded nanoparticles with the same dose are injected into the abdominal cavity at the dose of 20mg/kg, blood is taken from the right ventricle, and the dopamine concentration is measured by a high performance liquid chromatography. It was found that a "burst" occurred after administration of PEO-PAA-LD. However, the increase in blood DA concentration caused by burst release did not exceed the peak concentration of LD group (P>0.05). Compared with the burst release amount of 50% -60% of some grafted nano-carriers, the LD-loaded nano-particles LD designed by the experimental group have less burst release and small influence, do not cause great change of blood DA concentration, and are very ideal nano-carriers, as shown in figure 1.

4) 48 Kunming mice (20 +/-2 g) are randomly divided into two groups, each group comprises 24 mice, LD aqueous solution and LD-loaded nanoparticles with the same dose are injected into the abdominal cavity with the dose of 20mg/kg, the head is cut off, the brain is taken, and the dopamine concentration is measured by a double-striatum measuring high performance liquid chromatography. Experiments show that the blood DA content of the nano group is obviously reduced (P is less than 0.05) compared with the peak value of the LD group after administration, as shown in figure 1, the brain striatum DA content is obviously increased, the peak value concentration is 500-600 times of that of the LD group, and the brain striatum DA content is still more than 100 times of that of the LD group 28 days after administration, as shown in figure 2. Showing significant enrichment and long in vivo circulation time (at least over 28 days). The particle size of the LD-carrying nano particles designed by the experimental group is about 100nm, so the LD-carrying nano particles are not easy to enter bone marrow and kidney to excrete, and are not easy to be phagocytized by a mononuclear cell system under the protection effect of the PEG shell, and organs such as liver and spleen are low in distribution, so the LD-carrying nano particles can circulate in blood for a long time, enter a central nervous system under the endocytosis effect when flowing through a blood brain barrier, and are decomposed or diffused to release LD. Thereby realizing the passive targeting of the brain and the high penetration rate of the blood brain barrier.

5) After administration for 5 days, in the case that the blood DA content of the nano-group is returned to a normal level, the striatum DA content is still 200-300 times higher than that of the LD group, as shown in FIG. 1 and FIG. 2. The result fully shows that the LD-loaded nanoparticles have obvious effects on reducing peripheral LD decarboxylation, reducing peripheral DA content and reducing adverse reactions. Meanwhile, a larger space is provided for reducing the consumption of LD.

Description of the drawings:

FIG. 1 comparison of blood DA levels after administration to two groups of mice

FIG. 2shows the striatal DA (polyamine) content of each group of mice

Claims

1. A method for preparing a levodopa nano-preparation is characterized by comprising the following steps: firstly, adopting a one-step method to synthesize an amphiphilic block copolymer PEO-b-PAA by taking PEO-Br as a macroinitiator according to the following route:

a) preparation of macroinitiator PEO-Br

Weighing 10g of PEO (Mn 5000), vacuum drying for 5 hours, dissolving in 150ml of toluene, adding 0.42ml of triethylamine (0.003mol), and performing ice-water bath at 0 ℃; CH is dripped within half an hour₃CH (Br) COBr 0.003ml. Stirring and reacting at room temperature overnight; filtering, precipitating, extracting, drying, reacting overnight to obtain white solid powder macroinitiator PEO-Br;

b) synthesis of amphiphilic block polymer PEO-b-PtBA

2.064g of macroinitiator and CuBr58.9mg are taken to be put into a reactor, nitrogen is filled into the reactor, 8ml of redistilled tBA monomer is added into the reactor, liquid nitrogen is added into the reactor for freezing, and the reactor is vacuumized and filled with nitrogen. Dissolving in water, and repeating for three times; adding catalyst PMDETA0.42ml, reacting in oil bath at 80 ℃ for half an hour, and quickly freezing by liquid nitrogen to stop the reaction; selecting 80 ℃ and reacting for 0.5 hour as reaction conditions to obtain a diblock polymer with the PAA end length of about 25 repeating units;

c) purification of the product

Washing with deionized water, filling a chromatographic column with 200-300-mesh neutral aluminum trioxide as a stationary phase for four times of chromatography, distilling a product, and drying in vacuum to obtain white solid powder PEO-b-PtBA;

d) hydrolysis

Weighing PEO-b-PtBA1.2g, dissolving with 15ml dichloromethane, bubbling nitrogen for half an hour, injecting 1.4ml of trimethyl iodosilane, reacting in an oil bath at 26 ℃ for 3 hours, after the reaction is finished, decompressing, draining, dissolving with 2-3ml of THF, dropping sodium metabisulfite solution with pH of 1 until yellow color is eliminated, and stirring for about one hour; dialyzing for 48 hours, freezing and drying to obtain PEO-b-PAA solid powder;

then preparing LD-loaded nano particles

Dissolving 10mgPEO-b-PAA in 6ml deionized water under continuous low temperature (0 ℃) by ultrasonic, dissolving 32.5mgLD in 10ml deionized water, adding PEO-b-PAA solution under low temperature ultrasonic drop by drop, dissolving to 10ml, obtaining LD by molecular self-assembly technology: levodopa (LD) -loaded nanoparticle (1 ‰) formulation with AA ═ 1.0.

2. The levodopa nano preparation prepared by the method is characterized in that: with PEO₁₁₃-b-PAA₂₅Is a carrier, and the molecular structural formula is as follows:

the structural formula of levodopa enclosed in the core is as follows: