CN100372524C - Amphotericin B nano preparation - Google Patents

Abstract

The present invention belongs to the technical field of medicine, which relates to nanometer preparation of amphotericin B (AmB) of antifungal medicine. The present invention is prepared by the carrier material of the AmB which uses poly-alkyl-alpha-cyanoacrylate (PBCA). An animal test shows that the present invention can permeate through a blood-brain barrier and measures that the medicine concentration of the present invention in brain tissue is higher than the AmB liposome preparation of clinical application. The experimental result of curative effect shows that the nanometer preparation can obviously prolong the lifetime of a mouse with cryptococcal meningitis and reduce the amount of infectious bacteria in the brain tissue; the curative effect of the present invention is better than the AmB liposome preparation.

Description

A kind of amphotericin B nano preparation

Technical field

The present invention relates to medical technical field, is a kind of nanometer formulation of antifungal agent amphotericin B.

Background technology

Amphotericin B (AmB) belongs to the macro ring polyene antibiotics, is the choice drug that is used for the treatment of deep fungal infection.Cryptococcal meningitis is a kind of common deep fungal infection disease, greatest problem with the AmB treatment is because existing injectable powder is difficult for seeing through blood brain barrier, need to merge direct injection in the sheath clinically, but intrathecal injection is not only operated inconvenience, and erious adverse reaction, the patient is difficult to accept.Though the Liposomal formulation of clinical practice can pass through blood brain barrier, limited widely-used because of costing an arm and a leg.

Summary of the invention

The present invention is the AmB carrier material with PBCA (PBCA), and the AmB nanometer formulation of a kind of energy by blood brain barrier is provided.Preparation method is as follows:

One, reagent:

1, amphotericin B (25mg/ props up, the new pioneer's Pharmaceutical in Shanghai);

2, α-Qing Jibingxisuanzhengdingzhi (Beijing Shunkang Medical Adhesive Co., Ltd);

3, dextran Dextran-70 (Pharmacia company);

4, poloxalkol Poloxamer188 (Beijing Century China woods bio tech ltd);

5, Tween-80 (Tween-80) (Shanghai chemical reagents corporation);

Two, concrete preparation method:

Stabilizing agent Dextran-70 and/or Poloxamer188 respectively are dissolved in distilled water by the concentration of 6mg/ml, add or do not add the cosolvent NaTDC simultaneously, concentration is 0.4mg/ml, be about in the ph value under 3 the condition and slowly add α-Qing Jibingxisuanzhengdingzhi while stirring, to final concentration be 1%, behind the stir about 4 hours, adjust pH is to 7.5-8.5, get blank α-poly-alkyl-alfa-cyanoacrylate nanoparticles colloid solution, ratio with blank nanoparticle colloid solution 10ml:20mg amphotericin B powder adds the amphotericin B powder then, be about at pH value under 7 the neutrallty condition and continued stir about 4 hours, get amphotericin B-poly-alkyl-alfa-cyanoacrylate nanoparticles (AmB-PBCA-NP) colloid solution, hatch in 37 ℃ of calorstats with 1% Tween-80 at last, amphotericin B nano preparation.

Laser particle size analyzer is measured size and distribution

It is an amount of to get above-mentioned amphotericin B nano preparation, adds the injection water and makes dispersion, puts in the Nano-S particle size determination instrument and measures particle diameter, and mean diameter is 74.38nm.

Transmission electron microscope is observed the nanoparticle form down

Amphotericin B nano preparation outward appearance of the present invention is faint yellow opaque emulsion liquid, gets 1-2 and drips solution and place on the copper mesh 2% Salkowski's solution negative staining, room temperature is dried, and transmission electron microscope is observed its form down, and nanoparticle is circle or similar round, size is comparatively even, does not have obviously and assembles.

Ultraviolet method is measured envelop rate and drug loading

It is an amount of to get amphotericin B nano preparation, centrifugal in 4 ℃, 35000r/min, gets supernatant and measures ultraviolet absorptivity, and gained concentration is counted C _b(mg/ml), press the initial concentration that dosage calculates AmB, count C (mg/ml), the initial concentration of the blank nanoparticle of PBCA-NP is counted M (mg/ml), computational envelope rate and drug loading as follows.

The envelop rate computing formula: $E=\frac{C-C_b}{C}\×100\%$

The drug loading computing formula is: $Y(\mathrm{mg}/\mathrm{mg})=\frac{C-C_h}{M}\×100\%$

Get the envelop rate scope at 23.10%-56.10%, drug loading is 25%-92%.

The extracorporeal releasing test of amphotericin B nano preparation

Selecting the aqueous solution of polyvinylpyrrolidone (PVP) is protecting colloid, and the PVP aqueous solution of preparation higher concentration adds and puts in right amount in the AmB-PBCA-NP liquid, makes that to contain PVP be 2% AmB-PBCA-NP liquid, sonic oscillation 5 minutes, room temperature jolting 12 hours.Get 1ml and place the bag filter of having handled well, carry out extracorporeal releasing experiment, the result shows that preparation of the present invention is time dependence and discharges, and discharges release in 40.9%, 10 hour release 93.28% in 47.45%, 24 hour, and has certain slow-releasing in 5 hours.

The specific embodiment

Now in conjunction with the embodiments, the present invention is described in detail:

Embodiment 1:

Take by weighing 300mg Dext ran-70, put in the beaker, the adding distil water stirring and dissolving, the pH value to 3.0 of the hydrochloric acid conditioning solution of usefulness 0.01mol/L is settled to 50ml.The BCA that under room temperature, electromagnetic agitation, slowly adds 0.5ml, to final concentration be 1%, stir after 4 hours, regulate pH to 8.5 with the sodium hydroxide of 0.1mol/L, blank PBCA nanoparticle colloid solution.Measure blank nanoparticle colloid solution 10ml, add the Amphotec of 20mg, measure pH value and be about 7.5, continue to stir 4 hours, get AmB-PBCA-NP colloid solution.Add 1% Tween-80 at last and in 37 ℃ of calorstats, hatched 2 hours, promptly get nanometer formulation of the present invention.After testing, envelop rate 56.10%, drug loading 82%, mean diameter is 79.9nm.

Embodiment 2:

Take by weighing 300mg Dextran-70, put in the beaker, the adding distil water stirring and dissolving, the pH value to 3.0 of the hydrochloric acid conditioning solution of usefulness 0.01mol/L is settled to 50ml.The BCA that under room temperature, electromagnetic agitation, slowly adds 0.5ml, to final concentration be 1%, stir after 4 hours, regulate pH to 7.5 with the sodium hydroxide of 0.1mol/L, blank PBCA nanoparticle colloid solution.Measure blank nanoparticle colloid solution 10ml, add the Amphotec of 20mg, measure pH value and be about 7.5, add cosolvent NaTDC 20mg again, continue to stir 4 hours, get AmB-PBCA-NP colloid solution.Add 1% Tween-80 at last and in 37 ℃ of calorstats, hatched 2 hours, promptly get nanometer formulation of the present invention.After testing, envelop rate 23.1%, drug loading 25%, mean diameter is 90.7nm.

Embodiment 3:

Take by weighing 300mg Dextran-70 and 300mgPoloxamer188, put in the beaker, the adding distil water stirring and dissolving, the pH value to 3.0 of the hydrochloric acid conditioning solution of usefulness 0.01mol/L is settled to 50ml.The BCA that under room temperature, electromagnetic agitation, slowly adds 0.5ml, to final concentration be 1%, stir after 4 hours, regulate pH to 8.5 with the sodium hydroxide of 0.1mo l/L, blank PBCA nanoparticle colloid solution.Measure blank nanoparticle colloid solution 10ml, add the amphotericin injectable powder of 20mg, measure pH value and be about 7.0, continue to stir 4 hours, get AmB-PBCA-NP colloid solution.Add 1% Tween-80 at last and in 37 ℃ of calorstats, hatched 2 hours, promptly get nanometer formulation of the present invention.After testing, envelop rate 38.4%, drug loading 92%, mean diameter is 83.4nm.

Embodiment 4:

Take by weighing 300mg Dext ran-70 and 300mgPoloxamer188, put in the beaker, the adding distil water stirring and dissolving, the pH value to 3.0 of the hydrochloric acid conditioning solution of usefulness 0.01mol/L is settled to 50ml.The BCA that under room temperature, electromagnetic agitation, slowly adds 0.5ml, to final concentration be 1%, stir after 4 hours, regulate pH to 8.5 with the sodium hydroxide of 0.1mol/L, blank PBCA nanoparticle colloid solution.Measure blank nanoparticle colloid solution 10ml, add the Amphotec of 20mg, measure pH value and be about 7.0, add cosolvent NaTDC 10mg again, continue to stir 4 hours, get AmB-PBCA-NP colloid solution.Add 1% Tween-80 at last and in 37 ℃ of calorstats, hatched 2 hours, promptly get nanometer formulation of the present invention.After testing, envelop rate 34.8%, drug loading 45%, mean diameter is 88.6nm.

Stabilizing agent among the embodiment 1 adopts Dex t ran T-70, and does not add the cosolvent NaTDC, and the result shows that particle diameter is less, and drug loading and envelop rate are all higher.

Animal experiment study

In order further to verify the brain targeting of nanometer formulation, we have carried out zoopery, adopt high efficiency liquid phase chromatographic analysis method (HPLC) to measure drug level in the mouse brain.Animal is a healthy kunming mice in 6-8 age in week, male and female half and half, body weight 25 ± 5g (purchasing the Experimental Animal Center in The 2nd Army Medical College).

Animal is divided into 4 groups, 21 every group:

1, AmB injectable powder group: tail vein injection Amphotec (1mg/kg)

2, AmB nanometer formulation group of the present invention: tail vein injection AmB-PBCA-NP (3mg/kg)

3, AmB liposome group: tail vein injection AmB-L (10mg/kg)

4, blank group: tail vein injection saline (volumetric injection 0.1ml)

Each organize mice respectively at administration after 0.5h, 1h, 3h, 6h, 12h, 24h and 48h extract eyeball and get blood, get cerebral tissue after disconnected at once marrow is put to death and continue to employ.Blood places and drips the anticoagulant tube that heparin sodium is arranged, and light shaking shakes up, and prevents to solidify, and 100 μ l are standby for the centrifuging and taking supernatant; The cerebral tissue normal saline is made homogenized, gets supernatant 100 μ l after centrifugal, add contain in target acetonitrile 100 μ l, vortex, centrifugal is got supernatant 100 μ l, gets supernatant after centrifugal again, and is to be measured.

Dikma-C18 chromatographic column (specification 200 * 4.6mm) is adopted in chromatography, pre-column adopts the general HPLC guard column of Dikma Ea syGuard (post core size 8 * 4mm) (DIKMA company), mobile phase consists of the acetic acid of acetonitrile, water (40: 60) and 4%, and flow velocity is 0.9ml/min; UV detection wavelength 405nm, 37 ℃ of column temperatures, sample size 20 μ l.

Fail to measure medicine in the AmB injectable powder group mouse brain tissue, this is consistent with bibliographical information and clinical observation result; And AmB nanometer formulation of the present invention demonstrates higher drug level in cerebral tissue, 30 minutes mean concentrations are 61.4930ng/g, reached 132.5203ng/g in 3 hours, concentration still is 66.1001ng/g after 48 hours, and Liposomal formulation just can be measured medicine after 6 hours, 12 hour concentrations are only up to 66.1400ng/g, and concentration slowly descends afterwards.The result shows that PBCA can carry AmB and arrive brain, and the brain targeting of AmB nanometer formulation of the present invention obviously is better than liposome.

The experiment of animal curative effect science

Choose ATCC30629B type neogenesis cryptococcus bacterial strain (Long March hospital dermatology department fungus preserving chamber provides) and BALB/c female mice (20 ± 5g, age in 6-8 week, purchase Shanghai Experimental Animal Center) in the Chinese Academy of Sciences, set up the mouse model of cryptococcal meningitis by intracerebral ventricle injection, model mice is divided into 4 groups, 10 every group.

One, animal grouping and processing:

1, AmB injectable powder treatment group: tail vein injection Amphotec (1mg/kg)

2, AmB-PBCA-NP treatment group: tail vein injection AmB-PBCA-NP (3mg/kg)

3, AmB liposome therapeutic group: tail vein injection AmB-L (10mg/kg)

4, blank group: tail vein injection saline (volumetric injection 0.1ml)

Two, observe life cycle

Observe the survival rate of respectively organizing mice and find that AmB injectable powder group mice and normal saline matched group do not have significant difference life cycle, all dead in 20 days; 10 days survival rates of AmB-PBCA-NP treatment group mice are that 100%, 20 day survival rate is 80%, and AmB liposome therapeutic group mice 10 days and 20 days survival rates are 80%, illustrate that the nanometer formulation short term efficacy is significantly better than Liposomal formulation.

Three, colony counting comparative study

Get and respectively organize mice treatment front and back cerebral tissue, homogenate, sabouraud culture medium is cultivated under 37 ℃ of conditions, observes the colony growth situation.The result shows that organizing colony counting through one day mouse brain of AmB injectable powder treatment is 308000cfu/g, rises to 549000cfu/g after the week, illustrates that the injectable powder intravenously administrable does not have therapeutical effect to cryptococcal meningitis.And brain tissue homogenate's colony counting of mice only is 9800cfu/g after one week of nanometer formulation treatment of the present invention, the colony counting of Liposomal formulation treatment mice under the equal conditions is 53400cfu/g, significant difference is remarkable, indirect proof AmB nanometer formulation of the present invention have the brain targeting, cryptococcal meningitis is had the obvious and effective therapeutical effect.

Claims (2)

1. amphotericin B nano preparation, preparation method is as follows:

Stabilizing agent dextran Dextran-70 and/or poloxalkol Poloxamer188 respectively are dissolved in distilled water by the concentration of 6mg/ml, add or do not add the cosolvent NaTDC simultaneously, the concentration of NaTDC is 0-0.4mg/ml, be slowly to add α-Qing Jibingxisuanzhengdingzhi while stirring under 3 the condition at pH value, to concentration be 1%, stir after 4 hours, adjust pH is to 7.5-8.5, get blank α-poly-alkyl-alfa-cyanoacrylate nanoparticles colloid solution, then with blank nanoparticle colloid solution 10ml: the ratio of 20mg amphotericin B powder adds the amphotericin B powder, be to continue to stir 4 hours under 7 the neutrallty condition at pH value, get amphotericin B-poly-alkyl-alfa-cyanoacrylate nanoparticles colloid solution, hatch in 37 ℃ of calorstats with 1% Tween-80 at last, amphotericin B nano preparation.

2. by the described amphotericin B nano preparation of claim 1, it is characterized in that said stabilizing agent is dextran Dextran-70 in the preparation method, be made into the aqueous solution of 6mg/ml, add α-Qing Jibingxisuanzhengdingzhi again, stir after 4 hours adjust pH to 8.5.