CN103483353B - Dithiole the nanoparticle of pyrrolidone compound and preparation method - Google Patents

Dithiole the nanoparticle of pyrrolidone compound and preparation method Download PDF

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CN103483353B
CN103483353B CN201210195057.2A CN201210195057A CN103483353B CN 103483353 B CN103483353 B CN 103483353B CN 201210195057 A CN201210195057 A CN 201210195057A CN 103483353 B CN103483353 B CN 103483353B
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aqueous phase
nanoparticle
dithiole
oil phase
pyrrolidone
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赵锋
栾瀚森
王浩
杨莉
徐凤兰
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Shanghai Modern Pharmaceutical Engineering Research Center Co Ltd
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Abstract

The invention discloses a kind of dithiole and the nanoparticle of pyrrolidone compound and preparation method and device, described dithiole the nanoparticle of pyrrolidone compound, comprise the component of following weight percent: dithiole pyrrolidone compound 0.007 ~ 20%, biodegradable polymers 0 ~ 89.85%, additive 0.07 ~ 95%, freeze-drying caffolding agent, 0.1-80%, the mean diameter 10nm-800nm of nanometer.The present invention adopts ultrasonic-static mixing device for the preparation of nanocrystal and nanoparticle.Dithiole of the present invention the nanoparticle of pyrrolidone compound, have significant nanoparticle characteristic features, particle diameter 60-300nm, Zeta potential is at-30-20mv, and can be used for oral or intravenously administrable, bioavailability is high, circulation time in vivo extends, and exploitation said preparation has broad prospects.

Description

Dithiole the nanoparticle of pyrrolidone compound and preparation method
Technical field
The present invention relates to dithiole and the preparation of pyrrolidone compound and preparation method.
Technical background
Dithiole pyrrolidone compound, 6-amino-4-(2 disclosed in CN101381371, US20100210856A1,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate (hereinafter referred to as ZL-004), the compound of transformation is modified by mother nucleus structure 1,2-dithiole [4,3-b] pyrroles-5 (4H)-one ring.Synthesized voluntarily by the flat researcher of kingdom of Shanghai Institute of Pharmaceutical Industry, this type of structural compounds has patent report, there is antibacterial, anti-tumor activity feature, as (HelveticaChimicaActa2002 such as (wo2003/080624), Stachel such as Webster, 85,4453), (JP63-284181, JP11-279179), Webster etc. (US6020360, WO99/12543), the Godfrey & Dell(GB2170498 such as river Tian Xiusi) report.
Through non-clinical pharmacology, toxicological experiment, ZL-004 shows the characteristic of stronger rising peripheral leukocytes quantity.There is wide potential applicability in clinical practice.
But because ZL-004 belongs to insoluble drug, in 37 DEG C of water, solubleness only has 90 – 120 μ gmL -1, only 0.90-1.50mgmL in methyl alcohol -1, belong to soluble,very slightly material.On microcosmic, its insoluble makes it with molecularity through microbial film, cannot be scattered in the water miscible recycle system, act on biological target cell.Macroscopically, add difficulty that is oral, mucosal route administration, previous experiments also confirms giving full play to of its specific physical character restriction drug effect, only has and improves its target cell activity ratio by the modification of self structure or the change of preparation aspect.
In view of its insoluble feature, the necessary technology of pharmaceutics method exploitation one that adopts can improve it through microbial film, improve the preparation of the active function usefulness of compound, for research is provided convenience from now on.
Summary of the invention
The object of this invention is to provide dithiole and the nanoparticle of pyrrolidone compound and preparation method, to overcome the defect that prior art exists, improve its action effect and range of application further.
Described dithiole the nanoparticle of pyrrolidone compound, comprise the component of following weight percent:
The mean diameter 10nm-800nm of described nanometer;
Preferably, the component of following weight percent is comprised:
The mean diameter of described nanometer is 90nm-180nm;
Described thia cyclopenta pyrrolidone compound is 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate; (being called for short ZL-004)
Described biodegradable polymers comprises poly(lactic acid), poly lactic coglycolic acid, lactic-co-glycolic acid-ethylene glycol copolymer, lactic-co-glycolic acid-polyethyleneglycol block copolymer, methylate in polyoxyethylene glycol-glycolide-lactide copolymer, polycaprolactone or polyacrylamide more than one;
Preferably methylate polyoxyethylene glycol-glycolide-lactide copolymer;
Preferably, methylate in polyoxyethylene glycol-glycolide-lactide copolymer, the scope of monomer molar ratio is 50: 50-25: 75;
Preferably, the weight-average molecular weight of biodegradable polymer is 5,000-60,000 dalton, wherein, 7,000-50,000 dalton is comparatively suitable;
Preferably, described additive is selected from polyethylene glycol stearate or polyoxyethylene hydrogenated castor oil;
Described freeze-drying caffolding agent is selected from N.F,USP MANNITOL, sucrose, lactose, sodium alginate.
Dithiole of the present invention the nanoparticle preparation method of pyrrolidone compound adopt following nanoparticle preparation facilities to realize:
Described nanoparticle preparation facilities comprises: aqueous phase reservoir, oil phase reservoir, ultrasonic generator, ultrasonication chamber, frame, the static mixer being provided with inner member, circulation tube, fluid accept cup and agitator;
Described static mixer by anchor vertical be fixed in described frame;
The outlet of described aqueous phase reservoir is connected by aqueous phase pipeline and is connected with described ultrasonication chamber, and the outlet of described oil phase reservoir is connected with described ultrasonication chamber by oil phase pipeline, and the probe of described ultrasonic generator is inserted in described ultrasonication chamber;
Described ultrasonic generator is mechanically fixed in frame;
Described fluid accepts the below that cup is arranged on the outlet bottom described static mixer;
Fluid described in the insertion of described circulation tube one end accepts in cup, and the other end is connected with described aqueous phase reservoir by handling equipment;
And method of the present invention comprises the steps:
(1) aqueous phase in the oil phase in oil phase reservoir and aqueous phase reservoir is sent into described ultrasonication chamber simultaneously, and start ultrasonic generator, ultrasonic wave is sent into ultrasonication chamber;
Described oil phase is the organic solvent containing thia cyclopenta pyrrolidone compound, biodegradable polymer and additive;
Described organic solvent is selected from tetrahydrofuran (THF), acetone, the alcohols of carbon atom 1 to 4, N, water-soluble or methylene dichloride, trichloromethane, ethyl acetate, the N such as N-dimethylformamide or dimethyl sulfoxide (DMSO), dioxane, acetonitrile, the water such as N-dimethyl pyrrolidone or phenylcarbinol are soluble solvent slightly;
Described aqueous phase is the aqueous solution containing polyvinyl alcohol;
In ultrasonic wave and static mixer inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer, enter fluid and accept cup, nanoscale emulsion fluid accepted in cup sends aqueous phase reservoir back to, move in circles said process, until nanoparticle particle diameter reaches requirement;
(2) then fluid is accepted the material in cup, centrifugal, collect solid matter, wash, add freeze-drying caffolding agent, freeze-drying, described in acquisition containing dithiole the nanoparticle of pyrrolidone compound;
When the add-on of biodegradable polymers is 0mg, what obtain is nano-crystal grains containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
Method of the present invention, from traditional prepare nanocrystal, nanoparticle method different, traditional preparation nanocrystal comprises the preparation methods such as solvent precipitation, emulsification-evaporation method, salting-out process, supercritical fluid technology, compound emulsion method, interface polycondensation.The present invention adopts ultrasonic-static mixing device for the preparation of nanocrystal and nanoparticle.The patent US2011/0237748A1 of nanoparticle prepared by Static mixing tank, although it uses static mixer, water miscible organic solvent can only be used in oil phase, as acetone, acetonitrile, tetrahydrofuran (THF), N-Methyl pyrrolidone etc., limit the use that water section dissolves organic solvent, as the application of methylene dichloride, trichloromethane, ethyl acetate etc., this kind solvent can increase solubleness, raising encapsulation rate, the drug loading of non-polar drug.And provided by the invention ultrasonic-organic solvent and APM can change into superfine drop by static mixing device before combination, can obtain the nanoparticle that high encapsulation, size distribution are homogeneous after by mixing tank, being easy to amplification quantity produces, and can break through nanoparticle and prepare bottleneck.
Dithiole of the present invention the nanoparticle of pyrrolidone compound, have significant nanoparticle characteristic features, particle diameter 60-300nm, Zeta potential is at-30-20mv, and can be used for oral or intravenously administrable, bioavailability is high, circulation time in vivo extends, and exploitation said preparation has broad prospects.
Accompanying drawing explanation
Fig. 1 is nanoparticle preparation facilities structural representation.
Fig. 2 is the grain size distribution (quantity footpath) of embodiment 3.
Fig. 3 is the grain size distribution (volumetric diameter) of embodiment 3.
Fig. 4 is extracorporeal releasing test.
Fig. 5 is the In vivo analysis ultrahigh pressure liquid phase chromatogram-dual level Four bar mass spectrum typical case mass spectrum of ZL-004.
Fig. 6 is zl-004UPLC-MS/MS color atlas.
Fig. 7 is the behavior of intravenous injection Internal pharmacokinetics.
Fig. 8 is oral Internal pharmacokinetics behavior.
Embodiment
See Fig. 1, nanoparticle preparation facilities of the present invention comprises: aqueous phase reservoir 1, oil phase reservoir 2, ultrasonic generator 6, ultrasonication chamber 9, frame 4, the static mixer 5 being provided with inner member 501, circulation tube 10, fluid accept cup 7, agitator 8;
Described static mixer 5 by anchor 401 vertical be fixed in described frame 4; The aspect ratio of static mixer 5 is 50-80;
Described inner member 501 adopts the universal product, and if model is the inner members such as SV, SL, SK, SX, SD, the structure of described inner member and correlation parameter, " pipeline static mixer handbook " is described later in detail, and the present invention repeats no more;
The outlet of described aqueous phase reservoir 1 is connected by aqueous phase pipeline and is connected with described ultrasonication chamber 9, the outlet of described oil phase reservoir 2 is connected with described ultrasonication chamber 9 by oil phase pipeline, the probe 601 of described ultrasonic generator 6 is inserted in described ultrasonication chamber 9, preferably, described aqueous phase pipeline is provided with handling equipment 11, as peristaltic pump with oil phase pipeline;
Described ultrasonic generator 6 is general-purpose equipment, power adjustable scope can be adopted to be the ultrasound probe of 30-200w, pop one's head in as cylindric, and head is coniform (cone diameter is 0.5-2.0cm, and long is 0.5-2.0cm), is mechanically fixed in frame 4;
Described fluid accepts the below that cup 7 is arranged on the outlet 503 bottom described static mixer 5;
Fluid described in the insertion of described circulation tube 10 one end accepts in cup 7, and the other end is connected with described aqueous phase reservoir 1 by handling equipment;
Preferably, also comprise agitator 8, described fluid accepts cup 7 and is placed on described agitator 8, and described agitator 8 is magnetic stirring apparatus preferably;
And method of the present invention comprises the steps:
(1) aqueous phase in the oil phase in oil phase reservoir 2 and aqueous phase reservoir 1 is sent into described ultrasonication chamber 9 simultaneously, and start ultrasonic generator 6, ultrasonic wave is sent into ultrasonication chamber 9;
Described oil phase is the organic solvent containing thia cyclopenta pyrrolidone compound, biodegradable polymer and additive;
Described organic solvent is selected from tetrahydrofuran (THF), acetone, the alcohols of carbon atom 1 to 4, N, water-soluble or methylene dichloride, trichloromethane, ethyl acetate, the N such as N-methyl nitrosourea or dimethyl sulfoxide (DMSO), dioxane, acetonitrile, the water such as N-dimethyl pyrrolidone or phenylcarbinol are soluble solvent slightly;
In described oil phase, total concentration of thia cyclopenta pyrrolidone compound, biodegradable polymer and additive is 7.0 ~ 680 grams per liters;
Described aqueous phase is the aqueous solution containing polyvinyl alcohol, wherein:
The concentration of polyvinyl alcohol is 0.1-15 grams per liter, comparatively suitable with 1.0 ~ 12 grams per liters;
With the volumeter of organic solvent and water, organic solvent: water=1: 9.0 ~ 1: 480;
The speed of oil phase is 1.0-4.0mlmin -1; The speed of aqueous phase is 150-350mlmin -1;
In described organic solvent, the residence time of oil phase is 20 ~ 70 seconds, preferably 27 ~ 66 seconds;
The residence time of oil phase is relevant with the effect of ultrasonication, and be Con trolling index, the residence time is defined as follows:
T=(S1+S2)/υ, unit is second;
Wherein: S1 is the length of static mixer cavity, unit is cm; S2 is ultrasonication chamber 9 length, and unit is cm; υ is the speed of material, and unit is cm/ second;
With described ultrasonication chamber 9 volume for benchmark, ultrasonic power is 4.0-235wcm -3;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator 8, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to by circulation tube 11, and move in circles said process, until nanoparticle particle diameter reaches requirement;
(2) then fluid is accepted the material in cup 7,3-20 DEG C, 10000-50000rmp is centrifugal, collect solid matter, wash, add freeze-drying caffolding agent, freeze-drying, described in acquisition containing dithiole and the nanoparticle of pyrrolidone compound.
Embodiment 1
Water-miscible organic solvent acetone is adopted to prepare the nano-crystal grains of 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate;
Prescription:
ZL-00410mg
Polyethylene glycol stearate 100mg
N.F,USP MANNITOL 22mg
The median size of nanocrystal is 95 ± 8nm;
Adopt the device of Fig. 1, the nano-crystal grains of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate;
The constitutional features of equipment:
Static mixer 5: be highly 60cm, internal diameter is 1.0cm, and aspect ratio is 60;
Ultrasonication chamber 9: diameter: 1.0cm; Highly; 6cm; Aspect ratio: 6; Volume: (1/2) 2× 3.14 × 10=0.785cm 3;
Ultrasonic power is: 30w; With ultrasonication chamber 9 upper volume for benchmark, ultrasonic power is: 38.22wcm -3;
Inner member 501 model is that Sk type is (long: 40cm; Internal diameter: 0.6cm);
6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyethylene glycol stearate are dissolved in organic solvent 10ml acetone, dissolve, obtain oil phase;
By 0.5 gram of polyvinyl alcohol dissolution in 300ml water liquid, it is aqueous phase;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
The speed of oil phase is 1.0mlmin -1; The speed of aqueous phase is 150mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(60+6)/1.0=66 second;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, and the said process 3 times of moving in circles is to until nanoparticle particle diameter reaches 95 ± 8nm.
10 DEG C, 12000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds N.F,USP MANNITOL, freeze-drying, obtains product.
Embodiment 2
Water-miscible organic solvent acetonitrile is adopted to prepare the nano-crystal grains of 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate
Prescription 2:
ZL-00440g
Polyethylene glycol stearate 400g
Sucrose 220g
Obtained nanocrystal median size is 180 ± 14nm;
Preparation method:
Adopt the device of Fig. 1, the nano-crystal grains of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 100cm, internal diameter is 2.0cm, and aspect ratio is 50;
Ultrasonication chamber 9: diameter: 1.5cm; Highly; 10cm; Aspect ratio: 6.7;
Ultrasonic power is: 100w; With the volume of above-mentioned static mixer 5 upper end for benchmark, ultrasonic power is: 5.66wcm -3;
Inner member 501 adopts model to be SV type, (long: 80cm; Internal diameter: 1.8cm)
6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyethylene glycol stearate are dissolved in organic solvent 10 liters of acetonitriles, dissolve, filter, obtain oil phase;
By 0.5 kilogram of polyvinyl alcohol dissolution in 95 premium on currency liquid, it is aqueous phase;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
The speed of oil phase is 4.0mlmin -1; The speed of aqueous phase is 350mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(100+10)/4.0=27.5 second;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, and the said process 3 times that moves in circles is until nanoparticle particle diameter reaches requirement.10 DEG C, 12000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds sucrose, freeze-drying, obtains product.
Embodiment 3
Containing the nano-crystal grains adopting water summary solubleness organic solvent methylene dichloride to prepare 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
ZL-004150mg
Polyoxyethylene hydrogenated castor oil 1500mg
Sodium alginate 495mg
Obtained nanocrystal median size is 123 ± 20nm;
Preparation method:
Adopt the device of Fig. 1, the nano-crystal grains of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 60cm, internal diameter is 1.0cm, and aspect ratio is 60, and effective volume is; 47.1cm -3
Ultrasonication chamber 9: diameter: 1.0cm; Highly; 6cm; Aspect ratio: 6; Volume: (1/2) 2× 3.14 × 10=0.785cm 3
Ultrasonic power is: 200w; With the volume in ultrasonication chamber 9 for benchmark, ultrasonic power is: 254.78wcm -3;
Inner member 501 adopts model to be Sk type, (long: 40cm; Internal diameter: 0.8cm)
By 6-amino-4-(2; 4-dimethoxy phenyl)-4-hydrogen [1; 2] dithiole [4; 3-b]-pyrrolidone-ethyl formate and polyoxyethylene hydrogenated castor oil be dissolved in 22ml mixed organic solvents (methylene dichloride: acetone=10:1; volume ratio) in; dissolve, filter, obtain oil phase;
By 6 grams of polyvinyl alcohol dissolution in 594ml water liquid, it is aqueous phase;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
The speed of oil phase is 1.0mlmin -1; The speed of aqueous phase is 150mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(60+6)/1.0=66 second;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, and the said process 3 times that moves in circles is until nanoparticle particle diameter reaches requirement.10 DEG C, 12000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds sodium alginate, freeze-drying, obtains product.Grain size distribution (quantity footpath) is shown in Fig. 2; Size distribution (volumetric diameter) is shown in Fig. 3.
Embodiment 4
Containing the nano-crystal grains adopting water summary solubleness organic solvent ethyl acetate to prepare 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
Nanocrystal prescription 4:
Preparation method:
Adopt the device of Fig. 1, preparation is containing 4-N(2,4-p-methoxy-phenyl) nano-crystal grains of-6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 100cm, internal diameter is 2.0cm;
Ultrasonication chamber 9: diameter: 1.5cm; Highly; 10cm; Aspect ratio: 6.7; Volume: (15/2) 2× 3.14 × 10=17.66cm 3
Ultrasonic power is: 160w; With ultrasonication chamber 9 volume for benchmark, ultrasonic power is: 9.06wcm -3;
Inner member 501 adopts SL type (long: 80cm; Internal diameter: 1.8cm).
By 6-N-ethyl ester formyloxy-1,2-dithiole and pyrrolidone and polyoxyethylene hydrogenated castor oil are dissolved in 400ml mixed organic solvents (ethyl acetate: acetone=10:1), dissolve, filter, obtain oil phase;
By 120 grams of polyvinyl alcohol dissolution in 11.88 premium on currency, it is aqueous phase;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
The speed of oil phase is 3mlmin -1; The speed of aqueous phase is 250mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(100+10)/3.0=36.67 second;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, and the said process 3 times that moves in circles is until nanoparticle particle diameter reaches requirement.10 DEG C, 12000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds sucrose freeze-drying, obtains product.
Embodiment 5
Containing adopting the preparation of water-miscible organic solvent tetrahydrofuran (THF), the polymer nanoparticle of 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
Polymer nanoparticle prescription 1:
In MPEG-PLGA, MPEG:LA:GA=10:67.5:22.5, weight-average molecular weight is 50000 dalton, and the median size of nanoparticle is 97 ± 7nm;
Adopt the device of Fig. 1, the polymer nanoparticle of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 100cm, internal diameter is 1.25cm, and aspect ratio is 80;
Ultrasonication chamber 9: diameter: 1.25cm; Highly; 8cm; Aspect ratio: 6.4; Volume: (1.25/2) 2× 3.14 × 8=9.81cm 3
Ultrasonic power is: 60w; With the volume of above-mentioned static mixer 5 upper end for benchmark, ultrasonic power is: 6.12wcm -3;
Inner member 501 adopts model to be that Sk type is (long: 80cm; Internal diameter: 1.10cm)
6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyethylene glycol stearate are dissolved in organic solvent 250ml tetrahydrofuran (THF), dissolve, filter, obtain oil phase;
By 0.6 kilogram of polyvinyl alcohol dissolution in 119.4 premium on currency, obtain aqueous phase;
The speed of oil phase is 4.0mlmin -1; The speed of aqueous phase is 350mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(100+8)/4.0=27 second;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, move in circles above-mentioned 3 times to until nanoparticle particle diameter reaches requirement.10 DEG C, 12000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds N.F,USP MANNITOL freeze-drying, obtains product.
Embodiment 6
Containing adopting the preparation of water-miscible organic solvent acetone, the polymer nanoparticle of 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
Polymer nanoparticle prescription 2:
In MPEG-PLGA, MPEG:LA:GA=20:40:40, weight-average molecular weight is 7000 dalton, and the median size of nanoparticle is 139 ± 23nm;
Adopt the device of Fig. 1, the polymer nanoparticle of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 100cm, internal diameter is 1.25cm, and aspect ratio is 80;
Ultrasonication chamber 9: diameter: 1.25cm; Highly; 10cm; Aspect ratio: 8; Volume: (1.25/2) 2× 3.14 × 10=12.27cm 3
Ultrasonic power is: 140w; With ultrasonication chamber 9 volume for benchmark, ultrasonic power is: 11.41wcm -3;
Inner member 501 adopts model to be that Sk type is (long: 40cm; Internal diameter: 0.6cm)
6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyoxyethylene hydrogenated castor oil are dissolved in organic solvent 1000ml acetone, dissolve, filter, obtain oil phase;
By 0.6 kilogram of polyvinyl alcohol dissolution in 119.4 premium on currency, it is aqueous phase;
The speed of oil phase is 4.0mlmin -1; The speed of aqueous phase is 150mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(100+10)/4.0=27.5 second;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, move in circles above-mentioned 3 times to until nanoparticle particle diameter reaches requirement.10 DEG C, 36000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds lactose freeze-drying, obtains product.
Embodiment 7
Containing the polymer nanoparticle adopting water summary solubleness organic solvent methylene dichloride to prepare 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
Polymer nanoparticle prescription 3:
In MPEG-PLGA, MPEG:LA:GA=10:45:45, weight-average molecular weight is 24000 dalton, and the median size of nanoparticle is 157 ± 23nm;
Adopt the device of Fig. 1, the nano-crystal grains of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 60cm, internal diameter is 0.75cm, and aspect ratio is 80;
Ultrasonic power is: 110w;
Ultrasonication chamber 9: diameter: 0.75cm; Highly: 6cm; Aspect ratio: 8; Volume: (0.75/2) 2× 3.14 × 6=0.84cm 3
With the volume of above-mentioned static mixer 5 upper end for benchmark, ultrasonic power is: 130.95wcm -3;
Inner member 501 adopts model to be that Sk type is (long: 40cm; Internal diameter: 0.65cm)
By 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyethylene glycol stearate be dissolved in 22ml mixed organic solvents (methylene dichloride: N, N-dimethyl pyrrolidone=10:1) in, dissolve, filter, obtain oil phase;
Be aqueous phase by 6 grams of polyvinyl alcohol dissolution in 594ml water liquid;
The speed of oil phase is 1.5mlmin -1; The speed of aqueous phase is 150mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(60+6)/1.5=44 second;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, and the said process 3 times that moves in circles is until nanoparticle particle diameter reaches requirement.10 DEG C, 45000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds sodium alginate, freeze-drying, obtains product.
Embodiment 8
Containing the polymer nanoparticle adopting water summary solubleness organic solvent methylene dichloride to prepare 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate.
In MPEG-PLGA, MPEG:LA:GA=10:45:45, weight-average molecular weight is 12000 dalton, and the median size of nanoparticle is 139 ± 23nm;
Adopt the device of Fig. 1, the polymer nanoparticle of preparation containing 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate:
The constitutional features of equipment:
Static mixer 5: be highly 100cm, internal diameter is 2.0cm, and aspect ratio is 50;
Ultrasonication chamber 9: diameter: 1.5cm; Highly; 10cm; Aspect ratio: 6.7; Volume: (1.5/2) 2× 3.14 × 10=17.66cm 3
Ultrasonic power is: 132w, and with the volume of above-mentioned static mixer 5 upper end for benchmark, ultrasonic power is: 7.47wcm -3;
Inner member 501 adopts model to be that Sk type is (long: 40cm; Internal diameter: 0.6cm)
By 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate and polyoxyethylene hydrogenated castor oil be dissolved in 88ml mixed organic solvents (methylene dichloride: phenylcarbinol=10:1), dissolve, organic membrane filter, obtains oil phase;
By 24 grams of polyvinyl alcohol dissolution in 2.4 premium on currency, it is aqueous phase;
The speed of oil phase is 2mlmin -1; The speed of aqueous phase is 200mlmin -1;
The oil phase residence time: T=(S1+S2)/υ=(100+10)/2.0=55 second;
Described aqueous phase and oil phase are placed in aqueous phase reservoir 1 and oil phase reservoir 2 respectively, then send into the upper end entrance 502 of described static mixer 5, start ultrasonic generator (6) simultaneously, by the static mixer 5 that ultrasonic wave is sent into;
In ultrasonic wave and static mixer 5 inner member acting in conjunction under, oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet bottom static mixer 5, enter fluid and accept cup 7, under the stirring of agitator, nanoscale emulsion fluid accepted in cup 7 sends aqueous phase reservoir 1 back to, move in circles above-mentioned 3 times to until nanoparticle particle diameter reaches requirement.10 DEG C, 36000rpm centrifugation, collect the solid of centrifugal acquisition, uses distilled water Eddy diffusion, three times repeatedly, adds lactose freeze-drying, obtains product.
Embodiment 9
Extracorporeal releasing test:
The measuring method of nanoparticle slowly-releasing:
Precision takes 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate bulk drug, zl-004 nanocrystal and MPEG-PLGA nano-granule freeze-dried powder, put in dialysis tubing, and (compound method of PBS gets potassium primary phosphate 27.2g, sodiumazide 0.2g, sodium lauryl sulphate 1g, sodium-chlor 4.32g to add physiology isotonic phosphate buffer liquid (pH6.8), sodium hydroxide 0.944g, be dissolved in water into 1000mL, shake up) form colloidal solution, make ZL-004 be 1mgmL -1, get wherein 1.5mL and be placed in dialysis tubing, after exhaust, tighten port, be put in the tool plug Erlenmeyer flask of 40ml phosphate buffered saline buffer.
Erlenmeyer flask is placed in 37 DEG C of water bath with thermostatic control concussion casees, timing sampling, mends same volume fresh dissolution medium after every sub-sampling, sample adopts HPLC-UV to measure the concentration of ZL-004, calculates burst size and the rate of release of ZL-004 nanoparticle.
Test-results is shown in Fig. 4, and as seen from Figure 4, ZL-004 nanoparticle discharges slowly in physiological conditions, meets long circulating in its body, reaches the characteristic of the slow release of target site.
Embodiment 10
6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate Internal pharmacokinetics research:
Get 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate (zl-004) bulk drug, the zl-004 lyophilized powder of embodiment 5, the ZL-004 nanocrystal of embodiment 2, embodiment 6 ZL-004 polymer nanoparticle appropriate, redissolve with physiological saline, make concentration be 3mgmL -1(calculating by ZL-004).
SD rat 15, is divided into 5 groups, at random by 3mgkg -1dosage give respectively first group of gavage bulk drug, second group of gavage ZL-004 nanocrystal, the 3rd group of gavage ZL-004 nanoparticle, the 4th group give tail vein injection ZL-004 nanocrystal, the 5th group of tail vein injection ZL-004 polymer nanoparticle.Get blood respectively at administration forward and backward 0.25,0.5,0.75,1,2,4,6,8,12,16,24,36,48 hour vena ophthalmica clump, in anticoagulant tube, centrifuging and taking blood plasma is-20 DEG C of preservations immediately, measure after extracting through UPLC-MS/MS.
The In vivo analysis ultrahigh pressure liquid phase chromatogram-dual level Four bar mass spectrum typical case mass spectrum of result: embodiment 5ZL-004 is shown in Fig. 5, and color atlas is shown in Fig. 6.Medicine moving curve is shown in Fig. 7,8, and compared with oral with bulk drug, the nanocrystal after preparation and nanoparticles oral administration, all can significantly improve C max, AUC 0 → tvalue, bioavailability significantly improves.And after intravenously administrable, compared with ZL-004 nanocrystal, ZL-004 polymer nanoparticle can improve AUC 0 → t, reduce C max, extend MRT.
After nanoparticle of the present invention, orally can improve ZL-004 bioavailability.After intravenously administrable, ZL-004 polymer nanoparticle can extend circulation time in vivo.
UPLC-MS/MS method: chromatographic column: shim-packXR-ODS III (1.6um), 2.0mm × 50mm;
Moving phase: methyl alcohol-damping fluid (5mM ammonium acetate, 0.1% acetic acid); Flow velocity: 0.3ml; Column temperature: 40 DEG C; Sample introduction: 10ul;
Gradient:
Embodiment 11
Raise peripheral leukocytes experimental study:
To 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1, the nanocrystal (embodiment 4) of 2] dithiole [4,3-b]-pyrrolidone-ethyl formate (zl-004) and the model mice leukocyte increasing of mPEG-PLGA nanoparticle (embodiment 8) and relevant cell effect are investigated.By endoxan modeling (modeling foundation: for peripheral leukocytes quantity is lower than 3.0 × 10 9), with ZL-004 nanocrystal (embodiment 2) and ZL-004mPEG-PLGA nanoparticle (embodiment 6), commercial preparation GM-CSF, non-administration model group and physiological saline group for study group, cultivate to be counted as index to evaluate rise white effect with periphery quantity of leucocyte and medullary cell, monocyte colony.Experimental group: get 45 mouse, be divided into 5 groups at random, often group intraperitoneal injection of cyclophosphamide (120mg/kg) respectively, once a day, continuous 4 days.Each group of murine interleukin sum is measured, by modeling according to determining selected mouse afterwards with blood analyser.Administration: ZL-004 nanocrystal and mPEG-PLGA nanoparticle calculate with ZL-004 by 3mg/kg() tail vein injection; GM-CSF presses 25ug/kg subcutaneous injection; Once a day, successive administration 7 days.With the 1st, 4,7 day often group get 3, eye socket gets blood 0.5ml, and through blood analysis-e/or determining quantity of leucocyte, femur marrow smear is got in rear execution, calculates medullary cell and monocytic colony number.Result shows: be changed to index by quantity of leucocyte, medullary cell and amount of mononuclear cells, and compared with the GM-CSF of commercial preparation, zl-004 nanocrystal group, zl-004-MPEG-PLGA nanoparticle group have significant leukocyte increasing function.Specifically in table 1,2.
The change (embodiment 11) of quantity of leucocyte after the different preparation intravenously administrable of table 1zl-004
Compare with endoxan group, " * " p<0.05.
The change (embodiment 11) of medullary cell and amount of mononuclear cells after the different preparation administration of table 2
Compare with endoxan group, " * " p<0.05.

Claims (6)

1. dithiole the nanoparticle of pyrrolidone compound, is characterized in that, comprise the component of following weight percent:
The per-cent sum of each component is 100%;
Described dithiole pyrrolidone compound is 6-amino-4-(2,4-dimethoxy phenyl)-4-hydrogen [1,2] dithiole [4,3-b]-pyrrolidone-ethyl formate;
The mean diameter 10nm-800nm of described nanometer;
Described biodegradable polymers is polyoxyethylene glycol-glycolide-lactide copolymer or the lactic-co-glycolic acid-ethylene glycol copolymer of methylating, methylate in polyoxyethylene glycol-glycolide-lactide copolymer, the scope of monomer molar ratio is 50: 50-25: 75, the weight-average molecular weight of polyoxyethylene glycol-glycollide-rac-Lactide or lactic-co-glycolic acid-ethylene glycol copolymer of methylating is 5,000-60,000 dalton;
Described additive is selected from polyethylene glycol stearate or polyoxyethylene hydrogenated castor oil;
Described freeze-drying caffolding agent is selected from N.F,USP MANNITOL, sucrose, lactose, glucose or sodium alginate.
2. preparing dithiole according to claim 1 and the nanoparticle preparation method of pyrrolidone compound, it is characterized in that, is adopt following nanoparticle preparation facilities to realize:
Nanoparticle preparation facilities comprises aqueous phase reservoir (1), oil phase reservoir (2), ultrasonic generator (6), ultrasonication chamber (9), frame (4), the static mixer (5) being provided with inner member (501), circulation tube (10), fluid accept cup (7), agitator (8);
Described static mixer (5) by anchor (401) vertical be fixed in described frame (4);
The outlet of described aqueous phase reservoir (1) is connected by aqueous phase pipeline and is connected with described ultrasonication chamber (9), the outlet of described oil phase reservoir (2) is connected with described ultrasonication chamber (9) by oil phase pipeline, and the probe (601) of described ultrasonic generator (6) is inserted in described ultrasonication chamber (9);
Described ultrasonic generator (6) is mechanically fixed in frame (4);
Described fluid accepts the below that cup (7) is arranged on the outlet (503) of described static mixer (5) bottom;
Fluid described in the insertion of described circulation tube (10) one end accepts in cup (7), and the other end is connected with described aqueous phase reservoir (1) by handling equipment.
3. method according to claim 2, is characterized in that, comprises the steps:
(1) aqueous phase in the oil phase in oil phase reservoir (2) and aqueous phase reservoir (1) is sent into described ultrasonication chamber (9) simultaneously, and start ultrasonic generator (6);
Described oil phase is the organic solvent containing thia cyclopenta pyrrolidone compound, biodegradable polymer and additive;
Described aqueous phase is the aqueous solution containing polyvinyl alcohol;
Oil phase and aqueous phase form oil-in-water nanoscale emulsion, flow out from the outlet of static mixer (5) bottom, enter fluid and accept cup (7), under the stirring of agitator (8), nanoscale emulsion fluid accepted in cup (7) sends aqueous phase reservoir (1) back to by circulation tube (10), move in circles said process, until nanoparticle particle diameter reaches requirement;
(2) then fluid is accepted the material in cup (7), 3-20 DEG C, 10000-50000rmp is centrifugal, collect solid matter, wash, add freeze-drying caffolding agent, freeze-drying, described in acquisition containing dithiole the nanoparticle of pyrrolidone compound.
4. method according to claim 3, it is characterized in that, described organic solvent is selected from tetrahydrofuran (THF), acetone, the alcohols of carbon atom 1 to 4, N, dinethylformamide or dimethyl sulfoxide (DMSO), dioxane, acetonitrile, methylene dichloride, trichloromethane, ethyl acetate, N, N-dimethyl pyrrolidone or phenylcarbinol.
5. method according to claim 3, is characterized in that, in described oil phase, total concentration of thia cyclopenta pyrrolidone compound, biodegradable polymer and additive is 7.0 ~ 680 grams per liters.
6. method according to claim 3, is characterized in that, with the volumeter of organic solvent and water, and organic solvent: water=1: 9.0 ~ 1: 480;
The speed of oil phase is 1.0-4.0mlmin -1; The speed of aqueous phase is 150-350mlmin -1;
In described organic solvent, the residence time of oil phase is 20 ~ 70 seconds;
With described ultrasonication chamber 9 volume for benchmark, ultrasonic power is 4.0-235wcm -3.
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