CN105030693A - Lung-targeting gelatin microsphere agent containing cefquinome sulfate and preparation method of lung-targeting gelatin microsphere agents - Google Patents
Lung-targeting gelatin microsphere agent containing cefquinome sulfate and preparation method of lung-targeting gelatin microsphere agents Download PDFInfo
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Abstract
The invention discloses a lung-targeting gelatin microsphere agent containing cefquinome sulfate and a preparation method of the lung-targeting gelatin microsphere agent. According to the lung-targeting gelatin microsphere agent, cefquinome sulfate is taken as the active ingredient, gelatin or gelatin-chitosan compound is taken as a carrier, and the weight ratio of the active ingredient to the carrier is 1 to 2-6. The invention further provides the preparation method of the lung-targeting gelatin microsphere agent. The encapsulation efficiency of the prepared microspheres is 65% or above, the particle sizes of 80% or more of the microspheres are distributed within 7-35 um, so that the microsphere agent can gather to the lung in a targeted manner, the therapeutic effect of the medicine is effectively improved, the toxic and side effects of the medicine are reduced, meanwhile, the retaining time of the medicine in the lug is prolonged, the concentration of the blood concentration is kept stable, and the long-term effect is achieved.
Description
Technical field
The present invention relates to veterinary formulations technical field, particularly a kind of lung targeting gelatin microsphere preparation and preparation method thereof of sulphuric acid cephalosporium quinol.
Background technology
Cefquinome is the 1st animal specific the 4th generation cephalosporins, is widely used in treating the respiratory system disease of domestic animal both at home and abroad because of its strong antibacterial activity, wide antimicrobial spectrum and low drug resistance.
The research and development of China to Cefquinome and preparation thereof are relatively late, only have sulphuric acid cephalosporium quinol crude drug and injection (suspension injection, injection powder injection) thereof to obtain Ministry of Agriculture's approval production and selling license at present, the type of dosage form matched with it is fewer.Cefquinome oral absorption is bad, and injection absorbs comparatively rapid, and the half-life is shorter in vivo, is only 1 ~ 3 hour.Effectively treating concentration for reaching, needing multiple injection administration, bring very large inconvenience to clinical practice.Therefore, the preparation with target slow-release performance is developed extremely urgent.
Gelatin lung-targeted microspheres can be trapped in pulmonary, makes the drug-rich of load in pulmonary, effectively improves therapeutic drug level, greatly reduces medicine to the toxic and side effects of non-target organ; Meanwhile, the sustained release performance of gelatine microsphere, can make the object that medicine reaches long-acting, and then avoids the inconvenience of frequent drug administration.
How to solve the limitation existing for domestic animal sulphuric acid cephalosporium quinol ordinary preparation, improve its using value, the novel formulation that deep development is raw material with it, and to be applied in production reality be the problem to be solved in the present invention.
Summary of the invention
Technical problem to be solved by this invention is lung targeting gelatin microsphere preparation providing a kind of sulphuric acid cephalosporium quinol and preparation method thereof, drug selectivity can be realized and concentrate on pulmonary, improve drug effect, reduce toxicity, medicine can be realized at pulmonary's slow releasing simultaneously, play long-acting effect.
In order to achieve the above object, the invention provides a kind of lung targeting gelatin microsphere preparation of sulphuric acid cephalosporium quinol, wherein, its preparation method is:
1) sulphuric acid cephalosporium quinol and carrier is got, carrier is added 40 ~ 50 DEG C of water and becomes 10-20% carrier solution, by sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 400-600W, ultrasonic 4-6s, stop being distributed in carrier solution in 5 circulations of 3-5s, form aqueous phase;
2) ratio being 20-50:1 according to volume ratio measures liquid paraffin and emulsifying agent, mixing becomes oil phase, get step 1) aqueous phase that obtains and oil phase with volume ratio for 2 ~ 5:20, be preheating to 50-60 DEG C, under 400 ~ 1100rpm rotating speed stirs, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 15-30min;
3) step 2) ice bath 20-40min at obtain emulsion 0-4 DEG C, stirring at low speed, slowly drips cross-linking agent 0.5 ~ 2mL, cross-linking reaction 1.5-2h;
4) by step 3) add 25-30mL isopropyl alcohol in the reactant that obtains, 20 ~ 30min, the 0.45-5 μm of membrane filtration that dewater obtains microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
Wherein, described carrier is gelatin, and the mass ratio of described crude drug and described carrier is 1:1 ~ 8.
Wherein, described carrier also can be the mixture of gelatin, chitosan, and wherein the ratio of quality and the number of copies of gelatin and chitosan is: 1:5-8.
Wherein, described carrier can also be gelatin, chitosan, N, N-dimethyl acetylamide, N-[3-(amino methyl) benzyl] t-butyl carbamate and Methanamide, its ratio of quality and the number of copies: 0.5-1:5-8:0.05-0.1:0.01-0.05:0.01-0.03.
Wherein, above-mentioned steps 2) middle emulsifying agent employing sorbitol anhydride oleate.
Wherein, above-mentioned steps 2) in emulsifying agent also adopt the mixture of sorbitol anhydride oleate, Tripolyglycerol monostearates and ten polyglycerol distearates, its mass fraction ratio is: 1-2:0.1-0.5:0.1-0.2.
Wherein, described cross-linking agent is the glutaraldehyde solution of 50%.
Wherein, described cross-linking agent can also be trimethylolpropane solution, the isocyanide ester solution of 10%, the mixed solution of 50% glutaraldehyde solution of 20%, and ratio of quality and the number of copies is: 0.1-0.3:0.05-0.1:0.5-1.
Wherein, the particle size range of described microsphere is 7 ~ 35um.
The invention has the beneficial effects as follows: the microsphere drug loading prepared by the present invention is greatly improved, envelop rate is more than 65%, the microspherulite diameter of more than 80% is distributed within the scope of 7 ~ 35um, therefore microball preparation can be enriched to pulmonary by targeting, the curative effect of effective raising medicine, reduces the toxic and side effects of medicine; Simultaneously can prolong drug in the holdup time of pulmonary, maintain blood drug level steady, play long-acting effect.
Figure of description
Fig. 1 is the mode of appearance of medicine carrying gelatine microsphere of the present invention: (A) 1:3 medicine carrying is than the gelatine microsphere (× 100 times) of preparation; (B) 1:3 medicine carrying is than the chitosan-gelatin complex microsphere (× 100 times) of preparation; And (D) medicine carrying gelatine microsphere and medicine carrying chitosan-gelatin complex microsphere SEM observed result (C);
Fig. 2-1 is Cefquinome and medicine carrying gelatine microsphere agent in vitro dissolution rate;
Fig. 2-2 is medicine carrying gelatine microsphere and the external dissolution rate of medicine carrying chitosan-gelatin complex microsphere;
Fig. 3 is the distribution that after single dose tail vein injection gelatine microsphere, mice respectively organizes Chinese medicine;
Detailed description of the invention
In order to achieve the above object, the invention provides a kind of lung targeting gelatin microsphere preparation of sulphuric acid cephalosporium quinol, wherein, its preparation method is:
1) sulphuric acid cephalosporium quinol crude drug and carrier is got, carrier is added 40 ~ 50 DEG C of water and becomes 10-20% carrier solution, by sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 400-600W, ultrasonic 4-6s, stop being distributed in carrier solution in 5 circulations of 3-5s, form aqueous phase;
2) ratio being 20-50:1 according to volume ratio measures liquid paraffin and emulsifying agent, mixing becomes oil phase, get step 1) aqueous phase that obtains and oil phase with volume ratio for 2 ~ 5:20, be preheating to 50-60 DEG C, under 400 ~ 1100rpm rotating speed stirs, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 15-30min;
3) step 2) ice bath 20-40min at obtain emulsion 0-4 DEG C, stirring at low speed, slowly drips cross-linking agent 0.5 ~ 2mL, cross-linking reaction 1.5-2h;
4) by step 3) add 25-30mL isopropyl alcohol in the reactant that obtains, 20 ~ 30min, the 0.45-5 μm of membrane filtration that dewater obtains microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
Wherein, described carrier is gelatin, and the mass ratio of described crude drug and described carrier is 1:1 ~ 8.
Wherein, described carrier also can be the mixture of gelatin, chitosan, and wherein the ratio of quality and the number of copies of gelatin and chitosan is: 1:5-8.
Wherein, described carrier can also be gelatin, chitosan, N, N-dimethyl acetylamide, N-[3-(amino methyl) benzyl] t-butyl carbamate and Methanamide, its ratio of quality and the number of copies: 0.5-1:5-8:0.05-0.1:0.01-0.05:0.01-0.03.
Wherein, above-mentioned steps 2) middle emulsifying agent employing sorbitol anhydride oleate.
Wherein, above-mentioned steps 2) in emulsifying agent also adopt the mixture of sorbitol anhydride oleate, Tripolyglycerol monostearates and ten polyglycerol distearates, its mass fraction ratio is: 1-2:0.1-0.5:0.1-0.2.
Wherein, described cross-linking agent is the glutaraldehyde solution of 50%.
Wherein, described cross-linking agent can also be trimethylolpropane solution, the isocyanide ester solution of 10%, the mixed solution of 50% glutaraldehyde solution of 20%, and ratio of quality and the number of copies is: 0.1-0.3:0.05-0.1:0.5-1.
Wherein, the particle size range of described microsphere is 7 ~ 35um.
One, the characteristic of Cefquinome lung-targeted microspheres is investigated
1.1 microsphere mode of appearance are observed
The mode of appearance of gelatine microsphere is observed by Optical microscope and SEM.
Sample treatment before observation by light microscope: take appropriate microsphere powder, is distributed in the normal saline of certain volume, and Glass rod dips one and drips on microscope slide, is covered gently by coverslip, be placed in basis of microscopic observation from drop side.
Sample treatment before scanning electron microscopic observation: paste two-sided conducting resinl on sample stage, after dipping microsphere sample powder with toothpick, conducting resinl rolls gently several under, a small amount of microsphere powder is made to be stained with on conducting resinl, after hair-dryer blows unstuck sample off, metal spraying process, upper machine is observed.
1.2 microspherulite diameters are measured
The foundation of microsphere drug content assaying method and microsphere drug carrying ability detect
Chromatograph working condition
Chromatographic column: AgilentC18 post (150mm × 4.6mm, 5 μm)
Mobile phase: acetonitrile-sodium perchlorate solution (12:88)
UV determined wavelength: 269nm
Sample size: 20 μ L
Flow velocity: 1.0mL/min
Column temperature: 25 DEG C
1.3 drug carrying abilities detect
Choose best preparation technology's continuous production 6 batches of medicine carrying gelatine microspheres, detect its drug loading and envelop rate respectively.
1.4 microsphere vitro releases are investigated
Vitro release is investigated with reference to the dialysis in pharmacopeia, and suitably change, concrete grammar is as follows: each 50.0mg of sulphuric acid cephalosporium quinol microsphere accurately taking continuous three batches, be respectively charged in three bag filters (molecular cut off 8000-14000Da), separately take the blank microsphere of homogenous quantities in bag filter, add appropriate amount of drug (suitable with medicine carrying microballoons drug content) to mix with blank microsphere, as blank, disperse with PBS solution (release medium) 2mL of ph7.4, and bag filter two ends rubber band is tightened, be placed in 250mL release medium, the research of drug-eluting speed is carried out under 150rpm rotating speed in 37 DEG C of constant-temperature tables.Sample 1mL respectively at 5min, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h and 36h, add the release medium (completing in this process 30s) of 37 DEG C of equivalent simultaneously.Sampling liquid is after 0.22 μm of membrane filtration, and 4 DEG C of preservations are to be measured.Afterwards, sample is entered HPLC and analyze, obtain the stripping percentage rate of medicine at each time point, draw cumulative release percentage rate-time drug release profiles figure.
1.5 polymeric microspheres stabilize are investigated
Stability test Primary Reference " People's Republic of China's veterinary drug allusion quotation " (2010 editions) first annex 246 pages of microsphere carries out about pharmaceutical preparation stability test guideline.
Influence factor tests
(1) high temperature experiment
(2) high humility experiment
(3) strong illumination experiment
1.6 accelerated test
2 results and discussion
2.1 microsphere mode of appearance observed results
Gelatine microsphere mode of appearance
Direct preparation some batches of medicine carrying gelatine microspheres, adopt Optical microscope and SEM to observe microsphere mode of appearance.
From microsphere picture A, the B of the microsphere photo of Fig. 1, medicine carrying gelatine microsphere is uniformly dispersed in normal saline, slightly adhesion, and this may be relevant with the character of medicine; Chitosan-gelatin complex microsphere is uniformly dispersed, and adhesion is less, and this may be higher relevant with its crosslinking degree.SEM photo C, D can find, medicine carrying gelatine microsphere surface ratio is brighter and cleaner, spherical rounding; The spherical rounding property of chitosan-gelatin complex microsphere is slightly poor, and microsphere surface covers more block, and suspection is the drug crystallization be not embedded.
2.2 microspherulite diameter measurement results
The particle diameter of the 9 batches of microspheres prepared according to following table and particle size distribution are in Table 1-1.
9 groups of microspherulite diameters prepared by orthogonal optimization test are in Table 1-1, and wherein No. 4 group scores are the highest, and the microsphere be distributed between 7-35 μm accounts for 85.56%, and particle size distribution is narrow, and homogeneity is better.
Table 1-1 orthogonal experiment respectively organizes gelatine microsphere particles size and distribution
Best preparation technology is adopted to compare the gelatine microsphere of preparation and the particle diameter of chitosan-gelatin complex microsphere and particle size distribution in Table 1-2 and table 1-3 according to different dispensings, when preparing gelatine microsphere according to 1:3 dispensing, microsphere encapsulation rate is the highest, mean diameter 24.02 μm, the microsphere of particle size distribution between 7-35 μm accounts for 81.53% of sum, and particle size distribution is more concentrated.
Each group of gelatine microsphere particles size and distribution prepared by the different dosage of table 1-2
| Experimental group | Dosage | Mean diameter (μm) | Particle size distribution (%) |
| 1 | 1:10 | 17.85 | 79.56 |
| 2 | 1:8 | 20.56 | 79.93 |
| 3 | 1:6 | 21.98 | 81.64 |
| 4 | 1:4 | 23.16 | 80.46 |
| 5 | 1:3 | 24.02 | 81.53 |
| 6 | 1:2 | 29.16 | 75.65 |
Each group of chitosan-gelatin complex microsphere particles size and distribution prepared by the different dosage of table 1-3
| Experimental group | Dosage | Mean diameter (μm) | Particle size distribution (%) |
| 1 | 1:8 | 23.45 | 75.24 |
| 2 | 1:6 | 25.68 | 75.87 |
| 3 | 1:4 | 28.87 | 71.36 |
| 4 | 1:2 | 30.56 | 70.85 |
| 5 | 1:1 | 34.32 | 39.58 |
2.3 microsphere drug carrying ability testing results
Adopt best preparation technology, according to 1:3 dosage continuous production 5 batches of medicine carrying gelatine microspheres, according to drug loading and the envelop rate of the detection method detection of drugs set up, the results are shown in Table 1-4.As can be seen from the table, stable preparation process, repeatability is good.
Table 1-4 gelatine microsphere drug carrying ability result
| Experimental group | Drug loading (%) | x±SD(％) | Envelop rate (%) | x±SD(％) |
| 1 | 19.89 | 76.56 | ||
| 2 | 19.28 | 74.21 | ||
| 3 | 20.12 | 19.71±1.05 | 77.46 | 75.85±4.05 |
| 4 | 20.57 | 79.18 | ||
| 5 | 18.31 | 70.49 |
2.4 microsphere vitro releases investigate result
The release in vitro of medicine carrying gelatine microsphere is shown in Fig. 2-1, and result shows, compares crude drug, and the slow releasing function of microball preparation in release medium is obvious.As seen from the figure, namely crude drug releases more than 90% at front 4h in bag filter, and rate of release is very fast; Gelatine microsphere preparation releases about 50% at front 4h, and when to 36h, just basic release completely.
Do a collection of medicine carrying gelatine microsphere and medicine carrying chitosan-gelatin complex microsphere continuously, observe its release in vitro situation, specifically see Fig. 2-2.Result shows.Add chitosan in gelatin after, its slow release effect slightly promotes, and this may be because the amino in chitosan structure adds the crosslinking degree of microsphere surface, causes the constraint ability of microsphere to medicine slightly to strengthen.
3 brief summaries
Overall merit has been carried out for prepared gelatine microsphere in this part.Carry out integrated survey from stability five aspects of the mode of appearance of microsphere, particle diameter and particle size distribution, drug carrying ability, release in vitro situation and microsphere, and set up liquid phase detection method.Result shows, the gelatine microsphere prepared by the technique of optimization, form rounding and dispersion; Gelatin mean diameter 24.02 μm, size meets the requirement of targeted microspheres, and particle size distribution mainly concentrates on 7 ~ 35 μm; The detection method set up is stablized feasible, and the drug loading of gelatine microsphere can reach 19.71% respectively, and average envelop rate can reach 75.85% respectively; Release in vitro result shows, gelatine microsphere has certain sustained release performance; Study on the stability result shows, microsphere at room temperature protection against the tide keep in Dark Place Nature comparison stablize.
Two, the tissue distribution research of Cefquinome lung targeting gelatin microsphere in Mice Body
1 experiment material
1.1 main agents
| Sulphuric acid cephalosporium quinol standard substance | Content 95% | The bright magnificent pharmacy in Shandong |
| Sulphuric acid cephalosporium quinol gelatine microsphere | Qualified | Laboratory is made by oneself |
| Phosphoric acid | Chromatographically pure | Tianjin Ke Miou chemical reagent factory |
| Concentrated hydrochloric acid | Analytical pure | Traditional Chinese medicines group |
| Acetonitrile | Chromatographically pure | U.S. TEDIA |
| Methanol | Chromatographically pure | U.S. TEDIA |
1.2 key instrument
The preparation of 1.3 solution
Sulphuric acid cephalosporium quinol lung-targeted microspheres preparation: under aseptic condition, according to the drug loading of sulphuric acid cephalosporium quinol in microball preparation, is mixed with the suspensoid injectio of certain medicament contg with sterile saline, now with the current;
Sulphuric acid cephalosporium quinol standard reserving solution (1600 μ g/mL): take 1.6mg sulphuric acid cephalosporium quinol standard substance in 1mL centrifuge tube, accurately add 1mL deionized water, make the mother solution of sulfur acid Cefquinome 1600 μ g/mL, 4 DEG C of Refrigerator stores;
Sulphuric acid cephalosporium quinol standard working solution: with deionized water dilute sulphuric acid Cefquinome standard reserving solution (1600 μ g/mL) to the solution of desired concn, be standard working solution, now with the current;
0.1% aqueous formic acid: get 1mL chromatograph formic acid, deionized water is diluted to 1000mL;
7.5% aqueous hydrochloric acid solution: get 75mL concentrated hydrochloric acid, adds deionized water and is diluted to 1000mL;
C18 post rinse liquid washing liquid: methanol: water (20:80);
C18 post eluent: methanol: 7.5% aqueous hydrochloric acid solution (5:95)
1.4 laboratory animal
Cleaning grade Kunming white mice, body weight 20 ± 2g, male and female dual-purpose, before experiment, within 3 days, to conform, institute's feed material containing any antibiotic, is not freely drunk water in pre-raising.
2 test methods
2.1 animals administers and tissue sample collection
Sulphuric acid cephalosporium quinol gelatine microsphere preparation group white mice often only presses the administration of 15mg/kgbw single dose tail vein injection.
Each experimental group white mice all after tail vein injection 0.25,0.5,2,6,12,24,48,72h cuts open and kills, gather mouse core, liver, spleen, lung and kidney five kinds of internal organs in 10mL centrifuge tube ,-80 DEG C of cryostates save backup.
2.2 tissue sample pre-treatments
Accurately taking 0.1g respectively organizes internal organs in 10mL centrifuge tube, adds 0.5% aqueous formic acid 1mL, homogenized, is placed in 1.5mL centrifuge tube, and add 150 μ l concentrated hydrochloric acid sedimentation albumen, in 4 DEG C of centrifuges, the centrifugal 15min of 12000rpm, gets supernatant.
C18 solid-phase extraction column with 5mL methanol, 5mLC18 post rinse liquid washing liquid, 5mL deionized water, the rinse of 3mLC18 post eluent; The supernatant of above-mentioned tissue extraction is added 0.5% aqueous formic acid, and be diluted to 2mL and cross post immediately, coutroi velocity is less than 1mL/min, effluent after collection post; Carry out eluting with 2mLC18 post eluent, collect eluent, to be merged into after above-mentioned post in effluent.
Above-mentioned is crossed post amalgamation liquid freeze overnight in-80 DEG C of cryostates, and lyophilizing, adds 0.5mL0.5% aqueous formic acid vortex dissolved residue, the centrifugal 10min of 12000rpm, gets supernatant and crosses 0.22 μm of filter membrane, detect with the sample size UPLC-MS/MS of 10 μ l.
2.3UPLC-MS/MS detect
2.3.1 chromatograph working condition and mass spectrometry parameters
Chromatographic column: AgilentSB-C8 post, 2.1 × 100mm, 1.8 μm
Mobile phase: A (0.1 aqueous formic acid), B (acetonitrile)
Mass spectrometer system: Infinity injection stream ion focusing series connection quadrupole rod 6460 mass spectrograph
Ionization pattern: positive ion mode (ESI+)
Scan type: MRM
Cefquinome quota ion (m/z): 134
Table 1 gradient elution program
Table 2 mass spectrograph major parameter
| Parameter | Parameter value |
| Precursor Ion(m/z) | 529 |
| Product Ion(m/z) | 134 |
| Dwell | 200 |
| Fragmentor | 65 |
| Collision Energy(eV) | 13 |
| Cell Accelerator Voltage | 3 |
| Gas Temp(℃) | 350 |
| Gas Fiow(l/min) | 8 |
| Nebulizer(psi) | 25 |
| Sheath Gas Temp(℃) | 350 |
| Sheath Gas Flow(l/min) | 7 |
| Capillary(V) | 4000 |
2.3.2 the formulation of organizational standard curve
Get the blank tissue 0.1g of each internal organs respectively, add the homogenate of 1mL0.1% aqueous formic acid, add the sulphuric acid cephalosporium quinol standard substance working solution of certain volume respectively, make homogenised tissue liquid drug concentration be respectively 5,25,50,100,250,500,2500ng/g, process, by liquid quality detection.Add concentration for abscissa (X) with the sulphuric acid cephalosporium quinol in tissue, the peak area (A) of liquid quality detection is vertical coordinate, drawing standard curve, obtains and linearly looks back equation and correlation coefficient.Sample concentration exceeds the range of linearity, detects with mobile phase dilution.
2.3.3 determination of recovery rates
Get the blank tissue 0.1g of each internal organs respectively, add the homogenate of 1mL0.1% aqueous formic acid, add the sulphuric acid cephalosporium quinol standard substance working solution of certain volume respectively, make homogenised tissue liquid drug concentration be respectively 25,100,500ng/g, according to method process under 4.2.2 item, parallel 5 times of each concentration, by liquid quality detection peak area (A1).
The another blank tissue 0.1g getting each internal organs respectively, add the homogenate of 1mL0.1% aqueous formic acid, after method process under 4.2.2 item, add the sulphuric acid cephalosporium quinol standard substance working solution of certain volume respectively, make solution final concentration be respectively 25,100,500ng/g, parallel 5 times of each concentration, by liquid quality detection peak area (A2).
The response rate=A1/A2 × 100%.
2.3.4 precision measures
Get the blank tissue 0.1g of each internal organs respectively, add the homogenate of 1mL0.1% aqueous formic acid, add the sulphuric acid cephalosporium quinol standard working solution of certain volume respectively, make sample drug concentration be respectively 25,100,500ngg, process, by liquid quality detection.Each concentration is parallel 5 parts (in a few days) within same working day, processing different operating day 5 batches (in the daytime), calculate in a few days and in the daytime relative standard deviation.
2.3.5 detectability and quantitative limit
Getting blank tissue, add certain density Cefquinome standard working solution, after process, carry out UPLC-MS/MS detection, be detectability (LOD), S/N=10 is quantitative limit (LOQ) by signal to noise ratio S/N=3.Ask detectability and the quantitative limit of Cefquinome in tissue.
3 results and discussion
3.1 result
3.1.1 chromatographic behavior
Under above-mentioned chromatographic condition, the appearance time of sulphuric acid cephalosporium quinol is greatly about 4.03min, and peak type is good, and free from admixture disturbs.
3.1.2 organizational standard Dependence Results
By measuring, Organs of Mice organizes Chinese medicine to add concentration between 5 ~ 2500ng/g, and drug entities concentration and peak area linear relationship well, the results are shown in Figure 3.The linear equation of each organs and tissues Chinese medicine and correlation coefficient are in table 3, and as seen from table, the correlation coefficient of each internal organs standard curve all can reach more than 0.9990.
Table 3 linear equation and correlation coefficient
| Sample | Standard curve equation | Correlation coefficient (R) |
| Heart | Y＝20.694X+10.781 | 0.9998 |
| Liver | Y＝21.105X-7.1858 | 0.9997 |
| Spleen | Y＝20.179X-96.843 | 0.9990 |
| Lungs | Y＝14.009X-15.962 | 0.9999 |
| Kidney | Y＝22.038X+84.284 | 0.9994 |
3.1.3 determination of recovery rates result
The response rate of table 4 heart Chinese medicine
The response rate of table 5 liver Chinese medicine
The response rate of table 6 spleen Chinese medicine
The response rate of table 7 lungs Chinese medicine
The response rate of table 8 kidney Chinese medicine
3.1.4 precision measurement result
The precision of table 9 heart Chinese medicine
The precision of table 10 liver Chinese medicine
The precision of table 11 spleen Chinese medicine
The precision of table 12 lungs Chinese medicine
The precision of table 13 kidney Chinese medicine
3.1.5 detectability and quantitative limit
Record the detection of Cefquinome in blank tissue and be limited to 0.5ng/g, be quantitatively limited to 2ng/g.
3.1.5 the determination of drug concentration result in tissue
After mice single dose tail vein injection, medicine organizes the concentration of different time points in table 14 in different organs.
Mouse tissue drug concentration after table 14 single dose tail vein injection gelatine microsphere preparation
After single dose tail vein injection, the scattergram of medicine in each internal organs is shown in Fig. 3.
3.1.6 pharmacokinetic parameters
Main pharmacokinetic parameters after table 15 single dose tail vein injection sulphuric acid cephalosporium quinol gelatine microsphere in different organs tissue
3.1.7 targeting analysis
The targeting of microball preparation is evaluated by this parameter of targeting efficiency.Targeting efficiency te=(AUC) target/(AUC) non-target, wherein, in formula, te represents that pharmaceutical preparation or drug solution are to the selectivity of target organ.Targeting efficiency value is greater than 1 expression pharmaceutical preparation to the selectivity of target organ higher than non-target organ; Targeting efficiency value is larger, proves that the selectivity of pharmaceutical preparation to target organ is stronger.For evaluating the targeting of microball preparation prepared by this problem to pulmonary, analyzing the targeting efficiency of lung compared to other organs, the results are shown in Table 16.
The targeting efficiency of mouse lung after table 16 single dose tail vein injection microball preparation
4 brief summaries
Table 16 is known, and after injection Cefquinome gelatine microsphere preparation, lung tissue is all greater than 1 relative to the targeting efficiency of other each internal organs, and targeting is obvious.
Sum up, the microsphere drug loading prepared by the present invention is greatly improved, and envelop rate is more than 65%, the microspherulite diameter of more than 80% is distributed within the scope of 7 ~ 35um, therefore microball preparation can be enriched to pulmonary by targeting, effectively improves the curative effect of medicine, reduces the toxic and side effects of medicine; Simultaneously can prolong drug in the holdup time of pulmonary, maintain blood drug level steady, play long-acting effect.
In order to further illustrate the present invention, state below by detailed description of the invention.
Embodiment 1
Accurately take 0.60g gelatin, be dissolved at 50 DEG C in 3mL water, obtain the aqueous solution that gelatin concentration is 20%; Accurate weighing 0.20g sulphuric acid cephalosporium quinol, is added in gelatin solution, and disperse 5 circulations with Probe Ultrasonic Searching, ultrasonic power 600W, ultrasonic 4s, stops 5s, as final aqueous phase; Measure 20mL liquid paraffin, add 0.4mL emulsifying agent span-80, mixing, is preheating to 50 DEG C, and above-mentioned aqueous phase (medicine gelatin solution) is slowly added drop-wise in this oil phase, stirring and emulsifying 15min under stirring by 800rpm rotating speed; Ice bath 20min at emulsion 4 DEG C, stirring at low speed, slowly drips 50% glutaraldehyde water solution 1.0mL, cross-linking reaction 1.5h; Add cold isopropanol 25mL, dehydration 25min, 0.45um membrane filtration, obtains microsphere; With washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains medicine carrying microballoons powder.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 24.02 μm, and the microsphere of 81.53% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 19.98%, and envelop rate is 76.90%, and vitro Drug stripping can reach 36h.
Embodiment 2
Accurately take 0.40g gelatin, 0.05g chitosan, be placed in 2% aqueous acetic acid water 3mL and dissolve at 50 DEG C, obtained carrier material concentration is the aqueous solution of 15%; Accurate weighing 0.225g sulphuric acid cephalosporium quinol, is added in carrier material solution, and disperse 5 circulations with Probe Ultrasonic Searching, ultrasonic power 600W, ultrasonic 4s, stops 5s, as final aqueous phase; Measure 20mL liquid paraffin, add 0.4mL emulsifying agent span-80, mixing, is preheating to 50 DEG C, and above-mentioned aqueous phase is slowly added drop-wise in this oil phase, stirring and emulsifying 15min under stirring by 800rpm rotating speed; Ice bath 20min at emulsion 4 DEG C, stirring at low speed, slowly drips 50% glutaraldehyde water solution 1.5mL, cross-linking reaction 1.5h; Add cold isopropanol 25mL, dehydration 25min, 0.45 μm of membrane filtration, obtains microsphere; With washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains medicine carrying microballoons powder.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.56 μm, and the microsphere of 70.85% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.51%, and envelop rate is 70.40%, and drug release in vitro can reach more than 36h.
Embodiment 3
1) 0.09g gelatin, 0.66g chitosan, 0.006gN is got, N-dimethyl acetylamide, 0.001gN-[3-(amino methyl) benzyl] t-butyl carbamate and 0.043g Methanamide, add 3.2ml40 DEG C of water and become 20% carrier solution, by 0.2g sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 600W, ultrasonic 5s, stop being distributed in carrier solution in 5 circulations of 5s, form aqueous phase;
2) get 20ml liquid paraffin and 0.67ml sorbitol anhydride oleate, mixing becomes oil phase, gets step 1) aqueous phase that obtains, be preheating to 50 DEG C, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 20min under stirring by 800rpm rotating speed;
3) step 2) ice bath 30min at the emulsion 4 DEG C that obtains, stirring at low speed, slowly drips the glutaraldehyde solution 2mL of cross-linking agent 50%, cross-linking reaction 1.5h;
4) by step 3) add 30mL isopropyl alcohol, dehydration 30min, 0.45 μm of membrane filtration in the reactant that obtains, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.66 μm, and the microsphere of 78.82% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.41%, and envelop rate is 70.50%, and drug release in vitro can reach more than 36h.
Embodiment 4
1) get gelatin 0.8g, body adds 2.4ml50 DEG C of water becomes 20% carrier solution, by 0.1g sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 500W, ultrasonic 5s, stops being distributed in carrier solution in 5 of 5s circulations, forms aqueous phase;
2) mixture of 20ml liquid paraffin and 0.56ml sorbitol anhydride oleate, 0.06ml Tripolyglycerol monostearates, 0.05ml ten polyglycerol distearate is got, mixing becomes oil phase, get step 1) aqueous phase that obtains, be preheating to 50 DEG C, under 800rpm rotating speed stirs, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 20min;
3) step 2) ice bath 30min at the emulsion 4 DEG C that obtains, stirring at low speed, slowly drips the glutaraldehyde solution 1mL of cross-linking agent 50%, cross-linking reaction 1.5h;
4) by step 3) add 30mL isopropyl alcohol, dehydration 20min in the reactant that obtains, 1 μm of membrane filtration obtains microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.56 μm, and the microsphere of 85.75% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.59%, and envelop rate is 71.20%, and drug release in vitro can reach more than 36h.
Embodiment 5
1) get 0.8g gelatin to add 3.2ml50 DEG C of water and become 20% carrier solution, by 0.1g sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 600W, ultrasonic 6s, stops being distributed in carrier solution in 5 circulations of 5s, forms aqueous phase;
2) get 30ml liquid paraffin and 1ml sorbitol anhydride oleate, mixing becomes oil phase, gets step 1) aqueous phase that obtains, be preheating to 60 DEG C, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 20min under stirring by 800rpm rotating speed;
3) step 2) ice bath 40min at the emulsion 4 DEG C that obtains, stirring at low speed, slowly drips trimethylolpropane solution, the isocyanide ester solution of 0.15ml10%, the mixed solution of 1.54ml50% glutaraldehyde solution of 0.31ml20%, cross-linking reaction 2h;
4) by step 3) add 30mL isopropyl alcohol, dehydration 30min, 5 μm of membrane filtrations in the reactant that obtains, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.42 μm, and the microsphere of 85.85% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.58%, and envelop rate is 72.50%, and drug release in vitro can reach more than 36h.
Embodiment 6
1) 0.05g gelatin, 0.5g chitosan, 0.005gN is got, N-dimethyl acetylamide, 0.001gN-[3-(amino methyl) benzyl] t-butyl carbamate and 0.001g Methanamide, add 50 DEG C of water and become 20% carrier solution, by 0.09g sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 600W, ultrasonic 6s, stop being distributed in carrier solution in 5 circulations of 5s, form aqueous phase;
2) 20ml liquid paraffin and 1ml emulsifying agent is got, the mixture of 0.83ml sorbitol anhydride oleate, 0.083ml Tripolyglycerol monostearates and 0.087ml ten polyglycerol distearate, mixing becomes oil phase, get step 1) aqueous phase that obtains and oil phase take volume ratio as 2:20, be preheating to 50 DEG C, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 20min under stirring by 800rpm rotating speed;
3) step 2) ice bath 40min at the emulsion 4 DEG C that obtains, stirring at low speed, slowly drips the glutaraldehyde solution 2mL of cross-linking agent 50%, cross-linking reaction 1.5h;
4) by step 3) add 30mL isopropyl alcohol, dehydration 30min in the reactant that obtains, 0.45 μm of membrane filtration obtains microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.73 μm, and the microsphere of 85.75% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.63%, and envelop rate is 71.71%, and drug release in vitro can reach more than 36h.
Embodiment 7
1) 0.05g gelatin, 0.44g chitosan, 0.005gN is got, N-dimethyl acetylamide, 0.002gN-[3-(amino methyl) benzyl] t-butyl carbamate and first 0.003g amide, add 2ml50 DEG C of water and become 20% carrier solution, by 0.1g sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 600W, ultrasonic 6s, stop being distributed in carrier solution in 5 circulations of 5s, form aqueous phase;
2) mixture of 50ml liquid paraffin and 0.59ml sorbitol anhydride oleate, 0.29ml Tripolyglycerol monostearates and 0.12ml ten polyglycerol distearate is got, mixing becomes oil phase, get step 1) aqueous phase that obtains and oil phase take volume ratio as 5:20, be preheating to 60 DEG C, under 800rpm rotating speed stirs, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 30min;
3) step 2) ice bath 40min at the emulsion 4 DEG C that obtains, stirring at low speed, slowly drips trimethylolpropane solution, the isocyanide ester solution of 0.15ml10%, the mixed solution of 1.54ml50% glutaraldehyde solution of 0.31ml20%, cross-linking reaction 2h;
4) by step 3) add 30mL isopropyl alcohol, dehydration 30min in the reactant that obtains, 5 μm of membrane filtrations obtain microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
The microsphere of above-mentioned preparation, observation by light microscope finds the even and less adhesion of microsphere size, mean diameter 30.32 μm, and the microsphere of 85.95% is distributed within the scope of 7 ~ 35 μm; Scanning electron microscope display outward appearance rounding, surface compact is bright and clean; Drug loading is 23.68%, and envelop rate is 70.80%, and drug release in vitro can reach more than 36h.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a lung targeting gelatin microsphere preparation for sulphuric acid cephalosporium quinol, is characterized in that, its preparation method is:
1) get sulphuric acid cephalosporium quinol and carrier, carrier being added 40 ~ 50 DEG C of water becomes 10-20% carrier solution, by sulphuric acid cephalosporium quinol with ultrasonic probe at ultrasonic power 400-600W, ultrasonic 4-6s, stops being distributed in carrier solution in 5 of 3-5s circulations, forms aqueous phase;
2) ratio being 20-50:1 according to volume ratio measures liquid paraffin and emulsifying agent, and mixing becomes oil phase, gets the aqueous phase that step 1) obtains, be preheating to 50-60 DEG C, described aqueous phase is slowly added drop-wise in described oil phase, stirring and emulsifying 15-30min under stirring by 400 ~ 1100rpm rotating speed; Described aqueous phase is 2 ~ 5:20 with oil phase volume ratio;
3) step 2) ice bath 20-40min at obtain emulsion 0-4 DEG C, stirring at low speed, slowly drips cross-linking agent 0.5 ~ 2mL, cross-linking reaction 1.5-2h;
4) add 25-30mL isopropyl alcohol in reactant step 3) obtained, 20 ~ 30min, the 0.45-5 μm of membrane filtration that dewater obtains microsphere, with washed with diethylether microsphere once, washing with acetone twice, normal-temperature vacuum is dry, obtains lung targeting gelatin microsphere.
2. the lung targeting gelatin microsphere preparation of sulphuric acid cephalosporium quinol according to claim 1, is characterized in that, described carrier is gelatin, and the mass ratio of described crude drug and described carrier is 1:1 ~ 8.
3. the lung targeting gelatin microsphere preparation of sulphuric acid cephalosporium quinol according to claim 1 and 2, is characterized in that, described carrier is the mixture of gelatin, chitosan, and wherein the ratio of quality and the number of copies of gelatin and chitosan is: 1:5-8.
4. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of claims 1 to 3, it is characterized in that, described carrier is gelatin, chitosan, N, N-dimethyl acetylamide, N-[3-(amino methyl) benzyl] t-butyl carbamate and Methanamide, its ratio of quality and the number of copies: 0.5-1:5-8:0.05-0.1:0.01-0.05:0.01-0.03.
5. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of Claims 1-4, is characterized in that, above-mentioned steps 2) middle emulsifying agent employing sorbitol anhydride oleate.
6. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of claim 1 to 5, it is characterized in that, above-mentioned steps 2) in emulsifying agent adopt the mixture of sorbitol anhydride oleate, Tripolyglycerol monostearates and ten polyglycerol distearates, its mass fraction ratio is: 1-2:0.1-0.5:0.1-0.2.
7. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of claim 1 to 6, is characterized in that, described cross-linking agent is the glutaraldehyde solution of 50%.
8. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of claim 1 to 7, it is characterized in that, described cross-linking agent is trimethylolpropane solution, the isocyanide ester solution of 10%, the mixed solution of 50% glutaraldehyde solution of 20%, and ratio of quality and the number of copies is: 0.1-0.3:0.05-0.1:0.5-1.
9. the lung targeting gelatin microsphere preparation of the sulphuric acid cephalosporium quinol according to any one of claim 1 to 8, is characterized in that, the particle size range of described lung targeting gelatin microsphere is 7 ~ 35um.
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