CN1255331A - Hollow prosomatic liposome, its preparing process and clinical application of its encapsulated matter - Google Patents
Hollow prosomatic liposome, its preparing process and clinical application of its encapsulated matter Download PDFInfo
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Abstract
A hollow prosomatic liposome can be used to envelope both biological macro-molecular or biotechnical products such as protein, nucleic acid medicines, etc., and chemical medicine, and is sutable for different medicinal-applying approaches such as intravenous injection, oral application and applying medicine via oral cavity, eyes, nose and skin.
Description
This invention relates to a kind of field of pharmaceutical preparations, more specifically say so a kind of add medicine solution after, form the unprecedented body liposome of isoosmotic liposome turbid liquor, be a kind of exsiccant product pharmaceutical preparation and use patent.
Liposome (Liposome) is as pharmaceutical carrier, the research in drug delivery system (Drug Delivery System) and use increasingly extensive expansion, but also have certain limitation.General liposome turbid liquor stores with aqueous dispersion, and the medicine stability of facile hydrolysis is subjected to very big influence.Because easily oxidized, the particle precipitation of phospholipid, gathering, fusion cause entrapped drug leakage etc., these factors make the liposome disperse system can not long-term storage.The instability problem of above-mentioned existence is the important topic of liposome research; In addition, the liposome of injection is at the production process high temperature sterilize, with its bimolecular structure of heavy damage and entrapped thermally labile, activated medicine.In recent years, carry out methods such as freeze thawing, lyophilizing and prepared liposome such as L-asparaginase liposome (EPA 0485 143A), but existed the active medicine L-asparaginase that adds in the preparation to have the unnecessary loss problem.The restriction in above-mentioned liposome itself and preparation, its application also is restricted, though as another kind of liquid lipidosome Lipofecetin entrapped drug not, only be fit to the cell in vitro gene transfection, because of toxicity can not be used greatly in vivo.Just because of these liposome weak points have limited the commercialization of liposome and have used.Be to improve the stability of liposome, the liposome preparation of liquid state is become the liposome of solid-state storage, add the water hydration before the use and form liposome and be employed, this will be avoided the problems relevant with liposome turbid liquor.And then, the research of pro-liposome aspect has appearred.
The external pro-liposome of conventional preparation at present is the preparation that comprises certain medicine, can form liposome though add water, in preparation process, often add medicine, as BYUNG-NAK AHN, et al., J.Microencapsulation, 1995,12 (4): 363-365.O.P.KATARE, et al., J.Microencapsutation, 1995,12 (5): the 487-493 report, this is unfavorable for preparing those unsettled liposomees with active medicine.In addition, because most of biological technology products are thermo-labile, conventional preparation technology easily causes this series products damage inactivation, therefore, is necessary to develop a kind of prospect novel formulation widely, and not principal cartridge not inside, it is important further to face the unprecedented body liposome that the time spent now joins.
The object of the present invention is to provide a kind of can not only encapsulating protein class medicine and nucleic acid drug, and can intravenous injection or other administration, can be widely used in clinical new proliposome preparation.
The technology basis that the present invention relates to homogenize method of liposome is in the past improved, and with the sterilization of centre product process, postlyophilization gets the milky product in preparation process.
According to the present invention, unprecedented body liposome basic composition is soybean phospholipid and cholesterol (mol ratio is 7: 3).
Implementing technological approaches of the present invention mainly contains:
(1), the preparation of class lipoprotein solution: with soybean phospholipid and cholesterol (mol ratio is 7: 3); under thermal condition, be dissolved in phosphate buffer (containing mannitol, polyvinylpyrrolidone); logical nitrogen protection is down through tissue mashing machine's homogenize; made lipoprotein solution in 5～10 minutes by high-pressure emulsification 30～60Mpa pressure again; lipid concentration 0.1-8% wherein; mannitol concentration 0.25-6.5%, polyvinylpyrrolidone 0.02-6%.
(2), above-mentioned class lipoprotein solution was pushed 0.45 μ m Merlon microporous filter membrane, lipid soln, embedding under nitrogen protection, 100 ℃ of 30 minutes circulation steam sterilizations.
(3), the sterilization lipid soln, under aseptic condition, be sub-packed in the ampoule, through lyophilization, sealing by fusing behind the inflated with nitrogen.Make the unprecedented body liposome of white.
Wherein mannitol can also be sucrose, lactose, dextran, trehalose or other polysaccharide.Prepared unprecedented body liposome is characterized in that adding drug solution and carries out hydration and seal and form the medicinal liposome suspension, has to keep in animal or human's body leukocyte in the ability of normal level.
Pro-liposome (Proliposome) is a kind of exsiccant product, after adding entry or pharmaceutical aqueous solution, disperses or dissolves grade of formation and oozes liposome turbid liquor, is applicable to the novel process goods of vein or other administration.
Adopt medicinal composition to prepare the not unprecedented body liposome of entrapped drug, both be different from the conventional pro-liposome of forming by medicine and lipid, overcome its entrapped drug stability problem (medicine itself degraded or oxidized); Be different from conventional liposome turbid liquor again, overcome the pro-liposome that its suspension stability problem (or medicine relevant with lipid leaks) is fit to seal multiple medicine from liposome.
The present invention carries out hydration by precursor blank liposome adding L-asparaginase liquid and seals, and forms L-asparaginase liposome turbid liquor, has kept higher enzymatic activity and stable envelop rate is arranged.The liposome of the prepared L-asparaginase of sealing than original L-asparaginase toxicity reduce, intravenously administrable (high, in) dosage antitumor curative effect improves.Be that L-asparaginase liposome (precursor blank liposome add) i.p. (7400,3700 KU/kg) can significant prolongation transplantability mouse lymphocyte leukemia L
1210, P
388Survival natural law (P＜0.01), its effect is better than the L-asparaginase.In addition, total white blood cells and platelet count in the mice peripheral blood there is not obvious influence; And L-asparaginase high dose group (1000KU/kg) is compared the effect (P＜0.05) that total white blood cells and platelet count in the remarkable reduction mice peripheral blood are arranged with matched group.L-asparaginase liposome is significantly less than the L-asparaginase to the acute toxicity of mouse mainline.Cytology research is the result show, effect L-asparaginase liposome all has significant change in the hematopathy cell of people or Mus through optical microscope and winding radio sem observation.Under the optical microscope, cell rounding, poor growth, structure are unclear.Electron microscopic observation to cancerous cell nuclear dwindle, heterochromatin increases, part karyopycnosis, the fracture of parts of fine after birth, cell are imperfect.Particularly analyze through the flow cytometer dna content: the G0/G1 phase cell of HL-60 cell line obviously increases, enters S phase cell and reduces; And L-asparaginase liposome group reduces 5-8% than L-asparaginase group S phase cell.P
815The cell proliferation index decreased of cell line is more remarkable, L-asparaginase liposome group PI=39.4%, matched group PI=65%; HL-60, L
1210Cell line L-asparaginase liposome group is compared with matched group, and the apoptosis ratio significantly increases, and is respectively 18.19%, 11.67%, and matched group is 2.68%, 2.81%; The former increases by 8 times, and the latter increases by 500.This has reflected that L-asparaginase liposome is more remarkable to the tumor cell in vitro effect.
The present invention makes an explanation with following embodiment, and the purpose of these embodiment is just in order to explain rather than limit by any way the present invention.
The embodiment that finishes in conjunction with the accompanying drawings
The unprecedented body liposome of Fig. 1 L-asparagine enzymatic solution forms
The unprecedented body liposome of the transmission electron microscope photo of liposome (* 250,000) Fig. 2 adds grain that ASP solution forms liposome and adds ASP solution through distribution map 3 unprecedented body liposomes and form the liposome grain and add ASP solution through the unprecedented body liposome of (2 months) Fig. 4 that distributes and form the liposome grain through distribution (3 months) Fig. 5 P815 cell control group Fig. 6 P815 cell experiment A picture group 7 P815 cell experiment C picture groups 8 P815 cell experiment B picture groups 9 HL-60 cell control group Figure 10 HL-60 cell experiment A picture groups 11 L1210 cell control group Figure 12 L1210 cell experiment A picture groups 13 K652 cell control group Figure 14 K652 cell experiment A picture groups 15 P815 cell control group Figure 16 P815 cell experiment A picture groups 17 P815 cell experiment C picture groups 18 HL-60 cell control group Figure 19 HL-60 cell experiment A picture groups 20 L1210 cell control group Figure 21 L1210 cell experiment A picture groups 22 cell proliferation index map
The pro-liposome of the present invention preparation to the envelop rate of L-asparaginase between 46.2 ± 0.99 (%)～52.2 ± 1.25 (%). the coefficient of variation of three lot numbers (RSD%) is all less than 3%.
Unprecedented body liposome forms L-asparaginase suspension through L-door winter enzyme amine enzymatic solution, obtain containing L-asparaginase liposome eluent by SephadexG 200 separation, mix with human serum, 37 ℃ of storages. measure enzyme activity at 0,6,12,18,24,30,36,42,48 hour, calculate the enzyme activity conservation rate.See Table 2
Through 48 hours, (37 ℃) L-asparagine enzyme activity conservation rate well was 90.3% in human serum
The grain of embodiment 3, L-asparaginase liposome is through distributing
Unprecedented body liposome is added L-asparagine enzymatic solution, and hydration obtains liposome turbid liquor, with transmission electron microscope photograph (Fig. 1) Coulter-counter counting (Fig. 2), shows the liposome grain through being distributed in 0.662 ± 0.0792um scope, and the coefficient of variation is 12%.(66141 particles are added up 95% fiducial limit: 0.661～0.662um) L-asparaginase liposome turbid liquor, 4 ℃ store 2 months after, grain is through being distributed in 0.684 ± 0.1um scope, and the coefficient of variation is that 14.6% (42508 particles are added up 95% fiducial limit: the unprecedented body liposome of 0.683～table 1 forms the average envelop rate coefficient of variation of envelop rate lot number envelop rate (RSD) the N=6 % % ± SD % 50.8 of L-asparaginase liposome
49.5
53.296072701??????????????????????????????????????????????????????51.0±1.21????????????????????2.37
50.6
50.9
51.0
49.2
48.4
47.296072702??????????????????????????????????????????????????????48.2±0.99????????????????????2.06
48.2
49.3
46.4
52.3
53.5
53.296072903??????????????????????????????????????????????????????52.2±1.25????????????????????2.39
50.2
52.9
51.4
Table 2 L-asparaginase liposome is vigor conservation rate (37 ℃) time (h) 06 12 18 24 30 36 42 48 vigor in human serum
653.1 644.3 637.0 634.5 629.6 618.5 612.3 600.1 664.2 (unit) vigor conservation rate
100 98.3 97.0 95.9 95.5 94.8 93.1 92.1 90.3 (%) 0.685um) Fig. 3.L-asparaginase liposome through 4 ℃ store 3 months after, grain in being distributed in 0.745 ± 0.179um scope, (the statistics number of particles: 55277,95% fiducial limit: 0.744～0.747um) Fig. 4, the coefficient of variation 24.1%
Embodiment 4.L-asparaginase pro-liposome is to transplantability mouse lymphocyte leukemia L
1210Life prolongation effect
Get DBA/2 mice 18～22g, half and half, 80 of male and female are pressed transplanted tumor organon inoculation L
1210Ascites tumor, by being divided into 8 groups after the kind at random, 10 every group, male and female half and half, respectively by following three dosed administrations: L-asparaginase pro-liposome is (by L-asparaginase 7400,3700,1850KU/kg), L-asparaginase injection (7400,3700,1850KU/kg); Other establishes normal saline blank group (0.9NaCl, 0.4ml/20g) and amycin injection (2mg/kg) group, in inoculation back 24h lumbar injection, once a day, administration is 10 days altogether, observe lotus tumor DBA/2 mice survival natural law (survival more than 60 days mice by 60 days) after the administration, the gained experimental result is carried out statistics (t check) and is handled. and test triplicate, experimental result sees Table 3.
Experimental result shows with normal saline blank table group compares L-asparaginase pro-liposome (L-ASNaseliposomes) and L-asparaginase (L-ASNase)) and amycin (DR) all can the leukemic survival natural law of significant prolongation transplantability mouse lymphocyte (P＜0.01), to L
1210Life prolongation effect DR effect the strongest, its increase in life span is 265.9%; Secondly be L-asparaginase liposome, its increase in life span is up to 184.7%.The L-ASNase effect is the most weak, and its increase in life span is up to 132.9%.L-asparaginase pro-liposome and compares with the L-asparaginase of same dose, and its height, middle dosage group (7400,3700KU/kg) significant prolongation L is arranged all
1210The survival natural law effect (P＜0.05) of mice, the test triplicate, the result is close, and increase in life span is calculated as follows formula:
Increase in life span=(T-C)/C * 100%
C is a matched group The average survival time natural law in the formula; T is that the The average survival time natural law is given in administration. the results are shown in Table 3
Embodiment 5 L-asparaginase pro-liposome are to transplantability mouse lymphocyte leukemia P
388Life prolongation effect
Get DBA/2 mice 18～22g, half and half, 80 of male and female are pressed transplanted tumor organon inoculation P
388Ascites tumor after the inoculation, is divided into 8 groups at random, and 10 every group, male and female half and half, respectively by following dosed administration: L-asparaginase pro-liposome is (by L-ASNase 7400,3700,1850KU/kg); L-asparaginase injection (7400,3700,1850KU/kg); Other establishes normal saline blank group (0.9%NaCl, 0.4ml/20g) and amycin injection (20mg/kg) group, in back 24 hours lumbar injections of inoculation, once a day, continuous 10 days, record lotus tumor DBA/2 mice mean survival time (survival more than 60 days mice by 60 days) after the administration calculated administration group increase in life span, and carrying out statistics (t check) respectively and handle. the test triplicate the results are shown in Table 4
Experimental result shows: compare with normal saline blank group, L-asparaginase liposome, L-asparaginase and amycin all can prolong significantly transplants mouse lymphocyte leukemia P
388Survival natural law (P＜0.01). to P
388The effect of life prolongation effect amycin the strongest, increase in life span is 253.4%, secondly be up to 183.3% for its increase in life span of L-asparaginase pro-liposome, the effect of L-asparaginase is the most weak, its increase in life span is up to 132.1%. and compares with the L-asparaginase injection of same dose, and L-asparaginase pro-liposome height, middle dosage group (7400,3700KU/kg) all have significant prolongation P
388The survival natural law of mice (P＜0.05). the test triplicate, the result is close. the results are shown in Table of the influence of 4. embodiment, 6 L-asparaginase pro-liposome to total white blood cells and platelet count in normal mouse body weight, the peripheral blood
Table 3 L-asparaginase pro-liposome is to transplantability mouse lymphocyte leukemia
L
1210Mean survival time influence (i.p.) (* ± SD, n=10) the experiment cnt preparation animal dosage The average survival time rate elongation lot number kind number KU/kg time (my god) (%)
Matched group 10 8.5 ± 1.57
L-asparaginase 10 7,400 24.2 ± 4.16
△ △ * *184.7
10?????????????1850??????????12.5±3.81
***??????????????47.096072701
The L-asparaginase
10?????????????7400??????????19.8±4.73
***??????????????132.9
Injection
10?????????????3700??????????15.1±3.84
***??????????????77.6
10?????????????1850??????????11.8±3.26
***??????????????38.8
Amycin
10??????????????2mg??????????31.1±7.87
***??????????????265.9
Matched group 10 8.6 ± 1.43
10?????????????7400??????????24.3±4.24
△△ ***???????????182.6
The L-asparaginase
10?????????????3700??????????18.6±3.43
△△ ***???????????116.3
Liposome
10?????????????1850??????????12.3±3.65
***??????????????43.096072702
L-asparaginase 10 7,400 19.5 ± 4.43
* *126.7
10?????????????1850??????????11.5±2.80
***??????????????33.7
Matched group 10 8.6 ± 1.52
10?????????????7400??????????23.9±4.12
△△ ***???????????177.9
The L-asparaginase
10?????????????3700??????????18.9±3.63
△△ ***???????????119.8
Liposome
10?????????????1850??????????10.6±3.14???????????????????23.396072703
L-asparaginase 10 7,400 19.3 ± 4.14
* *124.4
10?????????????1850??????????9.7±3.05????????????????????12.8
Table 4 L-asparaginase pro-liposome is to transplantability mouse lymphocyte leukemia
P
388Mean survival time influence (i.p) (* ± SD, n=10) the experimental preparation animal dosage The average survival time rate elongation lot number kind number KU/kg time (my god) (%)
Matched group 10 8.9 ± 1.10
10?????????????7400?????????24.5±4.97
△△ ***?????????175.3
The L-asparaginase
10?????????????3700?????????18.8±3.55
△△ ***?????????111.2
Liposome
10?????????????1850?????????12.7±3.59????????????????42.796072701
L-asparaginase 10 7,400 19.9 ± 4.75
* *123.6
10?????????????1850?????????11.9±3.01
***?????????????33.7
Matched group 10 8.4 ± 1.35
10?????????????7400?????????23.8±3.60
△△ ***??????????183.3
L-asparaginase 10 3,700 18.5 ± 3.44
△ △ * *120.2
10?????????????7400?????????19.5±4.50
***?????????????132.1
The L-asparaginase
10?????????????3700?????????15.3±3.37
***?????????????82.1
Injection
10?????????????1850?????????11.7±2.95
***?????????????39.3
Matched group 10 8.6 ± 1.43
L-asparaginase 10 7,400 23.4 ± 3.94
△ △ * *172.1
10?????????????1850?????????10.8±3.01?????????????????25.696072903
The L-asparaginase
10?????????????7400?????????19.5±4.09
***?????????????126.7
Injection
10?????????????3700?????????15.0±3.64
***?????????????74.4
10?????????????1850?????????10.4±2.87?????????????????20.9
* * p＜0.01 is compared with matched group
△ △ p＜0.05 is compared with the L-asparaginase injection with dosage
Table 5 L-asparaginase liposome to the influence (i.v.) of mice peripheral blood cells sum and platelet count (* ± SD, n=10)
Dosage mice average weight (gram) erythrocyte number of platelets preparation
(ku/kg) number/mm after the preceding administration of administration
310
9/ rise matched group 19.3 ± 1.3 25.8 ± 2.4 7696 ± 2,130 274 ± 74
10,000 19.2 ± 1.3 24.6 ± 1.8 5781 ± 2,016 256 ± 57L-asparaginase
5,000 19.5 ± 1.4 25.9 ± 2.0 7436 ± 2,083 267 ± 68 liposomees
2500????????19.0±1.1???????26.3±2.4?????????7854±2169????????279±81
10,000 19.3 ± 1.3 23.8 ± 1.9 5196 ± 0.23
*218 ± 61
*The L-asparaginase
5,000 18.9 ± 1.1 25.6 ± 2.1 6924 ± 2,007 269 ± 71 injection
2,500 19.0 ± 1.2 26.0 ± 2.2 7564 ± 2,148 270 ± 63
*P＜0.05 is compared with matched group
Get Kunming mouse 18～22g, 70, be divided into 7 groups at random, every group 10, male and female half and half, if normal saline blank group, experiment is by following dosage: L-asparaginase pro-liposome (by L-ASNase 10000,5000,2500KU/kg): L-asparaginase injection (10000,5000,2500KU/kg); The intraperitoneal injection volume be 0.4/20Gg once a day, continuous 10 days, after drug withdrawal, weighed in first day, and get blood by the eye socket vein, the gained data are carried out statistics (t check) and are handled, experimental result sees Table 5.
Experimental result shows: compare with the blank group, L-asparagine alcohol high dose group (1000KU/kg) has the effect (P＜0.05) of total white blood cells and platelet count in the remarkable reduction mice peripheral blood, and L-asparaginase liposome does not have obvious influence to total white blood cells and platelet count in the mice peripheral blood.L-asparaginase liposome group and L-asparaginase group do not have obvious influence to weight of mice.The mensuration of the acute toxicity testing-maximum tolerated dose of embodiment 7 L-asparaginase pro-liposome
Get 20 of 18～22g Kunming kind white mice, male and female half and half, tail vein injection administration (consistent) with clinical application, dosage is for giving maximum drug level (4000KU/ml), maximum intravenous injection volume (0.5ml/20g) is administered once, and observed and recorded mice activity every day, death condition were observed 14 days altogether after the administration, claimed the mice body weight, and calculate the body weight gain rate in the 15th day after administration.
Observed 14 days after the administration, dead and any toxic and side effects do not appear in mice, and body weight increases all to some extent, and male mice body weight gain rate is 44.39%.Female mice body weight gain rate is 31.28%, sees Table 6.
Table 6. L-asparaginase liposome to the influence (i.v.) of mice body weight (* ± sD, n=10)
| Sex animal number | Average weight (g) weight increase percentage rate |
| Begin to finish (%) | |
| | ?19.6±1.31??28.3±1.42??????44.39 ?19.5±1.27??25.6±1.01??????31.28 |
The maximum tolerated dose that shows mouse mainline L-asparaginase pro-liposome is 10.0 * 10
5KU/kg, maximum tolerated dose is equivalent to the clinical plan of people's every day 5000 times with dosage.It is 200KU/kg that clinical people intends consumption, 10.0 * 10
5KU/kg ÷ 200KU/kg=5000 (doubly).
L-asparaginase liposome (the precursor blank liposome face with before add L-asparagine enzymatic solution join i.p. (7400,3700KU/kg) can prolong transplantability mouse lymphocyte leukemia L significantly
1210And P
388The survival natural law (P 0＜.01) its effect is better than the L-asparaginase.To L
1210Mice and P
388The mice increase in life span reaches as high as 184.7% and 183.3%.20 of Kunming kind white mice, tail vein injection L-asparaginase liposome, dosage is with maximum drug level (40000KU/ml), maximum intravenous injection volume (0.5ml/20g) is administered once, observed 14 days, dead and any toxic and side effects do not appear in mice, and male mice body weight gain rate is 44.39%, male mice body weight gain rate is 31.1%, and the maximum tolerated dose of L-asparaginase liposome is 10.0 * 10
5KU/kg.Its acute toxicity is significantly less than the L-asparaginase.Embodiment 8 L-asparaginase pro-liposome are to the inhibitory action of tumor cell in vitro
The L-asparaginase is the active drug of treatment leukemia and malignant lymphoma.The research report of relevant L-asparaginase pro-liposome is still few.Situation behind this paper primary study tumor cell in vitro drug effect has been carried out morphological observation under optical microscope, the ultramicroscope, cell proliferation rate and the analysis of flow cytometer dna content.And then study the effect of this medicine, so that instruct clinical rational drug use.
Cell line: HL-60 human promyelocytic leukemia P
815Mice plasmocytoma, L
1210The mouse lymph leukemia; K
562The people is red
The leukaemia.
L-asparaginase liposome (forming) by pro-liposome.
Culture fluid RPMI 1640 U.S. DIBCO companies include 10% calf serum.Penicillin 100U/ML
Matched group: add culture fluid; Experiment A group: (L-asparaginase liposome) contains enzyme 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml respectively; Experiment B group: add blank liposome (forming) by pro-liposome; Experiment C group: add the L-asparaginase. embodiment 8.1. cell culture
To be in the cell of exponential phase, with 5 * 10
5Individual cell/ml suspension makes A, C group contain medicine 30 μ g/ml.A, B group and contains lipid 1mg/ml by planting in the 25ml Tissue Culture Flask.Place 37 ° of incubators to cultivate 24 hours, under inverted microscope, observe morphological change.Again cell is taken out, centrifugal 3000 rev/mins * 8 minutes, abandon supernatant, add 4% glutaraldehyde fixedly behind the 2hr.With distilled water flushing, gradient alcohol dehydration, resin Epon-812 embedding, LKB-V type ultramicrotome cuts out thin slice, acetic acid uranium, lead citrate dyeing, H-300 transmission electron microscope observing.Embodiment 8.2. tetramethyl azo azoles salt (Sigma) MTT colorimetry
To be in the cell of exponential phase, with 2 * 10
5Individual cell/ml concentration adds in the 40 hole ELISA Plate, and 100 μ l/ holes add the medicine to be measured of fresh RPMI1640 culture fluid doubling dilution again, 100 μ l/ hole culture supernatant, add MTT solution (its concentration 5mg/ml) 30 μ l/ holes, go up vibration 1 minute, place 5%CO in the vibration product
2Educated 4 hours for 37 ℃, take out to inhale and abandon supernatant, add 0.4mol/L hydrochloric acid isopropyl alcohol 100 μ l/ holes, vibration back 570nm on microplate reader reads the A value.Embodiment 8.3. flow cytometer dna content is analyzed
With the exponential phase cell, furnishing 5 * 10
5Individual cell/ml suspension is inoculated in the 25ml Tissue Culture Flask, and adding A, B, C organize medicine, A, C is formed contain medicine 30 μ g/ml, and A, B group contains lipid 1mg/ml.Put 5%CO
2Hatched 24 hours for 37 ℃, take out centrifugally, abandon supernatant, add fixative, DNA dyeing is carried out with iodate third ingot in the back, with flow cytometer (FCM), does DNA cell cycle sampling analysis (5000 cells of every duplicate samples survey).Data is all collected, is stored and analyze through Cellfit software.
9 four kinds of cells of embodiment are through testing A, B, three groups of drug effects of C 24 hours, and the morphocytology variation sees Table 7,8 optical microscope photographs and sees Fig. 5～14.
Table 7 P
815Cellular morphology
Matched group experiment A group experiment B group experiment C group
Cell shows epithelium shape, the cell poor growth, and cell shows epithelium shape, the effect of L-asparaginase
Garden shape adherent growth, most cells becomes the garden, and garden shape is pasted and is warded off growth, after, compare with the A group
Cell outline is clear, and cell is not of uniform size, and is little with the matched group difference.Divide cell to become the garden, growth
It is vigorous to grow.Loosely organized.Rapidly.
Table 8 HL-60, L
1210, K
562Cellular morphology cell line matched group experiment A group
The cell suspension growth, profile is clear, the cell poor growth, the growth of form size HL-60 cell is vigorous.Differ, nuclear chromatin concentrates.L
1210The cell suspension growth, profile is clear, and cellular morphology is irregular, structure
The garden is and bright loose, and nuclear chromatin concentrates.。K
562The cell suspension growth, profile is clear, and cell outline is not asked,
Garden and bright.Form is not of uniform size.Embodiment 10 transmission electron microscope observings the results are shown in Table 9,10, and attached ultrathin section photo is seen Figure 15～21.
Table 9 transmission electron microscope observing P
815The cell ultrathin section
Matched group experiment A group experiment B group cancerous cell form is irregular, normal coloured portions cancerous cell nuclear dwindles, the trend pyknosis, cancerous cell and matched group are relatively, matter is abundant, kernel is obvious, and cavity, the fat that occurs size in the kytoplasm in the kytoplasm drips does not have obviously this change.Contain a small amount of mitochondrion and rough endoplasmic reticulum in the kytoplasm, and marrow sample corpusculum, some cell membrane not rough endoplasmic reticulum slightly increases.Cell surface has short and small microvillus.Complete.
Table 10 transmission electron microscope observing HL-60, L
1210The cell ultrathin section
Cell line matched group experiment A group
HL-60 cancerous cell kernel form is irregular, the big cancerous cell of kernel and dwindle heterochromatin
And obviously, some limit collection, euchromatin increase, the part karyopycnosis.Kernel
Abundant, kytoplasm is few, contains a large amount of free not obviously, occurs a large amount of cavitys in the kytoplasm,
Ribosome.Other cell line is few, and cytolipin drips and marrow sample corpusculum, the parts of fine after birth
There is a small amount of short and small microvillus on the surface.Fracture.Cell is imperfect.
L
1210Cancerous cell is circular and irregular shape.Examine the heterochromatin of big cancerous cell nuclear and increase, some
Out-of-shape, euchromatin is abundant.Trend pyknosis, cracked.Occur in the kytoplasm
Contain in the kytoplasm that a large amount of free ribosomes, a small amount of cavity, matter drip, the crack, after birth is intact
Rough endoplasmic reticulum and mitochondrion, cell surface is intact whole, a large amount of cancer cellular necrosis, disintegrate.
There is a small amount of short and small microvillus in the crack.
After embodiment 11 tetramethyl azo azoles salt MTT colorimetric method for determining results showed that cell is subjected to the effect of L-asparaginase liposome, the cell growth obviously was suppressed.Along with A group L-asparaginase liposome concentration increases, the rate of increase reduces.But P
815Cell does not have obvious difference through (blank) liposome effect and matched group.The L-asparaginase is to P
815Cell (C group) is obvious not as adding L-asparaginase liposome group (A group).See Figure 22.Embodiment 12 cells behind drug effect, cell generation cycle phase analysis and apoptosis rate of change.See Table 11,12.
Table 11 phase analysis cell generation cycle
Cell line matched group experimental group
G
0/G
1??????S???????G
2M?????????G
0/G
1?????????S????????????G
2M
HL-60????????29.7????????61.6??????8.6??????????41.2????????????49.9??????????9
L
1210????????21.0????????67.5??????11.5?????????38.1????????????60.0??????????1.9
P815?????????34.6????????59.9??????5.5????a.????60.6????????????13.8??????????25.6
b.????16.9????????????75.9??????????7.2??????????????????????????????????????????????????????c.????59.0????????????19.6??????????21.4
A. P
815Experiment A group b. P
815Experiment B group c. P
815Experiment C group
From table 11 explanation, after medicine A, C group was L-asparaginase liposome (getting through the pro-liposome hydration) A group and L-asparaginase (C group) and tumor cell effect, G0/G1 phase cell concentration obviously increased, and obviously reduces and enter the cell of S phase.
Table 12 tumor cell proliferation exponential sum apoptosis ratio
Cell line matched group experimental group
PI?(％)???????????RA?(％)??????????PI?(％)??????????RA?(％)
HL-6????????69.6???????????????2.68????????????58.9?????????????18.19
L1210???????79.0???????????????2.81????????????61.9?????????????11.67
P815????????65.0???????????????1.6????????a.???39.4?????????????1.17
b.???83.1?????????????0.48
c.???41.0?????????????2.10
A.P
815Experiment A group b.P
815Experiment B group c.P
815Experiment C group
Proliferation index shown in the table 12 (proliferation index, PI), the propagation degree of expression tumor cell colony, S, G in the cell cycle
2The shared percent of M phase cell, according to formula:
PI=(S+G
2M)/(G
0/ G
1+ S+G
2M) can draw the significantly decline of PI value of proliferation index L-asparaginase liposome group and L-asparaginase group (A, C group).HL-60, L
1210The apoptosis ratio significantly increases.P
815Cell is compared difference with matched group not remarkable.
Above technical scheme has the following advantages: this product adds medicine solution, be solubilized, unprecedented body lipid physical efficiency is used in body, and stores with solid-state form, improved physics, the chemical stability of lipid body, room temperature is placed or 4 ℃ of storages of low temperature still had good appearance and stability in 2.0 years. Unprecedented body lipid body can not only encapsulating protein class medicine such as ASP, and can seal nucleic acid drug such as ASON and common small-molecule drug such as adriamycin; It is a kind of carrier formulation that can carry multi-medicament. And solved to a certain extent short problem of macromolecular drug injection agent half-life. Biological technology products is made the preparation of reduced immunogenicity, and then reduce allergic reaction, effect time, reduction dosage reduce side effect in the extension body. Simultaneously, unprecedented body lipid body suitability for industrialized production or clinical medicine application quality are further improved and become possibility.
Claims (10)
1. a unprecedented body liposome is characterized by a kind of white dried product, mainly is made up of soybean phospholipid and cholesterol, and lipid concentration is 0.1-8%, and mannitol concentration is 0.25-6.5%, and polyvinylpyrrolidone is 0.02-6%.
2. according to claims 1, wherein the optimum mole ratio of soybean phospholipid and cholesterol is 7: 3.
3. according to claims 1, wherein soybean phospholipid can also be Ovum Gallus domesticus Flavus lecithin or other lipid.
4. according to claims 1, cholesterol can also be the amino carboxyl cholesterol of dimethylaminoethyl, or other trim of cholesterol.
5. according to claims 1, mannitol can also be sucrose, lactose, dextran, trehalose or other polysaccharide.
6. according to the preparation method of the unprecedented body liposome of claims 1, it is characterized in that:
(1) preparation of class lipoprotein solution:, under thermal condition, be dissolved in phosphoric acid with soybean phospholipid and cholesterol (mol ratio is 7: 3)
Buffer (containing mannitol, polyvinylpyrrolidone), logical nitrogen protection is passed through down through tissue mashing machine's homogenize again
High-pressure emulsification 30～60Mpa pressure was made class liposoluble in 5～30 minutes, lipid concentration 0.1-8% wherein, mannitol concentration
0.25-6.5%, polyvinylpyrrolidone 0.02-6%.
(2) above-mentioned class lipoprotein solution was pushed 0.45 μ m Merlon microporous filter membrane, got lipid soln, under nitrogen protection, irritated
Envelope, 100 ℃ of 30 minutes circulation steam sterilizations.
(3) Mie Jun lipid soln is sub-packed in the ampoule under aseptic condition, through lyophilization, and sealing by fusing behind the inflated with nitrogen.Make
The unprecedented body liposome of white.
6. according to the unprecedented body liposome of claims 1, it is characterized in that adding drug solution and carry out hydration and seal and form the medicinal liposome suspension.
7. according to the unprecedented body liposome of claims 1, it is characterized in that adding drug solution and carry out hydration and seal and form the medicinal liposome suspension, have and keep in animal or human's body leukocyte in the ability of normal level.
8. according to the unprecedented body liposome of claims 1, it is characterized in that adding L-asparagine enzymatic solution and carry out hydration and seal the formation liposome turbid liquor.
9. according to claims 6 described L-asparaginase liposome turbid liquors, can encapsulating protein class medicine and nucleic acid drug, and can intravenous injection, route of administration such as oral, intramuscular injection is used for clinical.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98125058 CN1255331A (en) | 1998-11-30 | 1998-11-30 | Hollow prosomatic liposome, its preparing process and clinical application of its encapsulated matter |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98125058 CN1255331A (en) | 1998-11-30 | 1998-11-30 | Hollow prosomatic liposome, its preparing process and clinical application of its encapsulated matter |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1255331A true CN1255331A (en) | 2000-06-07 |
Family
ID=5229000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 98125058 Pending CN1255331A (en) | 1998-11-30 | 1998-11-30 | Hollow prosomatic liposome, its preparing process and clinical application of its encapsulated matter |
Country Status (1)
| Country | Link |
|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335124C (en) * | 2004-10-19 | 2007-09-05 | 上海新药研究开发中心 | IL-2 precursor liposome and its preparation method |
| CN101810610A (en) * | 2010-04-19 | 2010-08-25 | 海南美兰史克制药有限公司 | Amoxicillin sodium flucloxacillin sodium medicine compound liposome injection |
| CN101474155B (en) * | 2009-01-24 | 2011-03-16 | 重庆医科大学 | Lung-targeted medicine carrying precursor liposome for injection and method of use thereof |
-
1998
- 1998-11-30 CN CN 98125058 patent/CN1255331A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335124C (en) * | 2004-10-19 | 2007-09-05 | 上海新药研究开发中心 | IL-2 precursor liposome and its preparation method |
| CN101474155B (en) * | 2009-01-24 | 2011-03-16 | 重庆医科大学 | Lung-targeted medicine carrying precursor liposome for injection and method of use thereof |
| CN101810610A (en) * | 2010-04-19 | 2010-08-25 | 海南美兰史克制药有限公司 | Amoxicillin sodium flucloxacillin sodium medicine compound liposome injection |
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