CN1364644A - Immuno liposome therapeutic hepatitis B vaccine and its preparing method - Google Patents
Immuno liposome therapeutic hepatitis B vaccine and its preparing method Download PDFInfo
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Abstract
The therapeutic hepatitis B vaccine is liposome containing hepatitis B surface antigen with or without immunoregulation molecule. It may be used to treat chronic hepatitis B caused by HIV infection and to prevent HIV infection. The technological process of inverse phase evaporation and freeze drying to prepare the said vaccine is also provided.
Description
Technical field
The present invention relates to a kind of Hepatitis B virus vaccine, specifically be meant a kind of immuno liposome therapeutic hepatitis B vaccine, the present invention also relates to the preparation method of this vaccine simultaneously.
Background technology
Hepatitis B is one of pandemic infectious disease in the world, seriously threatens human beings'health.Chronic viral hepatitis B patient and HBV virus carrier are the main sources of infection.People mainly use preventive measures to hepatitis B at present, promptly inoculate hepatitis B vaccine and prevent.Vaccine is mainly used in the body that never infected, so the virus protein of natural structure can be directly as vaccine antigen.But developed the Hepatitis B virus vaccine of application, though can infect the good preventing effect of playing to HBV, to infecting the body of HBV, the effect that but is difficult to bring into play treatment and removes virus.Main cause is that body has produced specific immunologic tolerance to hepatitis B virus, and immune system can not be removed virus effectively.To the virus carrier of hepatitis B, or the infected, especially the patient of the such infection of chronic viral hepatitis B virus hepatitis does not still have effective medicine and means at present.Therefore, development can be played the good preventing effect to HBV, can infect the relevant disease that causes to HBV again and bring into play the focus that the therapeutic hepatitis B vaccine of therapeutical effect has become domestic and international research.
Summary of the invention
The object of the present invention is to provide a kind of immuno liposome therapeutic hepatitis B vaccine, it is the liposome that comprises hepatitis B surface antigen, it also can comprise or not comprise immune modulatory molecules, it can infect the hepatitis B that causes to HBV and bring into play effective therapeutical effect, can infect the good preventing effect of playing again to HBV again.
Another object of the present invention provides a kind of preparation method of immuno liposome therapeutic hepatitis B vaccine.
For reaching above-mentioned purpose the technical solution used in the present invention be; with liposome as carrier and adjuvant; with a kind of immuno liposome therapeutic hepatitis B vaccine that hepatitis B surface antigen combines and is prepared into, this vaccine can the excitating organism humoral immune reaction, produces protection antibody; remove the virus in the blood circulation; can excite the activity of CTL again, produce very strong cell immune response, eliminate the virus in the hepatocyte; thereby reach thorough removing virus, treatment chronic viral hepatitis B and hepatitis B virus carriers.
This vaccine can be with immunomodulator, as thymosin, interleukin II, LAK cell, immune ribonucleic acid, specific transfer factor, hepatitis spirit, polyporusum bellatus, Lentinula edodes mycelium polysaccharide, Poria, coenzyme Q10, levamisole, lipid A, MPL, QuilA, BCG etc.Also can be not with immunostimulant.
Feature of the present invention is to add antioxidant such as vitamin E in the described immunoliposome therapeutic hepatitis B vaccine, adds negative charge phospholipid, prevents the liposome cohesion, merges, and changes dosage form, and the preparation lyophilized preparation is avoided seepage, prolongs the liposome holding time.
Hepatitis B surface antigen of the present invention comprises S, any among the S2 before the S1+ before the S2 before the S2 before the S1 before the S+, preceding S1+, S+, S+.
The preferred hepatitis B surface antigen of the present invention comprises the preceding S2 of the preceding S1+ of S+ preceding S2, S+, and the hepatitis B surface antigen of the best of the present invention is the preceding S2 of S1+ before the S+.
The amount that contains hepatitis B surface antigen in the immunoliposome therapeutic hepatitis B vaccine unit dose of the present invention is: the 1-80 microgram.
The preferred amounts that contains hepatitis B surface antigen in the immunoliposome therapeutic hepatitis B vaccine unit dose of the present invention is: the 20-30 microgram.
Liposome involved in the present invention is made up of phospholipid, non-phospholipid, steroid and their derivant and film material.What be used to prepare liposome at present has lecithin, a fabaceous lecithin, and PHOSPHATIDYL ETHANOLAMINE, cholesterol, cephalin, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, cholesterol acetyl liposome, B-sitosterol, natrii tauroglycocholas, Yolk lecithin, two palmityls-DL-a phosphatidylcholine, phosphatidyl silk amino acid, phosphatidylinositols, sphingomyelin, sphingo, two Cetyl Phosphates, dimyristoyllecithine, stearmide, dioxy ethylene cetyl ether and four oxygen ethylene lauryl ethers etc. all can be used for the present invention.
Film material involved in the present invention includes but not limited to cholesterol, 18-amine., phosphatidic acid etc.Cholesterol can be regulated bilayer flowability, permeability etc., and 18-amine., phosphatidic acid can change the surface of liposome charge property.
The preferred liposome sphere diameter of the present invention is 20～5000nm, and the present invention more preferably liposome sphere diameter is 100～200nm and 950-1050nm.
Contain lipid scale of construction 0.3-1.5ml in the immunoliposome therapeutic hepatitis B vaccine unit dose of the present invention.
Containing in the immunoliposome therapeutic hepatitis B vaccine unit dose of the present invention that liposome preferably measures is 0.5-1.0ml.
The preparation technology of liposome is that those skilled in the art is known, its ultimate principle all is that oily material and the anti-material of aqueous are formed water in oil preparation through suitably handling the back, but the character of encapsulate substances may have bigger difference, as chemosynthesis material, organic substance; Biological substance such as albumen, polypeptide and nucleic acid etc.The latter is vulnerable to the influence of preparation condition in processing procedure, as the influence of factors such as temperature, time, vibration and acid-base value.Therapeutic hepatitis B vaccine belongs to the polypeptide in the biological preparation.Conventional method for preparing lipidosome resembles membrane process, reverse phase evaporation, pressurization extrusion molding, fusion method and lyophilizing aquation method or improves and all can be used for the present invention and prepare the immunoliposome therapeutic hepatitis B vaccine.
The preferable preparation technique of immuno liposome therapeutic hepatitis B vaccine of the present invention is reverse phase evaporation, lyophilizing aquation method.The preparation technology of the most preferred liposome of the present invention is a lyophilizing aquation method.
The lyophilizing aquation method preparation technology of immuno liposome therapeutic hepatitis B vaccine of the present invention comprises the steps:
(1) liposome, film material and additives or immune modulatory molecules are dissolved in the solvent with mol ratio at 1: 1: 1, place rotary evaporator, remove organic solvent, rotary evaporation forms immobilized artificial membrane;
(2) add phosphate buffer, 40-60 ℃, ultrasonic 3-8 minute, form the liposome suspension;
(3) the liposome suspension is mixed with mol ratio with hepatitis B antigen solution at 3: 1, add cryoprotective agent, carry out lyophilization;
(4) adopt method sterilizations such as wet heating or cobalt irradiation, obtain freeze dried powder;
(5) add sterilized water, concussion, rehydration fusion;
(6) obtain immuno liposome therapeutic hepatitis B vaccine.
The reverse phase evaporation preparation technology of immuno liposome therapeutic hepatitis B vaccine of the present invention comprises the steps:
(1) liposome, film material and additives or immune modulatory molecules are dissolved in the solvent with mol ratio at 1: 1: 1, add hepatitis B antigen solution, 40-60 ℃, ultrasonic in short-term 3-8 minute, obtain stable water-in-oil emulsion;
(2) 40-60 ℃, reduction vaporization obtains colloidal materials;
(3) add phosphate buffer, 40-60 ℃ of rotary evaporation, reduction vaporization 10-20 minute, remove micro-organic solvent, obtain the liposome aqueous suspension, placed 20-40 minute, dialysed 20-30 hour, remove the free hepatitis B antigen that does not wrap into liposome;
(4) adopt method sterilizations such as wet heating or cobalt irradiation;
(5) obtain immuno liposome therapeutic hepatitis B vaccine.
Advantage of the present invention is an immuno liposome therapeutic hepatitis B vaccine of the present invention, and it can play the good preventing effect to HBV, can infect the hepatitis B that causes to HBV again and bring into play effective therapeutical effect.This vaccine can the excitating organism humoral immune reaction, produces protection antibody, removes the virus in the blood circulation; can excite the activity of CTL again, produce very strong cell immune response, eliminate the virus in the hepatocyte; thereby reach thorough removing virus, treatment chronic viral hepatitis B and hepatitis B virus carriers.
The immuno liposome therapeutic hepatitis B vaccine of the present invention's preparation can adopt subcutaneous or intravenous administration.
In order to understand technical scheme of the present invention and beneficial effect better, the present invention is further elaborated below in conjunction with concrete experiment and experimental result.
Experiment one: the preparation technology of immuno liposome therapeutic hepatitis B vaccine is to the influence of vaccine-induced ability
We have groped several preparation technologies to the active influence of antigenic substance in experiment, especially monitor its immune induction activity, the method that the experimental result proof should select soft relatively, the no violent physico chemical factor of preparation process to change.We with the injected in mice vaccine after toes swelling degree as the index of judging immunoreation intensity.Its ultimate principle is toes after the mice vaccination for the first time, produces the immunological memory reaction in the body, then produces intensive immunne response by same antigenic substance once more.Therefore antigen locate to be rich in lymphoid tissue in the injection of sole position, and very easily inducing cell immunoreation is judged immunoreactive intensity by the swelling degree of injection site.
Experimental technique: behind the left front toes thickness of surveying record mice, in left front toes place inoculation therapeutic hepatitis B vaccine 0.5ml, inoculate the vaccine 0.5ml of same concentration after 10 days, measure left front toes thickness after for the second time inoculating 3 days, and calculating toes swelling index, promptly experiment back thickness is divided by the preceding thickness of experiment, and the swelling index is high more, and then the local immunoreation that causes of expression is strong more.
Experimental result sees Table 1
The preparation technology of table 1 immuno liposome therapeutic hepatitis B vaccine is to the influence of vaccine-induced ability
Experimental result shows: several method all can produce significant difference compared with the control, the immunoreation that lyophilizing aquation method causes is the strongest, it is most preferred method of the present invention, though the immunoreation that reverse phase evaporation causes is the most weak, but compare with normal control, still there were significant differences, and its biggest advantage is that technical process is simpler relatively, easily realize large-scale production, be still one of preferable methods of the present invention.Test of the influence of 2 liposome types to the hepatitis b vaccine immune inducibility
| Preparation method | Experimental group example number (only) | Toes swelling coefficient |
| Membrane process | 10 | 0.58±0.22 |
| Reverse phase evaporation | 10 | 0.24±0.09 |
| The pressurization extrusion molding | 10 | 0.67±0.21 |
| Fusion method | 10 | 0.43±0.16 |
| Lyophilizing aquation method | 10 | 0.75±0.12 |
The sealing folliculus that liposome is made up of one or more layers lipoid molecular layer, in its bilayer or interlayer can comprise medicine, its similar cell membrane.Can be divided into four classes (1) unilamelar liposome, sphere diameter≤100nm according to its sphere diameter size; (2) large unilamellar vesicle, sphere diameter 100-200nm; (3) multilamelar liposome, sphere diameter 200-1000nm; (4) macrovesicle, sphere diameter is about 1000-5000nm.The amount of the wrapping kmedicine by liposome of different sphere diameters is different, and liposome volume difference, and the distribution after it enters in the body also is different.As immunity therapeutic preparation, we expect that it is a macrophage phagocytic by immune accessory cell/antigen presenting cell preferentially after entering in the body.We connect with the enzyme of the liposome of different sphere diameters and the combined specificity antibody-to-surface antigen that is brought out of hepatitis B antigen of the present invention and adsorb immunoreation and experimentize as index.Wherein the surface antigen standard substance are provided by Shenzhen Kang Hua company.Mouse anti human IgG enzymic-labelled antibody is available from magnificent biotech firm.
Experimental result sees Table 2.
Table 2 liposome sphere diameter and immune induction ability
| Liposome sphere diameter (nm) | Specific antibody titre (HBsAb) |
| ≤100 | ?1∶1000 |
| 100-200 | ?1∶100,000 |
| 800-1200 | ?1∶100,000 |
| 950-1050 | ?1∶120,000 |
| 1200-5000 | ?1∶10,000 |
| Simple hepatitis B antigen contrast | ≤1∶1000 |
| The blank liposome contrast | ≤1∶10 |
Experiment showed, the liposome and 150 of sphere diameter about 1000 ± 200nm
+ 50The easiest about nm by macrophage phagocytic, and cause intensive immunoreation, preferable sphere diameter is 1000 ± 200nm and 150
+ 50Nm, optimum sphere diameter is 950-1050nm.Test of the influence of the holding time of 3 immuno liposome therapeutic hepatitis B vaccines to the Hepatitis B virus vaccine inducibility
Liposome class preparation is owing to itself be the conjugate of oil and water, and how inevitable pure seepage phenomenon at content delays, reduces the seepage of encapsulation object and dissociating of liposome in the preservation process, is the important technology index of the decision quality of the pharmaceutical preparations.By the immuno liposome therapeutic hepatitis B vaccine of the inventive method preparation, because the improvement of antioxidant and preparation technology and dosage form has prolonged the holding time greatly.We use the immuno liposome therapeutic hepatitis B vaccine immune mouse 0.5ml of the present invention of different holding times, inoculate the vaccine 1ml of same concentration after 10 days, inoculate 4 days for the second time, get its splenocyte In vitro culture, and add specific surfaces antigen.Can judge immune effect of vaccine according to its lymphopoiesis degree.It is index that lymphopoiesis adopts the infiltration rate of the tritium-labeled thymus pyrimidine of isotope.
Laboratory animal: the Balb/c mice is provided by preclinical medicine institute of Jilin University animal center.
Reagent: hepatitis B virus surface antigen is available from 5 of Military Medical Science Institutes
Experimental result is as follows:
The holding time of table 3 immuno liposome therapeutic hepatitis B vaccine is to the influence of its immune effect
| The holding time of immuno liposome therapeutic hepatitis B vaccine (my god) | Lymphocytic proliferation rate (SI) |
| 1 | 10.15 |
| 5 | 11.23 |
| 10 | 10.56 |
| ?30 | ?8.07 |
| ?60 | ?6.59 |
| ?90 | ?3.34 |
| ?180 | ?2.22 |
Experimental result shows that the immuno liposome therapeutic hepatitis B vaccine of method preparation of the present invention has the holding time of better curative effect can reach 30 days, even certain immune effect was also arranged in 60 days.Test the influence of different antigenic components contained in 4 Hepatitis B virus vaccines to the mouse immune reaction
Antigenic component contained in the Hepatitis B virus vaccine can comprise albumin, S albumen, preceding S2 and pre-s1 protein.The liposome of sphere diameter about 1000 ± 200nm combined with the hepatitis B antigen of various combinations respectively, give mouse inoculation, observe antibody titer and lymphocytic proliferation rate.Result such as table 4.
The different hepatitis B antigen of table 4 combines the influence to immune effect with liposome
Experimental result shows that antibody titer does not have significant difference between the 1-5 group; 1,2,3 groups and 4,5 groups relatively there were significant differences for the lymphocytic hyperplasia rate, and the lymphocytic hyperplasia rate does not have significant difference between 1,2,3 group, and the lymphocytic hyperplasia rate does not have significant difference between 4 and 5 groups, and the 1-5 group more all has significant difference with the 6th group.
| Group | Antigenic type | Lymphocytic proliferation rate (SI) | Antibody titer (HBsAb) |
| 1 | ?S | ?9.14 | ?1∶10,000 |
| 2 | S1 before the S+, | ?9.15 | ?1∶10,000 |
| 3 | S2 before the preceding S1+ | ?9.33 | ?1∶100,000 |
| 4 | S2 before the S+ | ?13.96 | ?1∶100,000 |
| 5 | S2 before the S1+ before the S+ | ?14.58 | ?1∶100,000 |
| 6 | Albumin | ?1.36 | ?＜1∶100 |
More all there were significant differences for the lymphocytic hyperplasia rate of 1-5 group and antibody titer and 6 groups.That wherein lymphocytic proliferation rate and antibody titer are higher is the preceding S2 of the preceding S1+ of S2, S+ before the S+, and the hepatitis B surface antigen of the best of the present invention is the preceding S2 of S1+ before the S+.Test the immune induction reaction that different hepatitis B antigen content and liposome content cause in the immuno liposome therapeutic hepatitis B vaccine of 5 unit dose.
Experiment combination below we have designed in experiment, 4 described immunology routine techniques lymphocytic proliferation rate determine that the unit dose immuno liposome therapeutic hepatitis B vaccine causes the amount ranges and the best amount ranges of immunoreactive different component combination by experiment.Experimental result such as table 5
Table 5 causes immunoreactive immuno liposome therapeutic hepatitis B vaccine
The amount ranges of different component combination and best amount ranges
| Hepatitis B antigen content ug in the liposome content lymphocytic proliferation rate unit dose in the unit dose | ?0.3ml | ?1ml | ?0.8ml | ?0.5ml | ?1.5ml |
| ?1 | ?8.33 | ?8.98 | ?9.01 | ?8.74 | ?8.98 |
| ?10 | ?10.02 | ?10.23 | ?10.23 | ?10.22 | ?10.27 |
| ?20 | ?10.15 | ?11.96 | ?12.26 | ?11.87 | ?12.08 |
| ?25 | ?10.21 | ?12.56 | ?12.58 | ?11.99 | ?12.37 |
| ?30 | ?10.27 | ?12.39 | ?12.44 | ?12.45 | ?12.36 |
| ?80 | ?11.03 | ?12.35 | ?12.45 | ?12.30 | ?12.34 |
Experimental result shows: immuno liposome therapeutic hepatitis B vaccine of the present invention can cause that the preferred content range of hepatitis B antigen is 1-80ug in the effective immune response unit dose, most preferred content range is 20-30ug, and the preferred content range of liposome is that the most preferred content range of 0.3ml-1.5ml is 0.5ml-1.0ml.Its relative liposome of the volume of hepatitis B surface antigen can be disregarded in the immuno liposome therapeutic hepatitis B vaccine unit dose of the present invention.Test the clinic trial result of 6 therapeutic hepatitis B vaccines
In order to verify the clinical effectiveness of therapeutic hepatitis B vaccine, we and domestic epidemic prevention station, hospital with complete test condition and technical force cooperate, and its effect is observed.
We select hepatitis B virus to carry the patient at random, and hepatitis B virus is in replicative phase in its body, and standard is that serum detects surface antigen positive, the e antigen positive, the cAg positive (claiming " great three positive " again), the course of disease is more than 2 years, no acute attack sign, liver function is normal.All patients all carry out quantitative PCR detection.Matched group is naked vaccination.For guaranteeing the objectivity of observed result, adopt paired sera to detect, promptly the serum before the patient treatment detects under the same conditions simultaneously with treatment back serum, to get rid of reagent, season and operator's subjective error.Experimental result is as follows
Table 6 immuno liposome therapeutic hepatitis B vaccine compares (Guangdong health and epidemic prevention station) before and after the treatment of hepatitis B virus labelling
*p＜0.05
| Group | The example number | The cloudy routine number (%) that changes of surface antigen | The cloudy routine number (%) that changes of surface antibody | The cloudy routine number (%) that changes of e antigen | The cloudy routine number (%) that changes of HBVDNA |
| Treatment finishes | 76 | ?12(15) * | 10(13) * | 40(52) * | 36(47) * |
| Treated back 3 months | 76 | ?22(28) * | 19(26) * | 48(63) * | 42(55) * |
| Treated back 6 months | 76 | ?23(30) * | 20(26) * | 49(64) * | 47(61) * |
| The contrast treatment finishes | 70 | ?3(4) | 3(4) | 17(24) | 14(20) |
| Contrast treatment 3 months | 70 | ?4(5) | 3(4) | 16(22) | 15(21) |
| Contrast treatment 6 months | 70 | ?4(5) | 4(5) | 16(22) | 15(21) |
Table 7 immuno liposome therapeutic hepatitis B vaccine compares (Linyi City the People's Hospital) before and after the treatment of hepatitis B virus labelling
*p＜0.05
| Group | The example number | The cloudy routine number (%) that changes of surface antigen | The cloudy routine number (%) that changes of surface antibody | The cloudy routine number (%) that changes of e antigen | The cloudy routine number (%) that changes of HBVDNA |
| Treatment finishes | 52 | ?10(19) * | 10(19) * | 22(56) * | 23(44) * |
| Treated back 3 months | 52 | ?18(34) * | 15(28) * | 31(59) * | 36(69) * |
| Treated back 6 months | 52 | ?19(36) * | 14(26) * | 30(58) * | 34(65) * |
| The contrast treatment finishes | 41 | ?2(3) | 2(3) | 8(15) | 9(17) |
| Contrast treatment 3 months | 41 | ?3(5) | 3(5) | 9(17) | 9(17) |
| Contrast treatment 6 months | 41 | ?3(5) | 2(3) | 7(13) | 8(15) |
Table 8 immuno liposome therapeutic hepatitis B vaccine compares (Shaodong epidemic prevention station) before and after the treatment of hepatitis B virus labelling
*p＜0.05
| Group | The example number | The cloudy routine number (%) that changes of surface antigen | The cloudy routine number (%) that changes of surface antibody | The cloudy routine number (%) that changes of e antigen | The cloudy routine number (%) that changes of HBVDNA |
| Treatment finishes | 142 | ?24(17) * | 19(14) * | 93(52) * | 88(62) * |
| Treated back 3 months | 142 | ?36(26) * | 37(26) * | 96(63) * | 82(58) * |
| Treated back 6 months | 142 | ?38(27) * | 37(26) * | 90(64) * | 86(61) * |
| The contrast treatment finishes | 65 | ?2(3) | 3(5) | 8(13) | 11(17) |
| Contrast treatment 3 months | 65 | ?3(5) | 3(5) | 9(15) | 10(16) |
| Contrast treatment 6 months | 65 | ?2(3) | 3(5) | 11(17) | 9(15) |
In order to understand essence of the present invention better, the present invention is done further detailed explanation below in conjunction with specific embodiment.
Specific embodiment embodiment 1 lyophilizing aquation legal system is equipped with immuno liposome therapeutic hepatitis B vaccine
Get (mol ratio are 1: 1: 1) such as soybean lecithin and cholesterol, phosphoric acid lipid As, be dissolved in alcoholic solution; Rotary evaporation is removed organic solvent, forms immobilized artificial membrane; (0.1M PH7.4), 45 ℃, ultrasonic 5 minutes, forms the small unilamellar vesicle suspension to add an amount of phosphate buffer; S2 solution before the S1+ before liposome turbid liquor and the hepatitis B surface antigen S+ is mixed, and mol ratio is 3: 1, adds cryoprotective agent mannitol again, and 7 ℃/minute, quick freezing is carried out lyophilization to-30 ℃; Cobalt 60 radiation sterilizations; Add sterilized water again, with oscillator concussion 5 minutes, aquation formed big monolayer or few layer liposome turbid liquor again, is immuno liposome therapeutic hepatitis B vaccine.Embodiment 2 lyophilizing aquation legal systems are equipped with immuno liposome therapeutic hepatitis B vaccine
Get (mol ratio are 1: 1) such as soybean lecithin and cholesterol, be dissolved in alcoholic solution; Rotary evaporation is removed organic solvent, forms immobilized artificial membrane; (0.1M PH7.4), 45 ℃, ultrasonic 5 minutes, forms the small unilamellar vesicle suspension to add an amount of phosphate buffer; S2 solution before the S1+ before liposome turbid liquor and the hepatitis B surface antigen S+ is mixed, and mol ratio is 3: 1, adds cryoprotective agent mannitol again, and 7 ℃/minute, quick freezing is carried out lyophilization to-30 ℃; Cobalt 60 radiation sterilizations; Add sterilized water again, with oscillator concussion 5 minutes, aquation formed big monolayer or few layer liposome turbid liquor again, is immuno liposome therapeutic hepatitis B vaccine.Embodiment 3 reverse phase evaporations prepare immuno liposome therapeutic hepatitis B vaccine
Get (mol ratio are 1: 1: 1) such as soybean lecithin and cholesterol, phosphoric acid lipid As, be dissolved in chloroform and the isopropyl alcohol mixed liquor; S2 aqueous solution before the S1+ before the hepatitis B surface antigen S+ is added in the above-mentioned mixed liquor, and the mol ratio of mixed liquor and antigenic solution is 2: 1, in the formula of bath Ultrasound Instrument, 45 ℃, handled 5 minutes, form stable water in oil emulsion, remove organic solvent in 45 ℃ of evaporations of pressurizeing down then, a stiff jelly, add an amount of phosphate buffer (0.1M, PH7.4), the dissolving jelly, continue 45 ℃ of rotary evaporations, reduction vaporization is 15 minutes then, removes micro-organic solvent.Place after 30 minutes, the liposome of gained is put into bag filter, (0.1M, PH7.4), at room temperature dialysis, is removed the free hepatitis B antigen that does not wrap into liposome at 24 hours total times with phosphate buffer.With 100 ℃ of gained liposome wet heating sterilizations, 30 minutes, promptly get immuno liposome therapeutic hepatitis B vaccine.Embodiment 4 reverse phase evaporations prepare immuno liposome therapeutic hepatitis B vaccine
Get soybean lecithin and cholesterol (mol ratio is 1: 1), be dissolved in chloroform and the isopropyl alcohol mixed liquor; S2 aqueous solution before the S1+ before the hepatitis B surface antigen S+ is added in the above-mentioned mixed liquor, and the mol ratio of mixed liquor and antigenic solution is 2: 1, in the formula of bath Ultrasound Instrument, 45 ℃, handled 5 minutes, form stable water in oil emulsion, remove organic solvent in 45 ℃ of evaporations of pressurizeing down then, a stiff jelly, add an amount of phosphate buffer (0.1M, PH7.4), the dissolving jelly, continue 45 ℃ of rotary evaporations, reduction vaporization is 15 minutes then, removes micro-organic solvent.Place after 30 minutes, the liposome of gained is put into bag filter, (0.1M, PH7.4), at room temperature dialysis, is removed the free hepatitis B antigen that does not wrap into liposome at 24 hours total times with phosphate buffer.With 100 ℃ of gained liposome wet heating sterilizations, 30 minutes, promptly get immuno liposome therapeutic hepatitis B vaccine.
The described method preparation of all available embodiment 1-4 of liposome involved in the present invention and hepatitis B surface antigen or immune modulatory molecules.
Claims (16)
1, a kind of immuno liposome therapeutic hepatitis B vaccine, it is the liposome that includes hepatitis B virus surface antigen.
2, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 1 is characterized in that it includes immune modulatory molecules.
3, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 1 and 2, wherein said hepatitis B virus surface antigen is to be selected from S, the preceding S2 of S1+ before S2 and the S+ before the S2 before the S1 before the S+, preceding S1+, S+.
4, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 3, wherein preferred hepatitis B virus surface antigen are to be selected from preceding S2 of S+ and the preceding S2 of the preceding S1+ of S+.
5, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 4, wherein most preferred hepatitis B virus surface antigen are the preceding S2 of S1+ before the S+.
6, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 1 and 2, wherein the hepatitis B virus surface antigen 1-80ug that contains in the vaccine of unit dose.
7, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 6, wherein the hepatitis B virus surface antigen preferred amounts that contains in the vaccine of unit dose is 20-30ug.
8, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 1 and 2, wherein said liposome are that phospholipid, non-phospholipid, steroid and their derivant and film material are formed.
9, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 8, wherein said liposome is selected from soybean lecithin, fabaceous lecithin, PHOSPHATIDYL ETHANOLAMINE, cholesterol, cephalin, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, cholesterol acetyl liposome, the B-sitosterol, natrii tauroglycocholas, Yolk lecithin, two palmityls-DL-a phosphatidylcholine, phosphatidyl silk amino acid, phosphatidylinositols, sphingomyelin, sphingo, two Cetyl Phosphates, dimyristoyllecithine, stearmide, dioxy ethylene cetyl ether and four oxygen ethylene lauryl ethers etc.
10, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 8, the sphere diameter of wherein said liposome is 20～5000nm.
11, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 10, the preferred sphere diameter of wherein said liposome is 100～200nm or 950-1050nm.
12, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 1 and 2, wherein the lipid scale of construction that contains in the vaccine of unit dose is 0.3-1.5ml.
13, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 12, wherein the liposome preferred amounts that contains in the vaccine of unit dose is 0.5-1.0ml.Vaccine in the liposome preferred amounts that contains be 0.5-1.0ml.
14, a kind of immuno liposome therapeutic hepatitis B vaccine according to claim 2, the immune modulatory molecules that wherein contains is selected from thymosin, interleukin II, LAK cell, immune ribonucleic acid, specific transfer factor, hepatitis spirit, polyporusum bellatus, Lentinula edodes mycelium polysaccharide, Poria, coenzyme Q10, levamisole, lipid A, MPL, QuilA, BCG etc.
15, a kind of method for preparing immuno liposome therapeutic hepatitis B vaccine comprises the steps:
(1) liposome, film material or immune modulatory molecules are dissolved in the solvent with mol ratio at 1: 1: 1, place rotary evaporator, remove organic solvent, rotary evaporation forms immobilized artificial membrane;
(2) add phosphate buffer, 40-60 ℃, ultrasonic 3-8 minute, form the liposome suspension;
(3) the liposome suspension is mixed with mol ratio with hepatitis B antigen solution at 3: 1, add cryoprotective agent, carry out lyophilization;
(4) adopt method sterilizations such as wet heating or cobalt irradiation, obtain freeze dried powder;
(5) add sterilized water, concussion, rehydration fusion;
(6) obtain immuno liposome therapeutic hepatitis B vaccine.
16, a kind of method for preparing immuno liposome therapeutic hepatitis B vaccine comprises the steps:
(1) liposome, film material or immune modulatory molecules are dissolved in the solvent with mol ratio at 1: 1: 1, add hepatitis B antigen solution, 40-60 ℃, ultrasonic in short-term 3-8 minute, obtain stable water-in-oil emulsion;
(2) 40-60 ℃, reduction vaporization obtains colloidal materials;
(3) add phosphate buffer, 40-60 ℃ of rotary evaporation, reduction vaporization 10-20 minute, remove micro-organic solvent, obtain the liposome aqueous suspension, placed 20-40 minute, dialysed 20-30 hour, remove the free hepatitis B antigen that does not wrap into liposome;
(4) adopt method sterilizations such as wet heating or cobalt irradiation;
(5) obtain immuno liposome therapeutic hepatitis B vaccine.
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| CN1364644A true CN1364644A (en) | 2002-08-21 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008104133A1 (en) * | 2007-02-28 | 2008-09-04 | Centro De Ingeniería Genética Y Biotecnología | Combination therapy for the treatment of chronic hepatitis b |
| CN107375921A (en) * | 2017-06-12 | 2017-11-24 | 商丘美兰生物工程有限公司 | A kind of glycyrrhizin liposome immunization adjuvant and preparation method thereof |
| CN111450245A (en) * | 2020-04-29 | 2020-07-28 | 艾棣维欣(苏州)生物制药有限公司 | Hepatitis B treating vaccine and preparation method thereof |
-
2002
- 2002-02-05 CN CN 02100482 patent/CN1364644A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008104133A1 (en) * | 2007-02-28 | 2008-09-04 | Centro De Ingeniería Genética Y Biotecnología | Combination therapy for the treatment of chronic hepatitis b |
| CN107375921A (en) * | 2017-06-12 | 2017-11-24 | 商丘美兰生物工程有限公司 | A kind of glycyrrhizin liposome immunization adjuvant and preparation method thereof |
| CN107375921B (en) * | 2017-06-12 | 2019-10-29 | 商丘美兰生物工程有限公司 | A kind of glycyrrhizin liposome immunization adjuvant and preparation method thereof |
| CN111450245A (en) * | 2020-04-29 | 2020-07-28 | 艾棣维欣(苏州)生物制药有限公司 | Hepatitis B treating vaccine and preparation method thereof |
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