CN102349996A - Human papilloma virus pharmaceutical composition and application thereof - Google Patents
Human papilloma virus pharmaceutical composition and application thereof Download PDFInfo
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- CN102349996A CN102349996A CN2011103135149A CN201110313514A CN102349996A CN 102349996 A CN102349996 A CN 102349996A CN 2011103135149 A CN2011103135149 A CN 2011103135149A CN 201110313514 A CN201110313514 A CN 201110313514A CN 102349996 A CN102349996 A CN 102349996A
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Abstract
The invention relates to a human papilloma virus pharmaceutical composition for preventing and treating related diseases caused by human papilloma virus. The pharmaceutical composition is characterized by comprising the following components: human papilloma virus virus-like particles (HPV VLPs) used as an active ingredient, an adjuvant for promoting immune response, a cell factor immune accelerant for activating cell immunity to durably and efficiently generate immune response, a salt solution for maintaining the integral spatial configuration and certain dissolubility of VLPs, a physiologically miscible buffer for maintaining a certain pH value, a non-ionic surfactant for protecting VLPs from violent shaking which can lead to activity decrease and the balance of water. The pharmaceutical composition has the characteristics that: HPV VLPs are prepared into a liquid pharmaceutical composition with integral spatial configuration, efficient immunity and stable storage property; the composition is used for preventing and treating various diseases caused by human papilloma virus; and the composition has the characteristics of safe use, convenience, economy, no side effect and the like.
Description
Technical field
The present invention relates to a kind of gene engineering method preparing technical field, particularly from pharmaceutical composition of the virus-like particle (VLPs) of human papilloma (HPV) and preparation method thereof.
Background technology
Human papillomavirus (HPV) is one type of double-chain small molecule DNA viruses, has strict species specificity, and the skin and the mucous membrane tissue of main infected person cause the proliferative lesion of corresponding site epithelial tissue.Clinically, vary in size according to HPV hypotype pathogenicity size or carcinogenic risk and HPV can be divided into two big types of low risk and high-risk-types.Low risk HPV mainly causes Intradermal tumor on exophytic wart class pathological changes and the low cervix uteri of skin of anus and male external genital organs, the big nympha of women, urethral orifice, vagina hypomere, and its virus subtype mainly contains HPV6,11,30,39,42,43 types and HPV44 type.High-risk HPV the more important thing is to cause Intradermal tumor on external genitalia cancer, cervical cancer and the height cervix uteri that its virus subtype mainly contains HPV16,18,31,33,35,45,51,52,56,58 types and HPV61 type except that can causing the external genitalia wart.
Current research shows HPV except directly causing cervical cancer, also with lung bronchogenic carcinoma, rectal cancer, oral cancer and skin carcinoma significant relationship is arranged.Show that according to the interrelated data of the WHO of WHO except causing cervical cancer, HPV infects and can also cause 60% skin carcinoma, 60% the esophageal carcinoma, 50% pulmonary carcinoma, 50% breast carcinoma, 25% human major diseases such as oral cancer.
HPV coding comprises eight early stage (E1～E7) and two late periods (genes of L1～L2); L1 albumen is main capsid protein; And has the molecular weight of 55～60KDa; L1 albumen or L1 and L2 protein combination are at yeast, and the expression in insect cell, mammalian cell or the antibacterial can cause the self assembly of virus-like particle (VLPs) (referring to Papillomaviruses Reviews:Current Research on Papillomaviruses; Lacey; Ed.Leeds; UK:Leeds Medical Information; Pp 101-12 (1996)); In a single day VLPs is similar with real virion in form, and gives the animal or human class, can cause the neutralizing antibody of high titre; Because VLPs does not comprise potential carcinogenic viral genome, therefore very high safety is arranged as the HPV vaccine., be well known in the art as potential vaccine prevention HPV with HPV virus-like particle (VLPs).
Verified at present; Inoculation VLPs bivalence HPV16 and HPV18 and tetravalence HPV6,11,16 and 18 vaccine produce immunne response in patient's body be effective; But successful vaccine is not only required in and produces immunne response in the body; But also excitating organism produces immunological memory effectively; When running into pathogen once more, produce rapidly and efficient immune.Immunological memory keep cellular immunization and the humoral immunization that depends on antigenic specificity.The T lymphocyte is the effector lymphocyte of antigenic specificity cell immune system, mainly comprises helper T lymphocyte (CD4+) and cytotoxic T cell (CTL) (CD8+), and the two plays an important role in the process of removing or control infection.CD4 + T cells play a major class of MHC-II-restricted help B lymphocytes (TH2) generating and maintaining antibody and antigen-specific memory CD8 + T cells (TH1) the role of its regulator activity, including: (1) supplementary role of B cells, B cells, promoting conversion ISOTYPE homologous chromosomal mutation and germinal center B cells to differentiate into plasma cells and memory cells; (2) CD4 + T cells
CD8 + T cells in a supporting role, to promote their effector cells and memory cells, and to maintain functional status; (3) CD4 + T cells and APCs on macrophages in vitro.Target cell is directly brought into play cytotoxic T lymphocyte (CTL) effect of MHC-I quasi-molecule restriction or through discharging growth and the survival that cytokine stops pathogen and have relatively more fugitive CD8+T cell response that should the stage; Its effector function is competence exertion in the presence of antigen only, and visible antigenic existence is the switch as the effector T cell of surviving with instant opening ways.Along with antigenic clean-up effect function stops and formation Memorability CD8+T cell, be positioned the mucosa position of pathogen invasion, when running into antigen once more, both having developed rapidly becomes the effector lymphocyte.The multiple composition of humoral immunoresponse(HI) is being brought into play different effects in the defence process of pathogenic infection.Plasma cell secretion multi-specificity antibody (natural antibody) by the growth of B1 crowd B cell differentiation; Constitute the pathogenic infection defence the first line of defence; These antibody have multiple effect: like the diffusion in the starting stage restriction pathogen that infects; Forming immune complex activation acquired immunity replys; Be enrolled into germinal center and activating complement, play the function served as bridge that connects the natural immunity and acquired immunity simultaneously; Memorability antibody by the long-life plasma cell secretion is defence line, second road, mainly is distributed in peripheral circulation and mucosa position, has high-affinity and high specific, can more effective defence pathogenic infection; The three lines of defence of Memorability B cellularity immune defence; Performance has two kinds of effects; When running into antigen once more, produce rapidly and effectively responsing reaction on the one hand,, keep the balance of long-life plasma cell quantity on the other hand as plasmacytic back-up system of long-life.Therefore; The target that present stage develops is under the prerequisite of the VLPs configuration that is kept perfectly; Prolong the pot-life of VLPs; Not only want simultaneously the immunne response of enhancing body to VLPs; But also will be through adding relevant composition; Excitating organism produces immunological memory to VLPs, and in patient's body, produces long-acting immunity.
Adjuvant is one type of material that share and can strengthen and regulate antigen immune response with antigen.In vaccine, add adjuvant and can make better play a role effect and improve protection efficient of vaccine, so adjuvant is important link in the vaccine research.So far the new adjuvant delivered of document has hundreds of, different adjuvants that the different immunoreactive mechanism that promote are arranged approximately.
Adjuvant can change functions of immune system, influences production of cytokines and regulatory function.The adjuvant that has can be strengthened whole functions of immune system; Most adjuvants then can be strengthened the secretion or the inhibition of some cytokine.The immune t-cell of existing known person and Mus has Th1 and Th2.Th1 mainly promotes complement fixation antibody and slow type anaphylaxis, and relevant cytokine has IFN-γ, IL-2, IL-12 etc.; Th2 promotes circulating antibody, and analytical type antibody comprises the generation of IgE, and relevant cell factor has IL-4, IL-5 and IL-10 etc.The adjuvant that has can be strengthened Th1, and the adjuvant that has is strengthened Th2.
Though adjuvant is of a great variety, not all adjuvant all is fit to human body to be used.The adjuvant that present stage goes through to go on the market is also only with several.And certain adjuvant also only is approved for certain particular vaccine, and do not mean that and can be used for other vaccines.Traditional adjuvant is used for new generation vaccine, also differs and play good immune effect surely, all novel vaccines and the compatibility of adjuvant must pass through strict zoopery and human clinical's experiment, could confirm whether it has practical value.
Cytokine is one group of heterogeneous peptide class regulatory factor with extensive biologic activity that immunocyte or non-immunocyte produce in the body, can activate and regulate the immunity cell in vivo, and the generation and the adjusting of immunne response had important function.The regulating action of every kind of cytokine all has multiformity.At present,, show that effectively irritation cell immunity of cytokine is united use with vaccine body is produced good immunologic enhancement through big quantity research though the mechanism of action that various cytokines are confirmed is still unintelligible.And there has been the various kinds of cell factor approval listing present stage, uses for many years clinically, and is distincter to its action effect and untoward reaction, and it as components in vaccines, is considered to safe and reliable.
But cytokine in vivo the half-life short and involve great expense, as using separately with targeting antigen, its action time and immune response asynchronism(-nization) step, so can't reach good immune effect.As uniting use with traditional adjuvant, can effectively avoid the drawback of self, prolong its half-life in vivo, and can reduce consumption.Simultaneously also can effectively replenish the cellular immunization defect of insufficient of traditional adjuvant self to body.
Therefore present stage need be found and a kind ofly can keep HPV VLPs stability for a long time and strengthen its immunogenic new pharmaceutical composition, clear and definite compatibility relationship, and production technology is simple, and production cost is lower, and safe and reliable to human body is vital.
Summary of the invention
The purpose of this invention is to provide pharmaceutical composition and application thereof that a kind of stable storing also can play human papilloma (HPV) virus-like particle (VLPs) of long-acting immunity.Repeatedly test to draw in VLPs solution according to us and use suitable salinity, buffer and pH value can effectively prevent the absorption of VLPs; Reduce the gathering of VLPs and increase its stability; Do not influence its immunogenicity; The immunogenicity that wherein adds this medicine of increase that relevant cytokine immunopotentiating agent can significance; The stimulation human body self immune system that this medicine that makes can efficiently also continue; Produce the neutrality antibody of high titre; And the generation immunological memory, thereby reach the prevention of relevant disease that HPV causes and the purpose of treatment.Therefore the pharmaceutical composition that uses of the present invention, plurality of advantages such as it is low to have a production cost, and technology is simple, and is safe and reliable.
The objective of the invention is to realize like this: the invention provides and a kind ofly be used to prevent and treat by HPV and infect and cause the human papillomavirus medicine compositions of relevant disease, it comprises that following composition and amount ranges form:
(1) HPV virus-like particle (VLPs) is adsorbed in adjuvant, and its VLPs concentration range is 10～60 μ g/ml;
(2) cytokine immunopotentiating agent, its field of activity are 1～5000 million international units;
(3) NaCl saline solution, its concentration range are 0.1M～0.4M;
(4) a kind of buffer of injection, its concentration range 5mM～20mM, the pH that keeps vaccine is 5.5～7.5;
(5) non-ionic surface active agent, its concentration range 0.0005%～0.05%;
(6) dissolve with water for injection.
The present invention also adds 0.1 of pharmaceutical composition total amount～10 ‰ pH regulator agent as required.
The selected immunogenic substance of the present invention comprises: a kind of, two or more combination in any of HPV6a, HPV6b, HPV11, HPV16, HPV18, HPV31 or HPV32; And the formed VLPs of other HPV hypotypes; Because it is identical to form mechanism, equally also be fit to this pharmaceutical composition.Show that according to research the optimum content of wherein various HPV VLPs is 10～60 μ g/ml, select content, can't produce the neutrality antibody of high titre in the body, thereby can't reach the purpose of long-acting immunity as being lower than 10 μ g/ml.As reaching identical immune effect, need frequent drug administration in a short time, so not only increase the patient suffering, immunity will reduce the immunity of body repeatedly, thereby destroy patient's autoimmune system.Select content as being higher than 60 μ g/ml, slight untoward reaction will appear in body, as be higher than 100 μ g/ml, and the body untoward reaction is more serious.
The selected adjuvant of the present invention comprises: aluminium adjuvant, Ca3 (PO
4)
2, MF59, SAF, QS-21,3-O-a kind of, two or more combination in any of removing acidylate monophosphoryl lipid (3D-MPL), immunostimulating complex (ISCOM), gathering third Acetic acid, hydroxy-, bimol. cyclic ester (PLG), AGP, lipid A derivant and CpG.Wherein preferred adjuvants is that aluminium adjuvant, ISCOM adjuvant, 3-O-remove acidylate monophosphoryl lipid (3D-MPL) adjuvant, Ca
3(PO
4)
2Adjuvant or MF59 adjuvant.
Aluminium adjuvant to comprise scope more extensive, comprise aluminium hydroxide (Al (OH)
3) adjuvant, aluminum phosphate (AlPO
4) adjuvant, Adju-Phos adjuvant, amorphous Adju-Phos sulfate (AAHS) adjuvant, Alumen (KAl (SO
4)-12H
2O) adjuvant or nano-class aluminum adjuvant.
For a long time, aluminium adjuvant is a most widely used adjuvant on the vaccine always, and when using with proper dosage, aluminium adjuvant is considered to the most safe and reliable.Existing most of in the world country ratifies, and aluminium adjuvant is used as the human adjuvant.Though present stage aluminium adjuvant Study on mechanism also be not very clear, it has been generally acknowledged that antigen is adsorbed on the aluminium adjuvant that aluminium adjuvant can effectively stimulate humoral immunization, produce IgG, the IgE of higher titre, activate the Th2 cell.
Cytokine immunopotentiating agent of the present invention is selected from interferon (IFN), interleukin (IL), a kind of, two or more combination in any of granulocyte-macrophage colony stimutaing factor (GM-CSF) or chemotactic factor (TCA).
Cytokine immunopotentiating agent of the present invention is selected from interleukin (IL), and its interleukin (IL) is preferably interleukin (II).
The application of pharmaceutical composition according to the invention, its route of administration are intradermal injection, subcutaneous injection, hypodermoclysis, intramuscular injection, intravenous injection or venous transfusion.
The selected cytokine immunopotentiating agent of the present invention be one type by activatory immunocyte (lymphocyte; Mononuclear phagocyte etc.) and relevant cell (fibrocyte; Endotheliocyte etc.) the high activity that produces with adjusting cell function; Multifunctional protein peptide molecule or glycoprotein; They interact through the specific receptor with high-affinity; Can activate and regulate immunologically competent cell in vivo and make it hypertrophy; Thereby strengthened humoral immunization; Cellular immunization and non-specific immunity make body rapidly and efficiently the HPV neutrality antibody and the immunological memory of the high titre of generation at short notice.Unite use with relevant adjuvant, can effectively avoid cytokine to lack action time in vivo, stimulate the impermanent end of keeping away.Prolong its retention time, play the effect of slow release in local organization.
Wherein IL-2 can act on various kinds of cell, comprises T, bone-marrow-derived lymphocyte, macrophage and NK cell etc., and immunne response is had rise effect widely.Show with in targeting antigen and the IL-2 intramuscular injection mice body resulting antibody titer and CD4 according to research
+The T cell proliferation all improves more than 100 times; The amount of the excretory various cytokines of vitro detection splenocyte; Find that IL-2 has strengthened the secretion of IFN-γ, IL-2 significantly; And IL-4 is had only slight reinforcement; Illustrate that IL-2 mainly is a Th1 type immunne response of having strengthened body; And help to break immunologic tolerance; It is generally acknowledged the prevention and the tumor treatment of most of infectious disease; The vaccine that can excitating organism produces Th1 type immunne response is only effectively, so the preferential selection of this patent is united use with aluminium adjuvant and IL-2.Can effectively remedy the defective that aluminium adjuvant can't activate body Th1 cell like this.
IL-12 mainly participates in cellullar immunologic response in vivo, and targeting antigen and IL-12 muscle are injected in the mice body, and the T cell obviously increases, and the CTL activity significantly improves.But humoral immunoresponse(HI) is played inhibitory action, show as antibody titer decline and B cell and reduce.
GM-CSF mainly comes enhance immunity to reply intensity through the quantity and the function of regulating antigen presenting cell in immune response.In targeting antigen injected the mice body, the positive rate of rotation of antibody obviously improved, through detecting its specific C D4
+The propagation of T cell and CTL activity all strengthen.
IFN-γ mainly breaks up to the Th1 type and regulates immunne response through participating in the Th cell, but humoral immunoresponse(HI) is inhibitory action or has no effect.
TCA3 is a member in the β chemoattracting cytoking family, can raise and activated mononuclear cell, macrophage and neutrophilic granulocyte.In targeting antigen injects the mice body, can the specific CTL effect significantly improve, IgG2a is slightly risen, illustrate that TCA3 can strengthen the former Th1 type immunne response that induces.
The selected saline solution of the present invention is NaCl; Being chosen as of this saline solution is well known in the art; Consider from the adaptability of body; The righttest salinity is 0.1～0.2M; But according to discovering; The ionic strength that increases saline solution can improve stability and the dissolubility of HPV VLPs, and then the mutual gathering between the VLPs that effectively prevents to cause owing to the temperature rising.Because high salt concentration is restricted, therefore consider that from stable aspect preferred salt concentration is 0.25～0.35M in injection.Can address the above problem though increase salinity, the injection of high salt concentration can cause the osmolality disturbance of blood plasma, can't carry out the intravenous route administration, will increase the pain of human body when subcutaneous equally, muscle, abdominal channels administration.Therefore the salinity of this patent preferred pharmaceutical compositions is: select for use when HPV VLPs concentration is lower than 100 μ g/ml etc. and to ooze NaCl solution, concentration is 0.15M.Selecting the NaCl solution concentration when HPV VLPs concentration is higher than 200 μ g/ml for use is 0.32M.
Selected solvent pH value is in 5.5～7.5 scope among the present invention, and its HPV VLPs could keep stable and form complete space conformation.Show that according to research low pH value can make between the high concentration HPV VLPs and be with identical charges, reduce gathering, avoid coagulation and the albuminous degeneration that causes, thereby make vaccine lose immunogenicity.Therefore the pH value of this patent preferred pharmaceutical compositions is: selecting pH value when HPV VLPs concentration is lower than 120 μ g/ml for use is 6.5～7.5.Selecting pH value when HPV VLPs concentration is higher than 180 μ g/ml for use is 5.5～6.5.
Selected buffer solution is phosphate buffer or histidine buffering liquid among the present invention, and the concentration of buffer is 5mM～20mM.Phosphate buffer is this area buffer system commonly used, and its buffering range is a pH value 6～8.The buffering range of histidine buffering liquid is a pH value 5.5～6.5.Show that according to research phosphate buffer causes the stability decreases of vaccine injecta because the ion of its generation can interact with aluminium adjuvant, so itself and aluminium adjuvant can not use simultaneously.Use simultaneously like need, need to increase the concentration of aluminium adjuvant in pharmaceutical composition.Phosphate buffer can cause phosphatic sucking-off when cryopreservation simultaneously, causes the drift of injection pH value.High concentration HPV VLPs meeting is coagulation owing to the instability of pH value.Thereby cause immunogenicity to descend.The buffering range of histidine buffering liquid is applicable to the preservation of high concentration HPV VLPs; Low temperature can not cause the instability of buffer system, so the buffer system of this patent preferred pharmaceutical compositions is: select phosphate buffer when HPV VLPs concentration is lower than 130 μ g/ml for use.When being higher than 200 μ g/ml, HPV VLPs concentration selects histidine buffering liquid for use.
Used in the present invention non-ionic surfactant surface-active agents, including Polysorbate 80 (eg: TWEEN
), polysorbate 20 (eg: TWEEN
).Wherein preferred activating agent is a polyoxyethylene sorbitan monoleate.Show that according to research polyoxyethylene sorbitan monoleate not only can play increases the dissolubility of HPV VLPs in pharmaceutical composition, and can also protect HPV VLPs aluminium adjuvant in the process of transportation, the reduction of the protein active of avoiding causing owing to violent concussion.But because the polyoxyethylene sorbitan monoleate of excessive concentrations can play slight blood pressure lowering and hemolytic effect in body, so the concentration range of the preferred polyoxyethylene sorbitan monoleate of this patent in pharmaceutical composition is 0.0005%～0.5% (weight/volume).
Characteristics of the present invention are to use method provided by the invention, can produce the HPV polyvalent vaccine that a kind of immunogenicity is good, the compatibility is good, the storage time is long and stable.The present invention also has characteristics such as method for preparing is simple, production cost is low, effect is good.
Description of drawings
Fig. 1 is for HPV16 VLPs is adsorbed on the different aluminum adjuvant, is kept under 2～8 ℃ the condition different pharmaceutical composition stable property comparable situation.
Fig. 2 is that HPV18 VLPs mixes with MPL, is adsorbed on the different aluminum adjuvant, is kept under 2～8 ℃ the condition different pharmaceutical composition stable property comparable situation.
Figure 3 shows the HPV6? VLPs with
mixed aluminum adsorbed on different adjuvants and stored in 2 ~ 8 ℃ under the conditions of stability of the pharmaceutical composition of different comparison.
Fig. 4 is adsorbed on respectively on the Alumen for HPV11 VLPs and QS-21 and MPL, is kept under 2～8 ℃ the condition different pharmaceutical composition stable property comparable situation.
Fig. 5 is adsorbed on Ca for HPV16 VLPs
3(PO
4)
2On the adjuvant, be kept under 2～8 ℃ the condition different pharmaceutical composition stable property comparable situation.
Fig. 6 is for HPV18 VLPs is adsorbed on the MF59 adjuvant, is kept under 2～8 ℃ the condition different pharmaceutical composition stable property comparable situation.
The specific embodiment
To further specify the present invention through embodiment below, but following instance only is the present invention's example wherein the interest field of not representing the present invention and being limited.
HPV6 VLPs, HPV11 VLPs, HPV16 VLPs, HPV18 VLPs (purity is more than 95%) are kept at 0.5M NaCl respectively; 0.003%Tween 80, in the solution of pH6.2, and aseptic filtration; Being diluted to concentration is 1mg/ml, under-70 ℃ condition, stores.
Embodiment 1: aluminium adjuvant compounding pharmaceutical compositions, the stability and the related application thereof of monitoring HPV VLPs long preservation.
The pharmaceutical composition study on the stability:
Preparation is with Al (OH)
3HPV16 VLPs as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 6.2 ± 0.1, is settled to 55ml.
This sterile pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV16 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Preparation is with the HPV16 VLPs of AAHS as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 6.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV16 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Will be with Al (OH)
3As the HPV16 VLPs pharmaceutical composition of adjuvant and with AAHS each 100 refrigerator that place 2～8 ℃ of HPV16VLPs pharmaceutical composition, regularly take a sample and carry out the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV16 VLPs to analyze) destination protein as adjuvant.During each the detection with the HPV6 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 1.
Embodiment 2: select novel MPL adjuvant compounding pharmaceutical compositions for use, the stability and the related application thereof of monitoring HPV VLPs long preservation.
The pharmaceutical composition study on the stability:
Preparation is with MPL/Al (OH)
3HPV18 VLPs as adjuvant:
Surplus is a water for injection.
Prepare by the aforementioned pharmaceutical compositions prescription, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV18 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Preparation is with MPL/AlPO
4HPV18 VLPs as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 7.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV18 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Will be with MPL/Al (OH)
3As the HPV18 VLPs pharmaceutical composition of adjuvant and with MPL/AlPO
4As each 100 refrigerator that place 2～8 ℃ of HPV18 VLPs pharmaceutical composition of adjuvant, the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV18 VLPs to analyze) destination protein is carried out in sampling regularly.During each the detection with the HPV18 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 2.
Example 3: Selection of new
adjuvant formulated pharmaceutical composition, monitoring HPV? VLPs long-term storage stability and related applications.
The pharmaceutical composition study on the stability:
Formulated to
/ AAHS as an adjuvant HPV6? VLPs:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 6.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV6 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Preparation with
/ AlPO
4HPV6 VLPs as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 6.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV6 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Will with
/ AAHS as the HPV6 VLPs pharmaceutical composition of adjuvant with
/ AlPO
4As each 100 refrigerator that place 2～8 ℃ of HPV6 VLPs pharmaceutical composition of adjuvant, the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV6 VLPs to analyze) destination protein is carried out in sampling regularly.During each the detection with the HPV6 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 3.
Embodiment 4: select novel QS-21 adjuvant compounding pharmaceutical compositions for use, the stability and the related application thereof of monitoring HPV VLPs long preservation.
The pharmaceutical composition study on the stability:
Preparation is with the HPV11 VLPs of QS-21/MPL/ Alumen as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 7.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds this pharmaceutical composition of 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV11 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Preparation is with the HPV11 VLPs of QS-21/ Alumen as adjuvant:
Surplus is a water for injection.
Prescription by aforementioned pharmaceutical compositions is prepared, and regulates pH value to 7.2 ± 0.1, is settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV11 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
To regularly take a sample and carry out the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV11 VLPs to analyze) destination protein with QS-21/MPL/ Alumen as the HPV11 VLPs pharmaceutical composition of adjuvant and with QS-21/ Alumen each 100 refrigerator that place 2～8 ℃ of HPV11 VLPs pharmaceutical composition as adjuvant.During each the detection with the HPV11 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 4.
Embodiment 5: select Ca for use
3(PO
4)
2Adjuvant preparation pharmaceutical vaccine compositions, the stability and the related application thereof of monitoring HPV VLPs long preservation.
The preparation related solution:
Preparation is with Ca
3(PO
4)
2HPV31 VLPs as adjuvant:
Surplus is a water for injection.
Prepare by the aforementioned pharmaceutical compositions prescription, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV31 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Will be with Ca
3(PO
4)
2As each 100 refrigerator that place 2～8 ℃ of HPV31 VLPs pharmaceutical composition of adjuvant, the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV31 VLPs to analyze) destination protein is carried out in sampling regularly.During each the detection with the HPV31 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 5.
Embodiment 6: select novel MF59 adjuvant preparation pharmaceutical vaccine compositions for use, the stability and the related application thereof of monitoring HPV VLPs long preservation.
The pharmaceutical composition study on the stability:
Preparation is with the HPV33 VLPs of MF59 as adjuvant:
Surplus is a water for injection.
Prepare by the aforementioned pharmaceutical compositions prescription, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition branch is filled in the aseptic cillin bottle, and every bottle adds 0.5ml, 100 of packing, and the nitrogen through aseptic filtration in whole process for preparation is processed HPV33 VLPs pharmaceutical composition to get rid of the bottle air, to roll at last to cover.
Will be with MF59 each 100 refrigerator that place 2～8 ℃ of HPV33 VLPs pharmaceutical composition as adjuvant, the percentage composition that BIAcore analyzes (utilizing the neutrality antibody of a kind of HPV33 VLPs to analyze) destination protein is carried out in sampling regularly.During each the detection with the HPV33 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is seen Fig. 6.
The immunogenicity experiments of embodiment 7:VLPs:
Immune detection and animal immune according to the foregoing description 1～4 described pharmaceutical composition formulated HPV16 VLPs pharmaceutical composition is used to be correlated with are tested.
The mouse immune experiment:
Choose 8～12 all BALB/c mouse, random packet, 12 every group; By following scheme (seeing table one) muscle place injecting immune before the right side of mice footpath.
The calibrating of immune serum antibody:
Back 14 days of immunity is for the second time extractd eyeball to mice and is got blood, collects blood with the disposable centrifuge tube of 15ml, and under 4 ℃ of conditions, the centrifugal 30min of 10000g collects serum, and-20 ℃ of preservations are subsequent use.Make envelope antigen with HPV16 VLPs, the ELISA method detects the HPV16 antibody titer in the immune serum.Wherein, A group immune serum does not all detect antibody activity, about 1: 1000 of the antibody titer of B group immune serum, about 1: 3000 of the antibody titer of C group immune serum; About 1: 5000 of the antibody titer of D, E, F group immune serum, about 1: 7000 of the antibody titer of G, H group immune serum.
The mouse red blood cell blood clotting suppresses experiment:
Because HPV virus has the ability of coagulation mouse red blood cell; But this characteristic can be suppressed by corresponding specific antibody; So we utilize its characteristic, contrived experiment detects the specific antibody and its content that whether has produced in the serum of immune mouse to VLPs.
HPV16 VLPs through detecting 1% mouse red blood cell suspension and doubling dilution interacts, and the result is when diluting for 1024 times, and VLPs makes the Mus erythrocyte be " ++ " agglutinative highly diluted multiple, promptly is defined as a viral unit.It is that VLPs dilutes by 1: 256 (618.54ng/ hole) that 4 viral units are chosen in blood clotting inhibition experiment.
Carry out red cell agglutination with 618.54ng/ hole VLPs with the mouse immune serum of different doubling dilutions and suppress experiment; Experimental result (seeing table two); It is 1: 8 that the blood clotting of B group immune serum suppresses to tire; It is 1: 16 that the blood clotting of C group immune serum suppresses to tire; It is 1: 32 that the blood clotting of D, E, F group immune serum suppresses to tire, and it is 1: 128 that the blood clotting of G, H group immune serum suppresses to tire.
The antibody capable that immune mouse produces correctly combines to stop VLPs and erythrocyte surface receptors bind with VLPs.This result of experiment has confirmed the antibody capable identification VLPs comformational epitope of mouse immune serum, and has blood clotting inhibition activity.
Embodiment 8: the influence that different proportionings change mice Th1/Th2 cytokines in the vaccine
According to the foregoing description 1 described drug regimen composition formula, adopt Al (OH)
3Adjuvant adds immune detection and the animal immune experiment that related immune promoter preparation HPV16 VLPs pharmaceutical composition is used to be correlated with.
The mouse immune experiment:
Choose 8～12 all BALB/c mouse, divide 6 groups at random, 25 every group.Wherein set up matched group (Al (OH)
3Adjuvant), IL-2 group, IL-12 group; The GM-CSF group, IFN-γ group, totally 6 groups of TCA3 groups; Each 150 μ l/ of mice leg muscle vaccinate, immunization protocol is 0,30 day; The immunity of 2 pins is respectively at 1,2 behind the initial immunity; 3; In the 2nd week behind 4 weeks and the booster immunization, all mices are all got blood through the eye socket vein, are stored in-20 ℃ behind the separation of serum.
Detect the Cytokine of Serum level:
Use BDTM Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine Kit and flow cytometer (FACS Calibur); Different blood serum samples is carried out 5 cytokines (TNF, IFN-γ, IL-5 simultaneously; IL-4, quantitative assay IL-2).
After experimental result confirms that immunopotentiating agent adds, can stimulate mice to produce stronger cellullar immunologic response, induce the Th1/Th2 cytokines of generation all to change over time.Five kinds of cytokines all reach maximum in the 4th week.Wherein IL-2 and IFN-γ group is showing that inducing mouse produces higher cytokine levels the 2nd, 3,4 weeks, apparently higher than matched group.Illustrate that this kind immunopotentiating agent initial immunity can strengthen the nonspecific immune response of vaccine, the immunogenicity that strengthens vaccine is had better action.
The clinical experiment in vitro of embodiment 9:HPV infected patient:
Cervical cancer patient's serum antibody ELISA detects:
The coating buffer 100 μ l/ holes of an amount of HPV16 VLPs PBS dilution are added in the 96 hole ELISA Plate, place 4 ℃ to encapsulate and spend the night.The liquid in the hole that inclines is with PBS washing liquid washing 3 times.Add 1%BSA-PBS 100 μ l/ holes subsequently, the washing of PBS washing liquid is 3 times behind the room temperature sealing 1hr.Add and use 0.1%BSA-PBS1: the patients serum of 3000 dilutions, the washing of PBS washing liquid is 3 times behind the incubated at room 1hr.Add afterwards and use 0.1%BSA-PBS1: the sheep anti-mouse igg 100 μ l/ holes of the HRP labelling of 3000 dilutions.The PBS washing liquid was washed 4 times after room temperature left standstill 1hr.Every hole adds 100 μ l OPD substrate buffer solutions, lucifuge reaction 10min.Last every hole adds 100 μ l stop buffer cessation reactions.Detect every hole optical density value with microplate reader and 490nm wavelength, patients serum's testing result is positive, and promptly patients serum's antibody titer is more than 1: 3000.
Detect the serum antibody and the HPV16 VLPs that draw the cervical cancer patient through ELISA and produce immunoreation, the result shows that patients serum's antibody titer is higher, and confirms that through experiment in vitro HPV16 VLPs can produce corresponding antibody in patient's body.Thereby corresponding eligible patients is reached the purpose of prevention and treatment.
Wherein, Infect and all kinds of diseases that cause by HPV, comprising by relevant diseases such as the caused cervicitis of HPV, cervical polyp, cervix uteri hypertrophy, condyloma acuminatum, breast carcinoma, lung bronchogenic carcinoma, rectal cancer, esophageal carcinoma, pharyngeal cancer, oral cancer, skin carcinomas.Can utilize said method, carry out clinical experiment in vitro but be not limited to said method.
Claims (10)
1. a human papillomavirus medicine compositions is used for prevention and treatment by the caused relevant disease of human papillomavirus, it is characterized in that: it comprises following composition and amount ranges composition:
(1) HPV virus-like particle (VLPs) is adsorbed in adjuvant, and its VLPs concentration range is 10～60 μ g/ml;
(2) cytokine immunopotentiating agent, its field of activity are 1～5000 million international units;
(3) NaCl saline solution, its concentration range are 0.1M～0.4M;
(4) a kind of buffer of injection, its concentration range 5mM～20mM, the pH that keeps vaccine is 5.5～7.5;
(5) non-ionic surface active agent, its concentration range 0.0005%～0.05%;
(6) dissolve with water for injection.
2. according to the pharmaceutical composition described in the claim 1, it is characterized in that: the HPV VLPs that is comprised is selected from a kind of, two or more combination in any of HPV6a, HPV6b, HPV11, HPV16, HPV18, HPV31 or HPV32.
3. according to the pharmaceutical composition described in the claim 1, it is characterized in that: adjuvant is selected from aluminium adjuvant, Ca
3(PO
4)
2, MF59, SAF, QS21,3D-MPL, ISCOM, PLG, AGP, lipid A derivant or CpG a kind of, two or more combination in any.
4. according to the pharmaceutical composition described in the claim 3, it is characterized in that: aluminium adjuvant is selected from aluminium hydroxide (Al (OH)
3) adjuvant, aluminum phosphate (AlPO
4) adjuvant, Adju-Phos adjuvant, amorphous Adju-Phos sulfate (AAHS) adjuvant, Alumen (KAl (SO
4)-12H
2O) adjuvant or nano-class aluminum adjuvant.
5. according to the pharmaceutical composition described in the claim 1; It is characterized in that: the cytokine immunopotentiating agent is selected from interferon (IFN); Interleukin (IL), a kind of, two or more combination in any of granulocyte-macrophage colony stimutaing factor (GM-CSF) or chemotactic factor (TCA).
6. according to the pharmaceutical composition described in the claim 5, it is characterized in that: described cytokine immunopotentiating agent is selected from interleukin (IL).
7. according to the pharmaceutical composition described in the claim 6, it is characterized in that: described interleukin (IL) be selected from interleukin (II),
8. according to the pharmaceutical composition described in the claim 1, it is characterized in that: buffer is selected from phosphate buffer or histidine buffering liquid.
9. according to the pharmaceutical composition described in the claim 1, it is characterized in that: non-ionic surface active agent is selected from polyoxyethylene sorbitan monoleate or polysorbate 20.
10. according to the application of pharmaceutical composition described in the claim 1, it is characterized in that: its route of administration is intradermal injection, subcutaneous injection, hypodermoclysis, intramuscular injection, intravenous injection or venous transfusion.
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| CN112439059A (en) * | 2019-09-02 | 2021-03-05 | 怡道生物科技(苏州)有限公司 | Recombinant human papilloma virus vaccine composition and application thereof |
| WO2022152204A1 (en) * | 2021-01-14 | 2022-07-21 | 神州细胞工程有限公司 | Stable preparation of human papillomavirus virus-like particle vaccine |
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