CN1293919C - Short corynebacteria pharmaceutics or short corynebacteria pharmaceutics without cells in application for preparing medicine of curing HIV infection or AIDS - Google Patents

Short corynebacteria pharmaceutics or short corynebacteria pharmaceutics without cells in application for preparing medicine of curing HIV infection or AIDS Download PDF

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CN1293919C
CN1293919C CNB031574548A CN03157454A CN1293919C CN 1293919 C CN1293919 C CN 1293919C CN B031574548 A CNB031574548 A CN B031574548A CN 03157454 A CN03157454 A CN 03157454A CN 1293919 C CN1293919 C CN 1293919C
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preparation
short corynebacteria
aids
acellular
corynebacteria
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CN1600323A (en
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高尚先
李守悌
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Shanghai Changrun Biotechnology Co ltd
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NATIONAL INSTITUTE FOR CONTROL OF PHARMACEUTICAL AND BIOLOGICAL PRODUCTS
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Abstract

The present invention discloses the application of a corynebactrium parvum preparation (CPP) or a non-cell corynebactrium preparation (NCP) on preparing medicaments for curing HIV infection or AIDS. The corynebactrium parvum preparation is a preparation which is made after carrying out formaldehyde inactivation on corynebacterium parvum (CP), and the non-cell corynebactrium preparation has the nanometer level, uniform particle size, improved absorptivity and diffusibility, low pyrogen and no residual formaldehyde; the CPP and the NCP of the present invention both have the bioactivity of spleen activation, tumor suppression, etc., and have obvious functions of resisting leukemia retrovirus in mice; additionally, the present invention can be used in treatment or adjunctive therapy of HIV infection or AIDS.

Description

The application in preparation treatment HIV infection or AIDS medicament of a kind of short corynebacteria preparation or acellular short corynebacteria preparation
Technical field
The present invention relates to a kind of short corynebacteria preparation (CPP) or the application of acellular short corynebacteria preparation (NCP) in pharmaceutical field, relate in particular to the application in preparation treatment HIV infection or AIDS (AIDS) medicament, belong to drug world.
Background technology
AIDS is that (acquied immunodeficiencysyndrome AIDS), is by HIV (human immunodeficiency virus) (human immunodeficiency virus, a kind of serious infectious disease that HIV) causes to acquired immune deficiency syndrome (AIDS).Virus-specific ground is invaded and loss lymphocyte differentiation group 4 +T lymphocyte (cluster of differentiation 4 +T cell), i.e. CD4 +The T cell claims the T4 cell again, causes the body cell immunocompromised host.Initial representation is the symptomless virus carrier clinically, continue to develop into persistence lymph nodes of body as a whole enlargement syndrome (persistent generalized lymphadenopathy, PGL) and AIDS AIDS-related complex AIDS (AIDS related complex, ARC), heating, asthenia, night sweat occur, become thin, feel sick, vomiting, stomachache, diarrhoea, the enlargement of whole body lymph, thrush, herpes labialis and herpes zoster etc., last concurrent various serious opportunistic infection and malignant tumor become AIDS (AIDS).Because of still not having specific short at present, case fatality rate is high, so be called as " pestilence in 20th century ".
AIDS is a kind of global infectious disease, and preventing and controlling this disease has become " a global political issue " that needs various countries to take seriously.According to UNAIDS's data, existing 6,000 ten thousand people have infected HIV (human immunodeficiency virus) by the end of the present whole world, and wherein 2,000 ten thousand people are devitalized by AIDS.At present, there are 4,000 ten thousand the infecteds in the whole world, and wherein 90% in developing country, and as not strengthening preventive measure, to the year two thousand twenty, global AIDS also will seize 6,800 ten thousand people's life.It is reported that simultaneously Chinese actual HIV (human immunodeficiency virus) number of the infected has surpassed 1,000,000 people at present, as effectively not controlling, will be by 2010 above 1,000 ten thousand people.AIDS has become the fourth-largest cause of death in the whole world.
The classification that the Center for Disease Control (CDC) Walter Reed is arranged that the diagnostic criteria of AIDS is commonly used in the world.Topmost condition is Kaposi's sarcoma and opportunistic infection.Laboratory diagnosis is that cd4 cell descends and the HIV (human immunodeficiency virus) antibody positive.The HIV of U.S. CDC in 1993 revision infects in classification and the AIDS diagnostic criteria, CD4 level and clinical disease are taken all factors into consideration interior, the important change of new classification be can count according to CD4＜200/ μ L determines to suffer from acquired immune deficiency syndrome (AIDS).
HIV (human immunodeficiency virus) (HIV) is a single strand RNA virus, can cultivate in external lymphocyte series, belongs to the Retroviridae lentivirus, divides two types, i.e. HIV-1 type and HIV-2 type, HIV-1 type selectivity infringement CD4 +The T lymphocyte also can infect mononuclear phagocyte, B cell, microgliacyte and bone marrow stem cell, is the main strain that causes AIDS; The virulence of HIV-2 type is strong not as the HIV-1 type.HIV-1 type and HIV-2 type all can cause AIDS, but the AIDS of general indication is due to the HIV-1 type.
The people is the source of infection of AIDS, and asymptomatic HIV the infected and AIDS VICTIMS all have infectiousness.Found that virus is present in blood, saliva, tear, milk, sperm and the vaginal secretions, prompting can cause propagation, still fails to detect HIV in the perspiration.
The route of infection of AIDS are: transmission through sex, injection propagation, the propagation of blood source and mother-to-baby transmission etc.Age of onset is mainly the person between twenty and fifty below 40 years old.
HIV is to CD 4 +T lymphocyte (comprising lymphocyte, mononuclear cell and macrophage etc.) has special close preferendum.According to present research, the morbidity of AIDS and HIV content, virulence, variation and CD 4 +T lymphocyte quantity, function are relevant with the immunity of organism situation.
1, after HIV intrusion and reproduction process HIV invade human body, at accessory receptor (chemokine receptors) CCR 5, CXCR 4Deng synergism under, virus surface gp120 and CD 4 +The lymphocytic CD of T 4The molecular specific receptors bind, after sloughing capsid by means of gp41, viral core protein and RNA enter cytoplasm, article two, the viral RNA chain is under the effect of reverse transcriptase, reverse transcription becomes single stranded DNA, is template repetition DNA under the effect of DNA polymerase with this DNA then, and part remains in the endochylema, partly the DNA with host cell integrates, and becomes the proviral DNA (proviral DNA) of latency.Proviral DNA can be activated by certain factor, duplicates, is transcribed into viral RNA and mRNA, and the translation virus protein is assembled into new virus, disengages in the blastogenesis mode, infects other cells again.
2, CD 4 +The mode and the performance of T lymphocyte damaged
(1) coup injury: HIV is massive duplication in cell, causes cytolysis or breaks.
(2) indirect injury: claim the amalgamation damage again, like the CD that infects 4 +T lymphocyte HIV-env gene height coding gp120 and gp41 make the cell surface that is contaminted have gp120 to express, can with the CD of contiguous uninfection 4 +The combination of T lymphocyte forms fused cell, makes permeability of cell membrane change cell generation dissolved destruction.
(3) bone marrow stem cell is impaired: HIV can infect and destroy stem cell, makes CD 4 +The T lymphocyte produces and reduces.
(4) immunologic injury: in the blood free gp120 can with CD 4 +The combination of T lymphocyte makes it to become target cell and is damaged by immunocyte, and its quantity is reduced.
CD4 +The T also function of appeal is unusual, mainly shows the recognition function obstacle, and lymphokine produce to reduce, and the interleukin-1 receptor expression decreased lowers the reactivity of allogenic antigen, to the miscellaneous function attenuating of B cell etc.
3, HIV is to the influencing HIV and can cause the impaired and dysfunction of monokaryon-macrophage of other cells, and there is CD on monokaryon-macrophage surface 4Molecule and accessory receptor CCR 5, CXCR 4Molecule etc., so HIV can infect monokaryon-macrophage, makes it to become the storage place of virus, and plays an important role in the diffusion of virus, carries virus by blood-brain barrier, causes central nervous system infection.
HIV can infect and destroy the monokaryon-huge stem cell system of biting of bone marrow.Macrophage has the cytopathic effect due to the anti-HIV infection, but along with virus is constantly duplicated, macrophage function occurs unusual, handles antigenic ability and weakens, and the ability of body antagonism HIV infection and other pathogenic infections is reduced.
4, the impaired and dysfunction bone-marrow-derived lymphocyte of bone-marrow-derived lymphocyte has low-level CD 4The expression of molecule, but can't determine whether CCR 5, CXCR 4Deng the existence of accessory receptor, so whether HIV can still have arguement by direct aggression B cell, but HIV the infected B cell function is sure unusually.Along with CD 4 +The lymphocytic dysfunction of T, the quantity and the function of B cell also change.Infect in early days at HIV, since the stimulation of virus and virus protein, PBA, peripheral blood B lymphocytosis, circulating immune complex occurs, and IgG, IgA level increase.Along with the progress of the state of an illness, the bone-marrow-derived lymphocyte dysfunction, the reactivity that neoantigen is stimulated reduces, and autoimmune phenomena can occur.
5, the unusual performance NK cell of natural killer cell (NK cell) damage has immunologic surveillance function, infection and antineoplastic action are arranged, though HIV the infected and patient AIDS NK cell counting are normal, functional defect loses the function of supervision to infection and antitumor cell.
6, body immune system collapse initial infection, body has produced fabulous immunoreation to HIV, and HIV is suppressed or is eliminated in the serum, CD 4 +T lymphocyte viral replication in is relative static conditions, does not therefore cause body's immunological function damage and exhaustion, and still keeps secular asymptomatic stage after the serum anti-HIV is changeed sun.But in course of infection, the HIV gene constantly produces variation, and antigen and virulence be constantly variation also, and antigenic variation can make HIV escape the body fluid of body and the attack of cellular immunization, and the virulence variation can influence the process and the seriousness of disease.Cause the new variant that continuous generation is duplicated soon, virulence is strong, make CD 4 +The T lymphocyte quantity reduces gradually, and immunologic function suffers damage, last CD 4 +The T lymphocyte reduces rapidly and exhausts, causes whole immune system collapse, makes the infected be developed to AIDS from asymptomatic stage at 2-10 in the year.
Owing to do not find the specific medicament of anti-HIV at present as yet, thereby emphasize Comprehensive Treatment, comprise the treatment of antiviral, immunomodulating, antitumor and controller opportunistic infections etc.The clinical treatment spininess of AIDS is to a certain particular stage in the viral biocycle, and the ability of the enzyme that duplicates, suppresses to play a crucial role by disturbing HIV RNA or the particulate assembling process of activity, break virus of regulatory factor and infection cell reaches the purpose of treatment.
Antiviral drugs commonly used has: (zidovudine, zidovudine are that present curative effect has been widely used in the anti-HIV medicine of the first clinical line preferably, AZT) to (1) neat Fu Duoding.It stops HIV (human immunodeficiency virus) to be duplicated by suppressing reverse transcriptase, can make CD 4 +Cell increases, and improves immunologic function and clinical symptoms, and the case that can also make short-term make progress into AIDS reduces.Main toxic action is bone marrow depression, and other side effect have myositis, headache, feel sick, vomiting etc.(2) dideoxycytidine (ddC) and didanosine (ddI) are to be used for the anti-HIV medicine of the second clinical line at present.Advantage is the bone marrow inhibition of no AZT, can effectively suppress virus P24 antigens, and the helper T lymphocyte absolute counting is increased, clinical manifestation improves, weight increase etc., and side effect is peripheral neuritis and pancreatitis etc., but side effect here is relevant with dosage.(3) foscarnet sodium (foscarnet sodium) reaches the purpose that stops HIV to duplicate by suppressing reverse transcriptase activity.Its side effect be feel sick, headache, weak, anemia and serum creatinine rising etc.(4) (α-interferon) has the anti-reverse transcription enzyme, suppress HIV duplicates and reduces virus antigen and produce isoreactivity alpha-interferon.Common adverse effect has heating, weak, influenza sample syndrome, leukopenia and thrombocytopenia etc., can with use in conjunction such as other antiviral agents such as AZT.(5) acyclovir (acyclovir) can suppress multiple virus and comprise duplicating of HIV, and side effect has heating, weak, local excitation etc., accidental renal damage.
HIV (human immunodeficiency virus) receptor blocking agent such as recombinant soluble CD4 +Prepared product (rsCD4) can prevent that HIV from combining with cd4 cell, can also stop HIV cell that infects and the CD4 that does not infect +The fusion of cell, thereby the intercellular diffusion of blocking virus.
Immune modulating treatment can strengthen HIV the infected's immunologic function, commonly used has: (1) thymosin (thymosin) cellular immunization reinforcing agent, be applicable to the viral and neoplastic disease due to immunodeficiency and the immune dysfunction, few side effects and light, accidental one crosses property dizziness, uncomfortable in chest etc.(2) lentinan (lentinan) excited body fluid of energy and cellular immunization have pharmacologically active widely, no obvious toxic-side effects.(3) interleukin-2 (IL-2) can strengthen the activity of T lymphocyte and natural killer cell (NK), and suppresses the active effect of viral dna polymerase, has excitatory cells immunity and antivirus action, and side effect has heating, shiver with cold, anorexia and feeling of fatigue.
Although said medicine is used in vivo or in vitro tests all shows certain antivirus action, can prolong survival time of patients, but effect is limited, the factors such as continuous appearance of the toxicity of medicine such as bone marrow depression and drug resistance Strain have still limited long-term heavy dose of use of these medicines, so emphasize therapeutic alliance at present, suitable drug combination is expected to reduce every kind of amount of drug and reduces the frequency that drug resistance strain produces.
Summary of the invention
The object of the present invention is to provide a kind of short corynebacteria preparation (CPP) or the new application of acellular short corynebacteria preparation (NCP) in pharmacy, i.e. application in preparation treatment HIV infection or AIDS medicament, described short corynebacteria preparation is the preparation of being made behind formalin-inactivated by short corynebacteria (CP), described NCP is a nanoscale, the granular size homogeneous, trap and diffusance improve, low pyrogen, formaldehydeless residual, have identical spleen activation with the whole cell preparation and reach biologic activity such as pressing down tumor, can reduce the fever of CPP, chest pain, the injection site redness, scleroma, side effect such as Liver and kidney toxicity; Short corynebacteria preparation (CPP) or acellular short corynebacteria preparation (NCP) all have the effect of tangible anti-mice reverse transcription leucovirus as a kind of nonspecific immunity regulator, can be used for the treatment or the auxiliary treatment of HIV infection or AIDS.
To achieve these goals, the technical solution used in the present invention is: the application of a kind of short corynebacteria preparation in preparation treatment HIV infection or AIDS medicament.
The application of a kind of acellular short corynebacteria preparation in preparation treatment HIV infection or AIDS medicament.
Short corynebacteria preparation of the present invention (CPP, 2000 editions " Chinese biological goods rules ") is a kind of nonspecific immunity regulator.It is the bacterin of being made behind formalin-inactivated by short corynebacteria (CP), and its preparation process is disclosed in 1995 editions and 2000 editions " Chinese biological goods rules ".The CPP preparation of using also has the inactivated vaccine that French Merleux institute is produced at present; The inactivated vaccine that Britain Wellcome pharmaceutical factory produces etc.
CPP has the ability of activated mononuclear phagocyte system.CPP mainly shows immune effect: the activated mononuclear macrophage, make the NK cytoactive strengthen, promote body that multiple antigen is produced IgG and IgM, inducement interferon, interleukin II etc., therefore have antitumor and anti-infectious function.
The antitumor action of CPP is confirmed by many experiments and clinical research.To using CPP before and after the laboratory animal inoculated tumour, can significantly suppress some solid tumor (as breast carcinoma, melanoma etc.), ascitic type tumor and leukemic growth.The animal of tumor regression has specific resistance for the tumor cell of attacking once more.CPP also has inhibitory action to neoplasm metastasis.
CPP antitumor and anti-infective core are by activating macrophage, and its quality and quantity is improved greatly, and activated macrophage can kill the microorganism of tumor cell and infection.
CPP is used for the history of the existing many decades of tumor and other treatment of diseases as immunomodulator, and it is generally acknowledged by activated mononuclear macrophage system antitumor, anti-infective effect.
02123571.6,02123570.8 and 02123572.4 applied for a kind of acellular short corynebacteria preparation, its preparation method and the purposes on preparation treatment tuberculosis medicine.
Acellular short corynebacteria preparation of the present invention (NCP) is that granularity is less than 100nm by the cell-free preparation of short corynebacteria (CP) through the fragmentation preparation.
Wherein said CP, formal name used at school are that propionibacterium acne (propionibacterium acnes) bacterium number is 65101 (76-27 or 7627), 65102 (H-84) or 65103 (77-1 or 771).
The pyrogen of described NCP is less than 160EU/ml, protein content 10-40% (g/g), and nucleic acid content 3-25% (g/g), all the other are polysaccharide.
The pyrogen of preferred NCP of the present invention is less than 60EU/ml, protein content 20-30% (g/g), and nucleic acid content 3-10% (g/g), all the other are polysaccharide.
The granularity of NCP of the present invention is preferably 10-50nm.
NCP of the present invention prepares by following step:
1) .CP work seed lot strain is continuous is inoculated in big bottle or nutrition jar after going down to posterity for 2-4 time, and cultivation is 5-7 days in culture medium;
2). collect the thalline of no living contaminants, obtained bacterium liquid in heated and boiled 15-60 minute;
3). the qualified bacterium liquid of sterility test is after washing, with the broken thalline of breaker;
4). the collecting precipitation after centrifugal of the suspension behind the bacterial cell disruption, suspension is made in washing precipitation;
5). with the suspension heat sterilization, obtain NCP.
Wherein the CP formal name used at school described in the step 1) is a propionibacterium acne, and bacterium number is 65101 (76-27 or 7627), 65102 (H-84) or 65103 (77-1 or 771).
Culture medium is to be selected from a kind of in THIOGLYCOLLIC ACID salt, peptone, liver or the yeast dialysis solution culture medium, and is low pyrogen culture medium.
Condition of culture is 36-37 ℃ of cultivation in the step 1).
Preferred described culture medium is not for containing the sulphur glycollate culture medium of agar.
Wherein the thalline described in the step 3) is an inactivated bacteria, and cell concentration is 50-500 hundred million/ml.
Described breaker is that bacterial cell disruption is arrived less than 100nm, and preferred size is the superhigh pressure water jet collider of 10-50nm.
Wherein said bacterial cell disruption is to carry out under aseptic condition.
Wherein the washing described in the step 3) is with precipitate physiological saline solution centrifuge washing at least 2 times; Washing described in the step 4) is with precipitate physiological saline solution centrifuge washing 0-5 time.
The centrifugal condition of suspension is 4 ℃-room temperature behind the step 4) bacterial cell disruption, the centrifugal 30-150 of 6000-12000rpm minute.
Being heated to be described in the step 5) 60-65 ℃ of heating 0.5-2 hour.
In the preparation method of the present invention, further comprise the NCP packing that step 5) is obtained after, ampoule 60+2 ℃ the heating 30 minutes.
Culture medium described in the present invention is preferably the low pyrogen culture medium through 0.22 μ m or μ m0.45 μ m membrane filtration.
In the preparation method of the present invention, also comprise the NCP lyophilization that step 5) is obtained, obtain the lyophilized formulations of NCP.
The NCP pyrogen of method for preparing is less than 160EU/ml, protein content 10-40% (g/g), and nucleic acid content 3-25% (g/g), all the other are polysaccharide.
The NCP pyrogen of preferred method for preparing is less than 60EU/ml, protein content 20-30% (g/g), and nucleic acid content 3-10% (g/g), all the other are polysaccharide.
The NCP granularity of method for preparing is less than 100nm, and preferred size is 10-50nm.
Steriling test described in the present invention is qualified to be meant must not check according to the method for " Chinese biological goods rules " regulation any bacterial growth.
Hang down pyrogen culture medium, particularly sulphur glycollate culture medium owing to adopting among the present invention, and in conjunction with strict Quality Control, and aseptic low grade fever origin operation in the whole process of production, guaranteed that final products hang down the pollution of pyrogen and the assorted bacterium of nothing.
The present invention is crushed to nanoscale by the superhigh pressure water jet collider with CP, by selecting different centrifugal force (rotating speed), determined centrifugal extraction conditions, the precipitation that obtains partly is NCP, prove through animal experiment repeatedly, said preparation has identical spleen activation with whole cell and reaches biologic activity such as pressing down tumor, and supernatant part then alienia activates and tumor-inhibiting action, shows that NCP of the present invention has good biologic activity.
Removing residue formaldehyde especially to the zest of responsive pleura, adopts heat inactivation to substitute formalin-inactivated to the zest of body among the CPP among the present invention in order to eliminate.Zoopery shows that heat-killed NCP, CPP have identical spleen activation and but biologic activity such as tumor than the CPP of formalin-inactivated.
The operation principle of superhigh pressure water jet collider is that (operating pressure is 10-3000kgf/cm to the liquid that will be mixed with machined object with high-pressure pump 2) be divided into two strands after the pressurization, entering two diameters with high-speed jet is that flow out the head-on collision back in opposite directions in the passage formed of 8MM diamond wafer.Because the speed of liquid is very high, when clashing in opposite directions, fluid produces very strong shock wave, and make diamond wafer produce high frequency, high intense ultrasonic wave.The high-power high-frequency ultrasound wave makes the moment pulverizing of machined object granule, ultramicronising.
In the preparation process of the present invention, being broken thalline better, adopting the superhigh pressure water jet collider, is 50-500 hundred million/ml at cell concentration, and operating pressure is 800-1500kgf/cm 2Condition under broken, thalline suspension after the fragmentation is under 4 ℃-room temperature, the centrifugal 30-150 of 6000-12000rpm minute, abandon supernatant, precipitation is made suspension with behind physiological saline solution solution washing 0-5 time, heats 0.5-2 hour at 60-65 ℃, the qualified back of sterility test merges, through preparation, packing, ampoule obtains the good purpose product of high-quality, granularity and homogeneity 60+2 ℃ of heating 15-60 minute after the packing.
NCP of the present invention is that cell breakage with CP is to nanoscale, through the invalid supernatant part of centrifugal removal, collecting precipitation partly prepares, product has low pyrogen, no living contaminants, formaldehydeless residual, granule has nanoscale, homogeneous grain diameter, trap and diffusance improve, and help absorption of human body and improve drug effect; Simultaneously can reduce fever, chest pain, side effect such as injection site redness, scleroma are a kind of promising immune formulations.
As required, NCP of the present invention can make the suspension formulation of physiological saline solution, and solids content is 1.0mg/ml or 2.0mg/ml, also can make freeze-dried powder or be fit to other medicinal dosage forms; CPP of the present invention can be dosage form commonly used at present, as suspension formulation, freeze-dried powder or suitable other medicinal dosage forms.
Used CPP, the NCP of the present invention is nonspecific immunity strengthening agent, NCP is by the cell-free preparation of CP through the fragmentation preparation, for nanoscale, granular size homogeneous, trap and diffusance improve, low pyrogen, formaldehydeless residual, have identical spleen activation with the whole cell preparation and reach biologic activity such as pressing down tumor, can reduce fever, the chest pain of CPP, injection site redness, scleroma, side effect such as Liver and kidney toxicity, therefore NCP has overcome the some shortcomings of CPP, and the nonspecific immunity strengthening agent that can be used as a kind of excellent performance is applied to treatment of diseases such as antitumor and infection.
The present invention is through the pharmacodynamic study to short corynebacteria preparation (CPP) or acellular short corynebacteria preparation (NCP), show that CPP, NCP have the effect of tangible mouse anti reverse transcription leucovirus, show the certain anti-AIDS effect of tool, can be applicable in preparation treatment HIV infection or the AIDS medicament; Because the NCP product has low pyrogen, no living contaminants, formaldehydeless residual, granule has nanoscale, homogeneous grain diameter, trap and diffusance height, be easy to absorption of human body, also reduce fever, chest pain simultaneously, side effect such as injection site redness, scleroma, so the effect of NCP in preparation treatment HIV infection or AIDS medicament is more obvious.
Because the intravital immune system of AIDS VICTIMS is subjected to catastrophic collapse, the few and dysfunction of T lymphocyte quantity, the patient dies because of the unmanageable infection of multiple cause of disease.The most common with pneumocystis carinii pneumonia in the infection, it is the activatory clinical marker of HIV latent infection.The clinical manifestation of AIDS is very complicated, and opportunistic infection and malignant tumor can be involved each system of whole body and organ, and multiple infection and tumor are often arranged and deposits, and often dies from these complication.Pneumonia, Ka Boqi sarcoma and the pulmonary tuberculosis etc. that cause such as opportunistic infection.
CPP of the present invention, NCP are nonspecific immunity strengthening agent, mainly act on M Φ, mononuclear phagocyte system had powerful and persistent stimulation, and can be by promoting that hematopoietic pluripotential stem cell is divided into mononuclear phagocyte, transfer the potentiality that body immune system is contained, bring into play anticancer, microbicidel function.
The anti-HIV effect of CPP, NCP mainly is by to the powerful and persistent stimulation of mononuclear phagocyte system, causes M Φ hypertrophy, activation, phagocytic function enhancing and secretion inducing interferon (IFN-γ), interleukin (IL-2) active oxygen (H 2O 2, NO) etc. killer factor and strengthen the NK cell killing activity, reach the purpose that suppresses and kill and wound HIV.
Along with the development to pathogenetic deeply understanding of HIV and molecular immunology theory, the effect of nonspecific immunity in anti-HIV more and more paid attention to by people.M Φ not only plays an important role in the body nonspecific immunity, and also plays important effect in specific immunity.Mononuclear phagocyte system is removed has phagocytic function, also can bring into play antigen presentation (antigen presentation) effect, also belong to antigen-presenting cell (antigen-presentation cell, APC), but helper T lymphocyte activation, and stimulate its function mutually, thereby amplify the specific immunity effect with lymphocyte.Yet have only the anti-HIV effect of activatory M Φ competence exertion, and constant HIV variation of the anti-HIV of activatory M Φ and chemical sproof influence.So give full play to body's immunological function, combined with chemotherapy has important effect in the treatment of AIDS.
A kind of short corynebacteria preparation of the present invention or acellular short corynebacteria preparation are when being used for preparation treatment HIV infection or AIDS medicament, its usage is: 1～7 time/week, each 1～20mg, intramuscular injection or other suitable route administrations, 1～12 month course of treatment or follow the doctor's advice.Can be at interval with or take continuously several courses of treatment.
Used CPP, the NCP of the present invention is a kind of immunomodulator, is used for preparation treatment HIV and infects or the AIDS medicament, has following advantage:
1. the invention provides the new medical usage of CPP, NCP, provide new approach treatment HIV infection or AIDS.
2. NCP preparation of the present invention has low pyrogen, no living contaminants, formaldehydeless residual, and granule has nanoscale, homogeneous grain diameter, and trap and diffusance improve, and help absorption of human body and improve drug effect; Simultaneously can reduce fever, chest pain, injection site redness, scleroma, side effect such as Liver and kidney toxicity are a kind of promising immune formulations, can be used as to be used for preparation treatment HIV infection or AIDS medicament.
3. preparation technology of the present invention is simple, can make oral, injection, tablet etc., and is easy to use.
4. show through drug efficacy study that CPP of the present invention, NCP have the spleen activation and reach biologic activity such as pressing down tumor, energy tuberculosis (TB) infects, anti-mice reverse transcription leucovirus infection effect, helps the state of an illness for HIV the infected and controls, and delays the state of an illness and shows effect; To AIDS with opportunistic infection or tumor inhibitory action is arranged, help the improvement of the AIDS state of an illness; Because NCP is nanoscale, have advantages such as good absorbing, side effect are little, its effect is better.
The specific embodiment
Be that specific embodiments of the invention are used to describe in detail the present invention below, described embodiment is used to describe the present invention rather than restriction the present invention.Wherein the CP formal name used at school is propionibacterium acne (propionibacterium acnes), bacterium number is that 65101 (76-27), 65102 (H-84) are disclosed in 1993 editions " Chinese medicine bacteria culture catalogues ", publishing house of Beijing Institute of Technology (ISBN7-81013-859-6/R.7), 65103 (77-1 or 771) are disclosed in 2000 editions " Chinese biological goods rules ", Chemical Industry Press.
Embodiment 1
Carry out foundation, calibrating and other calibrating of antibacterial seed lot with reference to the method for describing in 2000 editions " Chinese biological goods rules ".
The culture medium that adopts in the present embodiment is the sulphur glycollate culture medium (not containing agar) that Beijing three medicine scientific and technological development companies produce, and composition is: trypticase 15g/l, yeast soak powder 5g/l, glucose 5g/l, sodium chloride 2.5g/l, L-cystine 0.5g/l, sodium thioglycollate 0.5g/l.Before using with culture medium with 0.22 μ m membrane filtration.
Breakdown CP (formal name used at school propionibacterium acne, propionibacterium acnes) 771 (seeing " Chinese biological goods rules ") strain pipe, every pipe contains bacterium 6,000,000,000, draw about 0.2-0.3ml sulphur glycollate culture medium (not containing agar) with capillary tube, be added in strain pipe bottom and make it to dissolve fully the back sucking-off, in the pipe (capacity 38ml), mix in the adding, put 37 ℃ and cultivated 2 days with above-mentioned culture medium 8-10ml.(bacterium liquid: culture volume is than=1: 9) mix, put 37 ℃ and cultivated 2 days with above-mentioned culture medium to get bacterium liquid.The bacterium liquid of getting above-mentioned cultivation mixes with above-mentioned culture medium (by 1: 9), puts 37 ℃ and cultivates 3 days, results bacterium liquid.By the bottle microscopy, the person abandons it the living contaminants.Merge to collect the thalline heated and boiled 15 minutes of no living contaminants, with superhigh pressure water jet to clashing into (Japanese Nanonizer Inc. product, model NMG-75-200) at 1000kgf/cm 2Broken thalline is 3 times under the pressure; Suspension after the fragmentation was centrifugal 90 minutes of room temperature, 6000rpm, collecting precipitation, with physiological saline solution washing precipitation 3 times, make the suspension that solids content is 0.5mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions " The People's Republic of China's pharmacopeia ") are qualified, 60 ℃ were heated 1 hour, and obtained NCP.
CP wherein, formal name used at school is propionibacterium acne 65103 (77-1) or 771.
Detect (solids content 1mg/ml), NCP pyrogen 20EU/ml of the present invention through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China); Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", BJ University Press) is measured protein content 25% (g/g); Ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 10% (g/g); It is 10% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content.The measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide do not measure.It is 60-100nm that Electronic Speculum is measured granularity.
After the packing, ampoule was 60+2 ℃ of heating 20 minutes.
Embodiment 2
Method with reference to embodiment 1, with CP (formal name used at school propionibacterium acne, propionibacterium acnes) 65101 (76-27) are continuous is inoculated in the nutrition jar after going down to posterity for 4 times, in sulphur glycollate culture medium (not containing agar) (before using with culture medium with 0.45 μ m membrane filtration), 36-37 ℃ of cultivation 6 days; By the bottle microscopy, the person abandons it the living contaminants; The thalline heated and boiled of the no living contaminants of merging collection 60 minutes uses the superhigh pressure water jet collider at 1500kgf/cm 2Broken thalline is 5 times under the pressure; Centrifugal 1 hour of suspension 8000rpm after the fragmentation, with physiological saline solution washing precipitation 5 times, make the suspension that solids content is 2.0mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions " The People's Republic of China's pharmacopeia ") are qualified, 65 ℃ were heated 60 minutes, and obtained NCP.
Detect (solids content 1mg/ml), NCP pyrogen 160EU/ml through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China); Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", BJ University Press) is measured protein content 40% (g/g); Ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 5% (g/g); It is 20% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content.The measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide may not measure.It is 10-50nm that Electronic Speculum detects granularity, epigranular.
After the packing, ampoule was 60+2 ℃ of heating 30 minutes.
Embodiment 3
With reference to the method for embodiment 1, be inoculated in the nutrition jar after going down to posterity for 4 times with CP (formal name used at school propionibacterium acne, propionibacterium acnes) 65102 (H-84) are continuous, in protein culture medium, cultivated 7 days for 36-37 ℃; By the bottle microscopy, the person abandons it the living contaminants; The thalline heated and boiled of the no living contaminants of merging collection 30 minutes is with superhigh pressure water jet collider (Japanese Nanonizer Inc. product, model AQUA300) 800kgf/cm 2Broken thalline is 10 times under the pressure; Suspension 12000rpm after the fragmentation is centrifugal, with physiological saline solution washing precipitation 4 times, make the suspension that solids content is 1.0mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions Pharmacopoeias of the People's Republic of China) are qualified, 65 ℃ were heated 100 minutes, and obtained NCP.
Detect (solids content 1mg/ml) NCP pyrogen 60EU/ml through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China), Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", the BJ University Press) measures protein content 12% (g/g), ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 25% (g/g), it is 16% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content, the measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide do not measure.It is 40-80nm that Electronic Speculum detects particle mean size, epigranular.After the packing, ampoule was 60+2 ℃ of heating 30 minutes.
Embodiment 4
Other are with embodiment 1, and difference is that suspension after the fragmentation after 8000rpm is centrifugal 1 hour, uses the freeze dryer lyophilizing, obtains injectable powder.
Embodiment 5
Adopt the method for describing in 2000 editions " Chinese biological goods rules " with CP (formal name used at school propionibacterium acne, propionibacterium acnes) is inoculated in the nutrition jar after 771 continuous go down to posterity for 3 times, in sulphur glycollate culture medium (not containing agar), cultivated 5 days for 36-37 ℃; By the bottle microscopy, the person abandons it the living contaminants; The thalline of the no living contaminants of merging collection heated and boiled respectively carried out sterility test in 30,60 minutes, the qualified bacterium liquid of sterility test is after centrifugal, with fresh sterile normal saline solution washing precipitation 3 times and make suspension, 60-65 ℃ was heated 1 hour, the qualified back packing of aseptic detection, ampoule was 60+2 ℃ of heating 30 minutes, and to contain bacterium for every bottle be 6.0 * 10 to two kinds of samples after testing 9
Embodiment 6
Other are with embodiment 5, and difference is that the thalline of the no living contaminants gathered adds formalin to ultimate density and is about 0.5% (ml/ml) sterilization 48 hours, after testing every bottle to contain bacterium be 6.0 * 10 9, free formaldehyde content is 0.06g/L.
Experimental example 1
The residual formaldehyde that trace is arranged among the CPP, formaldehyde energy fixing protein, killing bacteria, but body is had stronger zest.Though residual formaldehyde seldom (should not be higher than 0.06g/l) among the CPP, but still can be to human body, especially the pleura to sensitivity produces zest.
The spleen of the CPP for preparing according to the method for stipulating in CPP manufacturing and the vertification regulation in the 240-241 page or leaf in 2000 editions " Chinese biological goods rules " among the present invention activates and presses down the tumor biologic activity and test, and the results are shown in Table 1.
Simultaneously, according to following method the CPP of embodiment of the invention 1-6, NCP spleen are activated among the present invention and press down the tumor biologic activity and test, the results are shown in Table 1.
Spleen activation experiment: with the purebred mice (C of body weight 18-20g 3H, 615 or C 57BL), each 10 of test group and matched groups, male and female half and half.Every mouse peritoneal of test group is injected CPP 0.5ml respectively and (is contained bacterium 1.5 * 10 9) and the NCP preparation 0.5ml (the NCP solids content of 1-3 is all adjusted to 1mg/ml) of embodiment 1-3, matched group is injected commensurability physiological sodium chloride solution.Put to death mice in 14 days, weigh respectively, spleen is heavy, calculate spleen index, its index should be not less than 2.0.
Press down tumor experiment (ehrlich ascites tumor): with the purebred mice (C of body weight 18-20g 3H, 615, C 57BL, K-LACA or Kunming mouse), each 10 of test group and matched groups, male and female half and half.Every oncocyte (5.0 * 10 that the mouse peritoneal injection is lived 6/ ml) 0.2ml (with the 5-8 days non-bloody ascites that go down to posterity).Test group is next day after preceding 1 day of oncocyte of injection and injection, and continuous abdominal cavity is injected CPP 0.25ml respectively and (contained bacterium 1.5 * 10 9) and the NCP 0.25ml (the NCP solids content of 1-3 is adjusted to 2mg/ml) of embodiment 1-3 5 times, each 1 day at interval.Matched group is injected commensurability physiological sodium chloride solution.Control group mice is all calculated test group mice survival rate because of cancer ascites is dead for starting point, and 30 days observation periods, its survival rate should be not less than 70%.
Table 1
Spleen index Press down the tumor experiment
CPP (formalin-inactivated, embodiment 6) ＞2.0 Qualified
CPP (heated and boiled deactivation 30 minutes, embodiment 5) ＞2.0 Qualified
CPP (heated and boiled deactivation 60 minutes, embodiment 6) ＞2.0 Qualified
NCP (embodiment 1) ＞2.0 Qualified
NCP (embodiment 2) ＞2.0 Qualified
NCP (embodiment 3) ＞2.0 Qualified
NCP (embodiment 4) ＞2.0 Qualified
Spleen activates and presses down tumor and test biologic activity and the drug effect that CPP or NCP can be described.The result of table 1 shows, the CPP and the NCP of the present invention of heated and boiled 30 minutes, heated and boiled 60 minutes, 0.5% formalin deactivation preparation, its mice spleen activation reaches but the tumor experiment effect is identical, no significant difference illustrates that heated and boiled deactivation and broken the extraction do not have obvious influence to biologic activity and the drug effect of CPP and NCP.
Experimental example 2
This experimental example relate to NCP absorbent properties and with the comparison of CPP absorbent properties.
Content: rabbit CPP and NCP Intradermal absorption experiment.
Rabbit: 1500-2000 gram, male and female half and half.
Grouping: be divided into CPP group and NCP group, every group each 4.
Administration: every rabbit back intradermal injection, matched group CPP group 0.2ml/ is (15,000,000,000/ml is equivalent to 2.5mg/ml) only, and NCP group 0.2ml/ is (2.5mg/ml) only, observes and the test injection part, the results are shown in Table 2.
Table 2
Time Rabbit number The NCP group The CPP group
24 hours 1# 0.2 * 0.2cm does not have redness, scleroma 0.8 * 0.8cm redness, scleroma
The result 2# 0.2 * 0.2cm does not have redness, scleroma 0.8 * 0.8cm redness, scleroma
3# 0.2 * 0.2cm does not have redness, scleroma 0.8 * 0.8cm redness, scleroma
4# 0.2 * 0.2cm does not have redness, scleroma 0.8 * 0.8cm redness, scleroma
48 hours results 1# 0.2 * 0.2cm does not have redness, scleroma 1.0 * 1.0cm redness, scleroma
2# 0.2 * 0.2cm does not have redness, scleroma 1.0 * 1.0cm redness, scleroma
3# 0.2 * 0.2cm does not have redness, scleroma 1.0 * 1.0cm redness, scleroma
4# 0.2 * 0.2cm does not have redness, scleroma 1.0 * 1.0cm redness, scleroma
72 hours results 1# 0.2 * 0.2cm does not have redness, scleroma 1.2 * 1.2cm redness, scleroma
2# 0.3 * 0.3cm does not have redness, scleroma 1.2 * 1.2cm redness, scleroma
3# 0.3 * 0.4cm does not have redness, scleroma 1.5 * 1.5cm redness, scleroma
4# 0.4 * 0.4cm does not have redness, scleroma 1.5 * 1.5cm redness, scleroma
Conclusion: observed 72 hours, NCP is easy to be absorbed, and does not produce obviously red and swollen, scleroma, and CPP is difficult for being absorbed, and has produced tangible redness, scleroma reaction.This presentation of results more helps the absorption of body with the cell breakage of CPP to the NCP of nanoscale preparation, and can reduce side effect, so the NCP effect is better, is preferred nonspecific immunity strengthening agent.
Experimental example 3
This experimental example relates to the animal acute toxicity test of NCP.
Medicine: adopt the method preparation of embodiment 1, but NCP content 31.7mg/ml is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Animal: Kunming mouse, male and female half and half are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute's animal center.
Maximum dosage-feeding is measured, get 40 of above-mentioned healthy mices, be divided into two groups at random, male and female half and half, an intramuscular injection Cmax of administration group mice (31.7mg/ml), the NCP of maximum volume (the every side of 0.1m/ * 2), an intramuscular injection equal-volume of matched group normal saline, the body weight and the clinical manifestation of the 3rd day and the 7th day the results are shown in Table 3 before and after the above two groups of administrations of record.
Table 3
Group Animal Dosage Body weight before the administration Body weight is the 3rd day the 7th day after the administration Suitable clinical application multiple
Only mg/kg g g
The NCP group 20 31.7 20.61+0.61 20.80+1.50 26.67+2.19 452.9
Matched group 20 0 20.10+0.48 22.00+1.70 27.41+1.84
The above results shows, the NCP 31.7mg/ml of an intramuscular injection maximal dose of mice, death does not appear in mice, do not find the clinical manifestation relevant with administration, do not find the influence of administration to body weight yet, 452.9 times of the suitable clinical application amount of dosage illustrate that the clinical plan dosage of people is a safe dose.
Experimental example 4
CPP of the present invention, NCP are nonspecific immunity strengthening agent, have the spleen activation and reach biologic activity such as pressing down tumor, can anti-mice reverse transcription leucovirus infection effect.Present embodiment selects preferred NCP that the pharmacodynamics of retrovirus murine leukemia infecting mouse model is estimated.
Interior animal experiment
Because the HIV species specificity is strong, can not infect the non-human primate animal, people manage by belonging to retroviral correlated virus together with HIV, or set up animal model by gene recombination method, and the most frequently used model is simian immunodeficiency virus (sinian immrnodeficiency virus, SIV) and mouse leukaemia virus (murineleukemia virus, MuLV), because the mouse model easy operating, dosage is few, low price is so the most normal being used.
1. experimental design is according to, purpose
According to Ministry of Public Health " new drug preclinical study guideline ", adopt retrovirus murine leukemia infecting mouse model that its anti-AIDS curative effect is studied, to estimate the drug action of the anti-AIDS of NCP.
2. materials and methods
2.1 animal, facility and raising: the Balb/c mice, female, in 6 ages in week, 16-19 gram is provided by Shandong University's Experimental Animal Center, the animal quality certification number: Shandong kinoplaszm [20021239]; Laboratory Animal Facility is qualified: Shandong rotating ring word 200101004; Conventional mouse is raised.
2.2 test sample
NCP (2mg/ml) is provided lot number: 020117 by Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Deposit time spent normal saline preparation for 4 ℃.
2.3 positive control drug
Combivir, tablet, every contains lamivudine 150mg, Qi Duofu pyridine 300mg; Glaxowellcom provides, lot number: MAR2001.
2.4 animal model
Mice reverse transcription leucovirus (SRS8), teaching and research room provides by Fudan University in Shanghai medical board biophysics; This Strain recovery back is the centrifugal supernatant that goes earlier, adds fresh 10% calf serum culture fluid subsequently and cultivates, the mouse peritoneal inoculation.
Virus virulence is measured: every mouse peritoneal injection 1 * 10 5～1 * 10 6Individual cell/0.3ml; The dead mouse situation is observed in the inoculation back.With animal dead 50% inoculum concentration is the model inoculum concentration.
2.5 dosage, grouping and administration
1) normal control group: 20
2) model control group: 20
3) positive controls: 20.The administration after 4 hours of contaminating, Combivir was irritated stomach, successive administration 14 days in 200mg/kg days.
4) NCP high dose group: 2.0mg/ time only, 10
5) dosage group among the NCP: 0.5mg/ time only, 20
6) NCP low dosage binder: 0.125mg/ time only, 10
The NCP medication: animal abdominal cavity contamination is administered intramuscular after 4 hours, the next day 1 time, totally 5 times.
2.6 observe, measure
Female Balb/c mouse peritoneal infected back 4 hours, by the dosage regimen administration; Weighed every 2 days; In the time of 14 days 1), 2), 3), 5) group respectively kills 10 mices, dissects and gets spleen, observes splenopathy kitchen range and counting; Claim spleen re-computation spleen coefficient; Get blood and survey routine blood test, compare with each test group; Remain animal and observe survival rate and average life day, observe, calculate protective rate and statistical significance to 60 days.
3. result
3.1 viral vitality test (seeing Table 4)
The viral vitality test of table 4
Inoculum concentration (individual cell/only) No 21 days survival rates (%)
1×10 6 5×10 5 3×10 5 1×10 5 10 10 10 10 0.0 0.0 0.0 40.0
Behind the mouse inoculation SRS8 Strain cell, the death time concentrates on 16～21 days, and inoculum concentration is 1 * 10 5The time, animals survived is about 50%, so mouse infection model inoculum concentration is 1 * 10 5Individual cell/only.
3.2 administration is the index variation in the time of 14 days
3.2.1 experimental result for the first time
In 14 days, each group of body weight changes not obvious (table 5) after the administration of mouse infection virus; The hematology changes obviously, Combivir matched group and NCP group WBC counting, lymph percentage ratio, and platelet count is apparently higher than model control group, and NCP group spleen index is apparently higher than normal control and model group (table 6), and significant difference.
3.2.2 the time-to-live
Counteracting toxic substances, when being administered to 22 days, model group ascites is obvious, other groups are not obvious;
30 days survival rates: NCP height, middle dosage group are apparently higher than matched group, model group, and demonstration doses effect relation; The model group survival rate is 0 in the time of 60 days, and NCP height, middle dosage group are greater than 40%, and low dose group only is 10%.
Table 5 mice administration body weight change (g)
Group N Time after the administration (d)
2 4 6 8 10 12 14
Normal control model group NCP organizes (0.5mg/ 10 10 10 22.17± 2.57 20.57± 1.10 19.89± 2.67 22.08± 2.88 21.68± 1.09 20.36± 2.32 21.80± 3.34 21.90± 0.96 20.62± 2.16 23.40± 3.24 22.36± 1.00 20.84± 2.39 23.36± 2.87 22.30± 1.41 21.31± 2.33 23.48± 2.72 22.43± 1.49 21.51± 2.26 23.02± 2.67 18.38± 2.15 20.95± 2.33
Only) Combivir 10 21.72± 1.53 19.76± 2.55 19.16± 3.71 21.8± 2.94 20.18± 2.15 20.68± 1.80 20.68± 2.39
Table 6 NCP administration is hematology's variation in the time of 14 days
Group WBC× 10 9/L The WBC classification PBC × 10 12/L HB g/l PLT× 10 9/L Spleen index (%)
N(％) L(％) M(％)
Normal control model group Combivir NCP group 12.71± 9.89 5.68± 3.23 12.37± 4.86** 13.29± 5.69** 79.5±7.9 70.9± 10.3 64.1± 10.2 64.8± 12.6 9.3±3.1 13.4±5.2 21.0± 6.7**ΔΔ 18.4± 5.7*ΔΔ 11.2±5.3 15.7±5.9 15.0±7.6 16.7±7.8Δ 7.46± 1.06 8.63± 0.98 7.99± 0.93 8.59± 1.22 141.7± 7.0 154.8± 12.9 133±10.7 143.7± 14.8 1788±572 1229±503 2967± 1061** 1785± 354** 0.60± 0.16 0.45± 0.12 1.35± 0.33 1.27± 0.18
N=10:NCP=0.5mg/ only, the next day 1 time totally 5 times; *P＜0.01, *P＜0.05 is compared with model group; Δ Δ P＜0.01 is compared with normal group.
WBC: leukocyte; N (%): neutrophilic granulocyte; L (%): lymphocyte; M (%): have a liking for acid, have a liking for alkali and mononuclear cell
RBC: erythrocyte; Hb; Hemoglobin; PLT: platelet
Conclusion:
The NCP result of the test shows that NCP has the effect of tangible anti-mice reverse transcription leucovirus, shows the certain anti-AIDS effect of tool.

Claims (10)

  1. Short corynebacteria preparation or acellular short corynebacteria preparation preparation treatment HIV infect or the AIDS medicament in application.
  2. 2. the application in preparation treatment HIV infection or AIDS medicament of short corynebacteria preparation according to claim 1 or acellular short corynebacteria preparation, it is characterized in that wherein said short corynebacteria preparation is the preparation of being made by short corynebacteria behind formalin-inactivated.
  3. 3. the application in preparation treatment HIV infection or AIDS medicament of short corynebacteria preparation according to claim 1 or acellular short corynebacteria preparation, it is characterized in that, wherein said acellular short corynebacteria preparation is that granularity is less than 100nm by the cell-free preparation of short corynebacteria through the fragmentation preparation.
  4. 4. according to claim 1 or 3 described short corynebacteria preparations or the application of acellular short corynebacteria preparation in preparation treatment HIV infection or AIDS medicament, it is characterized in that, wherein said acellular short corynebacteria preparation pyrogen is less than 160EU/ml, protein content 10-40% (g/g), nucleic acid content 3-25% (g/g), all the other are polysaccharide.
  5. 5. according to claim 1 or 3 described short corynebacteria preparations or the application of acellular short corynebacteria preparation in preparation treatment HIV infection or AIDS medicament, it is characterized in that the granularity of wherein said acellular short corynebacteria preparation is 10-50nm.
  6. 6. acellular short corynebacteria preparation according to claim 3 preparation treatment HIV infect or the AIDS medicament in application, it is characterized in that wherein said short corynebacteria is a propionibacterium acne, bacterium number is 76-27, H-84 or 77-1.
  7. 7. the application in preparation treatment HIV infection or AIDS medicament according to claim 3 or 6 described acellular short corynebacteria preparations is characterized in that wherein said short corynebacteria is 77-1.
  8. 8. the application in preparation treatment HIV infection or AIDS medicament according to claim 1 or 4 described acellular short corynebacteria preparations, it is characterized in that, wherein said acellular short corynebacteria preparation pyrogen is less than 60EU/ml, protein content 20-30% (g/g), nucleic acid content 3-10% (g/g), all the other are polysaccharide.
  9. 9. the application in preparation treatment HIV infection or AIDS medicament of short corynebacteria preparation according to claim 1 or acellular short corynebacteria preparation is characterized in that wherein said acellular short corynebacteria preparation prepares by following method:
    1). short corynebacteria work seed lot strain is continuous to be inoculated in big bottle or nutrition jar after going down to posterity for 2-4 time, cultivates 5-7 days in culture medium;
    2). collect the thalline of no living contaminants, obtained bacterium liquid in heated and boiled 15-60 minute;
    3). the qualified bacterium liquid of sterility test is after washing, with the broken thalline of breaker;
    4). the collecting precipitation after centrifugal of the suspension behind the bacterial cell disruption, suspension is made in washing precipitation;
    5). with the suspension heat sterilization, obtain acellular short corynebacteria preparation.
  10. 10. the application in preparation treatment HIV infection or AIDS medicament of short corynebacteria preparation according to claim 9 or acellular short corynebacteria preparation is characterized in that wherein said condition of culture is 36-37 ℃; Described culture medium is not for containing the sulphur glycollate culture medium of agar;
    Described thalline is an inactivated bacteria, and described breaker is that bacterial cell disruption is arrived less than 100nm, and preferred size is the superhigh pressure water jet collider of 10-50nm, and fragmentation is carried out under aseptic condition; Washing described in the step 3) is with precipitate physiological saline solution centrifuge washing at least 2 times;
    Washing described in the step 4) is with precipitate physiological saline solution centrifuge washing 0-5 time; The centrifugal condition of suspension is under 4 ℃-room temperature behind the bacterial cell disruption, the centrifugal 30-150 of 6000-12000rpm minute;
    Being heated to be described in the step 5) 60-65 ℃ of heating 0.5-2 hour.
CNB031574548A 2003-09-22 2003-09-22 Short corynebacteria pharmaceutics or short corynebacteria pharmaceutics without cells in application for preparing medicine of curing HIV infection or AIDS Expired - Fee Related CN1293919C (en)

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