CN101810583B - HIV (Human Immunodeficiency Virus) fusion inhibitor slow-release microsphere - Google Patents
HIV (Human Immunodeficiency Virus) fusion inhibitor slow-release microsphere Download PDFInfo
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Abstract
The invention discloses an HIV (Human Immunodeficiency Virus) fusion inhibitor slow-release microsphere. An HIV fusion inhibitor is selected from Enfuvirtide, Sifuvirtid and Albuvirtide. The HIV fusion inhibitor slow-release microsphere adopts a biodegradable polymer as a microsphere matrix, and the grain diameter is 1-1000 microns. The HIV fusion inhibitor slow- release microsphere can be used for injection and can be slowly released in a human body for 0.5-3 months or more time. The invention also discloses a method for preparing the HIV fusion inhibitor slow-release microsphere.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to sustained-release micro-spheres that is used for the treatment of the HIV infection medicine and preparation method thereof.
Background technology
Acquired immune deficiency syndrome (AIDS) (AIDS) is the pestilence of modern society.Whole world HIV (human immunodeficiency virus) (HIV) the infected was 4,000 ten thousand in 2004, dead 3,000,000,2005 newly-increased the infecteds 5,000,000.Official statistics result shows that the existing HIV the infected of China is about 650,000, wherein about 7.5 ten thousand, 2005 New Development the infecteds about 6～80,000 of acquired immune deficiency syndrome (AIDS) patient.Present China is in a HIV and infects quick build phase, and epidemic situation spreads to the general population from the high-risk group.Prevention, control and treatment that HIV infects are very urgent.
HIV-1 is when infecting body, and at first its peplos and host's target cell membrane merge, and then enter target cell.The medicine that the HIV-1 fusion inhibitor designs at this initial course of infection just.HIV-1 merges the key step that enters target cell: (1) HIV-1 surface glycoprotein gp120 is with CD4 receptors bind on the host CD4+T cell membrane; (2) gp120 is incorporated into surface of cell membrane auxiliary receptor CCR 5 and CXCR4, and occurred conformation changes; (3) HIV-1 strides film subunit gp41 conformational change and forms six leptospira structures, exposes fusogenic peptide (FP), and peplos and host cell membrane are merged.Any one link that suppresses this process all can suppress HIV-1 and enter target cell, thereby prevents infections.According to the mechanism of action that virus is merged, the HIV-1 fusion inhibitor can be divided into the HIV-1 fusion inhibitor that the HIV-1 that disturbs the gp120 combination adheres to inhibitor, HIV-1 accessory receptor inhibitor and stops the gp41 core texture to form.
Gp41 is the membrane portions of striding of HIV-1 envelope glycoprotein, gp120 in conjunction with accessory receptor after its protein structure conformation transform, and with the little ring interaction of the conservative on the gp41, elongate little ring both sides, expose gp41 and go up fusogenic peptide, fusogenic peptide inserts in the host cell membrane, and at this moment gp41 is activated, and meanwhile gp120 goes up loose or dislocation from peplos and CD4.Striding of fusogenic peptide and gp41 2 sections seven hydrophobic amino acid residue repetitive sequences are arranged between the membrane portions, that close N holds is NHR, and near the CHR that is that strides membrane portions, NHR and CHR can form α 2 helical structures.After the gp41 activation, NHR spiral and CHR spiral begin mutually to the opposite bending, it is adjacent to each other to drive host cell and virus surface formation parallel surface, be hairpin structure, the trimeric form of gp41 then can form 3 hairpin structures simultaneously and closely rearrange six aggressiveness helical bundles, the coiled coil at 3 NHR spiralization centers wherein, and 3 CHR spirals are combined in the drain tank on coiled coil surface with antiparallel, form outer trimer spiral, dark cave is arranged in the drain tank.This six aggressiveness helical bundle makes virus be connected side by side with cell membrane, finally causes film to merge, and viral content incorporates in the host cell.The variability of gp120 is very big, as the action target spot of HIV vaccine and medicine significant limitation is arranged, might the normal immune defense function of interference body and act on the chemical compound of receptors such as CD4, CXCR4, CCR5.The composition sequence of gp41 is more conservative than gp120, and is the albumen that virus forms self, does not have homology with human body self albumen, so gp41 is acknowledged as the target spot that is more suitable for as the research of HIV entry inhibitor.The dark cave structure that drain tank on the gp41 core texture and hydrophobic residue constitute plays a crucial role for conformational activation and the film fusion of gp41, therefore can be with they desired target as anti-fusion drug design.
Early stage the discovering nineties in 20th century, the C2 polypeptide SJ22176 that is derived from the gp41CHR zone can suppress HIV-1 very effectively to be infected, and its IC50 reaches every liter of nanomole level.After this, company of Roche Group develops artificial synthesis peptide T20 (En Fuwei peptide, enfuvirtide, trade name Fuzeon).This product went through to go on the market in March, 2003, became the 3rd class inverase that first acts on HIV and target cell fusion process, i.e. the HIV fusion inhibitor.T20 can be combined with the coiled coil surface hydrophobicity ditch that the gp41NHR trimer forms, and stops that 3 gp41CHR adhere in the coiled coil periphery, suppress the formation of six aggressiveness helical bundles.The very short intermediate of time-to-live that its action site exposes when being the gp41 activation, and can disturb the interaction of gp120 and CXCR4 in conjunction with gp120, antagonism X4 type Strain infects.But the En Fuwei peptide injection needs every day at present injects twice, very inconvenient.
Based on patent (Chinese invention patent, 01130985.7,02146006.X) and Safeway's peptide of preparation is the polypeptide that 36 aminoacid are formed, be slightly soluble in water, being insoluble to organic solvent, is a kind of novel HIV fusion inhibitor, and the potency ratio T20 of its anti-HIV is high 20 times.But half-life weak point in the body of Safeway's peptide, the common lyophilized injectable powder of development at present needs inject 1～2 time every day, and reaches for 52 weeks a course for the treatment of, patient's poor compliance, treatment cost height.The calm and peaceful Safeway of Ai Bowei peptide based on patent (Chinese invention patent, 03816434.5) preparation is similar, also is a polypeptide, the potency ratio T20 height of its anti-HIV, but there is the problem of Safeway's peptide in agreement.
Aforementioned polypeptides class HIV fusion inhibitor all exists frequency injection many, the problem that dosage is big.If be prepared into long-acting sustained-release dosage form, will be conducive to administration and patient and accept.
Microsphere is the solid skeletal thing of the small spherical entity of matrix type, medicine dissolution and/or be dispersed in the macromolecular material substrate.Microball preparation has the advantage that surmounts ordinary preparation as a kind of pharmaceutical preparation of high degree of dispersion, as dosage homogeneous, targeting, controlled capability etc.Injection back microsphere is by constantly degraded in vivo, discharges the medicine of parcel and has slow releasing function.But aforementioned polypeptides class HIV fusion inhibitor poorly water-soluble is difficult to obtain the high concentration solution, therefore is difficult to be prepared into sustained release microsphere agents.
Biodegradable material is nearly brand-new material that developed rapidly in 30 years.Biodegradation typically refers to polymer (under the effects such as water, enzyme, microorganism) macromolecular integrity in biotic environment and is damaged, and produces the phenomenon of fragment or other catabolite.The feature of degraded is molecular weight and molecular weight.Biodegradable process comprises reactions such as hydrolysis, oxidation, enzymolysis.After in the implanted body, Biodegradable polymer material can resolve into nontoxic material automatically after finishing mission, discharges in body, does not therefore need the taking-up of having an operation again.
Summary of the invention
The present invention creatively prepares the HIV fusion inhibitor slow-release microsphere, therefore the invention discloses a kind of HIV fusion inhibitor slow-release microsphere and preparation method thereof.
HIV fusion inhibitor slow-release microsphere among the present invention can be used for injection, nasal-cavity administration, lung inhalation, implantation, preferably drug administration by injection.The mode of drug administration by injection is selected from subcutaneous injection, intramuscular injection, intravenous injection, preferably subcutaneous injection.The HIV fusion inhibitor slow-release microsphere is compared with traditional injection (comprising injection powder pin or normal injection), has outstanding advantage.Can in human body, keep discharging medicine for a long time after the injection of HIV fusion inhibitor slow-release microsphere, thereby significantly reduce frequency injection, good patient compliance, the treatment cost descends greatly.
HIV fusion inhibitor slow-release microsphere among the present invention, in vivo slow-release time extend to 0.5 month, 1 month, 2 months, 3 months, 6 months or 9 months, preferably slow-release time extend to 0.5 month, 1 month, 2 months or 3 months, more preferably slow-release time extends to 0.5 month or 1 month.
HIV fusion inhibitor slow-release microsphere among the present invention, its particle diameter are 1～1000 micron, preferably 5～200 microns, and more preferably 10～50 microns.
HIV fusion inhibitor slow-release microsphere among the present invention, to the amount of the amount of HIV fusion inhibitor wherein, adjuvant without limits, as long as to produce corresponding slow releasing function just passable, preferably contain other of polymer, 0～10% weight ratio of HIV fusion inhibitor, 60～99.5% weight ratios of 0.5～40% weight ratio in the sustained-release micro-spheres in acceptable accessories.Other is selected from adsorbent, solubilizing agent, cosolvent, antiseptic, stabilizing agent, freeze drying protectant, the surfactant one or more in acceptable accessories.
HIV fusion inhibitor slow-release microsphere among the present invention, wherein the HIV fusion inhibitor without limits, preferably from many peptides HIV-1s fusion inhibitor, more preferably from En Fuwei peptide, Safeway's peptide, Ai Boweitai, most preferably Safeway's peptide.
HIV fusion inhibitor slow-release microsphere among the present invention, wherein microsphere substrate is selected from the various materials that can play slow releasing function, specifically is selected from polymer, the molten inorganic material of shipwreck, preferred autohemagglutination compound.Polymer is selected from biodegradable polymer and biological difficult or degradation polymer not, preferably from the biodegradable polymer.The slightly water-soluble inorganic material is selected from calcium carbonate, calcium phosphate, calcium sulfate, calcium lactate, silicate, silicon dioxide.Biodegradable polymer is selected from natural polymer, synthetic polymer, preferably synthetic polymer.Natural polymer is selected from polysaccharide, polypeptide, protein.Polysaccharide is selected from glucosan, chitosan, alginate, starch, microcrystalline Cellulose.Protein is selected from gelatin, bovine serum albumin.Synthetic polymer is selected from polyester, polyanhydride, polyphosphazene, polyamide, preferred autopolyester, polyanhydride, more preferably polyester.Polyester is selected from polylactic-co-glycolic acid (PLGA), polylactide Acetic acid, hydroxy-, bimol. cyclic ester, polylactic acid, polyglycolic acid, poly--the 3-butyric ester, poly (lactic acid-glycolic acid), poly-adjacent ester, polylactone, polyanhydride, poly butyric ester hydroxyl pentanoate copolymer, polypropylene glucosan, hydroxyacetic acid, polylactic-co-glycolic acid Polyethylene Glycol (PLGA-PEG), polylactic acid poly ethylene glycol (PLA-PEG), polyglycolic acid Polyethylene Glycol; Preferably from polylactic acid hydroxyacetic acid, polylactide Acetic acid, hydroxy-, bimol. cyclic ester, polylactic acid, poly (lactic acid-glycolic acid), poly butyric ester hydroxyl pentanoate copolymer, polylactic-co-glycolic acid Polyethylene Glycol; More preferably from polylactic acid hydroxyacetic acid, polylactic-co-glycolic acid Polyethylene Glycol; Polylactic-co-glycolic acid most preferably.Polylactic-co-glycolic acid preferably molecular weight 3,000～50, between 000 dalton, lactic acid and the polymer of hydroxyacetic acid polymerization ratio between 10: 90～90: 10.
HIV fusion inhibitor slow-release microsphere among the present invention contains additive, is selected from the adsorbent that can adsorb the HIV fusion inhibitor, the cosolvent that can increase the dissolving of HIV fusion inhibitor.The adsorbent that can adsorb the HIV fusion inhibitor is selected from gelatin, bovine serum albumin.The cosolvent that can increase the dissolving of HIV fusion inhibitor is selected from carbamide, cyclodextrin, Polyethylene Glycol, trehalose, lactose, sucrose, mannitol, acetic acid, preferably from carbamide, acetic acid.
HIV fusion inhibitor slow-release microsphere among the present invention, its preparation method is selected from single-emulsion-liquid drying method, emulsion-liquid drying method, spray drying method, cold nebulization extraction method, phase separation method, supercritical fluid technology, preferably from single-emulsion-liquid drying method, emulsion-liquid drying method, spray drying method, phase separation method, more preferably emulsion-liquid drying method.These preparation methoies can be finished by professional and technical personnel's design and operation with reference to relevant speciality books and document.The microsphere suspension for preparing, further drying can form powder.
HIV fusion inhibitor slow-release microsphere among the present invention, when adopting emulsion-liquid drying method, its preparation technology can comprise following step:
(1) uses the organic solvent dissolution biodegradable polymer, as oil phase;
(2) aqueous solution with additive dissolves the HIV fusion inhibitor, as interior water;
(3) oil phase and interior water are mixed, form Water-In-Oil (W/O) type emulsion by mechanical force or pressure;
(4) the W/O emulsion is added outer aqueous phase and form W/O/W (W/O/W) type emulsion;
(5) emulsion is volatilized organic solvent, microsphere is solidified, centrifugal, washing then, drying obtain the microsphere solid powder.
Above-mentioned preparation technology is a general process for preparing the HIV fusion inhibitor slow-release microsphere with emulsion-liquid drying method.In actual fabrication, as required, further the restriction preparation process condition specifically comprises following aspect.
The concentration of biodegradable polymer in organic solvent is 10～1000mg/mL in the oil phase among the above-mentioned preparation technology, and the HIV fusion inhibitor is 10～500mg/mL in the concentration of interior aqueous phase.Organic solvent is selected from a kind of in dichloromethane, chloroform, dimethyl formamide, dimethyl sulfoxide, ethyl acetate, dioxane, ether, acetone, oxolane, ethanol, acetonitrile, the triethylamine or two or more mixture wherein.Additive is selected from the adsorbent that can adsorb the HIV fusion inhibitor, the cosolvent that can increase the dissolving of HIV fusion inhibitor.The adsorbent that can adsorb the HIV fusion inhibitor is selected from gelatin, bovine serum albumin.The cosolvent that can increase the dissolving of HIV fusion inhibitor is selected from carbamide, cyclodextrin, Polyethylene Glycol, trehalose, lactose, sucrose, mannitol, acetic acid, preferably from carbamide, acetic acid.Outer aqueous phase is dissolved with following one or more materials, is selected from polyvinyl alcohol, polyvinylpyrrolidone, sodium polymethacrylate, sodium polyacrylate, poly-sodium carboxymethylcellulose pyce, sodium chloride, mannitol, trehalose, and concentration is 0.5～15% weight ratio.Implement the method for mechanical force or pressure and can utilize various plant equipment, if adopt high speed homogenizer, can change high-speed stirred 0.5～5 minute by per minute 5000～50000; If the employing ultra-sonic dispersion method, ultrasonic 1～5 minute of available 100W～1000W ultrasonic probe.Volatilize the method for organic solvent, can be by methods such as decompression volatilization, heating, room temperature volatilizations, if room temperature volatilization method can be changeed the organic solvent that volatilizees under the stirring at per minute 100～1000.
If adopt spray drying method for preparation HIV fusion inhibitor slow-release microsphere, with organic solvent the biodegradable polymer dissolving is obtained oil phase, wherein said organic solvent is selected from wherein a kind of of dichloromethane, chloroform, dimethyl formamide, dimethyl sulfoxide, ethyl acetate, dioxane, ether, acetone, oxolane, ethanol, acetonitrile, triethylamine or two or more mixture wherein, with the aqueous solution that contains additive the dissolving of HIV fusion inhibitor is formed water.Wherein the concentration of biodegradable polymer in organic solvent is 10～1000mg/mL, and the HIV fusion inhibitor is 10～500mg/mL in the concentration of aqueous phase.During preparation, adopt high speed homogenizer or ultrasonic probe, changeed high-speed stirred 0.5～5 minute or batch (-type) ultrasonic 1～3 minute in room temperature with per minute 5000～25000, form the W/O emulsion, adopt spray drying to make microsphere.Additive is selected from the adsorbent that can adsorb the HIV fusion inhibitor, the cosolvent that can increase the dissolving of HIV fusion inhibitor.
The form of the HIV fusion inhibitor slow-release microsphere among the present invention can be suspension or sterile injection powder.The suspension state is the state that HIV fusion inhibitor microsphere directly disperses in disperse medium, has sedimentation phenomenon after general the placement, and the energy suspendible is even after the jolting.Disperse medium is selected from water, aqueous solution, propylene glycol, and water generally adopts water for injection.Dissolve one or more compositions such as electrolyte, osmotic pressure regulator, pH regulator agent, suspensoid in the aqueous solution, be selected from sodium chloride, glucose, mannitol, phosphate buffer, acetate buffer, sodium carboxymethyl cellulose.The HIV fusion inhibitor slow-release microsphere of suspension state is called the sustained-release micro-spheres injection when being used for injection.Sterile injection powder is the drying regime of HIV fusion inhibitor slow-release microsphere, and is general through suitably handling, and obtains through spray drying or lyophilization.The HIV fusion inhibitor slow-release microsphere of pulverulence is called slow release microphere for injection when being used for injection.
Description of drawings
Fig. 1. multi-emulsion method prepares Safeway's peptide sustained-release micro-spheres microphotograph
Fig. 2. phase separation method prepares Safeway's peptide sustained-release micro-spheres microphotograph
Fig. 3. the outer release rate of Safeway's peptide microsphere acceleration bodies and time relation
Fig. 4. the outer release rate of Safeway's peptide microsphere and time relation
The specific embodiment
Embodiment 1. multi-emulsion methods prepare Safeway's peptide microsphere
100mg Safeway peptide is dissolved in the 1.5mL 30mg/mL aqueous solution of urea, 1.0g polylactic acid-glycolic guanidine-acetic acid (MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25) be dissolved in the 2mL dichloromethane, with the two merging, at room temperature use the ultrasonic 1min of ultrasonic probe (output 100W) batch (-type), form the W/O emulsion, pour the W/O emulsion into outer aqueous phase that 1000mL contains 0.2% polyvinyl alcohol, form the W/O/W emulsion, under the stirring of 500rpm, stir 4h, the volatilization organic solvent, distilled water wash 3 times are used in centrifugalize, lyophilization 20h, obtain the white powder of good fluidity, mean diameter 48.9 μ m, outward appearance is the sphere of rounding.
Embodiment 2. spray drying method for preparation Safeway peptide sustained-release micro-spheres
1.0g Safeway peptide is dissolved in 15mL human serum albumin (HSA) aqueous solution, 10g polylactic acid-glycolic guanidine-acetic acid (MW=20000, lactic acid: hydroxyacetic acid mol ratio=50: 50) be dissolved in the 20mL dichloromethane, with the two merging, high speed homogenizer high-speed stirred 5 minutes (10000rpm) under the condition of ice bath forms the W/O emulsion.Adopt spray drying device to prepare microsphere after being chilled to room temperature.The spray process parameter is: the air ports temperature is respectively 41～46 ℃ and 34～35 ℃, feed liquid speed 7.5mL/min, stream pressure 73Psi, air velocity 400NL/h.
Embodiment 3. phase separation methods prepare Safeway's peptide sustained-release micro-spheres
100mg Safeway peptide and 30mg lactose is soluble in water as water, 1g polylactic acid-glycolic guanidine-acetic acid (MW=10000, lactic acid: hydroxyacetic acid mol ratio=50: 50) be dissolved in the 1.5mL dichloromethane as oil phase.Under condition of ice bath, make the W/O emulsion with the ultrasonic 1min of ultrasonic probe (output 100W) batch (-type); Silicone oil is at the uniform velocity splashed in the W/O emulsion that is stirring, because the intersolubility of dichloromethane and silicone oil, PLGA is precipitated out the formation medicine carrying microballoons very soon.Add normal hexane/ethanol (1: 1) and solidify, stir 2h under 4 ℃ of conditions, vacuum drying obtains the microsphere powder.
Embodiment 4. solvents remove legal system and are equipped with Safeway's peptide sustained-release micro-spheres
1g polylactic acid-glycolic guanidine-acetic acid (MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25) be dissolved in the 30mL acetonitrile, add 100mg Safeway peptide, after ultrasonic (100W) is uniformly dispersed, slowly join in the mineral oil that contains 2% (W/W) Span40 and make the O/O emulsion, stir 30min with 3000rpm speed under 55 ℃ of water-baths, continue to stir 60min with the organic solvent of evaporation residue with 440rpm again.Emulsion is chilled to 35 ℃ (preventing that Span40 from separating out), makes the microsphere sclerosis, separate microsphere and drying under vacuum condition.
Embodiment 5. multi-emulsion methods prepare the Ai Boweitai sustained-release micro-spheres
50mg Ai Boweitai is dissolved in the 1.5mL 25mg/mL aqueous solution of urea, 1.0g polylactic acid-glycolic guanidine-acetic acid (MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25) be dissolved in the 2mL dichloromethane, with the two merging, at room temperature use the ultrasonic 1min of ultrasonic probe (output 100W) batch (-type), form the W/O emulsion, pour the W/O emulsion into outer aqueous phase that 1000mL contains 0.2% polyvinyl alcohol, form the W/O/W emulsion, under the stirring at low speed of 500rpm, stir 4h, the volatilization organic solvent, centrifugalize, with distilled water wash 3 times, lyophilization 15h obtains the white powder of good fluidity.
Embodiment 6. spray drying method for preparation En Fuwei peptide sustained-release micro-spheres
1.0g En Fuwei peptide is dissolved in 15mL human serum albumin (HSA) aqueous solution, 10g polylactic acid-glycolic guanidine-acetic acid (MW=20000, lactic acid: hydroxyacetic acid mol ratio=50: 50) be dissolved in the 20mL dichloromethane, with the two merging, high speed homogenizer high-speed stirred 5 minutes (10000rpm) under the condition of ice bath forms the W/O emulsion.Adopt spray drying device to prepare microsphere after being chilled to room temperature.The spray process parameter is: the air ports temperature is respectively 41～46 ℃ and 34～35 ℃, feed liquid speed 7.5mL/min, stream pressure 75Psi, air velocity 400NL/h.
It below is the experimental example about HIV fusion inhibitor slow-release microsphere slow release effect proof.
The acceleration extracorporeal releasing test of experimental example 1. Safeway's peptide sustained-release micro-spheres
Microball preparation slow-release time in vivo is very long, and the method that we adopt external acceleration to discharge is estimated its releasing effect, and predicts its sustained release profile in vivo test situation.
Test specimen: with Safeway's peptide microsphere of embodiment 1 described method preparation.
Test reagent: 0.02% Tween 80 aqueous solution.
Test apparatus: constant temperature water bath agitator, centrifuge.
Experimental condition: temperature, 50 ℃; Rotating speed, 160rpm.
Test method: precision takes by weighing the about 10mg microsphere of laboratory sample, and to place volume be the tool lid serum bottle of 100ml, adds 90ml release medium (0.02% Tween80 aqueous solution), places the constant temperature water bath agitator, keeps certain temperature and rotating speed to take a sample on time.
Sampling method: draw 5ml solution, the centrifugal 10min of 3000rpm adds 5ml release medium (0.02%Tween-80 aqueous solution) again, and supernatant HPLC detects.
Sampling time point (hour): 1,4,7,20,30.
Result of the test: 1 hour cumulative release rate of microspheres prepared acceleration release test of the present invention is that 9%, 30 hour cumulative release rate is 79%.Conventional tablet all discharged fully in 15 minutes with this understanding, and the release of the microsphere that we prepare reached more than 30 hours.Therefore prove that it has strong slow release effect, can reach the long-acting slow-release purpose in vivo.Microsphere accelerates the release test design sketch and sees Fig. 3.
The extracorporeal releasing test of experimental example 2. Safeway's peptide sustained-release micro-spheres
Test specimen: the microsphere that adopts embodiment 2 described method preparations.
Test reagent: 0.02% Tween 80 aqueous solution.
Test apparatus: constant temperature water bath agitator, centrifuge.
Experimental condition: temperature: 37 ℃, rotating speed: 100rpm.
Test method: precision takes by weighing the about 10mg of laboratory sample, and to place volume be the tool lid serum bottle of 100ml, adds 90ml release medium (0.02% Tween-80 aqueous solution), places the constant temperature water bath agitator, keeps certain temperature and rotating speed to take a sample on time.
Sampling method: precision is measured 5ml solution, and centrifugal 10min under the 3000 commentaries on classics conditions adds the release medium (0.02% Tween-80 aqueous solution) of 5ml again, and supernatant detects with HPLC.
Sampling time point (my god): 1,4,7,20,30.
Result of the test: the 1st day cumulative release rate of microspheres prepared acceleration release test of the present invention is that 12%, the 30 day cumulative release rate is 86%.Conventional tablet all discharged fully in 30 minutes with this understanding, and the release of the microsphere that we prepare reached more than 30 days.Therefore prove that it has strong slow release effect, can reach the long-acting slow-release purpose in vivo.Microsphere release test design sketch is seen Fig. 4.
The extracorporeal releasing test of experimental example 3. Ai Boweitai sustained-release micro-spheres
Test specimen: with the microsphere of embodiment 5 described method preparations.
Test method is with experimental example 2.
Result of the test: the 1st day cumulative release rate of microspheres prepared acceleration release test of the present invention is that 10%, the 30 day cumulative release rate is 89%.Prove that it has slow release effect, can reach the long-acting slow-release purpose in vivo.
The extracorporeal releasing test of experimental example 4. En Fuwei peptide sustained-release micro-spheres
Test specimen: with the microsphere of embodiment 6 described method preparations.
Test method is with experimental example 2.
Result of the test: the 1st day cumulative release rate of microspheres prepared acceleration release test of the present invention is that 12%, the 30 day cumulative release rate is 95%.Prove that it has slow release effect, can reach the long-acting slow-release purpose in vivo.
Pharmacodynamic experiment in experimental example 5. Safeway's peptide microspheres
Safeway's peptide microsphere of test drug: embodiment 1 preparation
Virus: HIV-1
Animal: immunodeficient mouse (SCID) mice) suppress immunity, intravenous injection human T lymphocyte through radiation.
Route of infection: tail vein injection HIV-1 viral infection is transplanted the human lymphocyte immune deficient mice.
Positive drug: Safeway's peptide normal injection agent.
Observation index: P24 antigen titre in the blood plasma, HIV-1 viral load (HIV-1RNA) and human lymphocyte.
Operation: will transplant the immunodeficient mouse grouping of human lymphocyte, and establish normal control group, virus control group, positive drug matched group and Safeway's peptide microsphere group.Use 1-10TCID
50HIV-1 virus tail vein injection infecting mouse infects back 2h by the peptide injection subcutaneous injection 1mg/kg of Safeway, every day 1 time, successive administration 30 days; The peptide microsphere 30mg/kg of Safeway, single subcutaneous injection.Get the anticoagulation separated plasma, measure the P24 titre, measure viral nucleic acid viral load (HIV-1RNA) with PCR method.Calculate virus control group and medication therapy groups blood human lymphocyte, judge therapeutic effect.
The result: Safeway's peptide microsphere shows and Safeway's peptide injection group identical treatment effect that virus titer and RNA are very low at the 20th day.
Claims (4)
1. Safeway's peptide sustained-release micro-spheres, step preparation below adopting: 100mg Safeway peptide is dissolved in the 1.5mL30mg/mL aqueous solution of urea, 1.0g polylactic acid-glycolic guanidine-acetic acid, MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25 is dissolved in the 2mL dichloromethane, with the two merging, at room temperature use ultrasonic probe, output 100W, the ultrasonic 1min of batch (-type) forms the W/O emulsion, pour the W/O emulsion into outer aqueous phase that 1000mL contains 0.2% polyvinyl alcohol, form the W/O/W emulsion, under the stirring of 500rpm, stir 4h, the volatilization organic solvent, centrifugalize, with distilled water wash 3 times, lyophilization 20h obtains the white powder of good fluidity, 48.9 microns of mean diameters, outward appearance are the sphere of rounding.
2. Safeway's peptide sustained-release micro-spheres, step preparation below adopting: 100mg Safeway peptide and 30mg lactose is soluble in water as water, 1g polylactic acid-glycolic guanidine-acetic acid, MW=10000, lactic acid: hydroxyacetic acid mol ratio=50: 50 is dissolved in the 1.5mL dichloromethane as oil phase; Under condition of ice bath, use ultrasonic probe, output 100W, the ultrasonic 1min of batch (-type) makes the W/O emulsion; Silicone oil is at the uniform velocity splashed in the W/O emulsion that is stirring, because the intersolubility of dichloromethane and silicone oil, PLGA is precipitated out the formation medicine carrying microballoons very soon; Add 1: 1 normal hexane/ethanol; Solidify, stir 2h under 4 ℃ of conditions, vacuum drying obtains the microsphere powder.
3. Safeway's peptide sustained-release micro-spheres, step preparation below adopting: get 1g polylactic acid-glycolic guanidine-acetic acid, MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25, be dissolved in the 30mL acetonitrile, add 100mg Safeway peptide, in the 100W ultra-sonic dispersion slowly joins the mineral oil of the 2%w/w Span40 that contains after evenly, make the O/O emulsion, stir 30min with 3000rpm speed under 55 ℃ of water-baths, continue to stir 60min with the organic solvent of evaporation residue with 440rpm again; Emulsion is chilled to 35 ℃, makes the microsphere sclerosis, separate microsphere and drying under vacuum condition.
4. Yi Zhong Ai Boweitai sustained-release micro-spheres, step preparation below adopting: 50mg Ai Boweitai is dissolved in the 1.5mL25mg/mL aqueous solution of urea, 1.0g the polylactic acid-glycolic guanidine-acetic acid, MW=10000, lactic acid: hydroxyacetic acid mol ratio=75: 25, be dissolved in the 2mL dichloromethane, with the two merging, at room temperature use ultrasonic probe, output 100W, the ultrasonic 1min of batch (-type), form the W/O emulsion, pour the W/O emulsion into outer aqueous phase that 1000mL contains 0.2% polyvinyl alcohol, form the W/O/W emulsion, under the stirring at low speed of 500rpm, stir 4h, the volatilization organic solvent, distilled water wash 3 times are used in centrifugalize, lyophilization 15h obtains the white powder of good fluidity.
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| CN103239712A (en) * | 2012-02-14 | 2013-08-14 | 天津市扶素生物技术有限公司 | HIV (human immunodeficiency virus) microbicide and application thereof |
| CN109692634B (en) * | 2019-01-31 | 2021-07-23 | 合肥工业大学 | Micro-polymer particles based on eutectic solvent emulsion and preparation method thereof |
| MX2021013634A (en) * | 2019-05-07 | 2022-01-31 | Frontier Biotechnologies Inc | Stable albuvirtide compositions. |
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| WO2001037896A2 (en) * | 1999-07-09 | 2001-05-31 | Trimeris, Inc. | Methods and compositions for administration of therapeutic reagents |
| WO2007021970A2 (en) * | 2005-08-15 | 2007-02-22 | Praecis Pharmaceuticals, Inc. | Stable pharmaceutical formulations and methods of use thereof |
| CN101400363A (en) * | 2006-01-18 | 2009-04-01 | Qps有限公司 | Pharmaceutical compositions with enhanced stability |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001037896A2 (en) * | 1999-07-09 | 2001-05-31 | Trimeris, Inc. | Methods and compositions for administration of therapeutic reagents |
| WO2007021970A2 (en) * | 2005-08-15 | 2007-02-22 | Praecis Pharmaceuticals, Inc. | Stable pharmaceutical formulations and methods of use thereof |
| CN101400363A (en) * | 2006-01-18 | 2009-04-01 | Qps有限公司 | Pharmaceutical compositions with enhanced stability |
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| 贾伟等.复乳－液中干燥法.《药物控释新剂型》.化学工业出版社,2005,第186－187页. * |
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| CN101810583A (en) | 2010-08-25 |
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