CN105816852B - Pharmaceutical composition, preparation method and use containing non-T cell conjugating peptide and lactic-glycolic acid block copolymer - Google Patents
Pharmaceutical composition, preparation method and use containing non-T cell conjugating peptide and lactic-glycolic acid block copolymer Download PDFInfo
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Abstract
The present invention provides a kind of pharmaceutical composition, and it includes non-T cell conjugating peptides and lactic-glycolic acid block copolymer (PLGA).Described pharmaceutical composition can be allowed to therefrom slow release, and have good biocompatibility as the storage cavern of non-T cell conjugating peptide, and encapsulation rate is high, and drugloading rate is high, the low advantage of burst release rate.The present invention also provides the preparation method of described pharmaceutical composition and its preparing the purposes in the drug for treating rheumatoid arthritis.
Description
Technical field
The invention belongs to pharmaceutical fields.Specifically, the present invention relates to a kind of biological agent, more particularly, to one kind containing non-
The pharmaceutical composition and preparation method thereof of T cell binding peptide and lactic-glycolic acid block copolymer (PLGA).
Background technique
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of special for significant pathology with erosive synovitis
It levies, using chronic destructive arthritis as the autoimmune disease of clinical characters.Studies have shown that RA is antigen driving, T cell Jie
The autoimmune disease led.Specifically, the starting link of RA is pathogenic antigens by the HLA-DR molecule of antigen presenting cell
Offer to cell surface, form three molecular complex of HLA- Antigenic Peptide-TCR in conjunction with T cell receptor (TCR), so that activation is caused a disease
Property T cell, activate downstream cytokine cascade reaction.Therefore, the formation of three molecular complex of HLA- Antigenic Peptide-TCR is blocked just
The response of RA pathological immune may be blocked from morbidity upstream, achieve the purpose that treat disease.
II Collagen Type VI (CII) is principal causative autoantigen relevant to RA, and wherein CII 263-272 is susceptible with RA
The Main Antigenic that gene HLA-DR β 1 is combined.Non-T cell conjugating peptide uses the amino acid in CII263-272 in conjunction with TCR
Alanine without side chain or glycine replacement, retain the amino acid combined with HLA-DR β 1, formed one kind only with HLA-DR β 1
Molecule combination, the Altered peptide without being identified by TCR.This feature enables non-T cell conjugating peptide Reverse transcriptase itself to cause
The combination of sick antigen and HLA-DR β 1 destroys the formation of three molecular complexes, to inhibit to be situated between by pathogenic antigens specific T-cells
The autoimmune response led and thus caused by the pathologic processes such as inflammatory mediator release, reach treatment RA and other T cells mediate
Autoimmunity disease purpose.Non-T cell conjugating peptide shows the treatment results of Collagen-Induced Arthritis (CIA) large and small mouse
Non-T cell conjugating peptide can inhibit the arthritic inflammation process of the large and small mouse of arthritis, and also have significantly to the osteoclasia in later period
Therapeutic effect, prompt it to can inhibit the degree of collagen induced arthritis and the course of disease.
However, non-T cell conjugating peptide is a small peptide, it is easy to be metabolized in vivo, half-life short, needs to inject repeatedly
It could work.Therefore, this field even needs the new formulation of non-T cell conjugating peptide, and the preparation needs that non-T cell can be used as
The storage cavern of binding peptide is allowed to therefrom slow, stable release, to overcome the Half-life in vivo of existing non-T cell conjugating peptide preparation
The poor disadvantages such as low with oral administration biaavailability of short, stability.
Summary of the invention
In order to solve the above-mentioned technical problem, the present inventor prepares non-T cell conjugating peptide and biodegradable material PLGA
At a kind of composition, this composition can be used as the storage cavern of non-T cell conjugating peptide, the drug as non-vein administration.In particular,
After the composition administration, non-T cell conjugating peptide can be slowly released from composition, to reduce administration frequency, increase
Add the compliance of patient, improve the quality of life of patient.
The term " non-T cell conjugating peptide " used herein includes amino acid sequence for Phe-Lys-Gly-Glu-Gln-
Peptide fragment (the NTAP of Ala-Gly-Ala-Gly-Glu；SEQ ID NO:1), while also the amino including the peptide fragment aminoterminal is logical
Cross the modified outcome (Ma-NTAP) of covalently key connection myristic acid group.Also, for simplicity, NTAP can also refer to Ma-
NTAP。
Specifically, it is an object of the present invention to provide a kind of non-vein administrations containing non-T cell conjugating peptide and PLGA
Pharmaceutical composition.
It is a further object of the present invention to provide the preparation methods of described pharmaceutical composition.
It is yet another object of the invention to provide the pharmaceutical applications of described pharmaceutical composition.
Technical solution for achieving the above object is as follows.
On the one hand, the present invention provides a kind of pharmaceutical composition, and it includes non-T cell conjugating peptides and PLGA.In the medicine group
It closes in object, Biodegradable polymer material PLGA is scattered in PLGA as host material, non-T cell conjugating peptide, to make
Non-T cell conjugating peptide therefrom slow release.
By weight percentage, it is preferable that described pharmaceutical composition include 3%~14% non-T cell conjugating peptide and
86%~97% PLGA.
Wherein, for the PLGA, molecular weight is 9.0 × 103~2.3 × 104Dalton, preferably 1.3 × 104Road
Er Dun；And preferably, in the PLGA, the mass ratio of polylactic acid and polyglycolic acid (PLA:PLG) be (50:50)~
(75:25), preferably 50:50.
Pharmaceutical composition of the invention is preferably microspheres form, and particle diameter distribution is 10~120 μm.The microsphere surface light
Sliding, regular particles are without adhesion (see Fig. 1).It is demonstrated experimentally that the microballoon can continue within 20-40 days time, stablize release
Non-T cell conjugating peptide；Preferably, the microballoon is sustainable in vivo, it is dense to stablize the effective non-T cell conjugating peptide of offer 30 days
Degree.
On the other hand, the present invention provides the preparation method of the pharmaceutical composition.
In short, pharmaceutical composition of the invention can pass through phase separation method, W/O/W double emulsion solvent volatility process or S/O/
It is prepared by W emulsion-solvent evaporation method.
Using phase separation method, it the described method comprises the following steps:
(1) PLGA is dissolved in organic solvent and oily phase is made, take non-T cell conjugating peptide that oily phase is added as solid phase；
(2) S/O suspension is formed after homogenizing the mixture that step (1) obtains, it is then that silicone oil is same what is stirred at low speed
When be added drop-wise in S/O suspension；
(3) after being added dropwise to complete, normal heptane is added and stirs at low speed with solidified microsphere, is saved after collecting microballoon.
Wherein, in step (1), organic solvent is the mixed liquor of methylene chloride or methylene chloride and acetone；Wherein when having
When solvent is the mixed liquor of methylene chloride and acetone, the volume ratio of methylene chloride and acetone is 75:35；Also, PLGA is having
Concentration in solvent is 100~250mg/ml, preferably 150mg/ml；
Preferably, the mass volume ratio of solid phase and oily phase is 3~40mg/ml, preferably 5~20mg/ml.
In step (2), obtained mixture is homogenized using ultrasonic cell disruptor or high-speed mixer；It is using
When ultrasonic cell disruptor, homogenized with 20~200s of low-power 80~120W ultrasound；When using high-speed mixer, with
10000~30000r/min, preferably 24000r/min stirring, 30~300s, preferably 60~240s is homogenized.
And in step (2), the volume ratio of silicone oil and the S/O suspension is 1:1~3:1；It is described stir at low speed for
It is stirred at low speed with magnetic stirring apparatus 300rpm.
In step (3), the volume ratio of normal heptane and the S/O suspension added with silicone oil is 11:1~30:1；It is described
It stirs at low speed and stirs at low speed 30min for 300rpm.
Using W/O/W double emulsion solvent volatility process, it the described method comprises the following steps:
(1) PLGA is dissolved in organic solvent and oily phase is made, take non-T cell conjugating peptide to be dissolved in Glycine-NaOH slow
Oily phase is added as inner aqueous phase in fliud flushing；
(2) stable W/O colostrum is formed after homogenizing the mixture that step (1) obtains, and then slowly drips the W/O colostrum
It is added in outer aqueous phase decentralized medium polyvinyl alcohol phosphate buffer, stirs at low speed obtained W/O/W lotion；
(3) phosphate buffer is added in the W/O/W lotion obtained to step (2), stirs at low speed 4~6h, make organic molten
Agent volatilization is complete, Collection and conservation after microballoon solidification.
Wherein, in step (1), organic solvent is the mixed liquor of methylene chloride or methylene chloride and acetone；Wherein when having
When solvent is the mixed liquor of methylene chloride and acetone, the volume ratio of methylene chloride and acetone is 75:35；Also, PLGA is having
Concentration in solvent is 100~250mg/ml, preferably 150mg/ml；
Concentration of the non-T cell conjugating peptide in inner aqueous phase is 25~75mg/ml, preferably 50mg/ml；
Preferably, the Glycine-NaOH buffer is preferably that the 0.3M Glycine-NaOH of pH8~10 is slow
Fliud flushing；
Preferably, the volume ratio of inner aqueous phase and oily phase is 0.02~0.1:1, preferably 0.05~0.1:1；
In step (2), obtained mixture is homogenized using ultrasonic cell disruptor or high-speed mixer；It is using
When ultrasonic cell disruptor, homogenized with 20~200s of low-power 80~120W ultrasound；When using high-speed mixer, with
10000~30000r/min, preferably 24000r/min stirring, 30~300s, preferably 60~240s is homogenized.
And in step (2), the concentration of polyvinyl alcohol phosphate buffer is 0.5%~5%, pH value is 6.0~
10.0；It is highly preferred that also including 0~5%, preferably 2~5%NaCl in the polyvinyl alcohol phosphate buffer.
Preferably, the volume ratio of polyvinyl alcohol phosphate buffer and W/O colostrum is 63~91:1, preferably 90.9:1；It is excellent
Selection of land stirs at low speed to stir 1min with the revolving speed of 500~600rpm；
In step (3), the concentration of phosphate buffer is 0.02M, pH value 5.8；
Preferably, the volume ratio of phosphate buffer and W/O/W lotion is 0.98~0.99:1, preferably 0.99:1；
Preferably, it stirs at low speed to stir 4~6h at room temperature with the revolving speed of 300rpm.
Using S/O/W emulsion-solvent evaporation method, it the described method comprises the following steps:
(1) PLGA is dissolved in organic solvent and oily phase is made, take non-T cell conjugating peptide that above-mentioned oily phase is added as solid phase；
(2) stable S/O suspension is formed after homogenizing the mixture that step (1) obtains, and then adds the S/O suspension
Enter in outer aqueous phase decentralized medium polyvinyl alcohol phosphate buffer, stirs at low speed obtained S/O/W lotion；
(3) phosphate buffer is added in the S/O/W lotion obtained to step (2), stirs at low speed, organic solvent is made to volatilize
Completely, Collection and conservation after microballoon solidification.
Wherein, in step (1), organic solvent is the mixed liquor of methylene chloride or methylene chloride and acetone；Wherein when having
When solvent is the mixed liquor of methylene chloride and acetone, the volume ratio of methylene chloride and acetone is 75:35；Also, PLGA is having
Concentration in solvent is 100~250mg/ml, preferably 150mg/ml；
Preferably, concentration of the PLGA in oily phase is 100~250mg/ml, preferably 150mg/ml；
Preferably, the mass volume ratio of solid phase and oily phase is 5~40mg/ml, preferably 5~20mg/ml；
In step (2), obtained mixture is homogenized using ultrasonic cell disruptor or high-speed mixer；It is using
When ultrasonic cell disruptor, homogenized with 20~200s of low-power 80~120W ultrasound；When using high-speed mixer, with
20000~30000r/min, preferably 24000r/min stirring, 30~300s, preferably 60~240s is homogenized.
And in step (2), the concentration of polyvinyl alcohol phosphate buffer is 0.5%~5%, pH value is 6.0~
10.0；It is highly preferred that also including 0~5%, preferably 2~5%NaCl in the polyvinyl alcohol phosphate buffer.
Preferably, the volume ratio of polyvinyl alcohol phosphate buffer and S/O suspension is 66~100:1, preferably 100:1；
Preferably, it stirs at low speed to stir 1min with the revolving speed of 500~600rpm；
In step (3), the concentration of phosphate buffer is 0.02M, pH value 5.8；
Preferably, the volume ratio of phosphate buffer and S/O/W lotion is 0.98~0.99:1, preferably 0.99:1；
Preferably, it stirs at low speed to stir 4~6h at room temperature with the revolving speed of 300rpm.
Another aspect, the present invention provides described pharmaceutical compositions in preparing the drug for treating rheumatoid arthritis
Purposes.
The present invention is prepared for containing using phase separation method, W/O/W double emulsion solvent volatility process and S/O/W emulsion-solvent evaporation method
The pharmaceutical composition of non-T cell conjugating peptide and PLGA, the specifically pharmaceutical composition of sustained-release micro-spheres form.Experiment shows
Pharmaceutical composition drugloading rate of the invention and encapsulation rate are high, and non-T cell conjugating peptide stability is good, and Zero order release reaches 20 days~40
It, and release the drug completely, it is suitable for non-vein administration.
In comparing three kinds of preparation methods, the inventors discovered that, in the mistake for preparing the pharmaceutical composition using phase separation method
A large amount of organic solvent is needed in journey, and the dispersed phase of W/O/W double emulsion solvent volatility process and S/O/W emulsion-solvent evaporation method is
Water, it is few using organic phase amount, it is more advantageous to environmental protection, and it is easier to industrialized production.
The present inventors have additionally discovered that using W/O/W double emulsion solvent volatility process and the preparation combination of S/O/W emulsion-solvent evaporation method
When object, after the NaCl of organic salt ion, 2%~5% is added in outer aqueous phase, microballoon external world osmotic pressure is improved, it is suppressed that
The exchange of inside and outside substance reduces the formation of microsphere surface aperture, contributes to form fine and close polymer backbone structure, improves micro-
Ball encapsulation rate reduces burst release rate.The release behavior of prepared composition is obviously improved, and burst release rate is substantially reduced, and is not had
There is the plateau during release, and discharges more complete.Therefore W/O/W double emulsion solvent volatility process and S/O/W emulsified solvent
Volatility process is more preferred preparation method.
The especially composition of S/O/W emulsion-solvent evaporation method preparation, surface is smooth, appearance uniform, inner skeleton knot
Structure is fine and close, and regular particles are without adhesion, and particle diameter distribution is between 10~100 μm, and drugloading rate is up to 2.88%, and encapsulation rate reaches
80%, it is external to be sustained the phase at 20~40 days.The non-T cell conjugating peptide PLGA microballoon release of S/O/W emulsion-solvent evaporation method preparation
Behavior is be more good.
For the composition using the preparation of S/O/W emulsion-solvent evaporation method, Pharmacokinetics in Rat reality has also been carried out
It tests.The results show that the drug slow release in preparation, is continued until 30 days or so.Said preparation can delay in this prompt 30 days
Slowly non-T cell conjugating peptide is smoothly discharged, effect can achieve the purpose for extending the drug action time.In addition, pharmacodynamics is ground
Study carefully the result shows that, microballoon group single SC is administered (20mg/kg) and intravenously administrable (1mg/kg, once every other day, totally 21 days) and is facing
Bed performance aspect does not have notable difference, prompts the controlled release composition preparation to have good slow release effect, single-dose can extend medicine
The action time of object.
Non-T cell conjugating peptide prepared by the present invention and PLGA composition, matrix PLGA is biodegradable, bio-compatible
Property it is good, can be used for the administration of the non-veins forms such as subcutaneous, muscle, especially in arthritic treatment.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows the electron microscope photo scanning of microspheres form pharmaceutical composition of the present invention made from embodiment 9.
Fig. 2 shows the cumulative in vitro release profiles of embodiment 13.
Fig. 3 shows the internal release profiles of embodiment 14.
Fig. 4 shows the internal arthritis clinical score comparison result of embodiment 15.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.Wherein, used non-T cell conjugating peptide by
Hebei Feinisi Biotechnology Co., Ltd.'s production, article No. 20120521, purity 97.79%, content 92.12%.
The pharmaceutical composition of microspheres form is characterized using following methods in embodiment:
1. using the partial size of scanning electron microscope measurement microballoon.
2. entrapment efficiency determination method:
Microballoon 20mg obtained is accurately weighed, 0.5ml methylene chloride is added and dissolves carrier material, 10000rpm centrifugation
5min removes organic phase, repeats this operation twice, keeps organic solvent volatilization complete.1ml is added containing 0.02% Tween-80
0.02M phosphate buffer (pH7.4) repeats this operation twice, non-T cell conjugating peptide is sufficiently extracted from organic phase, with height
Effect liquid phase chromatogram method method measures non-T cell conjugating peptide amount in water phase；Non-T cell conjugating peptide amount/non-T is thin in encapsulation rate=microballoon
Born of the same parents' binding peptide inventory × 100%.
3. the particle diameter distribution of granularmetric analysis instrument measurement microballoon.
4. the configuration of surface of scanning electron microscope scanning microballoon.
5. microballoon In-vitro release curves measuring method:
It takes pH7.4PBS buffer 1ml in centrifuge tube, microballoon is suspended wherein, set 36.5 DEG C, on 180r/min shaking table,
Daily sampling, when every sub-sampling, replace original buffer with fresh buffer；In high performance liquid chromatography measurement release sample
Non-T cell conjugating peptide content, accumulative non-T cell conjugating peptide is calculated after measurement and discharges percentage, draws tablets in vitro curve.It will
First day total volume is determined as burst release amount.
Embodiment 1Phase separation method prepares microballoon
The PLGA (wherein the mass ratio of PLA:PGA is 50:50) that 300mg molecular weight is 9000 is dissolved in 2ml organic solvent
Oily phase is made in (methylene chloride), takes 10mg non-T cell conjugating peptide powder that above-mentioned oily phase is added, uses high-speed mixer
30000r/min forms S/O suspension after being homogenized 60s, 4ml silicone oil is added drop-wise in S/O suspension, during dropwise addition
It is stirred at low speed using magnetic stirring apparatus 300rpm, after being added dropwise to complete, normal heptane 70ml is added in lotion, 300rpm is stirred at low speed
30min solidified microsphere collects microballoon.
Resulting composition average grain diameter is 60.7 μm, encapsulation rate 80.3%, drugloading rate 4.2%, and burst release rate is
18.2%, it stable in vitro can discharge 20 days, total drug release amount reaches 85.5%.
Embodiment 2Phase separation method prepares microballoon
The PLGA (wherein the mass ratio of PLA:PGA is 50:50) that 300mg molecular weight is 13000 is dissolved in 2ml organic solvent
Oily phase is made in (mixed liquor that the volume ratio of methylene chloride and acetone is 75:35), 40mg non-T cell conjugating peptide powder is taken to be added
Above-mentioned oil phase, forms S/O suspension after being homogenized 240s using high-speed mixer 20000r/min, 6ml silicone oil is added drop-wise to
It in S/O suspension, is stirred at low speed during dropwise addition using magnetic stirring apparatus 300rpm, after being added dropwise to complete, is added in lotion
Normal heptane 180ml, 300rpm stir at low speed 30min solidified microsphere, collect microballoon.
Resulting composition average grain diameter is 69.0 μm, encapsulation rate 75.3%, drugloading rate 3.8%, and burst release rate is
15.2%, it stable in vitro can discharge 30 days, total drug release amount reaches 60.5%.
By Examples 1 and 2 it is found that preparing the pharmaceutical composition of microspheres form of the invention using phase separation method, can obtain
The microballoon that drugloading rate and encapsulation rate are high, non-T cell conjugating peptide stability is good, and microballoon sustained release reaches 20 days or more.
Embodiment 3W/O/W double emulsion solvent volatility process
10mg non-T cell conjugating peptide powder is dissolved in the Glycine-NaOH buffer (pH10.0) of 0.2ml 0.3M
In, it is added to the PLGA (wherein the mass ratio of PLA:PGA is 50:50) two that the molecular weight that 2ml concentration is 150mg/ml is 9000
In chloromethanes solution, 120s is emulsified under conditions of stirring rate is 24000r/min with high-speed mixer, obtains W/O colostrum
Liquid；The emulsion is injected into 0.02M phosphate buffer (pH7.4) of the 200ml containing 2% polyvinyl alcohol with pipettor, with
W/O/W lotion is made in the revolving speed stirring 1min of 500rpm, and 200ml 0.02M phosphate buffer is added into the lotion
(pH5.8), 300rpm stirs 6h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, and distilled water washs three times,
It is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 4W/O/W double emulsion solvent volatility process
10mg non-T cell conjugating peptide powder is dissolved in the Glycine-NaOH buffer (pH10.0) of 0.2ml 0.3M
In, it is added to the PLGA (wherein the mass ratio of PLA:PGA is 50:50) two that the molecular weight that 2ml concentration is 150mg/ml is 9000
In chloromethanes solution, 120s is emulsified under conditions of stirring rate is 24000r/min, obtains W/O colostric fluid；By the emulsion
It is injected into pipettor in the 0.02M phosphate buffer (pH7.4) of 2% polyvinyl alcohol of the 200ml containing 5%NaCl, with
W/O/W lotion is made in the revolving speed stirring 1min of 500rpm, and 200ml 0.02M phosphate buffer is added into the lotion
(pH5.8), 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, and distilled water washs three times,
It is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 5W/O/W double emulsion solvent volatility process
10mg non-T cell conjugating peptide powder is dissolved in the Glycine-NaOH buffer (pH8.0) of 0.2ml 0.3M
In, it is added to the PLGA (wherein the mass ratio of PLA:PGA is 75:25) two that the molecular weight that 2ml concentration is 150mg/ml is 13000
In chloromethanes solution, 120s is emulsified under conditions of stirring rate is 24000r/min, obtains W/O colostric fluid；By the emulsion
It is injected into pipettor in the 0.02M phosphate buffer (pH7.4) of 0.5% polyvinyl alcohol of the 200ml containing 5%NaCl, with
W/O/W lotion is made in the revolving speed stirring 1min of 600rpm, and 200ml 0.02M phosphate buffer is added into the lotion
(pH5.8), 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, and distilled water washs three times,
It is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 6W/O/W double emulsion solvent volatility process
10mg non-T cell conjugating peptide powder is dissolved in the Glycine-NaOH buffer (pH8.0) of 0.15ml 0.3M
In, it is added to the PLGA (wherein the mass ratio of PLA:PGA is 75:25) two that the molecular weight that 3ml concentration is 100mg/ml is 23000
In chloromethanes solution, 240s is emulsified under conditions of stirring rate is 24000r/min, obtains W/O colostric fluid；By the emulsion
It is injected into pipettor in the 0.02M phosphate buffer (pH7.4) of 0.5% polyvinyl alcohol of the 200ml containing 5%NaCl, with
W/O/W lotion is made in the revolving speed stirring 1min of 600rpm, and 200ml 0.02M phosphate buffer is added into the lotion
(pH5.8), 300rpm stirs 6h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, and distilled water washs three times,
It is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 7S/O/W emulsion-solvent evaporation method
By 10mg non-T cell conjugating peptide particle addition 2ml concentration be 150mg/ml molecular weight be 9000 PLGA (wherein
The mass ratio of PLA:PGA is 50:50) in dichloromethane solution, 120s is emulsified under conditions of stirring rate is 24000r/min,
Obtain S/O suspension；The emulsion is injected into the 0.02M phosphate buffer that 200ml contains 2% polyvinyl alcohol with pipettor
(pH7.4) in, S/O/W lotion is made with the revolving speed stirring 1min of 500rpm, 200ml 0.02M phosphate is added into the lotion
Buffer, 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, and distilled water washs three times, presses
More solito freeze-drying, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 8S/O/W emulsion-solvent evaporation method
By 10mg non-T cell conjugating peptide particle addition 2ml concentration be 150mg/ml molecular weight be 9000 PLGA (wherein
The mass ratio of PLA:PGA is 50:50) in dichloromethane solution, 120s is emulsified under conditions of stirring rate is 24000r/min,
Obtain S/O suspension；The emulsion is injected into 0.02M phosphoric acid of the 200ml containing 5%NaCl and 2% polyvinyl alcohol with pipettor
In salt buffer (pH7.4), S/O/W lotion is made with the revolving speed stirring 1min of 500rpm, 200ml is added into the lotion
0.02M phosphate buffer, 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, distilled water
Washing three times, is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 9S/O/W emulsion-solvent evaporation method
PLGA (its for being 13000 by the molecular weight that 10mg non-T cell conjugating peptide particle addition 2ml concentration is 150mg/ml
The mass ratio of middle PLA:PLG is 50:50) in dichloromethane solution, emulsified under conditions of stirring rate is 24000r/min
120s obtains S/O suspension；The emulsion is injected into 0.02M of the 200ml containing 5%NaCl and 2% polyvinyl alcohol with pipettor
In phosphate buffer (pH7.4), S/O/W lotion is made with the revolving speed stirring 1min of 500rpm, 200ml is added into the lotion
0.02M phosphate buffer, 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, distilled water
Washing three times, is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 10S/O/W emulsion-solvent evaporation method
PLGA (its for being 13000 by the molecular weight that 10mg non-T cell conjugating peptide particle addition 2ml concentration is 150mg/ml
The mass ratio of middle PLA:PLG is 75:25) in dichloromethane solution, emulsified under conditions of stirring rate is 24000r/min
120s obtains S/O suspension；The emulsion is injected into 0.02M of the 200ml containing 2% polyvinyl alcohol, 5%NaCl with pipettor
In phosphate buffer (pH7.4), S/O/W lotion is made with the revolving speed stirring 1min of 600rpm, is added into the lotion
200ml0.02M phosphate buffer, 300rpm stirs 4h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification,
Distilled water washs three times, is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 11S/O/W emulsion-solvent evaporation method
The PLGA for being 23000 by the molecular weight that 10mg non-T cell conjugating peptide particle addition 3.0ml concentration is 100mg/ml
In (wherein the mass ratio of PLA:PLG is 75:25) dichloromethane solution, emulsified under conditions of stirring rate is 24000r/min
240s obtains S/O suspension；The emulsion is injected into 0.02M of the 200ml containing 5% polyvinyl alcohol, 5%NaCl with pipettor
In phosphate buffer (pH7.4), S/O/W lotion is made with the revolving speed stirring 1min of 600rpm, is added into the lotion
200ml0.02M phosphate buffer, 300rpm stirs 6h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification,
Distilled water washs three times, is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 12S/O/W emulsion-solvent evaporation method
PLGA (its for being 23000 by the molecular weight that 80mg non-T cell conjugating peptide particle addition 2ml concentration is 250mg/ml
The mass ratio of middle PLA:PLG is in 50:50) dichloromethane solution, in the suspension of ultrasonic cell disruptor ultrasound, ultrasonic time is
120s obtains S/O suspension；The emulsion is injected into 0.02M of the 200ml containing 5% polyvinyl alcohol, 2%NaCl with pipettor
In phosphate buffer (pH7.4), S/O/W lotion is made with the revolving speed stirring 1min of 600rpm, 200ml is added into the lotion
0.02M phosphate buffer, 300rpm stirs 6h at room temperature, and organic solvent is volatilized, and is collected by centrifugation after microballoon solidification, distilled water
Washing three times, is conventionally freeze-dried, obtains non-T cell conjugating peptide and PLGA composition.
Embodiment 13The pharmaceutical composition of microspheres form is characterized
The microballoon obtained to 3~embodiment of above-described embodiment 11 characterizes, partial size, drugloading rate, encapsulation rate and burst release rate
Comparing result is shown in Table 1, and In-vitro release curves are shown in Fig. 2.
The microballoon characteristic of the NTAP PLGA of 1 different process method of table preparation
Seen from table 1, with the increase of outer aqueous phase NaCl concentration, the encapsulation rate of obtained microballoon, drugloading rate is increased
Add, and burst release rate is reduced with the increase of NaCl concentration in outer aqueous phase.Identical PLA/PGA ratio, different molecular weight
NTAP microballoon prepared by PLGA, with the increase of PLGA molecular weight, encapsulation rate, drugloading rate, burst release rate all decrease.It is worth
It is noted that the encapsulation rate and drugloading rate of the PLGA NTAP microballoon of all molecular weight of preparation are all very low, and prepared by S/O/W method
PLGA NTAP microballoon encapsulation rate close to 80%, and drugloading rate is significantly improved compared with W/O/W method, close to theory drugloading rate
(3.23%).
From Figure 2 it can be seen that the time that NTAP microballoon totally discharges increases with the increase of PLGA molecular weight, entirely discharged
Release behavior is good in journey.The PLGANTAP microballoon of the 9K and 13K of the preparation of W/O/W method are persistently released respectively substantially at Zero order release
20 days and 30 days are put；And the PLGANTAP microballoon of 23K slightly shows plateau, overall sustained release in 5 to 15 days
40 days.The PLGA NTAP microballoon of 9K and 13K totally discharges the totality for all having reached the PLGA NTAP microballoon of 90% or more, 23K
Release has also reached 70%.The NTAP microballoon of the 9K (50:50) of S/O/W method preparation only releases 20 days, and Zero order release is presented；
The NTAP microballoon of 13K (50:50) releases 30 days, adds up release up to 90%, does not have plateau substantially, and 13K (75:25)
NTAP discharge microballoon early period it is also possible that but occur plateau after 15 days, overall sustained release 30 days, add up release not yet
Reach 60%.The microballoon release of 23K very slowly, begins with release behavior from the 10th talent, row is discharged in 10 days to 30 days always
To be good, overall sustained release 40 days, all there is plateau in early period and later period, adds up release not up to 60%.
By above data and analysis as it can be seen that the NTAP microballoon encapsulating with higher of the 13K (50:50) of S/O/W preparation
Rate and good release behavior are suitable for animal experiment.
Embodiment 14Internal pharmacokinetic studies
Following internal pharmacokinetic studies are carried out using microballoon made from embodiment 9.
Rat is randomly divided into 4 groups, i.e., blank group, NTAP water needle group (3mg/kg, intravenous injection), microballoon be low, high dose
Group (15mg/kg, 30mg/kg, intramuscular injection), every group of 8 rats.Vein water needle group take blood time point be 0.083,0.25,
0.75, it is 1 that 2,3,5,7,9, for 24 hours, microballoon group, which takes blood time point, 2,3,5,7,9,11,13,15,17,19,21,23,25,27,
29,30 days, blank group rat extracting blood is compareed as matrix blank at above-mentioned time point.
Blood point is taken in above-mentioned, takes blood 0.5ml into heparinised tubes from the interior eye corner of the eyes of rat, 3500r/min is centrifuged 10min
Separated plasma, for gained blood plasma as in silanization test tube, -20 DEG C of freezen protectives are to be measured, sample acquire to blood plasma separation 2h it
Interior completion.
The NTAP concentration in plasma sample is surveyed with high performance liquid chromatography, and according to NTAP concentration and time data, medication
The internal pharmacokinetic parameter of NTAP is calculated for dynamics software 3p87, and draws pharmaceutical concentration-time curve, is as a result seen
Table 2 and Fig. 3.
The pharmacokinetic parameter of 2 NTAP water needle of table and NTAP microballoon
| Sample | Kel(d-1) | MRT(d) | AUC0-t(ngd/ml)b | Bioavilability (%) |
| NTAP water needle 3mg/kg | 6.36±0.12 | 0.160±0.02 | 4227.1±313.2 | 100 |
| NTAP microballoon 15mg/kg | 0.51±0.09 | 13.9±2.1 | 18471.3±2546.8 | 87.4 |
| NTAP microballoon 30mg/kg | 0.56±0.08 | 14.7±1.8 | 36098.4±610.5 | 85.5 |
The results show that the mean residence time (MRT) of sustained release preparation group was from 0.16 day after 15mg/kg or 30mg/kg administration
It is extended for 14 days, and the drug slow release in preparation, is continued until 30 days or so.Said preparation can delay in prompt 30 days
Slowly NTAP is smoothly discharged, the effect of this slow release can achieve the purpose for extending the drug action time.
Embodiment 15Internal pharmacodynamic experiment
Following internal pharmacodynamic experiments are carried out using microballoon made from embodiment 9.
Using rat CIA model (Female Lewis rats, 6-7 weeks, with the ox II Collagen Type VI cream containing incomplete Freund's adjuvant
100 μ l of agent reinforces once after a week to rat intradermal injection immunity, establishes rat CIA model) to the microsphere composition of preparation
Pharmacodynamics studied:
Onset rat is randomly divided into 4 groups, i.e. model group (intravenous injection PBS, administered volume 5ml/kg), NTAP water needle group
(1mg/kg, intravenous injection, administered volume 5ml/kg), NTAP microballoon group (20mg/kg, intramuscular injection, administered volume 1ml/kg),
And microballoon solvent control group (intramuscular injection is used for the oil solvent of dispersion microsphere, administered volume 1ml/kg), model group and NTAP
Water needle group is administered once every other day, and totally 21 days, NTAP microballoon group and microballoon solvent control group single-dose.During administration, to rat
Clinical manifestation observed, the function and effect of NTAP are evaluated by arthritis clinical score.Each claw highest of rat
4 points are chosen as, every rat highest is chosen as 16 points, and specific standards are as follows: 0 point=swollen without redness and swelling of joints；1 point=joint is slightly red
It is swollen；2 points=joint moderate is red and swollen；3 points=joint severe is red and swollen；4 points=joint deformity cannot bear a heavy burden.It is specifically shown in Fig. 4.
Single SC administration (20mg/kg) as the result is shown is being faced totally with intravenously administrable (1mg/kg, once every other day, 21 days)
Bed performance aspect does not have notable difference, prompts the controlled release composition preparation to have good slow release effect, single-dose can extend medicine
The action time of object.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (21)
1. a kind of pharmaceutical composition, it includes non-T cell conjugating peptides and lactic-glycolic acid block copolymer PLGA, with weight hundred
Divide than meter, described pharmaceutical composition includes 3%~14% non-T cell conjugating peptide and 86%~97% PLGA, the PLGA
Molecular weight be 9.0 × 103~2.3 × 104Dalton, in the PLGA mass ratio of polylactic acid and polyglycolic acid be (50:
50)~(75:25)；
Wherein, described pharmaceutical composition is made by S/O/W emulsion-solvent evaporation method, and the S/O/W emulsion-solvent evaporation method includes
Following steps:
(1) PLGA is dissolved in organic solvent and oily phase is made, take non-T cell conjugating peptide that above-mentioned oily phase is added as solid phase；Wherein,
The mixed liquor of the organic solvent is methylene chloride or volume ratio is 75:35 methylene chloride and acetone, the PLGA is in oily phase
In concentration be 100~250mg/ml, the mass volume ratio of solid phase and oily phase is 3~40mg/ml；
(2) stable S/O suspension is formed after homogenizing the mixture that step (1) obtains, and then the S/O suspension is added outer
In Aqueous dispersions medium polyvinyl alcohol phosphate buffer, obtained S/O/W lotion is stirred at low speed；Wherein, the polyvinyl alcohol phosphorus
The concentration of phthalate buffer is 0.5%~5%, and pH value is 6.0~10.0, and also includes 2~5%NaCl, polyvinyl alcohol phosphoric acid
The volume ratio of salt buffer and S/O suspension is 66~100:1；
(3) phosphate buffer is added in the S/O/W lotion obtained to step (2), stirs at low speed, is evaporated completely organic solvent
Entirely, Collection and conservation after microballoon solidification；Wherein, the volume ratio of the phosphate buffer and S/O/W lotion is 0.98~0.99:1.
2. pharmaceutical composition according to claim 1, which is characterized in that the molecular weight of the PLGA is 1.3 × 104Dongle
.
3. pharmaceutical composition according to claim 1 or 2, which is characterized in that polylactic acid and poly- hydroxyl second in the PLGA
The mass ratio of acid is 50:50.
4. pharmaceutical composition according to claim 1 or 2, which is characterized in that described pharmaceutical composition is microspheres form, grain
Diameter is distributed as 10~120 μm.
5. pharmaceutical composition according to claim 4, which is characterized in that the microballoon can be within 20-40 days time
Continue, stablize release non-T cell conjugating peptide.
6. pharmaceutical composition according to claim 4, which is characterized in that the microballoon is sustainable in vivo, stablizes offer
30 days effective non-T cell conjugating peptide concentration.
7. pharmaceutical composition according to claim 1 or 2, which is characterized in that in the S/O/W emulsion-solvent evaporation method
In step (1):
Concentration of the PLGA in oily phase is 150mg/ml, and the mass volume ratio of solid phase and oily phase is 5~20mg/ml.
8. pharmaceutical composition according to claim 1 or 2, which is characterized in that in the S/O/W emulsion-solvent evaporation method
In step (2):
The volume ratio of the polyvinyl alcohol phosphate buffer and S/O suspension is 100:1.
9. pharmaceutical composition according to claim 1 or 2, which is characterized in that in the S/O/W emulsion-solvent evaporation method
In step (3):
The concentration of the phosphate buffer is 0.02M, and pH value 5.8, the volume ratio with S/O/W lotion is 0.99:1.
10. the preparation method of pharmaceutical composition according to any one of claims 1 to 9, the preparation method is that S/O/W is emulsified
Solvent evaporation method, the S/O/W emulsion-solvent evaporation method the following steps are included:
(1) PLGA is dissolved in organic solvent and oily phase is made, take non-T cell conjugating peptide that above-mentioned oily phase is added as solid phase；Wherein,
The mixed liquor of the organic solvent is methylene chloride or volume ratio is 75:35 methylene chloride and acetone, the PLGA is in oily phase
In concentration be 100~250mg/ml, the mass volume ratio of solid phase and oily phase is 3~40mg/ml；
(2) stable S/O suspension is formed after homogenizing the mixture that step (1) obtains, and then the S/O suspension is added outer
In Aqueous dispersions medium polyvinyl alcohol phosphate buffer, obtained S/O/W lotion is stirred at low speed；Wherein, the polyvinyl alcohol phosphorus
The concentration of phthalate buffer is 0.5%~5%, and pH value is 6.0~10.0, and also includes 2~5%NaCl, polyvinyl alcohol phosphoric acid
The volume ratio of salt buffer and S/O suspension is 66~100:1；
(3) phosphate buffer is added in the S/O/W lotion obtained to step (2), stirs at low speed, is evaporated completely organic solvent
Entirely, Collection and conservation after microballoon solidification；Wherein, the volume ratio of the phosphate buffer and S/O/W lotion is 0.98~0.99:1.
11. preparation method according to claim 10, which is characterized in that in the step (1):
Concentration of the PLGA in oily phase is 150mg/ml, and the mass volume ratio of solid phase and oily phase is 5~20mg/ml.
12. preparation method according to claim 10, which is characterized in that in the step (2):
Obtained mixture is homogenized using ultrasonic cell disruptor or high-speed mixer；Wherein crushed using supersonic cell
Machine is homogenized with 80~120W ultrasound, 20~200s；Or high-speed mixer is used, with 20000~30000r/min stirring
30~300s is homogenized.
13. preparation method according to claim 12, which is characterized in that in the step (2):
Using high-speed mixer, 60~240s is stirred with 24000r/min and is homogenized.
14. preparation method according to claim 10, which is characterized in that in the step (2):
The volume ratio of polyvinyl alcohol phosphate buffer and S/O suspension is 100:1.
15. preparation method according to claim 10, which is characterized in that in the step (2):
It stirs at low speed to stir 1min with the revolving speed of 500~600rpm.
16. preparation method according to claim 10, which is characterized in that in the step (3):
The concentration of phosphate buffer is 0.02M, pH value 5.8.
17. preparation method according to claim 10, which is characterized in that the volume of phosphate buffer and S/O/W lotion
Than for 0.99:1.
18. preparation method according to claim 10, which is characterized in that in the step (3):
It stirs at low speed to stir 4~6h at room temperature with the revolving speed of 300rpm.
19. preparation method described in any one of 0 to 18 according to claim 1, which is characterized in that pharmaceutical composition obtained is
Microballoon, the microballoon can continue within 20-40 days time, stablize release non-T cell conjugating peptide.
20. preparation method according to claim 19, which is characterized in that the microballoon is sustainable in vivo, stablizes offer
30 days effective non-T cell conjugating peptide concentration.
21. pharmaceutical composition according to any one of claims 1 to 9 is in preparing the drug for treating rheumatoid arthritis
Purposes.
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