TW201210610A - Method for controlled release of parathyroid hormone from encapsulated poly (lactic-co-glycolic acid) microspheres - Google Patents

Abstract

The present invention provides a method for producing a controlled release microsphere with me an average size greater than 50 μ m, comprising preparing a water-in-oil (w/o) emulsion comprising an inner aqueous layer containing a pharmaceutically effective amount of a biologically active polypeptide with activity similar to parathyroid hormone, and an oil layer containing a polymer substance of poly (lactic-co-glycolic acid) (PLGA), then adding the w/o emulsion into aqueous polyvinyl alcohol (PVA) solution to form a water-in-oil-in-water (w/o/w) double emulsion and then desorbing the solvent in the oil layer. The present invention also provides a controlled release microsphere prepared by the method and use thereof.

Description

201210610 VI. Description of the Invention: [Technical Field] The present invention relates to a method for producing controlled release microspheres, and a use of the method for controlling release of microspheres and the microspheres for controlling A pharmaceutically effective amount of the biologically active polypeptide is released and the polypeptide is similar to the activity of parathyroid hormone. [Prior Art] Parathyroid hormone (PTH) is a polypeptide composed of 84 amino acids, and its sequence is SEQ ID NO: 1 < Keutmann. HT, Sauer. MM, Hendy. GN, O Riordan. JLH , Potts. JT. Complete amino acid sequence of human parathyroid hormone, Biochemistry 17; 1978; 5723>, parathyroid hormone acts directly on bones and kidneys, and is the most important regulator of calcium balance in human body. Recent studies have shown that specific parathyroid hormone analogs can play a role in bone anabolism in humans. Podbesek R, Edouard C, Meunier PJ, Parsons JA, Reeve J, Stevenson RW, et al. Effects of two treatment regimes with Synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds. Endocrinology 1983; 112:1000-6>, and is of great concern in the treatment of bone diseases. The expression of PTH<1-34>, PTH<1-31> and PTH<1-38> in osteoblasts can be as complete as PTH<1-84>. In recent years, there have been many methods for the application of parathyroid hormone in clinical trials for disease treatment (Neer RM, Amaud CD, Zanchetta JR, 201210610).

Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH. Effect of parathyroid hormone &lt; 1 - 34 &gt; on fractures and bone mineral density in postmenopausal women with Osteoporosis. Engl J Med 2001;344:1434 - 41>. A recently reported method emphasizes that oral administration of PTH < 1-34> can exhibit biological activity. However, compared with subcutaneous injection, its bioavailability is only 5% and 2.1% (Leone-Bay A, Sato M, Paton D, Hunt AH,

Sarubbi D, Carozza M, Chou J, McDonough J, Baughman RA. Oral delivery of biologically active parathyroid hormone. Pharm Res 2001; 18 <7>: 964-70). On the other hand, transpulmonary perfusion or dry powder inhalation of PTH<1-34> and other lung pathways were 41% and 34% bioavailable, respectively (Codrons V, Vanderbist R, Verbeeck RK, Arras M, Lison D, Preat V, Vanbever R. Systemic delivery of parathyroid hormone (1-34) using inhalation dry powders in rats. J Pharm Sci 2003; 92 < 5>: 938 - 50>. In addition, intermittent PTH administration also includes the administration of osmotic pump and pulsatile transdermal administration (Suzuki γ, Nagase γ, Iga K, Kawase M, 〇ka M,

Yanai S, Matsumoto Y, Nakagawa S, Fukuda T, Adachi H, Higo N, Ogawa Y. Prevention of bone loss in ovariectomized rats by pulsatile transdermal iontophoretic administration of human PYH &lt; 1 - 34 &gt; . J Pharm Sci 2002; :350-61>. In the effect of parathyroid hormone on bone formation, both of the above administration methods have a considerable effect on subcutaneous administration, and human parathyroid hormone ρτΗ <138> has similar results. Relative to this, research work on local parathyroid hormone administration is rare. It is worth noting that 'these rare studies indicate that the local administration of para-adenosine directly via gene delivery is a financial benefit.<Bonadio J, 4 201210610

Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Medl999; 5: 753 - 9 &gt;. PTH < 1-34> also known as teriparatide, trade name FORTEO®, is marketed by EliLilly, Indianapolis, Ind Pharmaceuticals, and can be used in women with high risk group after menopause. 'Treatment of Osteoporosis <Zhang, S , Eli Lilly and company, Indianapolis, IA <US>. US patent No. US6590081-B1>. This drug is administered by subcutaneous injection of a PTH < 1-34> formulation (acetate buffer, mannitol, and m-cresol in water, pH 4) once daily. However, many people have an adverse reaction to the injection and therefore become a prescription dose that does not want to comply with PTH. Recently, the applicant found that PTH<1-34> acts on articular chondrocytes to inhibit their final differentiation' and also inhibits the effect of papain-induced osteoarthritis in rats (Chang. JK, Chang. LH, Hung. SH,

Wu. SC, Lee. HY, Lin. YS, Chen. CH, Fu. YC, Wang. GJ, Ho. ML,

Arthritis &amp; Rheumatism. 2009; 60; 3049-3060>. However, the treatment requires three days of administration, which will bring more pain and inconvenience to the patients receiving treatment. Therefore, in order to reduce the suffering of patients, it is necessary to develop a new parathyroid hormone peptide controlled release carrier formulation which has appropriate bioavailability to achieve a therapeutic concentration effective for treating parathyroid hormone related diseases. However, in general, 'proteins and peptides are unstable in the gastrointestinal tract, have a short half-life, and the bioavailability of their aqueous formulations is quite low. Fix, ja. 〇ral controlled release 201210610 technology for peptides: status and future prospects, Pharm Res. 1996 Dec;13<12>:1760-4 &gt; These characteristics make proteins and peptides challenging for clinically effective applications. SUMMARY OF THE INVENTION Natural and synthetic polymeric carriers <micron and nanospheres> have been developed as an effective method for controlling the release of coated proteins and avoiding their degradation. Lu L, Statamas GN, Mikas Ad Controlled release of transforming growth factor Beta from biodegradable polymer microparticles. J Biomed Mater Sci 2000; 50: 440-51. In these carriers, 'polylactic acid-glycolic acid (p〇ly (lactic_c〇_glyC〇iic anal, pLGA) and polylactic acid <PLA> have excellent biocompatibility and biodegradability when passed through natural pathways. Therefore, it is more suitable for the delivery of drugs. The present invention therefore provides a method for producing a stable controlled release of parathyroid hormone (PTH) for a long period of time, and maintaining its therapeutic concentration and bioavailability. The present invention discloses a manufacturing for control A method of releasing a PLGA microsphere coated with a biologically active and stable parathyroid hormone. The parathyroid hormone is successfully coated in two different compositions of PLGA. These compositions are invented for the purpose of Thyroxine can be stably stored in microspheres during long-term controlled release. The pLGA microspheres produced can release bioactive, non-aggregating parathyroid hormone and maintain effective therapeutic concentrations ranging from 1 X 10·7Μ to Between 5 X ΙΟ·9 , for 21 days. The released parathyroid hormone has been shown to be stable and biologically active during long-term controlled release. The present invention is coated on PLGA. ρΤΗ<134> for stable controlled release in the pellet is manufactured and studied. ΡΤΗ< 1-34> will become unstable under temperature and pH changes, so 6 201210610 The present invention provides that ΡΤΗ<1·34〉 can be long _Stabilization coating method. The invention says that the PLGA (four) method _ controls the local application of ρτΗ <134> and maintains a fixed concentration. The PLGa microsphere coated with PTH U_34 can continuously transmit the effective concentration finely for a certain period of time. PTH U_34>, so it can be used as a carrier for PTH &lt; 1-34> delivery system. ριχ} Α microspheres coated with pTH<(9)> can be used to treat parathyroid hormone deficiency, looseness, and bone. _Inflammation, etc. The meaning of the terms used herein is the same as commonly understood by those skilled in the art unless otherwise specifically defined. The meanings of the terms used in this application are as follows: PTH < 1-34> Means a polypeptide having 34 amino acid sequences consisting of the first to 34th amino acids in seqjj^nq: 1. More specifically, SEQ][DNa 2 is 疋PTH<1 -34> polypeptide sequence. "PTH<1 -31>" means having 31 The polypeptide of the amino acid sequence is composed of the first to the 31st amino acids in SEQ耵N〇: 1. "German 11<1-38>" means having 38 amino acid sequences. The polypeptide consists of 3 to 1 (51 〇 1 ^ 〇: 1 from the 1st to the 38th amino acid. "PTH < I-84>" means the complete human parathyroid hormone, and its polypeptide sequence is As shown by seq ID NO: 1. Degenerative bone disease means a series of diseases characterized by a decrease in bone mass and/or an increased risk of fracture due to structural incompleteness. Many degenerative bone diseases are caused by imbalances in bone formation and resorption. These imbalances may result from decreased osteogenic bone formation, increased resorption of fine radioactivity, or changes in the activity of both osteoblasts and cells. The lust of the lupus is a degenerative bone disease, characterized by a decrease in the quality of the bone path and a deterioration of the micro-structure of the bones. Her osteoporosis refers to the skeletal mass U associated with other diseases, only with aging and its associated hypogonadism. Osteoporosis reduction includes both post-menopausal and senile osteoporosis. ‘In addition, there is no latent surface element, and there is no other primary Μ that causes the cause of degeneration. The second Wei „ 赖 is caused by the system (4) Wei Wei, other diseases caused by osteoporosis ^ Boundary health group mosquito bone density is lower than the bone density reference standard < Guan 3 〇 years old health light for adults> 25 health Osteoporosis is the case below the cedar. "Bone necrosis" means a disease that collapses due to insufficient blood supply to the bone age. In living tissue, the threat also requires a defined blood flow for normal functioning. In the absence of sufficient blood flow, severe osteonecrosis can cause death of the bone. " Osteoarthritis" (abbreviated OA 'is degenerative arthritis or degenerative joint disease> is a disease-induced joint deterioration caused by some diseases and mechanical abnormalities, including articular cartilage and subchondral bones <subehondralbone> . The clinical specialties of osseous inflammation are joint pain, tenderness, stiffness, squeezing, joint atresia and sometimes local inflammation. As a strong and powerful protein matrix, cartilage can lubricate and alleviate joint impact. In osteoarthritis, many potential factors such as genetic, growth, metabolism and mechanical effects are guided by the process of cartilage loss. The present invention therefore provides a controlled release microsphere having an average particle size greater than 5 〇. The microspheres are produced by preparing a water-in-oil emulsion 201210610 <water-in-oil<w/o &gt; emulsion> containing an inner aqueous layer containing a pharmaceutically effective amount of biological activity. a polypeptide having a similar activity to parathyroid hormone; preparing an oil layer containing a polylactic acid-glycolic acid (PLGA) polymer; and then pouring the water-in-oil emulsion into an aqueous solution of polyvinyl alcohol (PVA) A water-in-oil-in-water double emulsion &lt;water-in-oil-in-water<w/o/w>double emulsion> is formed and then the solvent in the oil layer is desorbed. In a preferred embodiment, 'polylactic acid-glycolic acid is plga<50:50> or PLGA<65:35&gt;, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, which is immobilized in the In a stock solution of hydrochloric acid and bovine serum albumin. Preferably, the concentration of hydrochloric acid is from about 1 mM to about 8 mM, and the concentration of bovine serum albumin is from about 0.1% to about 5%. More preferably, the concentration of hydrochloric acid is from about 2 mM to about 6 mM, and the concentration of bovine albumin is from about 0.05% to about 0.15%. In a preferred embodiment, the polypeptide is released for at least 21 days at a therapeutic concentration ranging from about 1 X 1 〇-7 ]y [to about 5 X 10·9 Torr, and the weight percent concentration of the aqueous polyvinyl alcohol solution is about 0.1% to about 5%, and the encapsulation efficiency of the biologically active polypeptide is not less than about 6%. Preferably, the weight percentage of the aqueous polyvinyl alcohol solution is from about 0 5% to about 15 Torr. The present invention further provides a method for producing a controlled release microsphere having an average particle diameter of more than 5 Å, comprising preparing a water-in-oil <w/〇> emulsion comprising an inner water layer containing pharmaceutically An effective amount of a biologically active polypeptide having an activity similar to that of parathyroid hormone; preparing an oil layer containing a polylactic acid-glycolic acid polymer; and then slowly adding the water-in-oil emulsion to the aqueous polyvinyl alcohol solution to form water The oil is encapsulated in water <w/〇/w> and the solvent in the oil layer is then desorbed. Preferably, the polylactic acid-glycolic acid is pLGA <50:50> or PLGA<65:35>' and the amino acid sequence of the polypeptide is shown in SEQroN〇:2, 201210610, which is immobilized in containing hydrochloric acid and bovine serum white. Protein in the stock solution. Preferably, the concentration of hydrochloric acid is from about 1 mM to about 8 mM, and the amount of bovine serum albumin is from about % to about 5%. More preferably, the concentration of hydrochloric acid is from about 2 mM to about 6 mM and the concentration of bovine serum albumin is from about 0.05% to about 0.15%. In a preferred embodiment, the polypeptide is released for at least 21 days at an effective therapeutic concentration ranging from about 1 χ 1 〇 7 m to about 5 χ 1 〇 9 m, and the aqueous polyvinyl alcohol solution has a concentration by weight of about 0.1%. Up to about 5% 'and the encapsulation efficiency of the biologically active polypeptide is not less than about _. Preferably, the weight percentage of the aqueous polyvinyl alcohol solution is from about 0.5% to about 1.5%. The invention still further provides a method of controlled release of a therapeutic polypeptide to a subject comprising administering to the subject a controlled release microsphere having an average particle size greater than 50 Å, the microsphere being manufactured by: preparing - containing a water-in-oil emulsion having an inner aqueous layer containing a pharmaceutically effective amount of a biologically active polypeptide having a activity similar to that of parathyroid hormone; preparing an oil layer containing a polylactic acid-glycolic acid polymer; The water-in-oil emulsion is poured into a polyethylene terephthalate aqueous solution to form a water-in-oil-in-water Wei Wei and then dissolved in the oil layer.

The agent is detached. In a preferred embodiment, the polylactic acid-glycolic acid is PLGA < 50:50> or PU5A &lt;65:35>' and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, which is immobilized in the Hydrochloric acid and bovine serum are preserved from the protein. Preferably, the brewing concentration I is about! The concentration of _ to about 8 'bovine serum albumin is from about 0.01% to about 5%. More preferably, the acid concentration is from about 2 pm to about 6 mM, and the concentration of bovine serum albumin is from about _ % to about 〇 15 %. In a preferred embodiment, the polypeptide is in an amount ranging from about 1 X 1 〇 -7 Μ to about 5 χ 1 (the effective therapeutic concentration of γ 9 μ is at least U days, and the weight percent concentration of the aqueous solution of the polycytosine is 201210610 is about 〇 Preferably, the weight percentage of the aqueous solution of the polyethylene glycol is from about 1.5%. In the preferred embodiment, the contact system has parathyroidism, bone disease or softness. In the case of a better embodiment, the bone disease is osteoporosis or osteonecrosis; the soft moon disease rides arthritis. In another, more preferred embodiment, the subject is human. In conventional double emulsions In the technology, 250 to 400 mL of 1-5% PVA is used (Y.-Y. Yang et al., Biomaterials 22 (2001) 231-241 and G. Qin et al. / Biomaterials 25 (2〇〇4) 345-352), but the present invention uses only 1% of PVA as the aqueous phase. The usual double emulsion technique requires a preparation time of at least 4 hours (γ·_γ. Yang et al,

Biomaterials 22 (2001) 231-241), but the process of the invention requires only 2 hours of preparation time. The invention therefore also reduces the time required for preparation. The preparation of microspheres for extended release is disclosed in US Pat. No. 4, 954, 244, US Pat. No. 4, 954, 298, US Pat. No. 5, 271, 945, US Pat. Preparation time (within 2 hours): (2) The size of the microspheres is more suitable for intra-articular injection; (3) the repeatability is higher and more important; (4) the microspheres can release PTH<1-34> at least 21 Both days fall within the effective therapeutic concentration range.

Anthony et al. (Journal of Pediatric Surgery, 40, 81-85 (2005)) and

Fong et al. (Journal of Surgical Reserch, 143, 195-199 (2007)) revealed a controlled release system for parathyroid gland hypoxia, whose release concentration of PTH is 1〇〇〇_6〇〇〇 pico Gram (1 picogram = l (T12g) and very high explosive release 11 201210610 in the first day (24 hours). Their preparation method requires 4 hours and a large amount of PVA. The invention only takes 2 hours and A small amount of PVA. In addition, the PTH release concentration of the present invention is 20-400 micrograms (1 microgram = 1 (T6g) e. Therefore, the present invention is characterized by reducing the amount and percentage of the PVA solution, and using 700 ipm to produce A microsphere with a particle size greater than 50 μη is sized for intra-articular injection. The key step or discovery of the present invention is to use 20 mL of 1% PVA as the aqueous phase to produce a small size of appropriate size at a stirring rate of 700 rpm. Balls (according to Butoescu N, J〇rdan O, Doelker. Ε, Intra-articular drug delivery systems for the treatment of rheumatic diseases: A review of the factors influencing their performance European Journal of Pharmaceutics and Biopharmaceutics, 2 009; 73; 205-218, suitable for intra-articular injection of a diameter of 35 to i 〇 5) to release a PTH having a therapeutic concentration for more than three weeks. [Embodiment] The following examples are not intended to be limiting, only represent Various aspects and features of the invention. Example 1 Preparation and Characterization of Microspheres Two different PLGA compositions were used in this study: PLGA < 50:50 &gt; and PLGA <65:35>. Microspheres were w/o/w double Emulsion technology manufacturing <Figure 1>. In short, 90 ul of PTH <1-34> stock solution <PTH^HmMHCl/0.1% bovine serum albumin <BSA> solution> in 10% dichloromethane <DCM> polymer The solution was emulsified, and an ultrasonic probe (Virsonicl00, Cardiner, NY) with an output of 15 W was used in an ice bath for 20 seconds to form an initial water-in-oil (w/o) emulsion. The water-in-oil emulsion was gradually added to 2 〇mi 1% polyvinyl alcohol 12 201210610 <PVA> in an aqueous solution, and simultaneously forcefully mix (turn) to form a water-in-oil-in-water double emulsion. Stir the solution for 3 minutes at room temperature to harden the microspheres. Then, in the state of water absorption, the second gas m is then separated from the (10)_ship ball. The resulting microspheres were washed 3 times with steamed water and cooled; The overall shape of the microsphere will be studied in a gold sphere after the microsphere sample is coated on a microscope stage, and then the average size of the microsphere will be studied by a scanning electron microscope <SEM> &lt;Hitachi S3200, Tokyo, Japan. Measured by a particle size analyzer. 4% HCl and physiological saline < 〇.9% NaC1> and 〇1% BSA were used as stabilizers to protect PTH &lt;RTIgt; In order to control the release of the agent coated in the microsphere, the surface of the microsphere must be smooth. Observation by a scanning electron microscope confirmed that the PLGA microspheres were smooth and maintained during the degradation process. The literature report emphasizes that the appropriate size of the microspheres for intra-articular injection in rats is 351〇5 μηι (Butoescu. N, Jordan. O, Doelker. E, Intra-articular drug delivery systems for the treatment of rheumatic diseases: A review Of the factors influencing their performance European Journal of Pharmaceutics and

Biopharmaceutics, 2009; 73; 205-218 &gt;, data from a particle size analyzer show that more than 90% of microspheres with an average particle size of 45-80 μηη can be successfully produced by this method in this study (Fig. 2 and Figure 3a>. Example 2 Encapsulation and release kinetics Microspheres containing 10 mg of PTH<1-34> were suspended in 〇.9 % of 1 mL of sodium hydride and 200 uL of di-methane methane, and shaken at room temperature. Train for a few hours. pTH <丨_34> Standard solution &lt;0.1 ml&gt; was also prepared by adding 0.9% sodium hydride and 0.1% bsA. According to the manufacturer's instructions, the loading and encapsulation efficiency of the agricultural product of PXH<丨_34> according to the manufacturer's instructions is calculated by square and (2) (Table 1). 'Program <1> Equation (1): Protein loading <w/w%> = Total amount of protein in microspheres / Total amount of small spheres &lt;2>: Protein encapsulation efficiency <%> = <Measured protein Theoretical value of concentration/white matter concentration> XI quail egg Table 1 Encapsulation efficiency of PTH<1-;34> in PLGA microspheres PLGA PTH<ng/mL> PTH of concentration theoretical value measured <ng/mL> Concentration encapsulation Efficiency 50:50 1800 1319.0 73.3 % 65:35 1800 1129.4 62.7 % Example 3 Bio-external ρτΗ<1-34> Release The release curve of PTH released from PLGA microspheres in vitro was determined by the following method. The microspheres of ίο mg were suspended in 1 ml of PBS <pH = 74>, and the microsphere suspension was statically cultured at 4, 25, and 37 ° C, and '1 ml of the culture solution was collected by centrifugation at set time intervals'. And replace with the same amount of fresh pBS. The concentration of PTH < 1-34> in the released culture solution was measured from the manufacturer's recommendation using a PTH < 1-34> ELISA kit with a ρτΗ antibody-coated culture medium. San Clemente, CA&gt; Two cultures were used for each repetition at each time point. The absorbance was measured at 450 nm using a micr〇titer plate reader, and the concentration of PTG was calculated using the program i〇g_i〇git method with the standard curve <G mphpad s〇ftware, San Diego , CA>. According to a recent study by the inventors, the effective dose of PTH < 1-34> for the treatment of osteoarthritis induced by papain was 1〇·8 μ, and the frequency of injection was once every three days for five weeks. The release kinetic data from the ELISA<1-34> specific ELISA assay showed that at 37, the 'PLGA<65:35> microspheres released the desired concentration range ι〇-7_1〇_8 μ of ρτΗ <1-34> Two weeks <Figure 4>. Compared to 1&gt;1/(3-8<5〇:5〇>, 1)1^<65:35> where 37. °C simulated physiological conditions, a consistent release profile was shown after more than 17 days. 4 MTT (3- &lt;4,5-d^methylthiazo^2-yl&gt; -2,5-diphenyltetrazolium bromide) Assay When treating MC3T3-E1 <osteoblasts>, ρΐΉ<_> MTT assay. In short, as described earlier, by converting Μττ into formazan 'to detect the mitochondrial activity of MC3T3_m cultured in the culture grid, a large amount of yoghurt is released from the human Weiwei towel, and this is directly It is proportional to the number of viable cells in the cultured towel and can be measured by absorbance at 490 nm. Dilute the Μχτ in a standard medium at a volume ratio of 1: 5 <MTT: culture solution at a specified time interval, and add the freshly prepared sputum to the culture cell containing the cells, and then re-culture at 37%. Four hours. After additional culture, 100 ul of MTT, which was released into the culture medium and released by 15 201210610, was transferred to a 96-well culture plate, and the absorbance at 490 nm was recorded using a microplate reader <PathTech>. And use KCjunior software analysis <Figure 3b>. The biological activity of PTH<I-34> released in Example 5 was measured by measuring the release of PTH<l-> in the cells treated with the released PTH<1 - 34>. 34&gt; activity. For these experiments, MC3T3-E1 was cultured in α-ΜΕΜ supplemented with 1% fetal calf serum and 50 mg/mL ascorbic acid. The cells were cultured at a density of 5 cells per well on a 24-well plate of <24-well plates>. After incubation for six hours with ρτΗ < ^4> released from the microspheres, 〇1N hydrochloric acid and 0.5 mM cholera toxin <isobutylmethylxanthine> were added to allow the cells to be directly hydrolyzed in the medium to protect the cAMP produced. Intracellular cAMP was measured using a commercially available ELISA kit <Endogen/Pierce, Rockford, IL> according to the manufacturer's instructions. Bioactivity data showed that MC3T3-E1 cells treated with released PTH<1-34> increased cAMP production on the first and third days, indicating that the released PTH<1-34> was biologically active (Fig. 5). Example 6 Statistical Analysis Two samples were independently cultured for biochemical analysis. Each experiment was repeated at least three times and showed data for a representative experiment <expressed as mean ± standard deviation>. Statistical significance was assessed by single factor variance analysis (ANOVA) and multiple comparisons were performed using the Xuefei method. p &lt; 0.05 was considered significant. 201210610 Example 7 In vivo (/«vivo) experiment Method: Animal experiment Animal experiment was approved by the Animal Experiment Management Team of Kaohsiung Medical University. 54 12-week-old male Spmgue-Dawley rats <250-300 gm> were purchased from BioLASCO Taiwan and housed under standard laboratory conditions (temperature 24 °C). Day and night cycle 12 hours> and free access to food and water. The animals will be acclimated in the laboratory for a week before the experiment. Osteoarthritis Induction and PTH Treatment Each of the left knees, which were considered to be contralaterally controlled joints, was applied with a mediator that was not treated with PTH or induced by 〇a. The right knee was the joint being studied. Rats were divided into five groups: Non-OA+PTH group (treated with PTH<1-34> but not induced by 〇A><n=6>, Non-OA+PTH/PLGA group (with PTH) /PLGA microsphere treatment but not 〇A induced> <n=6>, OA group OA induced but not treated with PTH<1_34> <n=6>, OA+PTH group <PTH< 1-34 〉 After treatment, 're-inducing 〇A-induced> <n=6> and OA+PTH/PLGA group (treated with PTH/PLGA microspheres, then 〇A induced> <n=6>. In the OA group and the OA+PTH group, the right knee joint was injected with 20 μl of a 4% papain solution and 20 μl of 0.03 Μ cysteine to induce Α. These joints will be injected through the knee tendon with a 26-gauge needle on Days 4 and 7 of the experiment (US Patent No. US 659081). In the OA+PTH group, after the OA was induced, the rats were sacrificed 17 201210610 刖, and 4 μM of 10 nM PTH < 1-34> was injected every two days into the right knee joint. In the ρτΗ group, the same pTH<丨_34&gt; treatment was performed in the absence of OA induction. In the 0A+PTH7PLGA group, 〇4 mg of PTH/PLGA microspheres were injected into the right knee joint on the first and the 15th day after 〇A was induced. At the same time point after 5 weeks, the rats were sacrificed by inhalation of excess carbon dioxide. Histology After sacrifice, each rat's knee was taken and the tibial plateau was collected along with articular cartilage and fixed with 10% neutral buffered formalin prior to tissue preparation. The sample was then decalcified with 1% formic acid/PBS buffer, and the decalcified tibial joint sample was embedded in paraffin, and a coronal microsection of 5 was prepared. 〇 0 0 0 番 番 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〈 〉 Along the heart, M〇> staining. The local type 2 collagen and sputum collagen were immunostained. Histomorphometry study GAG was stained with red, with Image-Pro plus 5. 〇 software <Media Cybemetics

Inc. MD, USA> measures all areas in the articular cartilage near the proximal humerus and the reddish area (Fig. 6C). Calculate the ratio of the red-to-red in all areas (red/all) in each group. Immunohistochemistry The luminal joint sections were rehydrated (re seven yetted) and the endogenous peroxidase in the tissues of 201210610 was blocked with 3% hydrogen peroxide. Before the sample is allowed to stand still with the primary antibody, the epitope is recovered (epitoperetrieval) will be first decomposed by the enzyme. The method of decomposition of the enzyme is to modify the report of the pioneer (Butoescu. N, Jordan. 0, Doelker. E, Intra- Articular drug delivery systems for the treatment of rheumatic diseases: A review of the factors influencing their performance European Journal of Pharmaceutics and Biopharmaceutics, 2009; 73; 205-218). The best conditions for enzyme decomposition of type 2 collagen immunostaining '疋 372 (11 2.5% hyaluronidase <hyluronidase> and 1 mg / ml of the protease in PBS < pH 7.4> < Sigma, St. Louis, MO &gt; 1 hour. X-type collagen immunization The optimal conditions for staining were 1 hour in 〇1 u/ml of chondroitinase ABC> (Sigma, St Louis, MO) and 1 mg/ml tris-HCl (pH 3.0) at 37SC. 15 minutes in pepsin. The sections were then blocked with fetal bovine serum for 1 hour and at 37 SCTF with primary antibody against type 2 collagen <mice early antibody> <Chem Icon International, Temecula, CA> and primary antibody against type X collagen <multi-antibody of rat> <1:2〇〇> <C0SM0, Tokyo, Japan &gt; culture for 4 hours. Biotin-labeled goat anti-mouse immunoglobulin <DAKO, Carpinteria, CA> was cultured in secondary antibody, and type X collagen was biotinylated goat anti-rabbit immunoglobulin (Biocare medical, Walnut Creek, CA). Culture, and then add the bioactive enzyme <Streptravidin-HRP> <streptavidin coupled with horseradish peroxidase> (Biocare medical, Walnut Creek, CA>. The solution of 3,3,-diaminobenzidine <3,3'-diaminobenzidine> of hydrogen peroxide was stained brown. Finally, the sections were stained with hematoxylin as background (counterstained) and observed with a microscope. Relative density of immunostaining 19 201210610 <Community, degree/area' area 25.44± 2.77 mm2> Measurement was performed with Image-Pro plus 5.0 software <Media Cybernetics Inc. MD, USA> (Fig. 7C). Results: Histological and histomorphometric study of rat articular cartilage

On the opposite side, Νοη-0 A+PTH, Non-0 A+PTH/PLGA, OA, OA+PTH

Representative photographs of articular cartilage of OA+PTH/PLGA, saffron-stained rat joints in the group are shown in Fig. 6 <A&B>. The ratio of reddish-stained areas in all areas (red: all) was measured and compared in the group (Fig. 6C). Red between the groups, contralateral control joints: There was no significant difference in all ratios. The contralateral control of the red color of the cartilage in the joint: the total ratio 'and the red in the study joints of the Non-OA+PTH and Non-OA+PTH/PLGA groups: there is no significant difference in all ratios <Fig. 6 (:>.〇 In the eight groups, the red color of the cartilage of the joint was studied: the total ratio was significantly lower than the red: all ratio of the contralateral control cartilage induced by the sputum A for five weeks <P &lt; 〇.〇1> <circle 6C>. After five weeks of PTH < 1-34> treatment, the cartilage of the OA+PTH group did not differ significantly from the cartilage of the contralateral control group (Fig. 6C). After five weeks, the red color of the oa+PTH/PLGA group: all The ratio was significantly higher than that of the OA group <Ρ&lt;〇.〇1> <Fig. 6C>. In addition, the red color of the 〇A+PTH/pLGA group: all ratios were not significantly different from the contralateral control cartilage. At week, there was no significant difference between the OA+PTH group, the OA+PTH/PLGA group, and the contralateral control cartilage group (Fig. 6C). The immunohistochemical study of the π-type and proprotein of the rat articular cartilage segment was performed. Contralateral control, Non-OA+PTH, Non-OA+PTH/PLG A, OA, ΟΑ+ΡΊΉ 201210610 Spear OA+PTH/PLGA group of rat joints A representative photomicrograph of the second sputum protein staining of the articular cartilage (dyed to brown) is shown in Figure 7. <8, 师〇>. Measure the ratio of all areas of the second type collagen staining area <brown: all> and do each Comparison between groups <Fig. 7D>. There is no significant difference in the standard of the lateral control section between the groups; the color of the cartilage in the contralateral control: all ratios, and Ν(&gt;η_〇 There were no significant differences in the joints studied in the Α+ρτΗ and Non-OA+PTH/PLGA groups (Fig. 7D>c〇A study of brown in articular cartilage: all ratios were significantly lower than five weeks induced by 〇a The opposite side controls the brown color in the cartilage: the total ratio <p&lt;〇〇1> <Fig. 7D>. After 5 weeks of treatment with PTH<1-34>, the cartilage of the A+PTH group and the contralateral control cartilage were not significant. Difference <®7D>. After 5 weeks, the brown of the qa+pth/plgA group: all ratios are significantly different from the glare of the OA group: all ratios <p &lt;〇.〇】〉<图〉. , OA+PTH/PLGA group brown: all ratios were also not significantly different from contralateral control cartilage. In the fifth week' OA+PTH, OA+PTH/PL There was no significant difference between GA and contralateral control cartilage group (Fig. 7D). Immunohistochemical study of X-type Shengyuan in rat articular cartilage section. In the contralateral control cartilage, no obvious type X collagen stained chondrocytes were found. <Figs. 8A and 8B&gt; Immunolocalized type X collagen <stained brown> was mainly found in articular chondrocytes of group 0A, but in OA+FTH and OA+PTH/PLGA group cartilage after five weeks of treatment. 'The positive stained cells are rarely found (Figures 8A and 8B). Those skilled in the art will readily appreciate that the present invention makes it easy to achieve the results and advantages mentioned, as well as those that are present therein. The microspheres of the present invention and the scales and methods of their manufacture are representative of the miscellaneous, which are exemplary and not only earned money. Knowing the artist can be modified and used for other purposes. These modifications are intended to be within the spirit of the invention and are defined in the patent application. The descriptions and examples of the month of the month of the month reveal the details, so that any person skilled in the art can manufacture and manufacture Benja's. Even if the towel has various unique changes, materials, and advancements, it should be regarded as not departing. The spirit and scope of the present invention. All patents and publications mentioned in the New Moon are subject to the general technical requirements in the field of invention. All patents and wrinkles are referenced here, just like every individual edition. They are all referred to by the thief and side by side (4). The invention, as exemplified herein, may be implemented in the absence of any element, perhaps multiple elements, conditions, or the limitations disclosed herein. It is not intended to be used as a description of the book as a description of the book and not to limit such nouns and expressions that are equivalent to the features or parts thereof shown and described, but it is recognized that In the patent of the present invention, there may be various changes. Therefore, it is to be understood that the present invention has been specifically described in accordance with the preferred embodiments and the features of the present invention, and those skilled in the art will still be able to modify and change the invention disclosed herein, and such modifications and variations are still within the scope of the invention. 22 201210610 [Simple description of the diagram] Figure 1: Schematic diagram of the production of polylactic acid-glycolic acid <PLGA> microspheres coated with ΡΤΗ<1-34> Fig. 2: Polylactic acid coated with PTH<1-34> - Scanning electron microscope (SEM) image of a microbial acid microsphere. Figure 3: <a> is the size of a microsphere analyzed by a particle size analyzer; <b> PTH<1- analyzed by MTT 34> Cytotoxicity Figure 4: Cumulative release profile of PLGA<50:50> and PLGA<65:35> Figure 5: Bioactivity map of MC3T3-E1 cells in the release of PLGA microspheres <1-34> 6: Tissue analysis of the amount of GAG in articular cartilage stained with Saftanin-O. These figures are contralateral control group, Non-OA+PTH, Non-OA+PTH/PLGA, OA, OA+ Articular cartilage in the proximal tibia of rats in the PTH and OA+PTH/PLGA groups stained with Safranin-O. In the OA group The OA+PTH group was treated once every 3 days with PTH<1-34><10·8Μ>, while the 〇A+PTH/PLGA0.4mg group was treated twice every 15 days, and the joints of the rats studied were also listed. In the figure, each horizontal bar represents the mean soil standard deviation of eight samples. The data is analyzed by single factor variance (〇newayanalysis()fvarianee, ANOVA>' and multiple comparisons are performed using Scheffe's method. <**>P&lt;0.01' At each time point, the contralateral control group was compared with the experimental group samples. <##> p&lt;0.01 ' At each time point, the experimental group samples in the 0A group were compared. Figure 7: Type II collagen of stained articular cartilage in the contralateral control group, N〇n_OA+PTH group, Non-OA+PTH/PLGA group, OA group, ΟΑ+ΡΤΉ group, and OA+PTH/PLGA group <COL.II> _ woven analysis results. The representative of the sputum is the rat contralateral joint 23 201210610 proximal humerus immunostaining articular cartilage, OA + PTH group with PTH < 1-34> < 10-8M> The treatment was performed once a day, and the OA+PTH/PLGA 0.4 mg group in the OA group was processed every ls days. The graph shown in Fig. 2 is the rat joint studied. Color as positive to inhibit anti - staining of cartilage growth plate and a negative second BU fresh collagen stained brown. The average number of the eight sides of each job is ± the difference between the counties. Transfer to the Cui factor analysis and use the Xue Fei method for multiple comparisons. Ρ &lt;0·01’ is compared at each time point to the contralateral control group and the experimental group sample. <fine> ρ&lt;0.01' At each time point, the experimental group samples in the 〇Α group were compared. Round ". 恻 control group, Νοη_ΟΑ+ΡΊΉ group, Ν〇η_〇Α+ρ 遍 组 group, 〇α group, ΤΗ ΤΗ group and OA+PTH/PLGA group of stained articular cartilage 乂 type collagen _ _> analysis of the county. The proximal part of the coronal bone of the rats was immunostained for articular cartilage. The 0A+PTH group was treated with PTH(1)4>(10)8M> every 3 days, while the 0A+PTH/PLGAG 4mg group in the OA group was 2 times in 15 days. The growth plate is softly run, and the X-type collagen is dyed as a negative control. The arrows indicate that the soft f* of the type X collagen is dyed, and each of the bars represents the mean soil standard deviation of the eight samples. Data were analyzed as single factor variances and __ incoming weights were compared. <>P&lt;G.G1' At each time point, the contralateral control group was compared with the experimental group samples. <丝> P&lt;_' At each time point, the experimental group samples in the group A were compared. Annex 1 · Color diagram of Figure 1. 24 201210610 Annex 2: Color diagram of Figure 6A. Annex 3: Color diagram of Figure 6B. Attachment 4 • The color of the shirt of Figure 7A is not intended. Annex 5: Color diagram of Figure 7B. Annex 6: Color diagram of Figure 7C. Annex 7: Color diagram of Figure 8A. Annex 8: Color diagram of Figure 8B. 25 201210610 Sequence Listing &lt;110&gt; Kaohsiung Medical University &lt;120&gt; Method for controlling release of parathyroid hormone by P0LY (LACTIC-C0-GLYC0UC ACID) microspheres &lt;130&gt; 1183-KNU- US &lt;160&gt; 2 &lt;170&gt; Patentln version 3.4

&lt;210&gt; 1 &lt;211&gt; 84 &lt;212&gt; PRT &lt;213&gt; Human (Homo sapiens) &lt;220&gt;&lt;221&gt; PEPTIDE &lt;222&gt; (1)..(84) &lt;400&gt;

Ser Val Ser Glu lie Gin Leu Met His Asn Leu Gly Lys His Leu Asn 15 10 15 201210610

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His 20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45

Gin Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asn Val Asp Val Leu Thr Lys 65 70 75 80

Ala Lys Ser Gin &lt;210&gt; 2 &lt;211&gt; 34 &lt;212&gt; PRT &lt;213&gt; Human (Homo sapiens) &lt;220&gt;&lt;221&gt; MISC_FEATURE &lt;222&gt; (1)..(34) 201210610 &lt;400&gt; 2

Ser Val Ser Glu lie Gin Leu Met His Asn Leu Gly Lys His Leu Asn 15 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His 20 25 30

Asn Phe

Claims (1)

201210610 VII. Patent application scope: 1. - Control the release of microspheres, the average particle size is greater than 5 〇μπι. The microspheres are produced by: preparing a water-in-oil <w/o> emulsion comprising an inner aqueous layer comprising a pharmaceutically effective amount of a biologically active polypeptide having a activity similar to that of parathyroid hormone Preparing a polymeric polysaccharide layer containing polyglycoside (PLGA); the secret pure water emulsion is poured into a polyethylene glycol <PVA> aqueous solution to form a water-in-oil-in-water <w/Q/w> double emulsion and The solvent in the oil layer is then desorbed. 2. The microsphere according to item 1 of the patent application, wherein the polylactic acid monoglycolic acid <pLGA> is PLGA < 50:50> or PLGA < 65:35>. 3. The microsphere according to the scope of claim 帛i, wherein the polymorphic seric acid sequence is as shown in SEQ ID NO: 2. 4. According to the microsphere of the cap patent 丨 丨 丨 , ,, the polypeptide is finely defined in a stock solution containing hydrochloric acid and bovine serum albumin. 5. The microsphere according to the scope of claim 帛i, wherein the hydrochloric acid concentration is from about imM to about 8 mM 'bovine serum albumin at a concentration of from about 1% to about 5%. 6. The microsphere of claim i, wherein the aqueous solution of the polyethylene glycol has a concentration by weight of from about 0.1% to about 5%. 7. A method of producing controlled release microspheres having an average particle size greater than 50 microns, comprising: preparing a water-in-oil <w/o> emulsion comprising an inner aqueous layer comprising pharmaceutically effective a biologically active polypeptide having an activity similar to that of a parathyroid hormone; preparing an oil layer containing a polylactic acid-glycolic acid polymer; and then slowly adding the water-in-oil emulsion to the aqueous polyvinyl alcohol solution to form water The oil-in-water <w/o/w> double emulsion is then attached to the 201210610 gluten in the oil layer. 8.::::::: ―一醇一〉,川·50&gt; or PLGA<65:35 〉. 9. According to the patent application, it is still shown in muscle 2. The method wherein the amino acid sequence of the polypeptide is in the storage solution of the SEQ range, the method, and the bovine serum albumin. Clever and sour. In the method described, the concentration of bovine blood/month albumin is from about ο·οι% to about 5%. 12·minus_step tenderness, (4) 鸠 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约丨3. The method of claim 7, wherein the aqueous solution of the aqueous solution of the enasol has a concentration percentage of from about 0.1% to about 5%. 14. According to the method described in Section 7 of the application, the encapsulation efficiency of the suspected peptide is not less than about 60%. 15. A method of controlled release delivery of a therapeutic polypeptide to a subject, comprising administering to the subject an average particle size greater than 5 mm, the microsphere storage being manufactured by: preparing - containing a water-in-oil emulsion of an aqueous layer, the inner aqueous layer containing a pharmaceutically effective amount of a biologically active polypeptide, the activity of which is similar to that of gentamicin; preparing a layer of a polymer containing polylactic acid-glycolic acid; The aqueous emulsion of the aqueous solution of polyvinyl alcohol forms a water-in-oil-in-water double emulsion and then desorbs the solvent in the oil layer. 16. The method of claim 15, wherein the polymorphic amino acid sequence is as set forth in 201210610 SEQ ID NO: 2. 17. The method according to claim 15, wherein the subject is suffering from parathyroidism, bone disease or cartilage disease. 18. The method of claim 17, wherein the bone disease is osteoporosis or osteonecrosis. 19. The method of claim 17, wherein the cartilage disease is osteoarthritis. 20. The method of claim 15, wherein the subject is a human.