TWI395592B - Method for controlled release of parathyroid hormone from encapsulated poly (lactic-co-glycolic acid) microspheres - Google Patents

Description

Controlled by poly (lactic acid-glycolic acid) (POLY-CO-GLYCOLIC ACID) microspheres Method of releasing parathyroid hormone

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 release of a pharmaceutically effective amount of a biologically active polypeptide, and the polypeptide Similar to the activity of parathyroid hormone.

Human 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.

PTH<1-34>, PTH<1-31> and PTH<1-38> have the same intact activity in osteoblasts 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, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH. Effect of parathyroid hormone <1-34> 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 pulmonary 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 Y, Nagase Y, Iga K, Kawase M, Oka). 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 <1-34>. J Pharm Sci 2002; :350-61>. In the effect of parathyroid hormone on bone synthesis, both of the above administration methods have a considerable effect on subcutaneous administration, and human parathyroid hormone PTH <1-38> has similar results. In contrast, research work on local parathyroid hormone administration is rare. It is worth noting that these rare studies point out that topical administration of parathyroid hormone directly via gene delivery is equally beneficial in treating bone defects (Bonadio J, Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Med 1999; 5: 753-9>.

PTH<1-34> also known as teriparatide, trade name FORTEO®, is marketed by Eli Lilly, Indianapolis, Ind Pharmaceuticals, and can be used in women with high risk group after menopause to treat 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, Applicants have found that PTH<1-34> acts on articular chondrocytes, inhibiting their final differentiation, and inhibiting 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 & 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, there is a need to develop a new parathyroid hormone peptide controlled release carrier formulation which has appropriate biological effectiveness to achieve therapeutic concentrations effective in 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.Oral controlled release) Technology for peptides: status and future prospects, Pharm. Res. 1996 Dec; 13 <12>: 1760-4>. These properties make proteins and peptides challenging in clinically effective applications.

Natural and synthetic polymeric carriers <micron and nanospheres> have been developed as an effective method to control the release of coated proteins and to avoid their degradation. Lu L, Statamas GN, Mikos AG. Controlled release of transforming growth factor betal from biodegradable Polymer microparticles. J Biomed Mater Sci 2000; 50: 440-51. Among these carriers, since poly(lactic-co-glycolic acid, PLGA) and polylactic acid <PLA> have excellent biocompatibility and biodegradability through natural routes, Suitable for the delivery of drugs. The present invention therefore provides a method of making a stable controlled release of parathyroid hormone (PTH) for a prolonged period of time and maintaining its therapeutic concentration and biological effectiveness.

The present invention discloses a method of making a controlled release PLGA microsphere coated with a biologically active and stable parathyroid hormone. Parathyroid hormone was successfully coated in two different compositions of PLGA. These compositions were invented to allow parathyroid hormone to be stably stored in microspheres during long-term controlled release. The PLGA microspheres produced release bioactive, non-aggregating parathyroid hormone and maintain effective therapeutic concentrations ranging from 1 X 10 ^-7 M to 5 X 10 ^-9 M for 21 days. The released parathyroid hormone has been shown to be stable and biologically active during long-term controlled release.

The invention has been manufactured and studied for PTH<1-34> which is stably controlled release coated in PLGA microspheres. PTH<1-34> will become unstable under temperature and pH changes, so The present invention provides a method for stably coating PTH<1-34> for a long time.

The present invention demonstrates the feasibility of controlling the local administration of PTH < 1-34 > and maintaining a fixed concentration by PLGA coating. The PLGA microsphere coated with PTH<1-34> can continuously deliver PTH<1-34> in the effective concentration range for a certain period of time, so it can be used as a carrier for the PTH<1-34> delivery system. PLGA microspheres coated with PTH<1-34> can be used to treat parathyroid hormone deficiency, osteoporosis, and osteoarthritis.

The meaning of the terms used herein is the same as commonly recognized by those skilled in the art, unless specifically defined otherwise. 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 of SEQ ID NO: 1. More specifically, SEQ ID NO: 2 is the polypeptide sequence of PTH < 1-34>.

"PTH<1-31>" means a polypeptide having 31 amino acid sequences consisting of the 1st to 31st amino acids in SEQ ID NO: 1.

"PTH<1-38>" means a polypeptide having 38 amino acid sequences consisting of the first to 38th amino acids of SEQ ID NO: 1.

"PTH<1-84>" means intact human parathyroid hormone, the polypeptide sequence of which is shown in 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 be due to reduced osteogenic bone formation, increased bone resorption of osteoblasts, or both osteoblasts and osteoblasts. The activity changes.

"Osteoporosis" means a degenerative bone disease characterized by a decrease in bone mass and a deterioration in the microstructure of bone tissue, resulting in bone fracture and fracture. Primary osteoporosis refers to the loss of bone mass associated with other diseases and only associated with aging and the resulting loss of gonadal function. Primary osteoporosis includes both postmenopausal osteoporosis and senile osteoporosis. It also includes no underlying factors and no other primary osteoporosis that causes secondary causes of bone deterioration. Secondary osteoporosis refers to osteoporosis caused by other diseases in addition to bone deterioration caused by aging. The World Health Organization defines bone mineral density as lower than the bone density reference standard (based on healthy young adults of about 30 years old) > 2 standard deviations or less is osteoporosis.

"Bone necrosis" means a disease in which bone collapses due to insufficient supply of blood to bone tissue. In living tissue, bones also require a sufficient amount of blood flow to maintain normal function. In the absence of sufficient blood flow, severe osteonecrosis can cause death of bone tissue.

" Osteoarthritis" (abbreviated OA, that is, degenerative arthritis or degenerative joint disease) refers to joint deterioration caused by some diseases and mechanical abnormalities, including articular cartilage and subchondral bone. The clinical features of osteoarthritis include 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 heredity, growth and development, metabolism and mechanical effects, can lead to the process of cartilage loss.

Therefore, the present invention provides a controlled release microsphere having an average particle diameter of more than 50 μm, wherein the microsphere comprises: an inner aqueous layer containing a pharmaceutically effective amount of a biologically active polypeptide, and the activity of the biologically active polypeptide a secondary thyroxine; an intermediate layer comprising a polymeric layer of polylactic acid-glycolic acid (PLGA), the oil layer coating the inner aqueous layer to form a water-in-oil (w/o) emulsion; and an outer layer An aqueous layer comprising a polyvinyl alcohol (PVA) for coating the intermediate layer, wherein the microspheres control release of the biologically active polypeptide system in a range from about 1 X 10 ^-7 M to about 5 X 10 The effective concentration of ^-9 M is at least 18 days. . In a preferred embodiment, the polylactic acid-glycolic acid is PLGA <50:50> or PLGA<65:35>, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, which is immobilized in the presence of hydrochloric acid. And a stock solution of 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 0.01% 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 X 10 ^-7 M to about 5 X 10 ^-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 60%. Preferably, the weight percentage of the aqueous polyvinyl alcohol solution is from about 0.5% to about 1.5%.

The invention further provides a method for producing a controlled release microsphere having an average particle diameter of more than 50 micrometers, comprising: (a) providing an oil layer containing a polylactic acid-glycolic acid (PLGA) polymer; (b) preparing a oil pack a water <w/o> emulsion comprising an inner aqueous layer comprising a pharmaceutically effective amount of a biologically active polypeptide, the biologically active polypeptide having activity similar to that of parathyroid hormone; and (c) gradually slowing down The water-in-oil emulsion is added to an aqueous solution of polyvinyl alcohol (PVA) to form a water-in-oil-in-water <w/o/w> double emulsion and then the solvent in the oil layer is desorbed, wherein the microsphere controls release of the biologically active polypeptide The effective concentration is in the range of about 1 X 10 ^-7 M to about 5 X 10 ^-9 M for at least 18 days. Preferably, the polylactic acid-glycolic acid is PLGA<50:50> or PLGA<65:35>, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, 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 concentration of bovine serum albumin is from about 0.01% 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 X 10 ^-7 M to about 5 X 10 ^-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 60%. 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 [mu]m, the microsphere being manufactured by: (a) providing a polymeric layer comprising polylactic acid-glycolic acid (PLGA); (b) preparing a water-in-oil <w/o> emulsion comprising an inner aqueous layer comprising a pharmaceutically effective amount a biologically active polypeptide having a activity similar to that of parathyroid hormone; and (c) gradually adding a water-in-oil emulsion to an aqueous solution of polyvinyl alcohol (PVA) to form a water-in-oil water-in-water <w/o/w Double emulsion and then desorbing the solvent in the oil layer, wherein the microspheres control release of the biologically active polypeptide at an effective concentration ranging from about 1 X 10 ^-7 M to about 5 X 10 ^-9 M for at least 18 days. In a preferred embodiment, the polylactic acid-glycolic acid is PLGA <50:50> or PLGA<65:35>, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, which is immobilized in the presence of hydrochloric acid. And a stock solution of 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 0.01% 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 X 10 ^-7 M to about 5 X 10 ^-9 M, and the aqueous polyvinyl alcohol solution has a concentration by weight of about 0.1%. It is about 5%. Preferably, the weight percentage of the aqueous polyvinyl alcohol solution is from about 0.5% to about 1.5%.

In a preferred embodiment, the subject is suffering from parathyroidism, bone disease or cartilage disease. In a more preferred embodiment, the bone disease is osteoporosis or osteonecrosis; the cartilage disease is osteoarthritis. In another more preferred embodiment, the subject is a human.

In the traditional double emulsion technique, 250 to 400 mL of 1-5% PVA solution is used as the aqueous phase (Y.-Y. Yang et al., Biomaterials 22 (2001) 231-21 and G. Wei et al. /Biomaterials 25 (2004) 345-352), but the invention uses only 20 mL of 1% PVA as the aqueous phase.

Usually the double emulsion technique requires a preparation time of at least 4 hours (Y.-Y. 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.

Methods for preparing microspheres for extended release are disclosed in US 4, 652, 441, US Pat. No. 4, 954, 298, US Pat. No. 5, 271, 945, US Pat. Preparation time (within 2 hours): (2) The size of the microsphere is more suitable for intra-articular injection; (3) the repeatability is higher and more important; (4) the microsphere 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)) disclose a controlled release system for parathyroid hypoenergy, The release concentration of PTH was 1000-6000 pg (1 pg = 10 ^-12 g) and there was a very high explosive release on the first day (24 hours). Their preparation method requires 4 hours and a large amount of PVA. The invention requires only 2 hours and a small amount of PVA. In addition to this, the PTH release concentration of the present invention is 20-400 micrograms (1 microgram = 10 ^-6 g).

Accordingly, the present invention is characterized by reducing the amount and percentage of the PVA solution and using 700 rpm to produce microspheres having a particle size greater than 50 μm, which are sized for intra-articular injection. A key step or finding of the present invention is to use 20 mL of 1% PVA as the aqueous phase to still produce microspheres of appropriate size at a stirring speed of 700 rpm (according to 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, suitable for intra-articular injection with a diameter of 35 to 105 um) The PTH with therapeutic concentration is released for more than three weeks.

The following examples are not intended to be limiting, but merely represent various aspects and features of the invention.

Example 1 Preparation and characteristics of microspheres

Two different PLGA compositions - PLGA <50:50> and PLGA <65:35> were used in this study. The microspheres are manufactured using the w/o/w double emulsion technique (Fig. 1). Briefly, 90 ul of PTH<1-34> stock solution <PTH in 4 mM HCl/0.1% bovine serum albumin <BSA> solution> It was emulsified in a 10% dichloromethane <DCM> polymer solution, and an ultrasonic probe (Virsonic 100, Cardiner, NY) with an output of 15 W was used in an ice bath for 20 seconds to form the initial water-in-oil (w/ o) Emulsions. The water-in-oil emulsion was gradually added to 20 ml of a 1% polyvinyl alcohol <PVA> aqueous solution while vigorously stirring (700 rpm) to form a water-in-oil-in-water double emulsion. The solution was stirred at room temperature for 30 minutes to harden the microspheres, then the dichloromethane was evaporated under water absorption, and then the solid microspheres were collected by centrifugation. The generated microspheres were washed 3 times with distilled water and lyophilized. The overall shape of the microsphere will be studied by scanning electron microscopy (SEM) <Hitachi S3200, Tokyo, Japan after the microsphere sample is coated with gold on the microscope stage (Stub>, and the average size of the microsphere will be Particle size analyzer measurement.

4 mM hydrochloric acid and physiological saline <0.9% NaCl> and 0.1% BSA were used as stabilizers to protect PTH<1-34> encapsulated in microspheres. 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 this phenomenon during the degradation process. The literature report emphasizes that the appropriate size of microspheres for intra-articular injection in rats is 35-105 μm <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>, the data of the particle size analyzer shows that more than 90% of the average particle size can be successfully produced by the method in this study. -80 μm microspheres <Fig. 2 and Fig. 3a>.

Example 2 Encapsulation and release kinetics

The microspheres containing 10 mg of PTH<1-34> were suspended in 0.9% of a mixture of 1 mL of sodium chloride and 200 uL of dichloromethane, and incubated at room temperature for 1 hour with shaking. The PTH <1-34> standard solution <0.1 ml> was also prepared by adding 0.9% sodium chloride and 0.1% BSA. The concentration of PTH < 1-34> was measured according to the manufacturer's instructions using a commercially available immunoreagent kit. The protein loading and encapsulation efficiency <Encapsulation efficiency> is calculated by the equations <1> and <2> respectively (Table 1).

Equation <1>: Protein loading <w/w%> = total amount of protein in the microspheres / total number of microspheres

Equation <2>: Protein Encapsulation Efficiency <%> = <Measured Protein Concentration / Theoretical Value of Protein Concentration> X100

Example 3 PTH<1-34> release in vitro ( In vitro )

The release profile of PTH < 1-34> released from PLGA microspheres outside the body was determined by the following method. Suspend 10 mg of microspheres in 1 ml PBS <pH=7.4>, microsphere suspension The float was statically cultured at 4, 25, and 37 ° C, and 1 ml of the culture solution was collected by centrifugation at a set time interval, and replaced with an equal amount of fresh PBS. The concentration of PTH<1-34> in the released culture solution was measured in accordance with the manufacturer's recommendations using a PTH<1-34> ELISA kit (Immutopics, San Clemente, CA) with a PTH antibody-coated culture. Three cultures were used for each trip at each time point. The absorbance was measured at 450 nm using a microtiter plate reader, and the concentration of PTG was calculated by a log-logit method with a standard curve <GraphPad Software, San Diego, CA> using a GraphPad Prisms program.

A recent study by the inventors suggests that the effective dose of PTH <1-34> for the treatment of osteoarthritis induced by papain is 10 ^-8 M, and the frequency of injection is once every three days for five consecutive weeks. The release kinetic data from the PTH<1-34> specific ELISA assay showed that PLGA<65:35> microspheres released PTH<1-34 in the desired concentration range of 10 ^-7 -10 ^-8 M at 37 °C. 〉Two weeks <Figure 4>. Compared to PLGA <50:50>, PLGA <65:35> showed a consistent release profile after more than 17 days in simulated physiological conditions at 37 °C in PBS.

Example 4 MTT(3-<4,5-dimethylthiazol-2-yl>-2,5-diphenyltetrazolium bromide) analysis

When treating MC3T3-E1 <osteoblasts>, the toxicity range of PTH<1-34> was tested by MTT assay. Briefly, as described previously, by converting MTT to formazan to detect the mitochondrial activity of MC3T3-E1 cultured in the culture, a large amount of the Jiaji product is released into the culture solution, and This is directly proportional to the number of viable cells in the culture grid and can be measured by absorbance at 490 nm. MTT was diluted in standard medium at a volume ratio of 1:5 <MTT: culture medium> at a specified time interval, and the freshly prepared MTT was added to the cell containing cells, followed by 5% at 37 °C. The CO _{2 was} further cultured for four hours. After additional culture, in each culture cell, the culture solution was released and the converted 100 ul MTT was transferred to a 96-well culture plate, and the absorbance at 490 nm was recorded using a microplate reader <PathTech>, and KC junior software analysis was used (Fig. 3b).

Example 5 Bioactivity of released PTH<1-34>

The activity of the controlled release PTH < 1-34> was measured by measuring the cyclic adenosine <cAMP> contained in the cells treated with the released PTH<1-34>. For these experiments, MC3T3-E1 was cultured in α-MEM supplemented with 10% fetal bovine serum and 50 mg/mL ascorbic acid. The cells were cultured at a density of 50,000 cells per well on 24-well plates <24-well plates>. Six hours after incubation with PTH<1-34> released from microspheres, 0.1 N 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. The bioactivity data showed that the released PTH<1-34> treated MC3T3-E1 cells increased cAMP production on the first and third days, indicating that the released PTH<1-34> has biological activity (Fig. 5).

Example 6 Statistical Analysis

Three 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>. Single Factor variation analysis (ANOVA) assessed statistical significance and multiple comparisons were performed using the Xue Fei method. p < 0.05 was considered significant.

Example 7 In vivo experiment

method:

Animal experiment

Animal experiments were approved by the Animal Experiment Management Team of Kaohsiung Medical University. 54 12-week-old male Sprague-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 experimental animals will be acclimated in the laboratory for a week before the experiment.

Osteoarthritis induction and PTH treatment

Each of the left knee, which was considered to be the contralateral control joint, was applied with a mediator that was not treated with PTH or with OA, and the right knee was the joint being studied. Rats were divided into five groups: Non-OA+PTH group (treated with PTH<1-34> without OA induction><n=6>, Non-OA+PTH/PLGA group <PTH/ PLGA microsphere treatment but no OA induction><n=6>, OA group (for OA induction but not for PTH<1-34>>n=6>, OA+PTH group <PTH<1-34 〉 After treatment, OA induced><n=6> and OA+PTH/PLGA group (treated with PTH/PLGA microspheres, then OA induced><n=6>). In the OA group and the OA+PTH group, 20 μl of 4% papain solution and 20 μl of 0.03 M cysteine were injected into the right knee joint to induce OA. These off The section was injected through a knee tendon with a 26-gauge needle on days 1, 4 and 7 of the experimental day (US Patent No. US 659081). In the OA+PTH group, 40 μl of 10 nM PTH <1-34> was injected into the right knee joint every three days after the OA was induced until the sacrifice of the rat. In the PTH group, the same PTH <1-34> treatment was performed in the absence of OA induction. In the OA+PTH/PLGA group, 0.4 mg of PTH/PLGA microspheres were injected into the right knee joint on days 1 and 15 after induction of OA. 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 samples were then decalcified with 10% formic acid/PBS buffer, and the decalcified tibial joint samples were embedded in paraffin and 5 μm coronal microsections were prepared. GAG is stained with Safranin-O-Fast-Green (1% Safranin with 0.75% hematoxylin followed by 1% fast green for contrast staining) <Sigma, St. Louis, MO>. Collagen and type X collagen are immunostained.

Histomorphometric study

GAG was stained with saffron, and all regions in the articular cartilage of each proximal humerus and red-stained regions were measured with Image-Pro plus 5.0 software <Media Cybernetics Inc. MD, USA> (Fig. 6C). Calculate the ratio of the areas dyed red in each group in all areas (red/all).

immunochemistry

The tibial articular section was re-hydrated and the endogenous peroxidase in the tissue was blocked with 3% hydrogen peroxide. Before the sample is statically cultured with the primary antibody, the enzyme will be decomposed by the enzyme for the epitope return. The method for the decomposition of the enzyme is to modify the previous report (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). The best conditions for enzyme decomposition of type 2 collagen immunostaining are 2.5% hyaluronicase <hyluronidase> at 37oC and 1 mg/ml chain protease in PBS<pH 7.4><Sigma, St.Louis, MO> 1 hour in the mixture. The optimal conditions for x-type collagen immunostaining were 1 hour in 0.1 U/ml of chondroitinase ABC (Sigma, St. Louis, MO) and 1 mg/ml tris-HCl at 37 °C. Pepsin in (pH 3.0) for 15 minutes. The sections were then blocked with fetal bovine serum for 1 hour and at 37 °C with primary antibody against mouse type 2 collagen < mouse monoclonal antibody <Chemicon International, Temecula, CA> and primary antibody against type X collagen. <Multibody antibodies of rats><1:200><COSMO, Tokyo, Japan> cultured for 4 hours. The second type collagen was cultured with biotin-labeled goat anti-mouse immunoglobulin <DAKO, Carpinteria, CA>, and the type X collagen was labeled with biotin-labeled goat anti-rabbit immunoglobulin (Biocare medical, Walnut). Creek, CA> was cultured with secondary antibody, and then added with bioactive enzyme <Streptravidin-HRP><streptavidin coupled with horseradish peroxidase> (Biocare medical, Walnut Creek, CA>. Then, it was stained with a solution of 3,3'-diaminobenzidine <3,3'-diaminobenzidine> containing 0.01% hydrogen peroxide. Finally, the sections were stained with hematoxylin as backgrounds and observed by a microscope. The relative density of the immunostaining <density/area; area 25.44±2.77 mm ² > was measured by Image-Pro plus 5.0 software <Media Cybernetics Inc. MD, USA> (Fig. 7C).

result:

Histological and histomorphometric study of rat articular cartilage

Representative photographs of articular cartilage of saffron-stained rat joints in the contralateral control, Non-OA+PTH, Non-OA+PTH/PLGA, OA, OA+PTH, and OA+PTH/PLGA groups are shown in Figure 6 <A&B>. The ratio of reddish-stained regions in all regions (red: all) was measured and compared in the grouping (Fig. 6C). The red color in the contralateral control joint between the groups: the ratio was not significantly different. 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 was no significant difference in all ratios (Fig. 6C). In the OA group, the red ratio 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 OA for five weeks <P < 0.01> <Fig. 6C>. After 5 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: the overall ratio was significantly higher than that of the OA group <P<0.01> <Fig. 6C>. In addition, the red color of the OA+PTH/PLGA group: all ratios were not significantly different from the contralateral control cartilage. At the fifth week, there was no significant difference between the OA+PTH group, the OA+PTH/PLGA group, and the contralateral control cartilage group (Fig. 6C).

Immunohistochemical study of type II collagen in rat articular cartilage

Type 2 collagen staining of articular cartilage of rat joints in contralateral control, Non-OA+PTH, Non-OA+PTH/PLGA, OA, OA+PTH and OA+PTH/PLGA groups Representative photomicrographs of 〉 are shown in Figure 7 <A, B and C>. The ratio of the second type collagen stained area to the entire area <brown: all> was measured and compared between the groups (Fig. 7D). Contralateral control of joint brown: all ratios were not significantly different between groups; contralateral control of cartilage in the joints of brown: all ratios, and in the Non-OA+PTH and Non-OA+PTH/PLGA groups There were no significant differences in the joints (Fig. 7D). Brown in articular cartilage in the OA group: The overall ratio was significantly lower than that in the contralateral control cartilage induced by OA for five weeks: all ratios <P < 0.01 > <Fig. 7D>. There was no significant difference between the cartilage of the OA+PTH group and the contralateral control cartilage after 5 weeks of treatment with PTH<1-34> (Fig. 7D). After five weeks, the brown color of the OA+PTH/PLGA group: the overall ratio was significantly higher than that of the OA group: all ratios <P<0.01> <Fig. 7D>. In addition, the brown color of the OA+PTH/PLGA group: all ratios were also not significantly different from the contralateral control cartilage. At the fifth week, there was no significant difference between the OA+PTH, OA+PTH/PLGA, and the contralateral control cartilage group (Fig. 7D).

Immunohistochemical study of type X collagen in rat articular cartilage

In the contralateral control cartilage, no obvious type X collagen-stained chondrocytes were found (Figs. 8A and 8B). Immunolocalized type X collagen (stained brown) was mainly found in articular chondrocytes of the OA group, but positive staining was rarely observed in the OA+PTH and OA+PTH/PLGA group cartilage after five weeks of treatment. <positive stained> cells <Fig. 8A and 8B>.

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The microspheres of the present invention and the processes and methods for their manufacture are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope.

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it will be appreciated that while the invention has been specifically described in accordance with the preferred embodiments and any features, those skilled in the art will Modifications and variations are still within the scope of the invention as claimed.

Figure 1: Schematic diagram of the production of polylactic acid-glycolic acid <PLGA> microspheres coated with PTH <1-34>.

Figure 2: Scanning electron microscope (SEM) image of polylactic acid-glycolic acid microspheres coated with PTH <1-34>.

Figure 3: <a> is the size of the microspheres analyzed by the particle size analyzer; <b> PTH <1-34> cytotoxicity by MTT analysis.

Figure 4: Cumulative release profiles for PLGA <50:50> and PLGA <65:35>.

Figure 5: Biological activity of PTH<1-34> released from PLGA microspheres on MC3T3-E1 cells

Figure 6: Tissue analysis of the amount of GAG seen in articular cartilage stained with Safranin-O. These images are the contralateral control group, Non-OA+PTH, Non-OA+PTH/PLGA, OA, OA+PTH, and OA+PTH/PLGA groups. The Safranin-O-stained rat contralateral tibia proximal Articular cartilage. The OA+PTH group in the OA group was treated once every 3 days with PTH<1-34><10 ^-8 M>, while the OA+PTH/PLGA 0.4 mg group was treated twice every 15 days, and the study was large. Mouse joints are also listed in the figure. Each bar represents the mean ± standard deviation of eight samples. The data was analyzed by one way analysis of variance (ANOVA) and multiple comparisons were performed using the Scheffe's method. <**>p<0.01, at each time point, the contralateral control group was compared with the experimental group sample. <##>p<0.01, at each time point, the experimental group samples in the OA group were compared.

Figure 7: Type II collagen of stained articular cartilage in the contralateral control group, Non-OA+PTH group, Non-OA+PTH/PLGA group, OA group, OA+PTH group, and OA+PTH/PLGA group Results of histological analysis of <COL.II>. In the OA group, the proximal humerus of the contralateral joint of the rat was immunostained for articular cartilage, and the OA+PTH group was treated once every 3 days with PTH<1-34><10 ^-8 M>, while in the OA group. The OA+PTH/PLGA 0.4 mg group was treated twice every 15 days. The graph shown is the rat joint studied. The growth plate cartilage was stained for positive control, and no growth plate stained with primary antibody and articular cartilage were used as a negative control. The second type of collagen is stained brown. Each bar represents the mean ± standard deviation of eight samples. Data were analyzed as single factor variances and multiple comparisons were performed using the Xue Fei method. <**>p<0.01, at each time point, the contralateral control group was compared with the experimental group sample. <##>p<0.01, at each time point, the experimental group samples in the OA group were compared.

Figure 8: Type X collagen of stained articular cartilage in the contralateral control group, Non-OA+PTH group, Non-OA+PTH/PLGA group, OA group, OA+PTH group, and OA+PTH/PLGA group Results of histological analysis of COL.X>. In the OA group, the proximal humerus of the contralateral joint of the rat was immunostained for articular cartilage, and the OA+PTH group was treated once every 3 days with PTH<1-34><10 ^-8 M>, while in the OA group. The OA+PTH/PLGA 0.4 mg group was treated twice every 15 days. The growth plate cartilage was stained for positive control, and no growth plate stained with primary antibody and articular cartilage were used as a negative control. Collagen type X is stained brown. The cartilage in which type X collagen is stained is indicated by an arrow. Each bar represents the mean ± standard deviation of eight samples. Data were analyzed as single factor variances and multiple comparisons were performed using the Xue Fei method. <**>p<0.01, at each time point, the contralateral control group was compared with the experimental group sample. <##>p<0.01, at each time point, the experimental group samples in the OA group were compared.

Annex 1: Color diagram of Figure 1.

Annex 2: Color diagram of Figure 6A.

Annex 3: Color diagram of Figure 6B.

Annex 4: Color diagram of Figure 7A.

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.

<110> Kaohsiung Medical University

<120> Method for controlling release of parathyroid hormone by microspheres of poly(lactic acid-glycolic ACID)

<130> 1183-KNU-US

<160> 2

<170> PatentIn version 3.4

<210> 1

<211> 84

<212> PRT

<213> Human (Homo sapiens)

<220>

<221> PEPTIDE

<222> (1)..(84)

<400> 1

<210> 2

<211> 34

<212> PRT

<213> Human (Homo sapiens)

<220>

<221> MISC_FEATURE

<222> (1)..(34)

<400> 2

Claims (16)

A controlled release microsphere having an average particle diameter of more than 50 μm, wherein the microsphere comprises: an inner aqueous layer containing a pharmaceutically effective amount of a biologically active polypeptide, the activity of the biologically active polypeptide and parathyroid hormone a similar intermediate layer comprising a polymeric layer of polylactic acid-glycolic acid (PLGA), the oil layer coating the inner aqueous layer to form a water-in-oil (w/o) emulsion; and an outer layer comprising An aqueous layer of polyvinyl alcohol (PVA) for coating the intermediate layer, wherein the microspheres control release of the biologically active polypeptide system in a range from about 1 X 10 ^-7 M to about 5 X 10 ^-9 M The effective concentration is at least 18 days. The microsphere of claim 1, wherein the polylactic acid-glycolic acid (PLGA) is PLGA (50:50) or PLGA (65:35). The microsphere of claim 1, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2. The microsphere of claim 1, wherein the polypeptide is immobilized in a stock solution containing hydrochloric acid and bovine serum albumin. The microsphere of claim 4, wherein the hydrochloric acid concentration is from about 1 mM to about 8 mM, and the concentration of bovine serum albumin is from about 0.01% to about 5%. The microsphere of claim 1, wherein the aqueous polyvinyl alcohol solution has a concentration by weight of from about 0.1% to about 5%. A method for producing a controlled release microsphere having an average particle diameter of more than 50 micrometers, comprising: (a) providing an oil layer containing a polylactic acid-glycolic acid (PLGA) polymer; (b) preparing a water-in-oil <w/ o> an emulsion comprising an inner aqueous layer comprising a pharmaceutically effective amount of a biologically active polypeptide, the biologically active polypeptide having activity similar to that of parathyroid hormone; and (c) gradually slowing the water-in-oil emulsion Adding to an aqueous solution of polyvinyl alcohol (PVA) to form a water-in-oil-in-water <w/o/w> double emulsion and then desorbing the solvent in the oil layer, wherein the microspheres control release of the biologically active polypeptide system to a range 1 X 10 ^-7 M to an effective concentration of about 5 X 10 ^-9 M for at least 18 days. The method of claim 7, wherein the polylactic acid-glycolic acid <PLGA> is PLGA (50:50) or PLGA (65:35). The method of claim 7, wherein the amino acid sequence of the biologically active polypeptide is as shown in SEQ ID NO: 2. The method of claim 7, wherein the biologically active polypeptide is immobilized in a stock solution containing hydrochloric acid and bovine serum albumin. The method of claim 10, wherein the hydrochloric acid concentration is from about 1 mM to about 8 mM, and the concentration of bovine serum albumin is from about 0.01% to about 5%. The method of claim 7, wherein the aqueous polyvinyl alcohol solution has a concentration by weight of from about 0.1% to about 5%. The method of claim 7, wherein the bioactive peptide has an encapsulation efficiency of not less than about 60%. The method of claim 7, wherein the biologically active polypeptide is for the treatment of parathyroid dysfunction, bone disease or cartilage disease. The method of claim 14, wherein the bone disease is osteoporosis or osteonecrosis. The method of claim 14, wherein the cartilage disease is osteoarthritis.