WO2005105058A1 - Formulation pouvant etre injectee a liberation soutenue pour le traitement ou la prevention de maladies osseuses comprenant des micro-particules polymeriques qui contiennent du disphosphonate - Google Patents

Formulation pouvant etre injectee a liberation soutenue pour le traitement ou la prevention de maladies osseuses comprenant des micro-particules polymeriques qui contiennent du disphosphonate Download PDF

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WO2005105058A1
WO2005105058A1 PCT/KR2005/001306 KR2005001306W WO2005105058A1 WO 2005105058 A1 WO2005105058 A1 WO 2005105058A1 KR 2005001306 W KR2005001306 W KR 2005001306W WO 2005105058 A1 WO2005105058 A1 WO 2005105058A1
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bone
injectable formulation
bisphosphonate
drug
microparticles
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PCT/KR2005/001306
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English (en)
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Woo Jeong Choi
Hyeok Lee
Jeong Hwa Yang
Jung Ju Kim
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Amorepacific Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers

Definitions

  • the present invention relates to an injectable formulation comprising bisphosphonate-containing polymeric microparticles, and more particularly, to a sustained- release injectable formulation for treating or preventing bone-related diseases / comprising bisphosphonate-containing polymeric microparticles prepared by a W/O/ multiple emulsion method.
  • Bone tissue is a dynamic tissue where bone is remolded through continuous cycles of bone resorption and bone formation to maintain bone homeostasis. Bone resorption is mediated by osteoclasts, and bone formation is mediated by osteoblasts. Bone resorption and bone formation are tightly coupled, and bone homeostasis is maintained under normal states. However, the imbalance between bone resorption and bone formation accelerates bone resorption or reduces bone formation, leading to osteoporosis. Bone tissue, as a major reservoir of calcium, is affected by several hormones participating in calcium metabolism. Parathyroid hormone stimulates bone resorption, reduces the loss of calcium through urine, and promotes the production of active vitamin D in the kidneys. Vitamin D increases intestinal absorption of calcium and participates in bone calcification.
  • Breakdown of the balance between bone resorption and bone formation is caused by nutritional factors, for example, by dietary calcium and protein deficiency, reduced estrogen levels after menopause, smoking, hyperthyroidism, caffeine, which inhibits calcium absorption, vitamin deficiency, genetic factors, chronic stress, and the like.
  • nutritional factors for example, by dietary calcium and protein deficiency, reduced estrogen levels after menopause, smoking, hyperthyroidism, caffeine, which inhibits calcium absorption, vitamin deficiency, genetic factors, chronic stress, and the like.
  • NASH National Institutes of Health
  • osteoporosis is a bone disease entailing an increased risk of bone fracture resulting from decreased bone strength.
  • the physical strength of bone cannot be measured accurately but is known to be mainly determined (about 70%) by bone mineral density, and the quality of bone is also known to be important.
  • Normal bone has a net-like compact structure.
  • Osteoporosis in contrast, in osteoporosis, gaps are widen in the net-like structure, and fine structures become thin and weak. In this case, despite the same size and volume of bone, bone mass per unit volume is reduced, and thus, bone is easily fractured even upon slight impact. Histologically, there is a reduction in the cortical bone thickness and in the number and size of trabeculae of cancellous bone, leading to decreased trabecular connectivity and, finally, skeletal fragility. Osteoporosis, as described above, may be classified into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and secondary osteoporosis.
  • type I postmenopausal osteoporosis
  • type II senile osteoporosis
  • secondary osteoporosis secondary osteoporosis
  • Postmenopausal osteoporosis (type I) usually occurs in women at the onset of menopause when the rate of bone loss accelerates (2-3% every year) . Type I is responsible for increased vertebral crush fractures and wrist fractures. Senile osteoporosis (type II) develops slowly in men and women older than age 70 (0.5-1% every year) and is associated with gradual bone loss in hip bones and vertebrae. Secondary osteoporosis occurs regardless of age.
  • causes of secondary osteoporosis include disorders (endocrine diseases, gastrointestinal diseases, malignant tumors, etc.), drugs (adrenocortical hormone, anticancer chemotherapy, thyroid hormone, anticonvulsants, anticoagulants, methotrexate, cyclosporine, GnRH, etc.), alcohol, smoking, and accidents.
  • the diagnosis of osteoporosis is performed by measuring bone mineral density or detecting bone biochemical markers.
  • Bone mineral density (BMD) is an indicator of bone strength.
  • a high BMD value means stronger bones.
  • the World Health Organization (WHO) defines osteopenia as a bone mineral density 10-24% lower, and osteoporosis as a bone mineral density 25% or more lower, than that of healthy young adults.
  • the following drugs have been used in the treatment of osteoporosis: female sex hormones, such as estrogen; bone resorption inhibitors, such as calcitonin and bisphosphonates; bone formation stimulators, such as fluoride and parathyroid hormone; agents inhibiting bone resorption and stimulating bone formation, such as menatetrenone; active vitamin D and ipriflavone; and calcium supplements.
  • female sex hormones such as estrogen
  • bone resorption inhibitors such as calcitonin and bisphosphonates
  • bone formation stimulators such as fluoride and parathyroid hormone
  • agents inhibiting bone resorption and stimulating bone formation such as menatetrenone
  • active vitamin D and ipriflavone active vitamin D and ipriflavone
  • calcium supplements such as calcium supplements.
  • bisphosphonates which resemble naturally occurring inorganic pyrophosphate (PPi) in structure, have a P-C-P bond instead of the P-O-P bond of PPi, the oxygen being replaced by
  • bisphosphonates are stable substances resistant to degradation by pyrophosphatase.
  • Bisphosphonates are classified into first, second and third-generation agents according to the structure of two side chains binding to the carbon atom.
  • the mechanism of inhibition of bone resorption by bisphosphonates is as follows. Bisphosphonates, binding to hydroxyapatite on the bone surface, are released from bone tissue during osteoclast-mediated bone resorption.
  • osteoclasts are locally exposed to high concentrations of bisphosphonates, and bisphosphonates enter osteoclasts and induce several biochemical reactions, leading to apoptosis of osteoclasts and eventually inhibiting differentiation and functions of osteoclasts (Fleisch H, 1995, Bisphosphonates In Bone Disease, From The Laboratory To The Patient, 2nd Edition, Parthenon Publishing) .
  • bisphosphonates can be used in all diseases having increased bone resorption, which are exemplified by osteoporosis, Pagef s disease, hypercalcemia induced by malignant tumors, metastatic bone diseases induced by malignant tumors, multiple myeloma, bone loss caused by immobilization, bone loss after artificial joint replacement, childhood osteoporosis of unknown cause, rheumatoid arthritis, hyperparathyroidism, and periodontal disease.
  • osteoporosis Pagef s disease
  • hypercalcemia induced by malignant tumors metastatic bone diseases induced by malignant tumors
  • multiple myeloma multiple myeloma
  • bone loss caused by immobilization bone loss after artificial joint replacement
  • childhood osteoporosis of unknown cause rheumatoid arthritis
  • hyperparathyroidism and periodontal disease.
  • etidronate is the first drug to be clinically applied.
  • the continuous administration of etidronate is known to have a risk of
  • second and third bisphosphonates having enhanced inhibitory effects on bone resorption while not affecting bone calcification, have been applied to prevent and treat osteoporosis.
  • These bisphosphonates include pamidronate, alendronate, risedronate and ibandronate.
  • representative drugs applied to treat osteoporosis are pamidronate, alendronate, risedronate, etc.
  • Bisphosphonates have low bioavailability in the gastrointestinal tract when administered orally. The consequent high dosage may cause side effects in the stomach, the intestine and the esophagus.
  • alendronate which has a very low absorptivity of 1-3% in the gastrointestinal tract due to its polar property and non-lipophilicity, is known to have a bioavailability below 1% upon oral administration, 0.7% in women and 0.59% in men.
  • patients are recommended to take the drug on an empty stomach and fast for 30 min or more afterwards.
  • many patients consider fasting every day to ' be inconvenient.
  • pamidronate is associated with esophageal ulcers (E.G.
  • WO02/062352 discloses a device and a method for increasing bone mineral density, which are based on implanting or injecting a drug delivery device comprising a bisphosphonate.
  • the present inventors prepared an injectable formulation comprising sustained release polymeric microparticles into which a bisphosphonate is encapsulated using a W/O/W multiple emulsion method, and found that this injectable formulation continuously releases the bisphosphonate for a period of 4 weeks or more and maintains an appropriate serum concentration of the bisphosphonate, effectively increases bone mineral density even at a dosage less than that of oral administration, and has much lower local toxicity than does the direct injection of bisphosphonate, thereby leading to the present invention.
  • FIG. la is an electron microscopic image of a cross section of polymeric microparticles prepared in Comparative Preparation Example 1
  • FIG. lb is an electron microscopic image of a cross section of polymeric microparticles prepared in Preparation Example 1-3
  • FIG. 2 is a graph showing release rates of a drug from microparticles according to mixing ratios of dichloromethane and acetone in an organic solvent containing poly (lactic acid) and a hydrophobic surfactant
  • FIG. la is an electron microscopic image of a cross section of polymeric microparticles prepared in Comparative Preparation Example 1
  • FIG. lb is an electron microscopic image of a cross section of polymeric microparticles prepared in Preparation Example 1-3
  • FIG. 2 is a graph showing release rates of a drug from microparticles according to mixing ratios of dichloromethane and acetone in an organic solvent containing poly (lactic acid) and a hydrophobic surfactant
  • FIG. 3 is a graph showing release rates of a drug from microparticles according to mixing ratios of dichloromethane and acetone in an organic solvent containing poly (lactic acid-co-glycolic acid) and a hydrophobic surfactant;
  • FIG. 4 Is a graph showing drug release rates according to mixing ratios of poly (lactic acid-co-glycolic acid) and poly (ethylene glycol) ;
  • FIG. 5 is a graph showing drug release rates according to mixing ratios of dichloromethane and acetone in an organic solvent when chitosan instead of sodium hyaluronate was used as a water-soluble polymer;
  • FIG. 6 is a graph showing release rates of a drug according to changes in internal aqueous phase having viscosity;
  • FIG. 7 is a graph showing plasma levels of a bisphosphonate for a period of 4 weeks after bisphosphonate-containing polymeric microparticles were injected;
  • FIG. 8 is a graph showing the trabecular bone area measured in the tibia of female rats injected with bisphosphonate-containing polymeric microparticles;
  • FIGS. 9a to 9g are photographs of stained tibia tissue specimens, which were taken by an image analyzer, after bisphosphonate-containing polymeric microparticles were injected into female rats; and
  • FIG. 10 is a graph showing bone mineral density of I the tibia of female rats injected with bisphosphonate- containing polymeric microparticles .
  • the present invention relates to a sustained-release injectable formulation for treating or preventing bone-related diseases, comprising bisphosphonate-containing polymeric microparticles prepared by a W/O/W multiple emulsion method.
  • Bisphosphonates as active drugs contained in the injectable formulation of the present invention which directly or indirectly act on osteoclasts and reduce the number and activity of osteoclasts, thereby inhibiting bone resorption and increasing total bone mass, are used for preventing or treating diseases caused by increased bone resorption.
  • Diseases caused by bone resorption, in which a bisphosphonate is used as a preventive or therapeutic drug are exemplified by osteoporosis (S.E.
  • the present injectable formulation comprising a bisphosphonate as an active drug may be used for preventing or treating the aforementioned diseases .
  • the bisphosphonate is selected from alendronate, cimadronate, clodronate, EB-1053, etidronate, ibandronate, nerid onate, olpadronate, pamidronate, risedronate, tiludronate, YH 529, incadronate, minodronate, zoledronate, and pharmaceutically acceptable salts, esters and acids thereof and mixtures thereof. These compounds have a water solubility of 0.1 ⁇ g/ml to 1,000 mg/ml, and preferably 10 mg/ml to 500 mg/ml.
  • etidronate is the first drug to be clinically applied.
  • etidronate is known to have a risk of osteomalacia .
  • second and third bisphosphonates having enhanced inhibitory effects on bone resorption while not affecting bone calcification, have been applied to prevent and treat osteoporosis.
  • These bisphosphonates include pamidronate, alendronate, risedronate and ibandronate.
  • representative drugs applied to treat osteoporosis are pamidronate, alendronate, and risedronate.
  • Alendronate is accumulated in osteoclasts, causing changes in the cytoskeleton, a reduction of the ruffled border and a decrease in enzymatic activity, which lead to apoptosis of osteoclasts, thereby reducing the activity and number of osteoclasts. Also, alendronate first acts indirectly on osteoblasts to inhibit the differentiation and aggregation of osteoclasts, thereby reducing the number and activity of osteoclasts.
  • the bisphosphonate is alendronate or a pharmaceutically acceptable salt, ester or acid thereof.
  • the bisphosphonate-containing polymeric microparticles contained in the injectable formulation of the present invention are capable of sustaining the release of a drug for a long period of time because they are prepared by a multiple emulsion method and thus solve the problems of conventional polymeric microparticles, namely, low drug encapsulation efficiency and high initial bursts.
  • These polymeric microparticles are preferably prepared by dissolving and dispersing a bisphosphonate in an aqueous solution containing a water-soluble polymer and a hydrophilic surfactant to provide a polymer solution; adding the polymer solution to another polymer solution, prepared by adding a secondary organic solvent to a primary organic solvent containing a biodegradable polymer and a hydrophobic surfactant, to provide a primary emulsion (water-in oil (W/O) type) ; and dispersing the primary emulsion in an external continuous phase.
  • W/O water-in oil
  • the bisphosphonate-containing polymeric microparticles are prepared by a preparation method comprising: (1) adding a secondary organic solvent to a primary organic solvent containing a biodegradable polymer and a hydrophobic surfactant to provide a polymer solution; (2) dissolving or dispersing a bisphosphonate in an aqueous solution containing a water-soluble polymer and a hydrophilic surfactant, and adding a resulting dispersion to the polymer solution prepared at Step (1) to provide a primary emulsion (water-in oil (W/O) type) , wherein an internal aqueous phase of the primary emulsion is dehydrated to form microcoagulated particles of the water- soluble polymer, encapsulating the bisphosphonate into the microparticles; and (3) dispersing the primary emulsion into an external continuous phase to solidify the polymeric microparticles.
  • the preparation method of polymeric microparticles may further include a conventional filtering and washing procedure in Step (3) .
  • Step 1 Preparation of polymer solution
  • a polymer solution is prepared by adding a secondary organic solvent to a primary organic solvent containing a biodegradable polymer and a hydrophobic surfactant.
  • a polyester polymer may be used as the biodegradable polymer.
  • the biodegradable polymer may be one ore more selected from the group consisting of poly (lactic acid (PLA) , poly (glycolic acid) (PGA), poly (lactic acid-co- glycolic acid (PLGA) and polycarprolactone (PCL) .
  • the polymers are known as polymers with excellent biocompatibility and biodegradability because they are decomposed into harmless water and carbon dioxide in vivo via the citric acid cycle that is a fundamental metabolic pathway (see, S.
  • the biodegradable polymer is not particularly limited but preferably has a mean molecular weight ranging from 5,000 to 210,000. Also, the biodegradable polymer may be added to 10 to 60% (w/v) based on the organic solvent contained in the polymer solution. In addition, in the present invention, a crystalline polymer may be added at Step (1) to prepare polymeric microparticles.
  • the crystalline polymer serves as a drug release modifier.
  • any injectable • biocompatible material may be used without particular limitation. Preferred is poly (ethylene glycol) (PEG) or poly (lactic acid), the more preferred of which is poly (ethylene glycol) .
  • Low molecular weight PEG is known as a biocompatible polymer that is clinically used for intraarticular injection.
  • Preferred PEG has a molecular weight of 200 to 5,000.
  • PEG has a molecular weight of less than 200, PEG is not formed into crystals, thus failing to act as a drug release modifier.
  • the molecular weight of PEG exceeds 5000, it cannot be excreted through the kidney (see, K. K. Huang, T. W. Chang and T. Tzeng, Int. J. Pharm. , 156, 9-15, 1997).
  • the mass ratio of the crystalline polymer to biodegradable polymer is 0.1:99.9 to 20:80, and preferably, 1:99 to 10:90.
  • the biodegradable polymer is a low molecular weight copolymer in which the molar fraction of poly (lactic acid) to poly (glycolic acid) is 50:50, as it is an amorphous polymer in a rubbery state, the formation of pores and water channels that are main release routes of a drug encapsulated in microparticles is interrupted, thus the overall release rate of drug tends to be too low.
  • the use of poly (ethylene glycol) as a drug release modifier in physical combination with an amorphous polymer facilitates the formation of pores and water channels via formation of a crystal area within the amorphous rubbery polymeric microparticles, thereby achieving desired control of drug release.
  • the hydrophobic surfactant may be one or more selected from the group consisting of fatty acids, olefin, alkyl carbons, silicon, sulfate esters, fatty alcohol sulfate, sulfated fats and oils, sulfonic acid salts, aliphatic sulfonate, alkylaryl sulfonate, ligminsulfonate, phosphoric acid esters, polyoxyethylene, polyglycerol, polyols, imidazoline, alkanolamine, hetamine, sulfomethamine, phosphatide and sorbitan fatty acid esters.
  • sorbitan fatty acid esters Preferred are sorbitan trioleate or sorbitan monopalmitate .
  • the hydrophobic surfactant may be added to 0.1 to 30% (v/v) based on the organic solvent contained in the polymer solution, and preferably, 5 to 20% (v/v) .
  • the primary organic solvent should be miscible with the biodegradable polymer and the hydrophobic surfactant and phase-separated with water.
  • the primary organic solvent is not particularly limited as long as it satisfies the above requirements, but may be one or more selected from dichloromethane, chloroform, cyclohexane and ethylacetate .
  • the secondary organic solvent should be miscible with the primary organic solvent and with the biodegradable polymer and hydrophobic surfactant contained in the solvent, and should be also water-miscible.
  • the secondary organic solvent is not particularly limited as long as it satisfies the above requirements, but may be one or more selected from acetone, acetonitrile, dimethylsulfoxide, tetrahydrofuran and dioxan.
  • the microparticles of the present invention are preferably prepared using a solvent mixture of a primary organic solvent containing a biodegradable polymer and a hydrophobic surfactant and a secondary organic solvent having satisfactory water miscibility.
  • a solvent mixture of dichloromethane and acetone is more preferred.
  • the volume ratio of the primary organic solvent to the secondary organic solvent is 95:5 to 50:50, and preferably, 75:25 to 55:45.
  • the total volume of the primary organic solvent and the secondary organic solvent is 1/500 to 1/100 based on the volume of an external continuous phase (e.g., aqueous polyvinyl alcohol solution) , and preferably, 1/400 to 1/200.
  • an external continuous phase e.g., aqueous polyvinyl alcohol solution
  • Step 2 Preparation of primary emulsion and primary encapsulation of drug via the formation of microcoagulated particles of water-soluble polymer
  • a drug at higher than a saturation concentration was dissolved and dispersed in an aqueous solution containing a water-soluble polymer and a hydrophilic surfactant.
  • the resulting dispersion is added to the polymer solution prepared at Step 1 and vigorously stirred to provide a primary emulsion (water-in-oil (W/O) type) .
  • W/O water-in-oil
  • an internal aqueous phase is rapidly dehydrated.
  • the water-soluble polymer used in the present invention is highly biocompatible and harmless to the body and has viscosity in an aqueous phase when dissolved in water.
  • the water-soluble polymer may be one or more selected from the group consisting of cellulose, hemicellulose, pectin, lignin, starch as a storage carbohydrate, chitosan, xanthan gum, alginic acid, pullulan, curdlan, dextran, levan, hyaluronic acid, glucan, collagen, and salts thereof. Preferred is hyaluronic acid or a salt thereof.
  • the viscosity of the water-soluble polymer in the aqueous solution before dehydration is 300 to 50,000 cp (centi-poise) , and preferably, 500 to 30,000 cp .
  • Step 2 of the present method of preparing polymeric microparticulates other components may be further added to increase water solubility of the water- soluble polymer.
  • the water-soluble polymer is chitosan
  • chitosan is preferably dissolved in an aqueous solution of an organic acid, such as formic acid, citric acid, acetic acid and lactic acid, or an inorganic acid such as hydrochloric acid.
  • the concentration of an acid to water is preferably 0.5 to 3.0% (w/v).
  • the hydrophilic surfactant is used for evenly dispersing the drug particles at higher than a saturation concentration.
  • the hydrophilic surfactant may be one or more selected from the group consisting of protein surfactants, such as bovine serum albumin (BSA) and carbopol, polyoxyethylene-polyoxypropylene block copolymers and polyoxyethylene sorbitan fatty acid esters (Tween series) . Preferred are polyoxyethylene sorbitan fatty acid ester surfactants, and more preferred is polyoxyethylene sorbitan monooleate (trade name: Tween 80) .
  • the hydrophilic surfactant may be added to 0.1 to 30% (w/w) based on water, and preferably, 1 to 20% (w/w) .
  • Step 3 Preparation of polymeric microparticles by dispersing primary emulsion in external continuous phase and solidifying the dispersion
  • An external continuous phase for dispersing the primary emulsion may be selected from aqueous solutions of sodium dodecyl sulphate (SDS) , cetyltrimethyl ammonium bromide (CTAB) , methyl cellulose (MC) , gelatin, polyoxyethylene sorbitan monooleate and polyvinyl alcohol (PVA) .
  • SDS sodium dodecyl sulphate
  • CTAB cetyltrimethyl ammonium bromide
  • MC methyl cellulose
  • PVA polyvinyl alcohol
  • Preferred is an aqueous solution of polyvinyl alcohol.
  • the aqueous solution of polyvinyl alcohol is used at a concentration of 0.1 to 5% (w/v), and preferably, 0.3 to 2% (w/v) .
  • the polyvinyl alcohol has a molecular weight of 10,000 to 100,000, and preferably, 13,000 to 23,000, and a degree of hydrolysis of 75 to 95%, and preferably, 83 to 89%.
  • other ingredients commonly added in multiple emulsion preparation for example, ethyl acetate, can be added to the continuous phase. In this case, ethyl acetate is added to 1-20% (w/v) based on a PVA aqueous solution, preferably 5-10% (w/v) .
  • the bisphosphonate-containing polymeric microparticles prepared as described above have a mean particle diameter of 0.1 to 200 ⁇ m, and preferably, 10 to 120 ⁇ m, which are spherical particles in which enormous pores and water channels are formed. Since the microparticles have a larger surface area than a film- or cylinder-type preparation having the same weight, controlled release of bisphosphonates is achieved.
  • sustained release formulations for maintaining a constant serum concentration of a drug have a problem of excessively releasing the drug at early stages . Bursting of a drug results in the drug being present in a greater amount than that capable of being processed by organs, thereby reducing the overall therapeutic efficacy of the drug, and also may causes toxicity in the body due to the high dose of the drug.
  • the bisphosphonate- containing polymeric microparticles prepared as described above contain microcoagulated particles of a water-soluble polymer, which are distributed in the pores and encapsulate a drug, resulting in the drug being doubly encapsulated by the. water-soluble polymer and the biodegradable polymer.
  • This double encapsulation may minimize the loss of the drug toward an external continuous phase during microparticle preparation, as well as minimize high initial bursts of the drug.
  • an injectable formulation comprising the bisphosphonate-containing polymeric microparticles prepared as described above was injected to the body and assessed for changes in serum concentration of a drug (FIG. 7) .
  • the present injectable formulation comprising the bisphosphonate-containing polymeric microparticles prepared as described above has an excellent sustaining effect on drug release.
  • the present injectable formulation may overcome the inconvenient oral administration of conventional bisphosphonate drugs and side effects due to excessive administration of the drugs, and achieves the effect obtained by daily oral administration of the drugs for a period of one month through only single injection according to the present invention. Therefore, in a preferred aspect, the present invention relates to a sustained-release injectable formulation displaying a pharmaceutical effect for four weeks or more using a single administration.
  • injectable formulation refers to an aseptic preparation acting in such a way that a commonly injected liquid-phase drug is directly introduced into the body via the skin, muscle, vein, etc., and preferably may be injected intravenously, subcutaneously, intramuscularly, etc, and most preferably, intramuscularly injected.
  • injectable formulations may be divided into an aqueous injection, a non-aqueous injection, an suspension injection, a solid injection, a freeze-dried injection, etc. according to their ' preparation methods.
  • An aqueous injection is prepared by completely dissolving a predetermined amount of a drug in an appropriate amount of injectable water.
  • a solution adjuvant may be added when a poorly soluble drug is dissolved.
  • Non-aqueous injections, suspension injections, solid injections and freeze-dried injections may also be prepared based on the preparation of aqueous injections.
  • suspension injections a process may be further included, by which an insoluble medicament is sufficiently pulverized and processed into micropowder of less than 150 ⁇ m.
  • a suspending agent sodium carboxymethylcellulose (1-3%), HCO-60 (1-3%), and the like may be generally used for the preparation of aqueous suspension injections, and aluminum monostearate (2%) for the preparation of oil-based suspension injections.
  • Solid injections and freeze-dried injections are suspended and/or dissolved immediately before being used.
  • the bisphosphonate- containing polymeric microparticles are suspended in injectable water along with sodium chloride, sodium carboxymethylcellulose, a surfactant, etc. to provide an suspension injection.
  • the bisphosphonate-containing polymeric microparticles of the present invention may be suspended in a solution containing sodium carboxymethylcellulose, a polyoxyethylene sorbitan fatty acid ester and mannitol to provide an suspension injection.
  • the injectable formulation may be supplemented with a solution adjuvant, a stabilizer, a buffering agent for pH control, a tonicity controller, a suspending agent, an emulsifier, and the like.
  • a buffering agent such as butyric acid, tartaric acid or sodium hydroxide
  • a buffering agent such as butyric acid, tartaric acid or sodium hydroxide
  • an anti-oxidant such as ascorbic acid, sodium hydrogen . sulfite, sodium pyrosulfite, BHA and tocopherol
  • a stabilizer e.g., a chelating agent such as EDTA
  • a preservative exemplified by phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, methyl paraoxy-benzoate, propyl paraoxy-benzoate and benzylalcohol, may be added.
  • the bisphosphonate contained in the present injectable formulation as an active drug has been used for preventing or treating osteoporosis, Paget's disease, hypercalcemia induced by malignant tumors, metastatic bone diseases induced by malignant tumors, multiple myeloma, bone loss caused by immobilization, bone loss after artificial joint replacement, childhood osteoporosis of unknown cause, rheumatoid arthritis, hyperparathyroidism, periodontal disease, and the like.
  • the present injectable formulation comprising bisphosphonate-containing polymeric microparticles may be useful for treating or preventing the aforementioned diseases .
  • osteoporosis is characterized by the breakdown of the balance between bone formation and bone resorption, leading to a reduction in bone mass and cortical thickness.
  • osteoporosis there is also a reduction in the number and size of trabeculae of cancellous bone, leading to decreased trabecular connectivity and bone mass and, finally, skeletal fragility.
  • the diagnosis of osteoporosis is achieved by trabecualr area measurement, bone mineral density measurement and biochemical assays. Several methods are currently available for measuring bone mineral density.
  • Dual energy X-ray absorptiometry (DXA or DEXA) , quantitative computed tomography (QCT) , quantitative ultrasonometry (QUS) , etc. are commonly used.
  • Test results are represented by bone mineral density (BMD) , and a BMD result is compared with BMD results from normal young adults (T score) or individuals of the same age (Z .score) .
  • BMD bone mineral density
  • T score normal young adults
  • Z .score normal young adults
  • WHO World Health Organization
  • a T score -1.0 to -2.5 standard deviations (SD) below that of normal young adults is defined as osteopenia
  • a T score of more than -2.5 SD below that of normal young adults is defined as osteoporosis.
  • biochemical assays are used for measuring the function of osteoclasts and osteoblasts in order to evaluate current states of bone formation and bone loss. For example, concentrations of osteocalcin, a biomarker of bone formation, and the activity of alkaline phosphatase as another bone formation biomarker are measured. Alternatively, levels of a bone resorption biomarker, N-telopeptide (NTx) , are measured.
  • NTx N-telopeptide
  • the administration of the present injectable formulation resulted in a great reduction in local toxicity compared to direct injection of a bisphosphonate.
  • the bisphosphonate, as an active drug of the present injectable formulation is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient for the treatment or prevention of diseases, which is commensurate with a reasonable benefit/risk ratio applicable for medical treatment or prevention.
  • An effective dosage of the present injectable formulation may be determined depending on the severity of the illness, the activity of a drug, the patient's age, body weight, health state and gender, the sensitivity of a patient to a drug, administration time, administration routes and an excretion rate of the specific extract used, duration of treatment, drugs used in combination or simultaneously with the specific extract used, and other factors known in medical fields.
  • 0.5-2.5 mg/kg (0.02-0.1 mg/kg based on alendronate) may be administered at a frequency from once/4 weeks to once/48 weeks. More preferably, 0.625-1.875 mg/kg (0.025-0.075 mg/kg based on alendronate) may be administered at a frequency of once/4 weeks to once/24 weeks.
  • the present injectable formulation must be administered in an amount capable of obtaining a maximal effect using a minimal amount, and such an amount may be readily determined by those skilled in the art.
  • a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limits of the present invention.
  • Example 1 An internal aqueous phase was obtained by dispersing 100 mg of sodium alendronate in 500 ⁇ l of an aqueous solution in which sodium hyaluronate (0. 75% (w/v) based on water) and polyoxyethylene sorbitan monooleate (Tween 80, 20% (w/v) based on water) were dissolved.
  • a polymer solution in an organic phase was obtained by dissolving 10 parts by weight of poly(lactic acid) (molecular weight 100,000) and 5 parts by weight of sorbitan trioleate in 100 parts by weight of a solvent mixture, which was a 9:1 mixture of dichloromethane and acetone.
  • An external continuous phase was obtained by dissolving 1 part by weight of ethyl acetate in 99 parts by weight of an aqueous solution (made by dissolving 0.5 part by weight of polyvinylalcohol in 100 parts by weight of distilled water)
  • the internal aqueous phase and the organic phase were stirred vigorously to prepare a W/O type emulsion.
  • the prepared W/O type primary emulsion was slowly added thereto in a volume ratio of the primary emulsion to the external continuous phase of 1:200, and dispersed by a homogenizer for 5 min, thus providing a W/O/W type multiple emulsion.
  • the organic solvent was removed by filtration, and the remaining product was dried in a vacuum oven for 24 hrs to obtain microparticles .
  • Preparation Example 1-1 Microparticles were prepared according to the same method as in Preparation Example 1 except that a solvent mixture of dichloromethane and acetone at a ratio of 8:2 was used as an organic solvent forming an organic phase.
  • Preparation Example 1-2 Microparticles were prepared according to the same method as in Preparation Example 1 except that a solvent mixture of dichloromethane and acetone at a ratio of 7:3 was used as an organic solvent forming an organic phase.
  • Preparation Example 1-3 Microparticles were prepared according to the same method as in Preparation Example 1 except that a solvent mixture of dichloromethane and acetone at a ratio of 6:4 was used as an organic solvent forming an organic phase.
  • Preparation Example 2 An internal aqueous phase was obtained by dispersing 100 mg of sodium alendronate in 500 ⁇ l of an aqueous solution in which sodium hyaluronate (0. 75% (w/v) based on water) and polyoxyethylene sorbitan monooleate (Tween 80, 20% (w/v) based on water) were dissolved.
  • An external continuous phase was obtained by dissolving 1 part by weight of ethyl acetate in 99 parts by weight of an aqueous solution (made by dissolving 0.5 part by weight of polyvinylalcohol in 100 parts by weight of distilled water) .
  • the internal aqueous phase and the organic phase were stirred vigorously to prepare a W/O type emulsion. While the external continuous phase was homogeneously dispersed using a homogenizer at 5,000 rpm, the prepared W/O type primary emulsion was slowly added thereto in a volume ratio of the primary emulsion to the external continuous phase of 1:200 and dispersed using a homogenizer for 5 min, thus providing a W/O/W type multiple emulsion. After mild stirring for 30 min, the organic solvent was removed by filtration, and the remaining product was dried in a vacuum oven for 24 hrs to obtain microparticles .
  • poly (ethylene glycol) molecular weight 3,350)
  • poly (ethylene glycol) molecular weight 3,350)
  • Preparation Example 3 An internal aqueous phase was obtained by dispersing 100 mg of sodium alendronate in 500 ⁇ l of an aqueous solution in which lactic acid (1.5% (w/v) based on water), chitosan (0.75% based on water) and polyoxyethylene sorbitan monooleate (10% based on water) were dissolved.
  • An external continuous phase was obtained by dissolving 1 part by weight of ethyl acetate in 99 parts by weight of an aqueous solution (made by dissolving 0.5 part by weight of polyvinylalcohol in 100 parts by weight of distilled water) .
  • the internal aqueous phase and the organic phase were stirred vigorously to prepare a W/O type emulsion.
  • the prepared W/O type primary emulsion was slowly added thereto in a volume ratio of the primary emulsion to the external continuous phase of 1:200 and dispersed using a homogenizer for 5 min, thus providing a W/O/W type multiple emulsion.
  • the organic solvent was removed by filtration, and the remaining product was dried in a vacuum oven for 24 hrs to obtain microparticles .
  • Preparation Example 3-1 Microparticles were prepared according to the same method as in Preparation Example 3 except that a solvent mixture of dichloromethane and acetone at a ratio of 6:4 was used as an organic solvent forming an organic phase.
  • Preparation Example 4 An internal aqueous phase was obtained by dispersing 200 mg of sodium alendronate in 500 ⁇ l of an aqueous solution in which sodium hyaluronate (0. 75% (w/v) based on water) and polyoxyethylene sorbitan monooleate (Tween 80, 20% (w/v) based on water) were dissolved.
  • aqueous solution was used, which was prepared by dissolving 0.5 part by weight of polyvinylalcohol in 100 parts by weight of distilled water.
  • the internal aqueous phase and the organic phase (volume ratio of 1: 10) were stirred vigorously to prepare a W/O type emulsion.
  • the prepared W/O type primary emulsion was slowly added thereto in a volume ratio of the primary emulsion to the external continuous phase of 1:200 and dispersed using a homogenizer for 4 min, thus providing a W/O/W type multiple emulsion.
  • the organic solvent was removed by filtration, and the remaining product was dried in a vacuum oven for 24 hrs to obtain microparticles.
  • Comparative Preparation Example 3 Microparticles were prepared according to the same method as in Preparation Example 3 except that dichloromethane was used alone as an organic solvent forming an organic phase. Comparative Preparation Example 4 An internal aqueous phase was obtained by dispersing
  • the prepared W/O type primary emulsion was slowly added thereto in a volume ratio of the primary emulsion to the external continuous phase of 1:200 and dispersed using a homogenizer for 5 min to provide a W/O/W type multiple emulsion.
  • the temperature of the external continuous phase was maintained at 25°C.
  • the organic solvent was removed by filtration, and the remaining product was dried in a vacuum oven for 24 hrs to obtain microparticles.
  • Test Example 1 Evaluation of drug loading efficiency of polymeric microparticles 30 mg of prepared polymeric microparticles were weighed accurately, placed in a test tube with a cap, completely dissolved in 5 ml of chloroform, supplemented with 20 ml of distilled water, and vigorously stirred for 30 min. The solution was then centrifuged for 5 min at 5000 rpm. A predetermined amount of the supernatant was subjected to HPLC analysis to measure the concentration of a drug and determine the amount of the drug loaded into microparticles. Drug loading amount and efficiency of the polymeric microparticles were calculated according to the following equations . The test results are given in Equation 1, below.
  • Drug loading efficiency (%) (drug loading amount/theoretical drug loading amount) x 100
  • theoretical drug loading amount (%) refers to the total amount of the drug used in preparing microparticles/ (the total amount of the drug used in preparing microparticles + the total amount of other materials used in preparing microparticles) , and means drug loading amount obtained based on the assumption that the drug used in preparing microparticles is completely (100%) encapsulated without any loss to an external continuous phase during microparticle preparation.
  • the "other materials used in preparing microparticles” refers to the sum of the total amount of materials constituting an organic phase, including a polyester polymer and a hydrophobic surfactant, and materials constituting an internal aqueous phase, including a water-soluble polymer and a hydrophilic surfactant.
  • microparticulates had a drug loading efficiency of about 20% like those prepared in Comparative Preparation Examples 1 and 2.
  • microparticles were prepared using a primary organic solvent in combination with a secondary organic solvent to induce microcoagulation of sodium hyaluronate, they were found to have remarkably increased drug loading efficiency.
  • tests using microparticles of Preparation Examples 1-1,1-2 and 1- 3 revealed that the amount of a drug loaded into the microparticles increases with increasing content of acetone in the solvent mixture.
  • FIGS, la and lb images of the cross section of the prepared polymeric microparticles, which were taken by a differential scanning electron microscope, are shown in FIGS, la and lb.
  • FIG. la is a differential scanning electron microscopic image of a cross section of the polymeric microparticles prepared in Comparative Preparation Example 1.
  • Fig. lb is a. differential scanning electron microscopic image of a cross section of the polymeric microparticles prepared in Preparation Example 1-3. As shown in the image, the inside of discontinuous internal pores of the polymeric microparticles was filled with microcoagulated particles of sodium hyaluronate.
  • Microparticles prepared in Preparation Examples 2, 2- 1 and 2-2 and Comparative Preparation Example 2 were assessed for drug loading amount and drug loading efficiency according to the increase of poly (ethylene glycol) content, when poly (ethylene glycol) as a drug release modifier was mixed with a polyester polymer. As shown in Table 1, the addition of poly (ethylene glycol) having features of water solubility and crystallinity causes free influx and outflow of an external water phase with respect to microparticles during microparticle preparation, resulting in a decrease in drug loading amount and drug loading efficiency.
  • the microparticles thus prepared had remarkably increased drug loading amount and loading efficiency.
  • Test Example 2 In vitro drug release test
  • 100 mg of the prepared polymeric microparticles were accurately weighed, and placed in a membrane tube (molecular weight cut-off: 3,500) which was sealed at both ends, put in a test tube containing 30 ml of phosphate buffer at pH 7.4, capped, and placed in a shaking water bath at 37°C and a 60 times/min speed to allow sustained release of a drug for a period of 28 days or more. 15 ml of the buffer was taken and subjected to HPLC analysis to measure the concentration of the drug released into the buffer.
  • FIGS. 2 and 3 show drug release rates of microparticles according to mixing ratios of dichloromethane and acetone in an organic solvent containing poly (lactic acid) (FIG. 2) or poly (lactic acid- co-glycolic acid) (FIG. 3) , respectively, and a hydrophobic surfactant.
  • FIG. 6 shows changes in the initial release rate of a drug according to changes in internal aqueous phase having viscosity. It is confirmed that as mentioned above, gelation of gelatin itself (Comparative Preparation Example 4) has almost no inhibitory effect on initial burst release of the drug compared to the induction of microcoagulation of sodium hyaluronate (Preparation Example 2) or chitosan (Preparation Example 3) .
  • Test Example 3 In vivo drug release test A 3% sodium carboxymethylcellulose solution containing sodium chloride and Tween 20 in injectable
  • - distilled water was used as an injection vehicle.
  • sodium chloride was added to be isotonic, and microspheres were effectively suspended and maintained at a homogeneous suspension during injection.
  • sodium carboxymethylcellulose was used as a thickener to maintain a viscosity of 200 to 400 cps.
  • An injection solution was used after being sterilized.
  • the following components were filled into a 1.0-ml ampoule according to a conventional method for an injection and sterilized.
  • the resulting injection composition was mixed with 30.0 mg of a polymeric microparticle-alendronate composition prepared in an aseptic state, and then administered.
  • alendronate- encapsulating polymeric microparticles 800 mg/8 ml (30 mg/8 ml based on alendronate) was intramuscularly injected into New Zealand White rabbits .
  • Blood samples were collected on Days 1, 2, 5, 7, 13, 20 and 28.
  • Blood samples of 4 ml were taken from ear veins of rabbits at given time points, and centrifuged.
  • the supernatants, which were blood plasma, were subjected to HPLC under the following conditions to measure sodium alendronate level in plasma.
  • FIG. 7 shows sodium alendronate level in plasma according to time after polymeric microparticles encapsulating sodium alendronate were intramuscularly injected into rabbits. Sodium alendronate level in plasma gradually increased until Day 7 post-administration and decreased thereafter. Also, sodium alendronate was found in detectable levels in the blood even at Day 28, indicating that the drug was continuously released from the polymeric microparticles four weeks after administration.
  • Test Example 4 Measurement of bone mineral density and trabecular area Postnatal 15-week-old matured female Sprague-Dawley rats were adjusted to a general feedstuff (Purina chow diet) for one week, and weighed to provide rats of similar weight to each group. The rats were ovariectomized to induce osteoporosis. After experimental animals were stabilized for one week, they were intramuscularly injected twice (once per four week) with 3.75 mg/kg (0.15 mg/kg based on alendronate) of sodium alendronate-encapsulating polymeric microparticles, which were suspended in a sodium carboxymethylcellulose solution containing sodium chloride and Tween 20.
  • tibiae were isolated from both legs of the female rats 8 weeks after drug administration. Tibia tissue specimens were fixed in 4% formalin at pH 7.4 and assessed for bone mineral density and trabecular bone area using a bone density analyzer and a quantitative image . analysis system. Histological observation of tibiae and trabecular area measurement: the fixed tibia tissues were decalcificated with 10% nitric acid for 6 hrs, and dehydrated through 10 steps from 80% to 100%.
  • the tibia tissues were embedded using xylene and paraffin twice for each case. Paraffin blocks were sectioned into a thickness of 4 ⁇ m using a microtome for bone slice-cut. The obtained 4- ⁇ m sections of the tibias were stained with hematoxylin-eosin. Thereafter, the proximal part of the tibia was observed under an inverted tissue culture microscope (Olympus, Japan) , and a photograph was taken while focusing on a central part about 1 mm below a growth plate.
  • an inverted tissue culture microscope Olympus, Japan
  • a trabecular area was measured with 4Ox magnification in a predetermined standard area (about 3.3 mm 2 ) about 1 mm below a growth plate in the middle region of the tibia, and a percentage for a standard area was calculated.
  • Bone mineral density measurement of the tibia bone mineral density was measured using DEXA in a predetermined area corresponding to about 1/4 of the entire tibia including the region below the growth plate of the tibia.
  • Statistic analysis and evaluation data were expressed as mean ⁇ SD. A statistically significant difference between groups was investigated using the SAS program. When a significant difference was found after an
  • Test Example 4-1 Bone mineral density and trabecular bone area were measured according to the same method as in Test Example 4 except that rats were intramuscularly injected with 7.5 mg/kg (0.3 mg/kg based on alendronate) of polymeric microparticles .
  • Test Example 4-2 Bone mineral density and trabecular bone area were measured according to the same method as in Test Example 4 except that rats were intramuscularly injected with 22.5 mg/kg (0.9 mg/kg based on alendronate) of polymeric microparticles .
  • Comparative Test Example 1-1 Bone mineral density and trabecular bone area were measured according to the same method as in Test Example 4 except that rats were orally injected every day with 1.0 mg/kg of alendronate dissolved in sterile saline.
  • Bone mineral density (BMD) and trabecular bone area were measured according to the same method as in Test Example 4 except that female rats were not ovariectomized but received an surgical operation in the gastrointestinal tract to cause the same stress, and were not administered with the drug.
  • FIG. 8 is a graph showing the percentage of trabecular bone area measured in a predetermined area of the tibia. Compared to rats of Comparative Test Example 2, rats of Comparative Test Example 3 showed a great bone loss in which the trabecular bone was reduced by about 50% in the tibia.
  • FIGS. 9a to 9g are photographs of stained tibia tissue specimens, which were taken using an image analyzer.
  • FIGS. 9a, 9b, 9c and 9d show the results of Comparative Test Example 2, Comparative Test Example 3, Comparative Test Example 1 and Comparative Test Example 1-1, respectively.
  • FIGS. 9e, 9f and 9g show the results of Test Example 4, Test Example 4-1 and Test Example 4-2, respectively.
  • FIG. 10 shows bone mineral density of the tibia, which was measured using a bone density analyzer (DEXA) .
  • Test Example 5 Local irritation test on administration sites Postnatal 12 to 16-week-old New Zealand rabbits (male) were adjusted to a general feedstuff, and weighed to provide rabbits of similar weight to each group. Both thighs of rabbits were shaved before drug administration. 0.2 mg of sodium alendronate were dissolved in 0.1 ml of distilled water and intramuscularly injected into both thighs of the rabbits . Experimental animals were bred under standard conditions (23 ⁇ 3°C, 50+10% relative humidity, 10-15 times/hr ventilation, 150-300 Lux illumination) with sufficient water and food supply.
  • the rabbits were dissected, and administration sites were excised, observed with the naked eye, fixed in 10% formalin (pH 7.4), subjected to a general tissue processing procedure, stained with hematoxylin-eosin, and subjected to histopathological studies. Tissues surrounding administration sites were observed under a microscope, and their images were captured. Estimation of local irritation was processed ' and analyzed according to the following "criteria.
  • Test Example 5-1 A local irritation test was performed according to the same method as in Test Example 5 except that 0.5 mg of sodium alendronate was dissolved in 0.1 ml of sterile distilled water and intramuscularly injected.
  • Test Example 5-2 A local irritation test was performed according to the same method as in Test Example 5 except that 1.0 mg of sodium alendronate was dissolved in 0.1 ml of sterile distilled water and intramuscularly injected.
  • Comparative Test Example 4 A local irritation test was performed according to the same method as in Test Example 5 except that 0.1 ml of physiological saline was intramuscularly injected.
  • Comparative Test Example 5 A local irritation test was performed according to the same ⁇ method as in Test Example 5 except that 0.1 ml of 0.425% acetic acid was intramuscularly injected.
  • Comparative Test Example 5-1 A local irritation test was performed according to the same method as in Test Example 5 except that 0.1 ml of 1.7% acetic acid was intramuscularly injected. The results of observation of administration sites with the naked eye and under a microscope 2 days and 7 days after intramuscular injection of the drug are given respectively in Tables 2 and 3, below.
  • Irritation responses no change (-) ; mild irritation (+, 5 mm in diameter) ; moderate irritation (++, 6-10 mm in diameter) ; severe irritation (+++, 11 mm or more in diameter) Numerals indicate the number of experimental animals
  • Irritation responses no change (-) ; mild irritation (+, 5 mm in diameter) ; moderate irritation (++, 6-10 mm in diameter) ; severe irritation (+++, 11 mm or more in diameter) Numerals indicate the number of experimental animals As a result of the local irritation tests, a mild irritation response was found in Test Example 5, which was almost the same as in Comparative Test Example 4 and thus considered a physical stimulus due to injection. In Test Example 5-1, a moderate irritation response, corresponding to an irritation of about Grade II, was found upon observation with the naked eye and pathological studies . In Test Example 5-2, a severe irritation response, corresponding to an irritation of about Grade IV, was found, and this irritation was the same as that in Comparative Test Example 5-1. On the whole, on Day 7, lesion sites were reduced and fibrosis was pathologically increased compared to on Day 2, indicating that lesion sites caused by stimulation of the administered materials were recovered.
  • Test Example 6 A local irritation test was performed according to the same method as in Test Example 5 except that 40 mg (3 mg based on alendronate) of sodium alendronate- encapsulating polymeric microparticles (particle size: below 106 ⁇ m) , which were prepared using poly (lactic acid- co-glycolic acid) (PLGA) (RG503H, molecular weight 36,000), were suspended in 1 ml of a mannitol solution, containing sodium carboxymethylcellulose and Tween 80, and intramuscularly injected into both thighs of rabbits, and that rabbits were dissected 9 days and 30 days after drug administration.
  • PLGA poly (lactic acid- co-glycolic acid)
  • Test Example 6-1 A local irritation test was performed according to the same method as in Test Example 5 except that 75 mg (3 mg based on alendronate) of sodium alendronate- encapsulating polymeric microparticles (no limitation in particle size), which were prepared using poly (lactic acid- co-glycolic acid) (PLGA) (RG504H, molecular weight 54,000), were suspended in 1 ml of a sodium carboxymethylcellulose solution, containing sodium chloride and Tween 20, and intramuscularly injected into both thighs of rabbits, and that rabbits were dissected 9 days and 30 days after drug administration.
  • PLGA poly (lactic acid- co-glycolic acid)
  • Comparative Test Example 6 A local irritation test was performed according to the same method as in Test Example 5 except that 75 mg of polymeric microparticles (not encapsulating a drug, particle size: below 106 ⁇ m) , which were prepared using poly (lactic acid-co-glycolic acid) (PLGA) (RG504H, molecular weight 54,000), were suspended in 1 ml of a t sodium carboxymethylcellulose solution, containing sodium chloride and Tween 20, and intramuscularly injected into both thighs of rabbits, and that rabbits were dissected 9 days and 30 days after drug administration.
  • PLGA poly (lactic acid-co-glycolic acid)
  • Comparative Test Example 7 A local irritation test was performed according to the same method as in Test Example 5 except that 1 ml of 0.425% acetic acid was intramuscularly injected and rabbits were dissected 9 days and 30 days after drug administration.
  • Comparative Test Example 7-1 A local irritation test was performed according to the same method as in Test Example 5 except that 1 ml of 1.7% acetic acid was intramuscularly injected and rabbits were dissected 9 days and 30 days after drug administration. The results of observation of administration sites with the naked eye and under a microscope 9 days and 30 days after intramuscular injection of the drug are given respectively in Tables 4 and 5, below.
  • Irritation responses no change (-) ; mild irritation (+, 5 mm in diameter) ; moderate irritation (++, 6-10 mm in diameter) ; severe irritation (+++, 11 mm or more in diameter) Numerals indicate the number of experimental animals
  • Irritation responses no change (-) ; mild irritation (+, 5 mm in diameter) ; moderate irritation (++, 6-10 mm in diameter) ; severe irritation (+++, 11 mm or more in diameter)
  • Numerals indicate the number of experimental animals Compared to Test Example 5-2, in which 1.0 mg of sodium alendronate, not encapsulated, was intramuscularly injected, Test Example 6-1 resulted in a slight reduction in local irritation, although microparticles contained sodium alendronate in a much larger amount. However, these microparticles induced moderate irritation, corresponding to an irritation of about Grade III, on administration sites, and thus, were considered to be difficult to use for injections .
  • the sodium carboxymethylcellulose solution containing sodium chloride and Tween 20 which was used as a suspension solvent in Test Example 6-1 and Comparative Test Example 6, may be inconvenient for drug administration and causes pain due to its high viscosity, as well as inducing local irritation on administration sites.
  • Test Example 6 using a different type of biodegradable polymers microparticles having a particular particle size and a mannitol solution containing sodium carboxymethylcellulose and Tween 80 as a suspension solvent, local irritation on administration sites was remarkably reduced although the microparticles contained sodium alendronate in the same amount as those of Test Example 6-1.
  • Test Example 6 in which 40 mg (3 mg based on alendronate) of sodium alendronate-encapsulating polymeric microparticles (particle s'ize: below 106 ⁇ m) , which were prepared using poly (lactic acid-co-glycolic acid) (PLGA) (RG503H, molecular weight 36,000), were suspended in 1 ml of a mannitol solution, containing sodium carboxymethylcellulose and Tween 80, and intramuscularly injected into both thighs of rabbits, resulted in a great reduction in local irritation of administration sites although the microparticles contained sodium alendronate in a much larger amount than those of Test Example 5-2.
  • PLGA poly (lactic acid-co-glycolic acid)
  • the injectable formulation comprising bisphosphonate- containing polymeric microparticles according to the present invention has an effect of sustaining drug release in vivo for a period of 4 weeks or more using only a single administration, thereby reducing administration frequency and total dosage compared to conventional oral formulations administered every day. Also, the present injectable formulation causes remarkably reduced local irritation compared to a directly injected non-encapsulated bisphosphonate. Therefore, the present injectable formulation is more useful in the treatment or prevention of diseases against which bisphosphonate drugs have therapeutic or preventive effects than conventional oral formulations and direct injections of bisphosphonates.

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Abstract

L'invention porte sur une formulation pouvant être injectée à libération soutenue pour le traitement ou la prévention de maladies osseuses, comprenant des micro-particules polymériques qui contiennent du disphosphonate préparées par dissolution et dispersion d'un disphosphonate dans une solution aqueuse contenant un polymère hydrosoluble et un agent de surface hydrophile afin d'obtenir une solution polymérique ; par ajout d'un solvant organique secondaire dans un solvant organique primaire contenant un polymère biodégradable et un agent de surface hydrophobe afin d'obtenir une émulsion primaire (de type inverse) ; et par dispersion de l'émulsion primaire au cours d'une phase continue externe. Cette solution pouvant être injectée possède un effet de libération de médicaments soutenue in vivo pendant au moins quatre semaines au moyen d'une seule administration, ce qui permet de réduire le nombre d'administrations et le dosage total en comparaison avec des formulations orales courantes administrées chaque jour, et de réduire aussi nettement l'irritation locale en comparaison avec du disphosphonate non-encapsulé directement injecté. Par conséquent, cette formulation pouvant être injectée à libération soutenue est plus utile dans le traitement ou la prévention de maladies contre lesquelles des médicaments de disphosphonate ont des effets thérapeutiques préventifs, que les formulations orales courantes et les injections directes de disphosphonate.
PCT/KR2005/001306 2004-05-04 2005-05-04 Formulation pouvant etre injectee a liberation soutenue pour le traitement ou la prevention de maladies osseuses comprenant des micro-particules polymeriques qui contiennent du disphosphonate WO2005105058A1 (fr)

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