WO2018043942A1 - Oral dosage form of parathyroid hormone comprising gastrointestinal absorption enhancer - Google Patents
Oral dosage form of parathyroid hormone comprising gastrointestinal absorption enhancer Download PDFInfo
- Publication number
- WO2018043942A1 WO2018043942A1 PCT/KR2017/008604 KR2017008604W WO2018043942A1 WO 2018043942 A1 WO2018043942 A1 WO 2018043942A1 KR 2017008604 W KR2017008604 W KR 2017008604W WO 2018043942 A1 WO2018043942 A1 WO 2018043942A1
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- WIPO (PCT)
- Prior art keywords
- pth
- parathyroid hormone
- lysdoca
- oral administration
- rhpth
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- 239000003797 essential amino acid Substances 0.000 description 1
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- JQOAQUXIUNVRQW-UHFFFAOYSA-N hexane Chemical compound CCCCCC.CCCCCC JQOAQUXIUNVRQW-UHFFFAOYSA-N 0.000 description 1
- 102000058004 human PTH Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003870 intestinal permeability Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- JZJOGMHGKPNPTO-VIFPVBQESA-N methyl (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoate Chemical compound COC(=O)[C@@H](N)CCCCNC(=O)OC(C)(C)C JZJOGMHGKPNPTO-VIFPVBQESA-N 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001009 osteoporotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 239000003961 penetration enhancing agent Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229940100467 polyvinyl acetate phthalate Drugs 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
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- 229940043267 rhodamine b Drugs 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
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- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
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- A61K9/0012—Galenical forms characterised by the site of application
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/554—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
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- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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Definitions
- the intestinal membrane permeability of PTH is formed by mixing a parathyroid hormone (PTH) or a fragment thereof, terry paraparatide (PTH (1-34)) with a deoxycholic acid derivative, which is an intestinal membrane permeation enhancer, to form a complex.
- PTH parathyroid hormone
- PTH (1-34) terry paraparatide
- the present invention relates to a formulation composition for oral administration of parathyroid hormone containing a gastrointestinal absorption enhancer which enhances the gastrointestinal absorption of the drug by improving the gastrointestinal absorption of the drug by preparing the complex in the form of a preparation coated with an enteric material.
- Parathyroid hormone is a polypeptide consisting of 84 amino acids secreted by the parathyroid gland and is one of the major hormones that controls the concentration of calcium ions in the blood. It functions as an assimilation (bone formation) and catabolism in bone, which depends on the duration and pattern of exposure to PTH. Assimilation is facilitated by intermittent PTH exposure and results in differentiation and proliferation of osteoblasts or reduction of apoptosis. Catabolism is promoted by continuous PTH exposure and is associated with increased expression of receptor activator of nuclear factor-kB ligand (RANKL) and decreased expression of osteoprotegerin.
- RTKL nuclear factor-kB ligand
- PTH terry paratide
- type 1 PTH receptors which are highly expressed in bone and kidneys. Binding of PTH (1-34) to the type 1 PTH receptor activates a series of signaling systems, including the G protein-dependent cAMP / protein kinase A pathway, Calcium concentration is controlled.
- Teriparatide is a recombinant PTH (1-34) (rhPTH (1-34); recombinant human PTH (1-34)), a clinically postmenopausal female osteoporosis and gonadotropin male.
- teriparatide stimulates the formation of new bone by activating osteoblasts more than osteoclasts and reduces the risk of fractures in osteoporosis
- Many other osteoporosis therapies alter the nature of catabolism. It is a prophylactic treatment that only inhibits the activity of osteoclasts, which include bisphosphonates, estrogen receptor modulators, and calcitonin.
- Paratide is used to treat osteoporosis in patients at risk of fracture and who do not respond to other bone resorption inhibitors. Can be.
- Parathyroid hormone is a polypeptide consisting of 84 amino acids secreted by the parathyroid gland and is one of the major hormones that controls the concentration of calcium ions in the blood. It functions as an assimilation (bone formation) and catabolism in bone, which depends on the duration and pattern of exposure to PTH. Assimilation is facilitated by intermittent PTH exposure and results in differentiation and proliferation of osteoblasts or reduction of apoptosis. Catabolism is promoted by continuous PTH exposure and is associated with increased expression of receptor activator of nuclear factor-kB ligand (RANKL) and decreased expression of osteoprotegerin.
- RTKL nuclear factor-kB ligand
- PTH terry paratide
- type 1 PTH receptors which are highly expressed in bone and kidneys. Binding of PTH (1-34) to the type 1 PTH receptor activates a series of signaling systems, including the G protein-dependent cAMP / protein kinase A pathway, Calcium concentration is controlled.
- Teriparatide is a recombinant PTH (1-34) (rhPTH (1-34); recombinant human PTH (1-34)), a clinically postmenopausal female osteoporosis and gonadotropin male.
- teriparatide stimulates the formation of new bone by activating osteoblasts more than osteoclasts and reduces the risk of fractures in osteoporosis
- Many other osteoporosis therapies alter the nature of catabolism. It is a prophylactic treatment that only inhibits the activity of osteoclasts, which include bisphosphonates, estrogen receptor modulators, and calcitonin.
- Paratide is used to treat osteoporosis in patients at risk of fracture and who do not respond to other bone resorption inhibitors. Can be.
- the present invention provides a formulation composition for oral administration of parathyroid hormone, characterized in that the nanocomposite containing PTH (parathyroid hormone) and bile acid derivatives is coated with an enteric material.
- the PTH may be any one of PTH (1-28), PTH (1-31), PTH (1-34), PTH (1-38) or PTH (1-41), and the PTH ( 1-34) is a polypeptide consisting of amino acid sequences 1 to 34 of the human parathyroid hormone, preferably prepared by the recombinant method rhPTH (1-34) or chemically synthesized hPTH (1-34). .
- the bile acid derivative is preferably a deoxycholic acid derivative, and lysine is most preferably chemically bonded to deoxycholic acid in the deoxincolic acid derivative.
- the PTH and the bile acid derivative are preferably combined at a molar ratio of 1: 1 to 1:10.
- the nanocomposite in the present invention is preferably a particle size of 10 to 50nm.
- an "entertaining substance” is not specifically limited, For example, Eudragit (Eudragit; methacrylic acid-ethyl acrylate copolymer), hydroxypropyl methylcellulose phthalate, acetyl succinate hydroxypropyl methylcellulose And at least one polymer component selected from cellulose acetate phthalate, polyvinyl acetate phthalate, carboxymethylethyl cellulose, shellac and the like.
- one or two or more selected from the group consisting of shellac, eudragit (Eudragit: methacrylic acid-ethyl acrylate copolymer), hydroxypropylmethylcellulose phthalate, acetyl amber hydroxypropylmethylcellulose, and cellulose acetate phthalate are mixed.
- shellac eudragit
- eudragit methacrylic acid-ethyl acrylate copolymer
- hydroxypropylmethylcellulose phthalate hydroxypropylmethylcellulose phthalate
- acetyl amber hydroxypropylmethylcellulose cellulose acetate phthalate
- the present invention 1) forming a nanocomposite (PTH / deoxycholic acid derivative) by the ionic bond of PTH and deoxycholic acid derivatives; 2) preparing a suspension by dissolving the nanocomposite in water and then dispersing it in a solution in which the enteric material is dissolved; 3) moving the suspension to the inner inlet of the dual axis ultrasonic spray nozzle with a syringe pump; 4) moving the enteric material solution to the outer inlet of the double side ultrasonic spray nozzle; 5) forming microdroplets in which the drug solution inside the nozzle is surrounded by the external enteric material solution by vibration spraying of the ultrasonic jet nozzle; 6) the microdroplets are injected into the liquid paraffin and stirred to harden the shell of the microdroplets; and provides a method for preparing a formulation composition for oral administration of parathyroid hormone, characterized in that it comprises a.
- the PTH and the deoxycholic acid derivatives are preferably mixed at a molar ratio of 1: 1 to 1:10.
- the PTH and the deoxycholic acid derivatives are recovered through a vacuum filter and washed with hexane and dried. You can add more.
- the present invention is to mix the deoxycholic acid derivative having the effect of promoting the intestinal membrane permeability to the PTH and to coat the enteric material to enhance the permeability of the intestinal membrane to increase the absorption of the gastrointestinal tract, a problem that is conventionally used as a subcutaneous injection formulation
- By changing the route of administration to oral administration has the effect of enhancing the ease of taking the patient.
- rhPTH 10 ⁇ M rhPTH (1-34) in water or in an aqueous solution of 20% (v / v) 2,2,2-trifluoroethanol (TFE; 2,2,2-trifluoroethanol) according to an embodiment of the present invention.
- TFE 2,2,2-trifluoroethanol
- Dichroism spectroscopy of LysDOCA (deoxycholic acid-lysine), and PTH / LysDOCA (1:10) nanocomposites are shown.
- Figure 2 shows the rhPTH (1-34) particle size distribution and transmission electron microscope (TEM) image according to an embodiment of the present invention.
- FIG. 3 shows particle size distribution and transmission electron microscopy (TEM) images of PTH / LysDOCA (1:10) nanocomposites.
- Figure 4 shows the intracellular cAMP content after incubating MC3T3-E1 cells with rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites according to an embodiment of the present invention.
- Figure 5 is a subcutaneous injection of rhPTH (1-34) (0.02 mg / kg) and rhPTH (1-34) in the jejumam administration of the rat (Rat) according to an embodiment of the present invention (0.1 mg / kg) Or intravenous plasma concentration-time profile of rhPTH (1-34) following in-plant administration of PTH / LysDOCA (1:10) nanocomposites (0.1 mg / kg as rhPTH (1-34)).
- Figure 6 is a scanning electron microscope (SEM) (A) image of the enteric particles containing the PTH / LysDOCA (1:10) nanocomposite according to an embodiment of the present invention and confocal laser scanning microscopy (CLSM) (B) shows an image.
- SEM scanning electron microscope
- Figure 7 shows the in vitro cumulative drug release rate of the rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites from the enteric microparticles in dissolution test solution of pH 1.2 and pH 6.8 according to an embodiment of the present invention.
- FIG. 8 to 15 shows 1 (OVX-PTH-SC): 10 ⁇ g / kg rhPTH (1-34) once daily subcutaneous injection, 2 (OVX-PTH-ORAL) for 2 weeks according to an embodiment of the present invention. : 50 ⁇ g / kg rhPTH (1-34) orally once daily, 3 (OVX-PTH / LysDOCA-ORAL): 50 ⁇ g / kg rhPTH (1-34) / LysDOCA complex oral as rhPTH (1-34) (OVX-PTH / LysDOCA-MP-ORAL): enteric-microparticle orally administered sham-operated containing 50 ⁇ g / kg rhPTH (1-34) / LysDOCA nanocomposite as rhPTH (1-34) Micro-computed tomography ( ⁇ -CT) analysis of the tibia bone of rats with SHAM; placebo surgery) or ovarian ablation (OVX).
- ⁇ -CT Micro-computed
- 16 to 19 shows 1 (OVX-PTH-SC): 10 ⁇ g / kg rhPTH (1-34) once daily subcutaneous injection, 2 (OVX-PTH-ORAL) for 12 weeks according to an embodiment of the present invention. : 50 ⁇ g / kg rhPTH (1-34) orally once daily, 3 (OVX-PTH / LysDOCA-ORAL): 50 ⁇ g / kg rhPTH (1-34) / LysDOCA complex oral as rhPTH (1-34) (OVX-PTH / LysDOCA-MP-ORAL): Enteric-microparticle orally administered sham-operated containing 50 ⁇ g / kg rhPTH (1-34) / LysDOCA nanoparticles as rhPTH (1-34) Micro-computed tomography ( ⁇ -CT) analysis of the cortex of SHAM; placebo surgery) or ovarian resection (OVX) rats.
- ⁇ -CT Micro-computed tomography
- FIG. 20 shows SHAM, OVX, OVX-PTH-SC, OVX-PTH-ORAL, OVX-PTH / LysDOCA-ORAL and OVX-PTH / LysDOCA-MP-ORAL administered drugs for 12 weeks in accordance with an embodiment of the present invention.
- the present invention relates to oral dosage forms of parathyroid hormones containing gastrointestinal absorption enhancers.
- the parathyroid hormone is also known as parathormone (parathormone) and when the calcium concentration in the body fluid is secreted from the parathyroid (parathyroid glands) to increase the calcium concentration in the blood, mainly bone (bone) ), Kidney and intestine.
- the teriparatide is a fragment of the recombinant DNA peptide of the parathyroid hormone, which is used to treat male and female osteoporosis.
- the deoxycholic acid derivative means a combination of deoxycholic acid and lysine.
- deoxycholic acid is a kind of bile acid, the chemical formula is C 24 H 40 O 4 , the molecular weight is 393 g / mol and may correspond to 3 ⁇ , 12 ⁇ -dihydroxycholan.
- Lysine (lysine) is one of the basic ⁇ -amino acid, the formula is H 2 N (CH 2 ) 4 (NH 2 ) COOH and has a molecular weight of 146.19 g / mol.
- lysine is one of the basic ⁇ -amino acid, the formula is H 2 N (CH 2 ) 4 (NH 2 ) COOH and has a molecular weight of 146.19 g / mol.
- Existing in animal protein and low in vegetable protein, essential amino acid is not synthesized in the body and is used for food processing.
- LysDOCA derivative may be bound to a PTH or PTH fragment at a molar ratio of 1: 1 to 1:10.
- N -methylmorpholine ( N -methylmorpholine, 7.4 mL, 67.24 mmoL) was added to deoxycholic acid (DOCA) (24 g, 61.13 mmoL) dissolved in dry THF (Tetrahydrofuran) (300 mL) in an ice bath. After the addition, 6.4 mL of ethyl chloroformate was added dropwise. After stirring the mixed solution for 30 minutes and reacting at room temperature for 2 hours, H-Lys (Boc) -OMe.HCl (20 g, 67.24 mmoL) was added to the mixed solution and refluxed for 5 hours. After cooling to room temperature, the solvent was evaporated.
- DOPA deoxycholic acid
- Lys (Boc) DOCA was obtained after evaporation to dryness using Na 2 SO 4 .
- Lys (Boc) DOCA was stirred in a mixture of acetyl chloride (23.4 mL, 329 mmoL) and methanol (methanol, 100 mL) for half a day under nitrogen. The mixture was evaporated to dryness and the residue dissolved in distilled water was washed with chloroform. The final LysDOCA was lyophilized to form a white powder.
- the PTH / LysDOCA nanocomposites were formed by chemical bonding of the rhPTH (1-34) and LysDOCA.
- the rhPTH (1-34) and LysDOCA were separately dissolved in distilled water at a concentration of 1 mg / mL, respectively, and the LysDOCA solution prepared above was mixed while slowly adding to the prepared rhPTH solution.
- the enteric particulates containing the rhPTH or PTH / LysDOCA nanocomposites were prepared using a coaxial ultrasonic atomizer (Sono-Tek Corp., USA).
- FITC-dextran Fluorescein isothiocyanate-dextran
- RVC rhodamine B isothiocyanate
- FIG. 1 is 10 ⁇ M rhPTH (1-34), LysDOCA, PTH / LysDOCA in water or in an aqueous solution of 20% (v / v) 2,2,2-trifluoroethanol (TFE; 2,2,2-trifluoroethanol) (1:10) Dichroism spectroscopy of a nanocomposite.
- Figure 1 confirms that the CD spectra in the TFE aqueous solution and water at 208 nm and 222 nm is the same. Therefore, it was confirmed that LysDOCA of PTH / LysDOCA did not affect the secondary structure of PTH.
- the maintenance of secondary structure of PTH / LysDOCA is important for the biological activity of PTH because the relatively stable ⁇ -helix segments present in the secondary structure of PTH are important for receptor binding and the action of PTH.
- the particle size, size distribution, and surface charge of the PTH / LysDOCA nanocomposites were measured using a dynamic light scattering analyzer (DLS analyzer, Nano ZS, Malvern Instruments, UK), and the negative stained PTH / LysDOCA nanocomposites Morphological evaluation of was performed by transmission electron microscope (JEM-200, JEOL, Japan).
- TEM 2 is a particle size distribution and transmission electron microscope (TEM) images of rhPTH (1-34) and PTH / LysDOCA (1:10) nanocomposites.
- the diameter of the PTH / LysDOCA nanocomposite particles is 33.3 nm and the zeta potential is 2.85.
- the mole fraction of rhPTH and LysDOCA was 1: 10 (rhPTH: LysDOCA)
- the surface negative charge of rhPTH was neutralized by positively charged LysDOCA (6.27) and the zeta potential was determined to be 14.0. This is consistent with the fact that at least five negatively charged amino acids in the rhPTH chain can electrostatically interact with LysDOCA.
- Transmission electron microscopy images of the negatively stained PTH / LysDOCA nanocomposites confirmed that rhPTH itself is spherical nanoparticles that are regularly well formed, unlike the formation of aggregates of various sizes.
- MC3T3-E1 Preosteoblast cells, subclone 4, ATCC
- FBS ⁇ -MEM 5% CO 2 , 37 ° C.
- Cells were added to 96-well plates by 1 ⁇ 10 4 cells / well and incubated at 37 ° C. for 24 hours.
- Permeability of the rhPTH or PTH / LysDOCA to Caco-2 cell membrane was evaluated as follows. Caco-2 cells were treated with a transwell filter insert (pore size 0.4 ⁇ m, surface area 1.12 cm 2 , Corning Coastar, USA) at a concentration of 3 ⁇ 10 5 cells / mL, followed by media (apical 0.5 mL, basolateral 1.5 mL). The cells were changed every 48 hours and a cell monolayer having a TEER value of> 350 ⁇ cm 2 was used for the experiment. After incubating the cells for 21 to 29 days in a 12-well transwell, the medium was removed using a filter, and the monolayer membrane was incubated for 20 minutes at 37 ° C.
- rhPTH or PTH / LysDOCA dissolved in 200 ⁇ M concentration in HBSS to 0.5 mL by the filter in real positive side compartment (apical compartment) it was taken for sample C 0. Samples were taken at 200 ⁇ L in the basolateral compartment every 0.5, 1, 2, 3, 4, 5 hours during incubation at 37 ° C. Samples were filtered using a membrane filter (0.45 ⁇ m, PVDF) and stored at 4 ° C. for analysis. The concentration of rhPTH or PTH / LysDOCA permeated through the monolayer membrane was analyzed by HPLC.
- Samples were dispensed in 50 ⁇ L increments and injected into HPLC equipped with Kromasil C18 column (250 ⁇ 4.6 mm, 5 ⁇ m). Samples were separated for 25 minutes using a mobile phase (0.1% w / v trifluoroacetic acid (TFA) with 51% -76% linear gradient acetonitrile containing 0.1% w / v TFA) at a flow rate of 1.0 mL / min. Peaks of rhPTH and PTH / LysDOCA were measured at 220 nm with background correction (removal), and cell membrane permeability (P app ) was calculated according to the following equation.
- TFA trifluoroacetic acid
- A surface area of the monolayer film (cm 2 ))
- Mole fraction is 1: 10 or in the long-side of the PTH / LysDOCA - basal side (apical-to-basal) P app in the direction was increased 2.87 fold when compared to PTH alone [10.4 ⁇ 2.41 vs 3.62 ⁇ 0.45 ( ⁇ 10 -7 , cm / s)]. Therefore, further studies were performed with PTH / LysDOCA (1:10) to confirm the efficacy of in vivo intestinal absorption and anti-osteoporosis in rats.
- Sprague-Dawley rats female, 200-250 g were purchased from Orient Co., Ltd. (Korea). Rats were anesthetized with intraperitoneal injections of ketamine (45 mg / kg) and xylazine (5 mg / kg). Anesthetized rats were incised to excise the small intestine and 400 ⁇ L of rhPTH (0.1 mg / kg) or PTH / LysDOCA (0.1 mg / kg as rhPTH) solution was administered to the proximal jejunum.
- rhPTH 0.02 mg / kg dissolved in distilled water was separately injected subcutaneously. After drug administration, blood samples (200 ⁇ L) were taken at scheduled times and immediately mixed with 50 ⁇ L sodium citrate (3.8%) solution. The samples were centrifuged at 2,500 xg, 4 ° C for 15 minutes and the separated plasma samples were stored at 70 ° C for analysis. The concentration of rhPTH in plasma was measured at 620 nm wavelength using the human PTH (1-34) ELISA kit (ALPCO Diagnostics, USA). Pharmacokinetic parameters were estimated using a non-compartment method using WinNonlin ® Software (ver. 5.3; Pharsight Corporation, USA).
- Figure 4 shows the subcutaneous injection of rhPTH (1-34) and rhPTH (1-34) in the rat (0.1 mg / kg) or the PTH / LysDOCA (1:10) nanocomposite in rats.
- the C max was 39.16 ⁇ 19.80 pg / mL after the 0.1 mg / kg rhPTH intrajejunal administration and the AUC last was 1,853.2 ⁇ 1,491.8 pg ⁇ min / mL, and the relative bioavailability compared to subcutaneous injection was 1.06.
- T max was 15.0 for PTH subcutaneous injection, 30.0 ⁇ 0.0 for in-plant PTH administration, 35.0 minutes for in-plant administration of PTH / LysDOCA, and plasma concentrations after administration in PTH / LysDOCA plant reached 120 min. (lower limit).
- AUC last the area under the plasma concentration-time curve from 0 to the last measurement of plasma concentration
- the size of the enteric particulates of the prepared PTH / LysDOCA was measured using a laser diffraction particle size analyzer (Mastersizer X, Malvern Instruments Ltd., UK).
- the surface morphology and distribution of the prepared enteric fine particles of PTH / LysDOCA were analyzed by scanning electron microscope (SEM; Scanning Electron Microscopy, JSM-5300, JEOL, Japan) and a confocal laser scanning microscope (argon / HeNe laser and a Zeiss Axio Observer). Inverted microscope; JSM 710 NLO and LSM 780 NLO, Carl Zeiss Microscopy GmbH, Germany). Confocal fluorescence images of FITC-dextran and RITC were measured using 20 ⁇ objective lenses and excitation wavelengths of 488 nm and 568 nm, respectively.
- FIG. 5 is a scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) image of enteric particulates containing PTH / LysDOCA (1:10) nanocomposites.
- Eudragit L100-55 polymer was mixed with rhodamine isothiocyanate (RTC), the core containing the PTH / LysDOCA (1:10) nanocomposite is fluorescein isothiocyanate Mixed with labeled dextran (FITC) (green, fluorescein isothiocyanate-labeled dextran, green).
- RTC rhodamine isothiocyanate
- FITC labeled dextran
- Enteric fine particles containing the PTH / LysDOCA nanocomposites were prepared using dual axis ultrasonic atomization for oral administration of the rhPTH.
- the inner flow rate drug solution
- the outer flow rate enteric polymer solution
- More homogeneous microparticles were obtained when the inner flow rate was less than 0.5 mL / min.
- the inner flow rate exceeded 0.6 mL / min, no core-shell structure of the enteric particulates was formed.
- more uniform forms of fine particles with a smooth surface were formed when the stirring speed was 800 rpm or higher. Referring to FIG.
- the enteric fine particles of PTH / LysDOCA were encapsulated with an efficiency of about 80% and the average diameter of the particles was 60.2 ⁇ m. Confocal images also suggest that most PTH / LysDOCA are located at the core of the particles and are homogeneously encapsulated by a shell of enteric polymer.
- the dissolution test of the drug from the enteric fine particles of the prepared PTH / LysDOCA was performed at 37 °C ⁇ 0.2 °C with 500 mL of medium containing 0.1 N HCl (pH 1.2) or phosphate buffer (pH 6.8), 100 rpm Tested with USP type 1 (basket) device.
- the enteric microparticles containing rhPTH or PTH / LysDOCA were encapsulated in hard gelatin capsules. Each capsule was to contain 50 mg as rhPTH. 3 mL samples were taken 15, 30, 45, 60, 90, 120 minutes after the start of the dissolution test. After filtering, the amount of rhPTH or PTH / LysDOCA released in the elution test solution was measured using the HPLC-UV detector as described above.
- Osteoporosis treatment effects of orally administered rhPTH and PTH / LysDOCA were evaluated in 60 Sprague-Dawley rats (female, 8 weeks old). Bilateral ovaries of 50 rats were excised to induce osteoporosis and placebo surgery without ovarian extraction (SHAM) was performed on 10 rats. Two weeks later, ovarian-dissected rats were randomly divided into the following five groups (eight of each).
- OVX-PTH / LysDOCA-ORAL (Once administered once daily, 200 ⁇ L 3% Mg (OH) 2 antacid, 50 ⁇ g / kg rhPTH / LysDOCA as rhPTH)
- the dose of the drug was adjusted to the body weight daily. Twelve weeks after the start of administration, the left and right tibiae were isolated from rats and fixed with neutral buffered 4% formalin.
- Tibia was scanned using a In- vivo ⁇ -CT (Skyscan 1076 , Bruker Corp., Germany) to assess its microstructure, and measuring the structural parameters and the bone mineral density (BMD).
- the X-ray source setting was beam filtration through a 1.0-mm-thick aluminum filter at 100 kV, 100 ⁇ A.
- a 2D projection of 35mm width x 17mm height was recorded at every 0.5 ° rotational step from 0 ° to 360 °.
- Images of 3D microstructures were reconstructed using the modified Feldkamp back-projection algorithm. After reconstruction, the 3D image was a low-pass filter (Gaussian filter) to extract the mineral phase and globally thresholded over a fixed range of 0.0 to 0.04 cross-section.
- VOI volume of interest
- the cortex in the diaphysis of the tibia was also evaluated.
- the VOI for the analysis of the cortex is 0.9 mm below 0.54 mm from the end of the VOI of the spongy bone in the tibia.
- the following structural parameters were also calculated for the cortex:
- tibia formalin-fixed tibia was decalcified in 10% EDTA and dehydrated through graded ethanol treatment. After that, it was placed in a paraffin wax block, cut into 5 ⁇ m thickness, and stained with hematoxylin and eosin (H & E). Histological sections were observed under an optical microscope (Bx41, Olympus, Japan) and photographed with focus on the center of the growth plate.
- the OVX-PTH / LysDOCA-MP-ORAL group showed significant improvement in the sponge bone microstructural parameters similar to the OVX-PTH-SC group: 175% (7.6194% ⁇ 1.4202%) in BV / TV compared to the OVX control group. , 174% (0.9673 ⁇ 0.1968 mm ⁇ 1 ) at Tb.N.
- the decreases in BV / TV and Tb.N observed in OVX rats were not suppressed to a significant level.
- Tb.Sp, Tb.Pf, and SMI increases in OVX rats were significantly inhibited in OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups, but not in OVX-PTH-ORAL groups. .
- ⁇ -CT was used to evaluate the cortex in the midshaft region of the tibia, and the results are shown in FIG. 8.
- all the parameters of the OVX control group decreased to a significant level compared to the SHAM group.
- 97% lower Ct.BMD, 88% lower Ct.BV, 96% lower Ct.Th, 77% lower MMI (polar) in contrast, 103% in the OVX-PTH / LysDOCA-MP-ORAL group compared to the OVX control group.
- Ct.BMD, Ct.BV, Ct.Th and MMI (polar) were high.
- FIG. 9 Representative 3D ⁇ -CT reconstructions and longitudinal cross-sections across the proximal tibia are shown in FIG. 9.
- the decline in spongy bone structure was observed in the OVX control group.
- the OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups there was a more fine spongy bone structure of the proximal tibia than in the OVX control group or the OVX-PTH-ORAL group. It suggests that there is a greater assimilation due to oral administration of LysDOCA containing enteric particulates.
- the SHAM group showed a dense and uniform form of cavernous bone, in contrast to the OVX control group, which showed collapsed cavernous connectivity.
- the OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups showed increased structural integrity and denser spongy bone microstructure than the OVX control and OVX-PTH-ORAL groups.
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Abstract
The present invention provides a complex formed by mixing parathyroid hormone (PTH) or the fragment teriparatide (PTH(1-34)) thereof, which both account for an osteopromotive mechanism, and a deoxycholic acid derivative (deoxycholic acid and lysine associate), which is an intestinal membrane permeability enhancer, at a molar ratio of 1 to 10 through ion-ion interaction therebetween, and an oral dosage form of parathyroid hormone comprising a gastrointestinal absorption enhancer, which is the complex in the form of enteric coated microparticles. Therefore, the oral dosage form enhances the intestinal membrane permeability and gastrointestinal absorption of PTH and thus is effective for the treatment of osteoporosis.
Description
본 발명은 골형성 촉진 기전의 부갑상선 호르몬(PTH) 혹은 이의 단편인 테리파라타이드(PTH(1-34))에 장관막 투과증진제인 데옥시콜산 유도체를 혼합하여 복합체를 형성시킴으로써 PTH의 장관막 투과율을 증진시키고, 상기 복합체를 장용성 물질로 코팅한 미립자 형태의 제제로 제조하여 약물의 위장관 흡수를 증진시킨 위장관 흡수증진제를 함유하는 부갑상선 호르몬 경구 투여용 제제 조성물에 관한 것이다.According to the present invention, the intestinal membrane permeability of PTH is formed by mixing a parathyroid hormone (PTH) or a fragment thereof, terry paraparatide (PTH (1-34)) with a deoxycholic acid derivative, which is an intestinal membrane permeation enhancer, to form a complex. The present invention relates to a formulation composition for oral administration of parathyroid hormone containing a gastrointestinal absorption enhancer which enhances the gastrointestinal absorption of the drug by improving the gastrointestinal absorption of the drug by preparing the complex in the form of a preparation coated with an enteric material.
부갑상선 호르몬(PTH; parathyroid hormone)은 부갑상선에서 분비되는 84개의 아미노산으로 이루어진 폴리펩타이드로 혈중 칼슘이온의 농도를 조절하는 주요 호르몬 중 하나이다. 이것은 뼈에서 동화작용(뼈 형성)과 이화작용의 기능을 하며 이러한 작용은 PTH에 노출되는 기간 및 패턴에 의해 달라진다. 동화작용은 간헐적인 PTH 노출에 의해 촉진되며 조골세포의 분화와 증식 또는 세포자멸사의 감소를 야기한다. 이화작용은 지속적인 PTH 노출에 의해 촉진되며 RANKL (receptor activator of nuclear factor-κB ligand)의 발현 증가 및 오스테오프로테게린(osteoprotegerin)의 발현 감소와 관련이 있다.Parathyroid hormone (PTH) is a polypeptide consisting of 84 amino acids secreted by the parathyroid gland and is one of the major hormones that controls the concentration of calcium ions in the blood. It functions as an assimilation (bone formation) and catabolism in bone, which depends on the duration and pattern of exposure to PTH. Assimilation is facilitated by intermittent PTH exposure and results in differentiation and proliferation of osteoblasts or reduction of apoptosis. Catabolism is promoted by continuous PTH exposure and is associated with increased expression of receptor activator of nuclear factor-kB ligand (RANKL) and decreased expression of osteoprotegerin.
PTH의 N-말단으로부터 34개의 아미노산 잔기, 즉 테리파라타이드(PTH(1-34))는 뼈와 콩팥에 많이 발현되어있는 type 1 PTH 수용체의 활성화에 필수적이다. PTH(1-34)가 type 1 PTH 수용체에 결합하면 G단백질-의존적 cAMP/단백질인산화효소 A 경로(G protein-dependent cAMP/protein kinase A pathway)를 포함하는 일련의 신호전달 체계가 활성화되고 혈장의 칼슘 농도가 조절된다. 테리파라타이드(teriparatide)는 유전자 재조합 PTH(1-34)(rhPTH(1-34); 인간 유전자 재조합(recombinant human PTH(1-34))로서 임상적으로 폐경 후 여성의 골다공증과 생식선 억제성 남성의 골다공증 치료에 사용된다. 간헐적인 테리파라타이드의 투여는 조골세포를 파골세포보다 더 활성화시킴으로써 새로운 뼈의 형성을 자극하며 골다공증에서의 골절 위험을 감소시킨다. 다른 많은 골다공증 치료법들은 이화작용의 성질을 가지지 않고 오로지 파골세포의 활성을 억제시키는 예방적인 성격의 치료법이다. 이러한 골흡수억제제(antiresorptives)에는 비스포스포네이트(bisphosphonate), 에스트로겐 수용체 조절제(estrogen receptor modulator), 칼시토닌(calcitonin)이 포함된다. 반면, 테리파라타이드는 골절의 위험이 있으며 다른 골흡수억제제에 반응하지 않는 환자의 골다공증 치료에 쓰일 수 있다.Thirty-four amino acid residues from the N-terminus of PTH, terry paratide (PTH (1-34)), are essential for the activation of type 1 PTH receptors, which are highly expressed in bone and kidneys. Binding of PTH (1-34) to the type 1 PTH receptor activates a series of signaling systems, including the G protein-dependent cAMP / protein kinase A pathway, Calcium concentration is controlled. Teriparatide is a recombinant PTH (1-34) (rhPTH (1-34); recombinant human PTH (1-34)), a clinically postmenopausal female osteoporosis and gonadotropin male. Intermittent administration of teriparatide stimulates the formation of new bone by activating osteoblasts more than osteoclasts and reduces the risk of fractures in osteoporosis Many other osteoporosis therapies alter the nature of catabolism. It is a prophylactic treatment that only inhibits the activity of osteoclasts, which include bisphosphonates, estrogen receptor modulators, and calcitonin. Paratide is used to treat osteoporosis in patients at risk of fracture and who do not respond to other bone resorption inhibitors. Can be.
테리파라타이드는 현재 넓적다리나 복부에 2년간 1일 1회 피하주사 하는 형태로 투여되고 있다. 그러나 이러한 피하주사 방식은 환자에게 통증을 유발하고 환자들이 올바른 주사 방법을 습득해야 하는 불편함을 수반한다. 따라서 환자의 복용 순응도를 높이기 위해 PTH 펩타이드에 대한 경구투여, 경피투여, 비강투여 등의 대체 투여전달시스템의 개발이 시도되고 있다. 경구 투여의 장점으로는 환자의 편리함과 PTH(1-34)의 농도가 Tmax에 도달하는 데까지 걸리는 시간이 증가한다는 점이 있다. 그러나 PTH 경구 전달의 가장 큰 문제점 중 하나는 펩타이드 자체의 경구 생체이용률이 낮다는 것이다. 이러한 문제의 원인으로는 펩타이드의 짧은 반감기, 위산과 소화관의 단백질 분해효소에 의한 분해, 높은 분자량(약 4117.72 Da)에 기인한 낮은 위장관 세포막 투과성이 있다.Terriparatide is currently administered in the thigh or abdomen in a subcutaneous injection once a day for two years. However, this subcutaneous injection method causes pain in the patient and entails the inconvenience that the patient must learn the correct injection method. Therefore, development of alternative delivery systems such as oral, transdermal, and nasal administration of PTH peptides has been attempted to increase patient compliance. Advantages of oral administration include increased patient convenience and the time it takes for the concentration of PTH (1-34) to reach T max . However, one of the biggest problems of PTH oral delivery is the low oral bioavailability of the peptide itself. These problems include short half-life of peptides, degradation by gastric acid and digestive proteinases, and low gastrointestinal membrane permeability due to high molecular weight (about 4117.72 Da).
따라서 환자의 복용 순응도를 높이기 위해 테리파라타이드의 새로운 투여전달시스템의 개발이 시급한 실정이다.Therefore, it is urgent to develop a new dose delivery system of teriparatide in order to increase patient compliance.
부갑상선 호르몬(PTH; parathyroid hormone)은 부갑상선에서 분비되는 84개의 아미노산으로 이루어진 폴리펩타이드로 혈중 칼슘이온의 농도를 조절하는 주요 호르몬 중 하나이다. 이것은 뼈에서 동화작용(뼈 형성)과 이화작용의 기능을 하며 이러한 작용은 PTH에 노출되는 기간 및 패턴에 의해 달라진다. 동화작용은 간헐적인 PTH 노출에 의해 촉진되며 조골세포의 분화와 증식 또는 세포자멸사의 감소를 야기한다. 이화작용은 지속적인 PTH 노출에 의해 촉진되며 RANKL (receptor activator of nuclear factor-κB ligand)의 발현 증가 및 오스테오프로테게린(osteoprotegerin)의 발현 감소와 관련이 있다.Parathyroid hormone (PTH) is a polypeptide consisting of 84 amino acids secreted by the parathyroid gland and is one of the major hormones that controls the concentration of calcium ions in the blood. It functions as an assimilation (bone formation) and catabolism in bone, which depends on the duration and pattern of exposure to PTH. Assimilation is facilitated by intermittent PTH exposure and results in differentiation and proliferation of osteoblasts or reduction of apoptosis. Catabolism is promoted by continuous PTH exposure and is associated with increased expression of receptor activator of nuclear factor-kB ligand (RANKL) and decreased expression of osteoprotegerin.
PTH의 N-말단으로부터 34개의 아미노산 잔기, 즉 테리파라타이드(PTH(1-34))는 뼈와 콩팥에 많이 발현되어있는 type 1 PTH 수용체의 활성화에 필수적이다. PTH(1-34)가 type 1 PTH 수용체에 결합하면 G단백질-의존적 cAMP/단백질인산화효소 A 경로(G protein-dependent cAMP/protein kinase A pathway)를 포함하는 일련의 신호전달 체계가 활성화되고 혈장의 칼슘 농도가 조절된다. 테리파라타이드(teriparatide)는 유전자 재조합 PTH(1-34)(rhPTH(1-34); 인간 유전자 재조합(recombinant human PTH(1-34))로서 임상적으로 폐경 후 여성의 골다공증과 생식선 억제성 남성의 골다공증 치료에 사용된다. 간헐적인 테리파라타이드의 투여는 조골세포를 파골세포보다 더 활성화시킴으로써 새로운 뼈의 형성을 자극하며 골다공증에서의 골절 위험을 감소시킨다. 다른 많은 골다공증 치료법들은 이화작용의 성질을 가지지 않고 오로지 파골세포의 활성을 억제시키는 예방적인 성격의 치료법이다. 이러한 골흡수억제제(antiresorptives)에는 비스포스포네이트(bisphosphonate), 에스트로겐 수용체 조절제(estrogen receptor modulator), 칼시토닌(calcitonin)이 포함된다. 반면, 테리파라타이드는 골절의 위험이 있으며 다른 골흡수억제제에 반응하지 않는 환자의 골다공증 치료에 쓰일 수 있다.Thirty-four amino acid residues from the N-terminus of PTH, terry paratide (PTH (1-34)), are essential for the activation of type 1 PTH receptors, which are highly expressed in bone and kidneys. Binding of PTH (1-34) to the type 1 PTH receptor activates a series of signaling systems, including the G protein-dependent cAMP / protein kinase A pathway, Calcium concentration is controlled. Teriparatide is a recombinant PTH (1-34) (rhPTH (1-34); recombinant human PTH (1-34)), a clinically postmenopausal female osteoporosis and gonadotropin male. Intermittent administration of teriparatide stimulates the formation of new bone by activating osteoblasts more than osteoclasts and reduces the risk of fractures in osteoporosis Many other osteoporosis therapies alter the nature of catabolism. It is a prophylactic treatment that only inhibits the activity of osteoclasts, which include bisphosphonates, estrogen receptor modulators, and calcitonin. Paratide is used to treat osteoporosis in patients at risk of fracture and who do not respond to other bone resorption inhibitors. Can be.
테리파라타이드는 현재 넓적다리나 복부에 2년간 1일 1회 피하주사 하는 형태로 투여되고 있다. 그러나 이러한 피하주사 방식은 환자에게 통증을 유발하고 환자들이 올바른 주사 방법을 습득해야 하는 불편함을 수반한다. 따라서 환자의 복용 순응도를 높이기 위해 PTH 펩타이드에 대한 경구투여, 경피투여, 비강투여 등의 대체 투여전달시스템의 개발이 시도되고 있다. 경구 투여의 장점으로는 환자의 편리함과 PTH(1-34)의 농도가 Tmax에 도달하는 데까지 걸리는 시간이 증가한다는 점이 있다. 그러나 PTH 경구 전달의 가장 큰 문제점 중 하나는 펩타이드 자체의 경구 생체이용률이 낮다는 것이다. 이러한 문제의 원인으로는 펩타이드의 짧은 반감기, 위산과 소화관의 단백질 분해효소에 의한 분해, 높은 분자량(약 4117.72 Da)에 기인한 낮은 위장관 세포막 투과성이 있다.Terriparatide is currently administered in the thigh or abdomen in a subcutaneous injection once a day for two years. However, this subcutaneous injection method causes pain in the patient and entails the inconvenience that the patient must learn the correct injection method. Therefore, development of alternative delivery systems such as oral, transdermal, and nasal administration of PTH peptides has been attempted to increase patient compliance. Advantages of oral administration include increased patient convenience and the time it takes for the concentration of PTH (1-34) to reach T max . However, one of the biggest problems of PTH oral delivery is the low oral bioavailability of the peptide itself. These problems include short half-lives of peptides, degradation by gastric acid and digestive tract proteases, and low gastrointestinal cell membrane permeability due to high molecular weight (about 4117.72 Da).
따라서 환자의 복용 순응도를 높이기 위해 테리파라타이드의 새로운 투여전달시스템의 개발이 시급한 실정이다.Therefore, it is urgent to develop a new dose delivery system of teriparatide in order to increase patient compliance.
본 발명은 PTH(부갑상선 호르몬) 및 담즙산 유도체이 포함된 나노 복합체를 장용성 물질로 코팅한 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물을 제공한다.The present invention provides a formulation composition for oral administration of parathyroid hormone, characterized in that the nanocomposite containing PTH (parathyroid hormone) and bile acid derivatives is coated with an enteric material.
본 발명에서 상기 PTH는 PTH(1-28), PTH(1-31), PTH(1-34), PTH(1-38) 또는 PTH(1-41) 중 어느 것이라도 가능하며, 상기 PTH(1-34)는 인간 부갑상선 호르몬의 1번(N-말단)부터 34번째 아미노산 서열로 구성된 폴리펩티드로서 유전자 재조합 방법 rhPTH(1-34) 혹은 화학적 합성 hPTH(1-34)에 의해 제조된 것이 바람직하다.In the present invention, the PTH may be any one of PTH (1-28), PTH (1-31), PTH (1-34), PTH (1-38) or PTH (1-41), and the PTH ( 1-34) is a polypeptide consisting of amino acid sequences 1 to 34 of the human parathyroid hormone, preferably prepared by the recombinant method rhPTH (1-34) or chemically synthesized hPTH (1-34). .
또한 상기 담즙산 유도체는 데옥시콜산 유도체인 것이 바람직하며, 데옥신콜산 유도체 중에 데옥시콜산에 라이신이 화학적으로 결합된 것이 가장 바람직하다.In addition, the bile acid derivative is preferably a deoxycholic acid derivative, and lysine is most preferably chemically bonded to deoxycholic acid in the deoxincolic acid derivative.
본 발명의 상기 조성물에서 상기 PTH와 상기 담즙산 유도체은 1:1 내지 1:10의 몰비율로 결합된 것이 바람직하다.In the composition of the present invention, the PTH and the bile acid derivative are preferably combined at a molar ratio of 1: 1 to 1:10.
또한 본 발명에서 상기 나노 복합체는 입자크기가 10 내지 50nm인 것이 바람직하다.In addition, the nanocomposite in the present invention is preferably a particle size of 10 to 50nm.
또한 본 발명에 있어서, "장용성 물질" 는 특별히 한정되지 않으며, 예를 들어 유드라짓(Eudragit; 메타크릴산-에틸 아크릴레이트 공중합체), 히드록시프로필메틸셀룰로오스 프탈레이트, 아세틸호박산하이드록시프로필메칠셀룰로오스, 셀룰로오스 아세테이트 프탈레이트, 폴리비닐 아세테이트 프탈레이트, 카르복시메틸에틸셀룰로오스, 셸락 등에서 선택되는 1 종 이상의 중합체 성분을 들 수 있다. 이들 중에서 쉘락, 유드라짓(Eudragit : 메타크릴산-에틸 아크릴레이트 공중합체), 하이드록시프로필메칠셀룰로오스 프탈레이트, 아세틸호박산하이드록시프로필메칠셀룰로오스, 셀룰로오스 아세테이트 프탈레이트로 이루어진 군에서 선택된 하나 또는 둘 이상 혼합된 것을 사용할 수 있다.In addition, in this invention, an "entertaining substance" is not specifically limited, For example, Eudragit (Eudragit; methacrylic acid-ethyl acrylate copolymer), hydroxypropyl methylcellulose phthalate, acetyl succinate hydroxypropyl methylcellulose And at least one polymer component selected from cellulose acetate phthalate, polyvinyl acetate phthalate, carboxymethylethyl cellulose, shellac and the like. Among them, one or two or more selected from the group consisting of shellac, eudragit (Eudragit: methacrylic acid-ethyl acrylate copolymer), hydroxypropylmethylcellulose phthalate, acetyl amber hydroxypropylmethylcellulose, and cellulose acetate phthalate are mixed. Can be used.
또한 본 발명은 1) PTH와 데옥시콜산 유도체의 이온결합에 의한 나노복합체(PTH/데옥시콜산 유도체)를 형성하는 단계; 2) 상기 나노복합체를 물에 용해시킨 후 장용성 물질이 용해된 용액에 분산시켜 현탁액을 제조하는 단계; 3) 상기 현탁액을 시린지 펌프로 이중축 초음파 분사 노즐의 내측 주입구로 이동시키는 단계; 4) 이중측 초음파 분사 노즐의 외측 주입구로 장용성 물질 용액을 이동시키는 단계; 5) 초음파 분사노즐의 진동분사에 의해 노즐 내측 약물 용액이 외측 장용성 물질 용액으로 둘러싸인 미세액적을 형성하는 단계; 6) 미세액적이 유동 파라핀에 분사 및 교반되어 미세액적의 껍질이 단단하게 굳어지는 단계;를 포함하는 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물의 제조방법을 제공한다. In another aspect, the present invention 1) forming a nanocomposite (PTH / deoxycholic acid derivative) by the ionic bond of PTH and deoxycholic acid derivatives; 2) preparing a suspension by dissolving the nanocomposite in water and then dispersing it in a solution in which the enteric material is dissolved; 3) moving the suspension to the inner inlet of the dual axis ultrasonic spray nozzle with a syringe pump; 4) moving the enteric material solution to the outer inlet of the double side ultrasonic spray nozzle; 5) forming microdroplets in which the drug solution inside the nozzle is surrounded by the external enteric material solution by vibration spraying of the ultrasonic jet nozzle; 6) the microdroplets are injected into the liquid paraffin and stirred to harden the shell of the microdroplets; and provides a method for preparing a formulation composition for oral administration of parathyroid hormone, characterized in that it comprises a.
상기 1) 단계에서 상기 PTH 및 상기 데옥시콜산 유도체는 1:1 내지 1:10의 몰비율로 혼합되는 것이 바람직하며, 상기 4)단계이후에 진공필터를 통해 회수하고 헥산으로 세척 및 건조하는 단계를 더 추가할 수 있다.In the step 1), the PTH and the deoxycholic acid derivatives are preferably mixed at a molar ratio of 1: 1 to 1:10. After step 4), the PTH and the deoxycholic acid derivatives are recovered through a vacuum filter and washed with hexane and dried. You can add more.
본 발명은 PTH에 장관막 투과증진 효과를 지닌 데옥시콜산 유도체를 혼합하여 장용성 물질로 코팅함으로써 장관막 투과울이 증진되어 위장관 흡수가 증진되는 효과가 있어, 기존에 피하 주사용 제제로 사용되는 문제점을 투여 경로를 경구 투여로 변경하여 환자의 복용 편의성을 증진시키는 효과가 있다.The present invention is to mix the deoxycholic acid derivative having the effect of promoting the intestinal membrane permeability to the PTH and to coat the enteric material to enhance the permeability of the intestinal membrane to increase the absorption of the gastrointestinal tract, a problem that is conventionally used as a subcutaneous injection formulation By changing the route of administration to oral administration has the effect of enhancing the ease of taking the patient.
도 1은 본 발명의 실시예에 따른 물 또는 20% (v/v) 2,2,2-트리플루오로에탄올(TFE; 2,2,2-trifluoroethanol) 수용액상에서 10 μM rhPTH(1-34), LysDOCA(데옥시콜산-라이신), 및 PTH/LysDOCA (1:10) 나노복합체의 원이색성 분광 스펙트럼을 나타낸 것이다.1 is 10 μM rhPTH (1-34) in water or in an aqueous solution of 20% (v / v) 2,2,2-trifluoroethanol (TFE; 2,2,2-trifluoroethanol) according to an embodiment of the present invention. Dichroism spectroscopy of LysDOCA (deoxycholic acid-lysine), and PTH / LysDOCA (1:10) nanocomposites are shown.
도 2는 본 발명의 실시예에 따른 rhPTH(1-34) 입자 크기 분포와 투과전자현미경(TEM) 이미지를 나타낸 것이다.Figure 2 shows the rhPTH (1-34) particle size distribution and transmission electron microscope (TEM) image according to an embodiment of the present invention.
도 3은 PTH/LysDOCA (1:10) 나노복합체의 입자 크기 분포와 투과전자현미경(TEM) 이미지를 나타낸 것이다.FIG. 3 shows particle size distribution and transmission electron microscopy (TEM) images of PTH / LysDOCA (1:10) nanocomposites.
도 4는 본 발명의 실시예에 따른 MC3T3-E1 세포를 rhPTH(1-34) 또는 PTH/LysDOCA (1:10) 나노복합체와 함께 배양한 후의 세포내 cAMP 함량을 나타낸 것이다.Figure 4 shows the intracellular cAMP content after incubating MC3T3-E1 cells with rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites according to an embodiment of the present invention.
도 5는 본 발명의 실시예에 따른 랫드(Rat)에서 rhPTH(1-34)의 피하주사 (0.02 mg/kg)와 rhPTH(1-34)의 공장(jejumam) 내 투여 (0.1mg/kg) 또는 PTH/LysDOCA (1:10) 나노복합체 (rhPTH(1-34)로서 0.1mg/kg)의 공장 내 투여 후 rhPTH (1-34)의 정맥 혈장 내 농도-시간 프로파일을 나타낸 것이다.Figure 5 is a subcutaneous injection of rhPTH (1-34) (0.02 mg / kg) and rhPTH (1-34) in the jejumam administration of the rat (Rat) according to an embodiment of the present invention (0.1 mg / kg) Or intravenous plasma concentration-time profile of rhPTH (1-34) following in-plant administration of PTH / LysDOCA (1:10) nanocomposites (0.1 mg / kg as rhPTH (1-34)).
도 6은 본 발명의 실시예에 따른 PTH/LysDOCA (1:10) 나노복합체를 함유하는 장용성 미립자의 주사전자현미경(SEM) (A) 이미지와 공초점 레이저 주사 현미경(CLSM; confocal laser scanning microscopy) (B) 이미지를 나타낸 것이다.Figure 6 is a scanning electron microscope (SEM) (A) image of the enteric particles containing the PTH / LysDOCA (1:10) nanocomposite according to an embodiment of the present invention and confocal laser scanning microscopy (CLSM) (B) shows an image.
도 7은 본 발명의 실시예에 따른 pH 1.2와 pH 6.8의 용출 시험액에서 장용성 미립자로부터 rhPTH(1-34) 또는 PTH/LysDOCA (1:10) 나노복합체의 in vitro 누적 약물 방출률을 나타낸 것이다.Figure 7 shows the in vitro cumulative drug release rate of the rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites from the enteric microparticles in dissolution test solution of pH 1.2 and pH 6.8 according to an embodiment of the present invention.
도 8 내지 도 15는 본 발명의 실시예에 따른 2주 동안 ① (OVX-PTH-SC): 10 μg/kg rhPTH(1-34) 1일 1회 피하주사, ② (OVX-PTH-ORAL): 50 μg/kg rhPTH(1-34) 1일 1회 경구투여, ③ (OVX-PTH/LysDOCA-ORAL): rhPTH(1-34)로서 50 μg/kg rhPTH(1-34)/LysDOCA 복합체 경구투여, ④ (OVX-PTH/LysDOCA-MP-ORAL): rhPTH(1-34)로서 50 μg/kg rhPTH(1-34)/LysDOCA 나노복합체를 함유하는 장용-미세입자 경구투여한 sham-operated(SHAM; 플라세보 수술)된 랫드이거나 난소절제(OVX)된 랫드의 정강이뼈에 대한 미세-컴퓨터 단층촬영(μ-CT; micro-computed tomography) 분석을 나타낸 것이다. 8 to 15 shows ① (OVX-PTH-SC): 10 μg / kg rhPTH (1-34) once daily subcutaneous injection, ② (OVX-PTH-ORAL) for 2 weeks according to an embodiment of the present invention. : 50 μg / kg rhPTH (1-34) orally once daily, ③ (OVX-PTH / LysDOCA-ORAL): 50 μg / kg rhPTH (1-34) / LysDOCA complex oral as rhPTH (1-34) (OVX-PTH / LysDOCA-MP-ORAL): enteric-microparticle orally administered sham-operated containing 50 μg / kg rhPTH (1-34) / LysDOCA nanocomposite as rhPTH (1-34) Micro-computed tomography (μ-CT) analysis of the tibia bone of rats with SHAM; placebo surgery) or ovarian ablation (OVX).
도 16 내지 도 19는 본 발명의 실시예에 따른 12주 동안 ① (OVX-PTH-SC): 10 μg/kg rhPTH(1-34) 1일 1회 피하주사, ② (OVX-PTH-ORAL): 50 μg/kg rhPTH(1-34) 1일 1회 경구투여, ③ (OVX-PTH/LysDOCA-ORAL): rhPTH(1-34)로서 50 μg/kg rhPTH(1-34)/LysDOCA 복합체 경구투여, ④ (OVX-PTH/LysDOCA-MP-ORAL): rhPTH(1-34)로서 50 μg/kg rhPTH(1-34)/LysDOCA 나노입자를 함유하는 장용-미세입자 경구투여한 sham-operated(SHAM; 플라세보 수술)된 랫드이거나 난소절제(OVX)된 랫드의 골피질에 대한 미세-컴퓨터 단층촬영(μ-CT; micro-computed tomography) 분석을 나타낸 것이다.16 to 19 shows ① (OVX-PTH-SC): 10 μg / kg rhPTH (1-34) once daily subcutaneous injection, ② (OVX-PTH-ORAL) for 12 weeks according to an embodiment of the present invention. : 50 μg / kg rhPTH (1-34) orally once daily, ③ (OVX-PTH / LysDOCA-ORAL): 50 μg / kg rhPTH (1-34) / LysDOCA complex oral as rhPTH (1-34) (OVX-PTH / LysDOCA-MP-ORAL): Enteric-microparticle orally administered sham-operated containing 50 μg / kg rhPTH (1-34) / LysDOCA nanoparticles as rhPTH (1-34) Micro-computed tomography (μ-CT) analysis of the cortex of SHAM; placebo surgery) or ovarian resection (OVX) rats.
도 20은 본 발명의 실시예에 따른 12주 동안 약물을 투여한 SHAM, OVX, OVX-PTH-SC, OVX-PTH-ORAL, OVX-PTH/LysDOCA-ORAL 및 OVX-PTH/LysDOCA-MP-ORAL rats(각 그룹 당 n=10)의 원위 정강이뼈(distal tibia)에 대한 representative 2D, 3D 이미지를 타나낸 것이다.20 shows SHAM, OVX, OVX-PTH-SC, OVX-PTH-ORAL, OVX-PTH / LysDOCA-ORAL and OVX-PTH / LysDOCA-MP-ORAL administered drugs for 12 weeks in accordance with an embodiment of the present invention. Representative 2D and 3D images of the distal tibia of rats (n = 10 in each group) are shown.
도 21은 본 발명의 실시예에 따른 12주 동안 약물을 투여한 랫드(각 그룹 당 n=10)의 H&E 염색된 근위 정강이뼈(proximal tibia)에 대한 representative 종단 단면(longitudinal cross-sectional) 이미지를 나타낸 것이다.FIG. 21 is a representative longitudinal cross-sectional image of H & E stained proximal tibia of rats (n = 10 per group) administered drug for 12 weeks in accordance with an embodiment of the present invention. It is shown.
본 발명은 위장관 흡수증진제를 함유하는 부갑상선 호르몬의 경구 투여 제형에 관한 것이다.The present invention relates to oral dosage forms of parathyroid hormones containing gastrointestinal absorption enhancers.
상기 부갑상선 호르몬(PTH; parathyroid hormone)은 파라토르몬(parathormone)으로도 알려져 있으며 체액에서 칼슘 농도가 저하되면 부갑상선(parathyroid glands)으로부터 분비되어 혈액 속의 칼슘 농도를 증가시키는 작용을 하고, 주로 뼈(bone), 신장(kidney) 및 장(intestine)에서 작용한다.The parathyroid hormone (PTH) is also known as parathormone (parathormone) and when the calcium concentration in the body fluid is secreted from the parathyroid (parathyroid glands) to increase the calcium concentration in the blood, mainly bone (bone) ), Kidney and intestine.
상기 테리파라타이드(PTH(1-34))는 부갑상선 호르몬의 유전자 재조합 DNA 펩타이드 조각으로, 남성과 여성 골다공증 치료에 사용된다.The teriparatide (PTH (1-34)) is a fragment of the recombinant DNA peptide of the parathyroid hormone, which is used to treat male and female osteoporosis.
상기 데옥시콜산 유도체는 데옥시콜산과 라이신의 결합체를 의미한다.The deoxycholic acid derivative means a combination of deoxycholic acid and lysine.
상기 데옥시콜산(deoxycholic acid)은 담즙산의 일종으로서 화학식은 C24H40O4, 분자량은 393 g/mol이며 3α,12α-디히드록시콜란에 해당하는 것일 수 있다.The deoxycholic acid (deoxycholic acid) is a kind of bile acid, the chemical formula is C 24 H 40 O 4 , the molecular weight is 393 g / mol and may correspond to 3α, 12α-dihydroxycholan.
상기 라이신(lysine)은 염기성 α-아미노산의 하나로 화학식은 H2N(CH2)4(NH2)COOH이며 분자량은 146.19 g/mol이다. 동물성 단백질에 많이 존재하고 식물성 단백질에는 그 함유량이 적으며, 필수아미노산으로 체내에서 합성되지 않으며, 식품의 가공에도 이용된다.Lysine (lysine) is one of the basic α-amino acid, the formula is H 2 N (CH 2 ) 4 (NH 2 ) COOH and has a molecular weight of 146.19 g / mol. Existing in animal protein and low in vegetable protein, essential amino acid is not synthesized in the body and is used for food processing.
상기 PTH/LysDOCA 나노복합체는 PTH 혹은 PTH 단편에 LysDOCA 유도체가 1 : 1 내지 1 : 10의 몰비율로 결합될 수 있다.In the PTH / LysDOCA nanocomposite, LysDOCA derivative may be bound to a PTH or PTH fragment at a molar ratio of 1: 1 to 1:10.
이하, 본 발명의 이해를 돕기 위하여 구체적인 실시예를 통하여 본 발명의 구성 및 효과를 보다 상세하게 설명하나, 하기 실시예는 본 발명을 보다 명확하게 이해시키기 위한 것일 뿐 본 발명의 권리범위가 하기 실시예에 의해 한정되는 것은 아니고, 특허청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리 범위에 포함되는 것으로 해석되어야 한다.Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to specific examples in order to help understand the present invention, but the following examples are only intended to more clearly understand the present invention, and the scope of the present invention is given below. It should not be limited to the examples, but should be interpreted by the claims, and all technical ideas within the equivalent scope should be construed as being included in the scope of the present invention.
제조예 1: rhPTH(1-34)와 LysDOCA의 이온 결합에 의한 PTH/LysDOCA 나노복합체의 제조Preparation Example 1 Preparation of PTH / LysDOCA Nanocomposites by Ion Bonding of rhPTH (1-34) and LysDOCA
1. LysDOCA(데옥시콜산-라이신)의 합성1. Synthesis of LysDOCA (Deoxycholic Acid-Lysine)
얼음 수조 (Ice bath)에서 dry THF (Tetrahydrofuran) (300 mL)에 용해된 데옥시콜산 (DOCA) (24 g, 61.13 mmoL)에 N-메틸몰폴린 (N-methylmorpholine, 7.4 mL, 67.24 mmoL)을 넣은 뒤, 클로로포름산에틸 (ethyl chloroformate) 6.4 mL를 점적하여 첨가하였다. 상기 혼합용액을 30분간 교반 (stirring)한 뒤 상온에서 2시간 동안 반응시킨 후 상기 혼합용액에 H-Lys(Boc)-OMe·HCl (20 g, 67.24 mmoL)를 넣은 뒤 5시간 동안 환류시켰으며 상온으로 냉각시킨 후 용매를 증발시켰다. 최종 15%가 되도록 NaOH (20 mL)를 넣은 뒤 클로로포름 (chloroform)으로 추출하였다. Na2SO4를 사용해 증발건조한 뒤 Lys(Boc)DOCA를 얻었다. 상기 Lys(Boc)DOCA를 염화아세틸 (acetyl chloride, 23.4 mL, 329 mmoL)과 메탄올 (methanol, 100 mL)의 혼합액에서 질소 하에 반나절 동안 교반시켰다. 상기 혼합물을 증발건고 후 증류수에 용해된 잔여물은 클로로포름으로 씻어냈다. 최종 LysDOCA는 동결건조하여 하얀색 가루의 형태로 만들어졌다. N -methylmorpholine ( N -methylmorpholine, 7.4 mL, 67.24 mmoL) was added to deoxycholic acid (DOCA) (24 g, 61.13 mmoL) dissolved in dry THF (Tetrahydrofuran) (300 mL) in an ice bath. After the addition, 6.4 mL of ethyl chloroformate was added dropwise. After stirring the mixed solution for 30 minutes and reacting at room temperature for 2 hours, H-Lys (Boc) -OMe.HCl (20 g, 67.24 mmoL) was added to the mixed solution and refluxed for 5 hours. After cooling to room temperature, the solvent was evaporated. NaOH (20 mL) was added to the final 15% and extracted with chloroform. Lys (Boc) DOCA was obtained after evaporation to dryness using Na 2 SO 4 . Lys (Boc) DOCA was stirred in a mixture of acetyl chloride (23.4 mL, 329 mmoL) and methanol (methanol, 100 mL) for half a day under nitrogen. The mixture was evaporated to dryness and the residue dissolved in distilled water was washed with chloroform. The final LysDOCA was lyophilized to form a white powder.
2. PTH/LysDOCA 나노복합체의 합성2. Synthesis of PTH / LysDOCA Nanocomposites
상기 PTH/LysDOCA 나노복합체는 상기 rhPTH(1-34)와 상기 LysDOCA의 화학적 결합으로 형성되었다. 상기 rhPTH(1-34) 및 LysDOCA는 각각 따로 1 mg/mL의 농도로 증류수에 용해되었고 상기 제조된 LysDOCA 용액을 상기 제조된 rhPTH 용액에 서서히 첨가하면서 혼합하였다. 상기 두 물질의 결합 몰비율은 rhPTH(1-34) : LysDOCA = 1 : 10이며, PTH/LysDOCA 나노복합체를 얻기 위해 용액을 최종 원심분리한 뒤 곧바로 침전물을 동결건조하였다.The PTH / LysDOCA nanocomposites were formed by chemical bonding of the rhPTH (1-34) and LysDOCA. The rhPTH (1-34) and LysDOCA were separately dissolved in distilled water at a concentration of 1 mg / mL, respectively, and the LysDOCA solution prepared above was mixed while slowly adding to the prepared rhPTH solution. The binding molar ratio of the two materials is rhPTH (1-34): LysDOCA = 1: 10, and the precipitate was lyophilized immediately after the final centrifugation of the solution to obtain a PTH / LysDOCA nanocomposite.
제조예 2: 이중축 초음파 미립화(coaxial ultrasonic atomization)를 통한 PTH/LysDOCA의 장용성 미립자 제조Preparation Example 2 Preparation of Enteric Fine Particles of PTH / LysDOCA by Coaxial Ultrasonic Atomization
상기 rhPTH 또는 PTH/LysDOCA 나노복합체를 함유하는 장용성 미립자는 이중축 초음파 미립화 장치(coaxial ultrasonic atomizer, Sono-Tek Corp., USA)를 사용하여 제조하였다. 상기 rhPTH 또는 PTH/LysDOCA 나노복합체를 물에 용해시킨 뒤 (0.05 g/5 mL), 이를 에탄올(ethanol) : 아세톤(acetone) = 30 : 60 (w/w)을 용매로 하는 95 g Eudragit L100-55 용액 (4 mg/mL)에 분산시켰다. 상기 결과로 생긴 현탁액은 실린지 펌프(syringe pump)를 거쳐 0.5 mL/min의 속도로 내측 주입구(inner inlet; top feed channel)로 운반시켰고, 에탄올(ethanol) : 아세톤(acetone) = 32 : 64 (w/w)을 용매로 하는 4 mg/mL 유드라짓(Eudragit L100-55) 용액은 실린지 펌프를 거쳐 1.5 mL/min의 관류속도로 외측 주입구(outer inlet; side feed channel)로 운반시켰다. 또한 공초점 현미경을 통한 형광 이미지를 얻기 위해, 0.05% (w/w) FITC-dextran(Fluorescein isothiocyanate-dextran)과 RITC(rhodamine B isothiocyanate)를 각각 내측(inner) 현탁액과 외측 장용성 고분자 코팅(outer enteric polymer coating) 용액에 첨가하였다. 120 kHz 초음파진동 분사(atomizer vibration)에 의해 형성된 미세 액적 (microdroplet)은 350 rpm으로 교반시키면서 1% Span 80을 함유하는 유동 파라핀에 수집했다. 그 결과로 생긴 상기 용액은 장용성 미립자를 굳히기 위해 800 rpm으로 3시간 동안 교반하였다. 그 후 상기 제조된 장용성 미립자는 진공 필터(vacuum filtration)를 통해 회수하고 헥산(n-hexane)으로 4회 세척한 후 상온에서 건조하였다.The enteric particulates containing the rhPTH or PTH / LysDOCA nanocomposites were prepared using a coaxial ultrasonic atomizer (Sono-Tek Corp., USA). The rhPTH or PTH / LysDOCA nanocomposites were dissolved in water (0.05 g / 5 mL), and 95 g Eudragit L100- using ethanol: acetone = 30: 60 (w / w) as a solvent. It was dispersed in 55 solution (4 mg / mL). The resulting suspension was transferred to an inner inlet (top feed channel) at a rate of 0.5 mL / min via a syringe pump, and ethanol: acetone = 32: 64 ( 4 mg / mL Eudragit L100-55 solution with w / w) as a solvent was delivered to the outer inlet (side feed channel) via a syringe pump at a perfusion rate of 1.5 mL / min. In addition, 0.05% (w / w) Fluorescein isothiocyanate-dextran (FITC-dextran) and rhodamine B isothiocyanate (RITC) were added to the inner suspension and outer enteric polymer coating to obtain fluorescence images through confocal microscopy. polymer coating). Microdroplets formed by 120 kHz ultrasonic vibrations were collected in flowing paraffin containing 1% Span 80 with stirring at 350 rpm. The resulting solution was stirred at 800 rpm for 3 hours to solidify the enteric particulates. Thereafter, the prepared enteric fine particles were recovered through vacuum filtration, washed four times with hexane (n-hexane), and dried at room temperature.
실시예 1: rhPTH(1-34)와 LysDOCA의 화학적 결합에 의한 PTH/LysDOCA 나노복합체의 물리화학적 성질 평가Example 1 Evaluation of Physicochemical Properties of PTH / LysDOCA Nanocomposites by Chemical Combination of rhPTH (1-34) and LysDOCA
1. 원이색성스펙트럼 측정1. Dichroism Spectrum Measurement
상기 PTH/LysDOCA의 2차구조를 분석하기 위해, 20℃에서 수용액(또는 20% v/v TFE)상의 10 μM PTH와 PTH/LysDOCA에 대한 원이색성 분광학을 측정했다. 측정값은 10-mm 큐벳과 JASCO 분광편광계 (spectropolarimeter) (Easton, MD, USA)를 사용해 180 nm ~ 260nm 범위에서 얻었다. 모든 스펙트럼은 배경-보정(background-corrected)이 이루어졌고, mean residue ellipticity를 통해 계산되었으며, 이것들은 10번의 스캔 값의 평균을 대표한다. 상기 PTH/LysDOCA의 2차 구조는 물 또는 20% trifluoroethanol (TFE) 수용액에서 원이색성 분광학(Circular dichroism spectroscopy; CD spectroscopy)을 통해 측정하였다.To analyze the secondary structure of the PTH / LysDOCA, dichroism spectroscopy for 10 μM PTH and PTH / LysDOCA in aqueous solution (or 20% v / v TFE) was measured at 20 ° C. Measurements were taken from 180 nm to 260 nm using a 10-mm cuvette and JASCO spectropolarimeter (Easton, MD, USA). All spectra were background-corrected and calculated through mean residue ellipticity, which represents the average of 10 scans. The secondary structure of PTH / LysDOCA was measured by circular dichroism spectroscopy (CD spectroscopy) in water or 20% trifluoroethanol (TFE) aqueous solution.
도 1은 물 또는 20% (v/v) 2,2,2-트리플루오로에탄올(TFE; 2,2,2-trifluoroethanol) 수용액상에서의 10 μM rhPTH(1-34), LysDOCA, PTH/LysDOCA (1:10) 나노복합체의 원이색성 분광 스펙트럼이다. 도 1을 확인하면 208 nm와 222 nm에서 TFE 수용액상 및 물에서의 CD spectra가 동일하다. 따라서 상기 PTH/LysDOCA의 LysDOCA가 PTH의 2차 구조에 영향을 주지 않음을 확인하였다. PTH의 2차 구조에 존재하는 상대적으로 안정한 α-나선 분절은 수용체 결합과 PTH의 작용에 중요하기 때문에 PTH/LysDOCA의 2차 구조의 유지는 PTH의 생물학적 활성에 있어 중요하다.1 is 10 μM rhPTH (1-34), LysDOCA, PTH / LysDOCA in water or in an aqueous solution of 20% (v / v) 2,2,2-trifluoroethanol (TFE; 2,2,2-trifluoroethanol) (1:10) Dichroism spectroscopy of a nanocomposite. Figure 1 confirms that the CD spectra in the TFE aqueous solution and water at 208 nm and 222 nm is the same. Therefore, it was confirmed that LysDOCA of PTH / LysDOCA did not affect the secondary structure of PTH. The maintenance of secondary structure of PTH / LysDOCA is important for the biological activity of PTH because the relatively stable α-helix segments present in the secondary structure of PTH are important for receptor binding and the action of PTH.
2. 동적광산란법(DLS; dynamic light scattering) 및 투과전자현미경(TEM; transmission electron microscope) 측정2. Dynamic light scattering (DLS) and transmission electron microscope (TEM) measurements
상기 PTH/LysDOCA 나노복합체의 입자 크기, 크기 분포, 표면 전하는 동적광산란법 분석기(DLS analyzer, Nano ZS, Malvern Instruments, UK)를 사용하여 측정하였으며, 상기 음성 염색(negative staining)된 PTH/LysDOCA 나노복합체의 형태적 평가는 투과전자현미경 (JEM-200, JEOL, Japan)을 통해 수행하였다.The particle size, size distribution, and surface charge of the PTH / LysDOCA nanocomposites were measured using a dynamic light scattering analyzer (DLS analyzer, Nano ZS, Malvern Instruments, UK), and the negative stained PTH / LysDOCA nanocomposites Morphological evaluation of was performed by transmission electron microscope (JEM-200, JEOL, Japan).
도 2는 rhPTH(1-34) 및 PTH/LysDOCA (1:10) 나노복합체의 입자 크기 분포와 투과전자현미경(TEM) 이미지이다. 도 2에서 보는 바와 같이, 상기 PTH/LysDOCA 나노복합체 입자의 직경은 33.3 nm이며 제타 전위(zeta potential)는 2.85이다. 상기 rhPTH 및 LysDOCA의 몰분율이 1 : 10(rhPTH : LysDOCA)일 때, 상기 rhPTH의 표면 음전하는 양전하의 LysDOCA(6.27)에 의해 중화되었고 제타 전위는 14.0으로 측정되었다. 이것은 rhPTH 사슬에 존재하는 최소한 다섯 개의 음전하를 띈 아미노산이 LysDOCA와 정전기적으로 상호작용 할 수 있다는 사실과 일치했다. 음성 염색(negative staining)된 상기 PTH/LysDOCA 나노복합체의 투과전자현미경 이미지는 rhPTH 자체가 다양한 크기의 응집을 형성하는 것과 달리 규칙적으로 잘 형성된 구형의 나노입자임을 확인하였다.2 is a particle size distribution and transmission electron microscope (TEM) images of rhPTH (1-34) and PTH / LysDOCA (1:10) nanocomposites. As shown in FIG. 2, the diameter of the PTH / LysDOCA nanocomposite particles is 33.3 nm and the zeta potential is 2.85. When the mole fraction of rhPTH and LysDOCA was 1: 10 (rhPTH: LysDOCA), the surface negative charge of rhPTH was neutralized by positively charged LysDOCA (6.27) and the zeta potential was determined to be 14.0. This is consistent with the fact that at least five negatively charged amino acids in the rhPTH chain can electrostatically interact with LysDOCA. Transmission electron microscopy images of the negatively stained PTH / LysDOCA nanocomposites confirmed that rhPTH itself is spherical nanoparticles that are regularly well formed, unlike the formation of aggregates of various sizes.
실시예 2: PTH/LysDOCA 나노복합체의 in vitro 생리학적 활성 평가Example 2: In vitro Physiological Activity Evaluation of PTH / LysDOCA Nanocomposites
MC3T3-E1 (전조골세포; Preosteoblast cell, subclone 4, ATCC)는 10% FBS α-MEM을 배지로 하여 5% CO2, 37℃의 조건에서 배양하였다. 세포는 96-well plate에 1x104 cells/well 만큼씩 첨가한 후 37℃에서 24시간 동안 배양하였다. 생장배지를 제거한 뒤 200 μL PBS (0.5 mM 아이소부틸메틸잔틴; IBMX; isobutylmethylxanthine)로 씻은 후, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM 농도로 PBS (0.5 mM IBMX)에 녹인 rhPTH와 PTH/LysDOCA를 100 μL/well씩 처리하였다. 37℃에서 1시간 동안 배양한 뒤 100 μL 0.1 M HCl/well을 처리하고 10분 동안 배양하여 세포를 용해시켰다. 이어서 상온에서 600 g으로 10분 동안 원심분리 하였고 각각의 상층액에 존재하는 cAMP의 양은 cAMP competitive ELISA kit (Thermo Scientific, USA)를 사용해 정량하였다.MC3T3-E1 (Preosteoblast cells, subclone 4, ATCC) were cultured at 10% FBS α-MEM at 5% CO 2 , 37 ° C. Cells were added to 96-well plates by 1 × 10 4 cells / well and incubated at 37 ° C. for 24 hours. After growth medium was removed and washed with 200 μL PBS (0.5 mM isobutylmethylxanthine; IBMX; isobutylmethylxanthine), rhPTH dissolved in PBS (0.5 mM IBMX) at 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM PTH / LysDOCA was treated at 100 μL / well. After incubation for 1 hour at 37 ℃ treated with 100 μL 0.1 M HCl / well and incubated for 10 minutes to lyse the cells. Subsequently, centrifugation was performed at 600 g for 10 minutes at room temperature, and the amount of cAMP present in each supernatant was quantified using a cAMP competitive ELISA kit (Thermo Scientific, USA).
도 3은 MC3T3-E1 세포를 상기 rhPTH(1-34) 또는 상기 PTH/LysDOCA (1:10) 나노복합체와 함께 배양한 후의 세포내 cAMP 함량이다. 각 값은 평균±표준편차(각 그룹 당 n=5)를 의미한다.FIG. 3 shows the intracellular cAMP content after MC3T3-E1 cells were incubated with the rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites. Each value represents the mean ± standard deviation (n = 5 for each group).
in vitro 생물학적 활성 평가는 MC3T3-E1 세포의 용해물(lysate)에 존재하는 cAMP 레벨을 측정함으로써 이루어졌다. PTH 수용체 발현 세포에서 상기 rhPTH와 PTH/LysDOCA (1:10) 나노복합체의 cAMP 생산 유도 능력을 평가하였다. 도 3을 확인하면, 0.01 μM 내지 0.1 μM 농도범위 내에서, 상기 PTH/LysDOCA (1:10) 나노복합체에 반응해 나타나는 PTH 수용체 매개 cAMP 생산은 상기 rhPTH에 의한 것과 비슷했다. 이는 도 1에서 나타난 온전한 형태의 2차 구조와 같은 맥락의 결과였다. 1.0 μM PTH/LysDOCA는 분화된 조골세포 내에서의 cAMP 촉진작용을 0.01 μM PTH/LysDOCA에 비해 5.7배 더 높게 유도하였다. 반면, 10 μM 또는 100 μM 농도의 rhPTH, PTH/LysDOCA를 처리하였을 때의 cAMP 수준은 1.0 μM에서보다 감소하였다.In vitro biological activity assessments were made by measuring cAMP levels present in the lysates of MC3T3-E1 cells. The ability of the rhPTH and PTH / LysDOCA (1:10) nanocomposites to induce cAMP production in PTH receptor expressing cells was evaluated. 3, within the concentration range of 0.01 μM to 0.1 μM, PTH receptor mediated cAMP production in response to the PTH / LysDOCA (1:10) nanocomposites was similar to that by rhPTH. This was the result of the same context as the intact secondary structure shown in FIG. 1.0 μM PTH / LysDOCA induced cAMP promotion in differentiated osteoblasts 5.7 times higher than 0.01 μM PTH / LysDOCA. On the other hand, cAMP levels of 10 μM or 100 μM concentrations of rhPTH and PTH / LysDOCA were lower than those at 1.0 μM.
실시예 3: 장관 세포막 투과도 평가Example 3: Intestinal Cell Membrane Permeability Evaluation
Caco-2 세포막에 대한 상기 rhPTH 또는 상기 PTH/LysDOCA의 투과도는 다음과 같이 평가되었다. Caco-2 세포를 Transwell filter insert (pore size 0.4 μm, surface area 1.12 cm2, Corning Coastar, USA)에 3×105 cells/mL의 농도로 처리한 후 배지(apical 0.5 mL, basolateral 1.5 mL)는 매 48시간 마다 교환하였으며 TEER 값이 >350 Ω·cm2인 세포 단층막(cell monolayer)을 실험에 사용하였다. 12-well transwell에서 21일 내지 29일 동안 세포를 배양한 뒤 filter를 사용해 배지를 제거하였고 세포단층막을 0.5 mL HBSS에서 37℃ 20분간 배양하였다. TEER를 다시 측정한 뒤 filter를 사용해 HBSS를 제거하였다. 이후 HBSS에 200 μM 농도로 용해한 rhPTH 또는 PTH/LysDOCA를 0.5 mL씩 filter에 처리하고 즉시 정측 구획(apical compartment)에서 C0 샘플을 취하였다. 37℃에서 배양하는 동안 0.5, 1, 2, 3, 4, 5시간째마다 기저측 구획(basolateral compartment)에서 200 μL씩 샘플을 취하였다. 샘플들은 membrane filter (0.45 μm, PVDF)를 사용해 여과하였고 분석을 위해 4℃에 보관하였다. 단층막을 통해 투과한 상기 rhPTH 또는 PTH/LysDOCA의 농도는 HPLC로 분석하였다. 샘플은 50 μL씩 분주하여 Kromasil C18 column (250×4.6 mm, 5 μm)이 장착된 HPLC에 주입하였다. 샘플들은 이동상(0.1% w/v trifluoroacetic acid(TFA) with 51%~76% linear gradient acetonitrile containing 0.1% w/v TFA)을 사용하여 1.0 mL/min의 유속으로 25분간 분리되었다. rhPTH와 PTH/LysDOCA의 피크(peak)는 배경 보정(제거)을 하여 220 nm에서 측정하였고, 세포막 투과도(Papp)는 다음의 방정식에 따라 계산하였다.Permeability of the rhPTH or PTH / LysDOCA to Caco-2 cell membrane was evaluated as follows. Caco-2 cells were treated with a transwell filter insert (pore size 0.4 μm, surface area 1.12 cm 2 , Corning Coastar, USA) at a concentration of 3 × 10 5 cells / mL, followed by media (apical 0.5 mL, basolateral 1.5 mL). The cells were changed every 48 hours and a cell monolayer having a TEER value of> 350 Ωcm 2 was used for the experiment. After incubating the cells for 21 to 29 days in a 12-well transwell, the medium was removed using a filter, and the monolayer membrane was incubated for 20 minutes at 37 ° C. in 0.5 mL HBSS. The TEER was measured again and HBSS was removed using a filter. After processing the rhPTH or PTH / LysDOCA dissolved in 200 μM concentration in HBSS to 0.5 mL by the filter in real positive side compartment (apical compartment) it was taken for sample C 0. Samples were taken at 200 μL in the basolateral compartment every 0.5, 1, 2, 3, 4, 5 hours during incubation at 37 ° C. Samples were filtered using a membrane filter (0.45 μm, PVDF) and stored at 4 ° C. for analysis. The concentration of rhPTH or PTH / LysDOCA permeated through the monolayer membrane was analyzed by HPLC. Samples were dispensed in 50 μL increments and injected into HPLC equipped with Kromasil C18 column (250 × 4.6 mm, 5 μm). Samples were separated for 25 minutes using a mobile phase (0.1% w / v trifluoroacetic acid (TFA) with 51% -76% linear gradient acetonitrile containing 0.1% w / v TFA) at a flow rate of 1.0 mL / min. Peaks of rhPTH and PTH / LysDOCA were measured at 220 nm with background correction (removal), and cell membrane permeability (P app ) was calculated according to the following equation.
Papp = dQ/dt x 1/(A x C0)P app = dQ / dt x 1 / ( A x C 0 )
(dQ/dt: 기저측 부분(basolateral side)의 linear appearance rate of mass (μmoL/h)(dQ / dt: linear appearance rate of mass (μmoL / h) of the basolateral side
C0: 정측 부분(apical side)의 rhPTH, PTH/LysDOCA 초기농도(μM)C 0 : Initial concentration of rhPTH and PTH / LysDOCA at the apical side (μM)
A: 단층막의 표면 면적 (cm2)) A : surface area of the monolayer film (cm 2 ))
| Test materialTest material | Apparent permeability (Papp, cm/s)a Apparent permeability (P app , cm / s) a |
| rhPTH (1-34)rhPTH (1-34) | 3.62 ± 0.45 (× 10-7)c,e 3.62 ± 0.45 (× 10 -7 ) c, e |
| PTH/LysDOCA (1:1) complexPTH / LysDOCA (1: 1) complex | 2.93 ± 1.09 (× 10-7)c,e 2.93 ± 1.09 (× 10 -7 ) c, e |
| PTH/LysDOCA (1:3) complexPTH / LysDOCA (1: 3) complex | 4.12 ± 0.76 (× 10-7)b,e 4.12 ± 0.76 (× 10 -7 ) b, e |
| PTH/LysDOCA (1:5) complexPTH / LysDOCA (1: 5) complex | 7.35 ± 1.12 (× 10-7)d 7.35 ± 1.12 (× 10 -7 ) d |
| PTH/LysDOCA (1:10) complexPTH / LysDOCA (1:10) complex | 10.4 ± 2.41 (× 10-7)10.4 ± 2.41 (× 10 -7 ) |
a; rhPTH(1-34)와 PTH/LysDOCA 나노복합체의 Caco-2 세포 단층막에 대한 Papp(apparent permeability coefficient). 각 값은 평균±표준편차(n=6)를 의미한다.a; Apparent permeability coefficient (P app ) for Caco-2 cell monolayer of rhPTH (1-34) and PTH / LysDOCA nanocomposites. Each value represents the mean ± standard deviation (n = 6).
b; p < 0.01 PTH/LysDOCA (1:5) 복합체와 비교b; p <0.01 compared to PTH / LysDOCA (1: 5) complex
c; p < 0.001 PTH/LysDOCA (1:5) 복합체와 비교c; p <0.001 compared with PTH / LysDOCA (1: 5) complex
d; p < 0.01 PTH/LysDOCA (1:10) 복합체와 비교d; p <0.01 compared with PTH / LysDOCA (1:10) complex
e; p < 0.001 PTH/LysDOCA (1:10) 복합체와 비교e; p <0.001 compared with PTH / LysDOCA (1:10) complex
통계: one-way ANOVA 후, Tukey’multiple-comparison testStatistics: After one-way ANOVA, Tukey’multiple-comparison test
상기 표 1에 나타낸 바와 같이 상기 rhPTH와 상기 PTH/LysDOCA의 Caco-2 세포 단층막에 대한 in vitro 투과도를 시험한 결과, 상기 rhPTH : LysDOCA의 몰분율이 1 : 1 내지 1 : 10까지 증가함에 따라, Papp는 2.93±1.09에서 10.4±2.41 (×10-7, cm/s)으로 증가했다. 상기 LysDOCA와 상기 rhPTH의 몰분율이 5이상으로 증가함에 따라, 상기 rhPTH의 투과도가 단독 rhPTH(free rhPTH)의 투과도에 비해 유의하게 개선되었다. 몰분율이 1 : 10인 PTH/LysDOCA에서의 장측-기저측(apical-to-basal) 방향으로의 Papp는 PTH 단독일 때에 비해 2.87배 증가하였다 [10.4±2.41 vs 3.62±0.45(×10-7, cm/s)]. 따라서 이후 랫드에서의 in vivo 장관흡수와 항-골다공증 효능을 확인하기 위해 PTH/LysDOCA (1:10)를 가지고 추가적인 연구를 수행하였다.As shown in Table 1, in vitro permeability of the rhPTH and PTH / LysDOCA to Caco-2 cell monolayer was tested, and as the mole fraction of rhPTH: LysDOCA was increased from 1: 1 to 1:10, P app increased from 2.93 ± 1.09 to 10.4 ± 2.41 (× 10 −7 , cm / s). As the molar fraction of LysDOCA and rhPTH increased to 5 or more, the permeability of rhPTH was significantly improved compared to that of free rhPTH (free rhPTH). Mole fraction is 1: 10 or in the long-side of the PTH / LysDOCA - basal side (apical-to-basal) P app in the direction was increased 2.87 fold when compared to PTH alone [10.4 ± 2.41 vs 3.62 ± 0.45 (× 10 -7 , cm / s)]. Therefore, further studies were performed with PTH / LysDOCA (1:10) to confirm the efficacy of in vivo intestinal absorption and anti-osteoporosis in rats.
실시예 4: 랫드에서 경구 투여 후 혈중 약물동력학적 실험Example 4 Blood Pharmacokinetic Experiments After Oral Administration in Rats
동물실험은 서울대학교 IACUC에서 승인하였으며 NIH guidelines for the Care and Use of Laboratory Animals와 IACUC guidelines에 따라 수행되었다. Sprague-Dawley 랫드 (암컷, 200~250 g)는 Orient Co., Ltd.(Korea)에서 구입하였다. 랫드는 케타민(ketamine, 45 mg/kg)과 자일라진(xylazine, 5 mg/kg)의 복강내주사로 마취시켰다. 마취된 랫드는 소장을 꺼내기 위해 복부를 절개하였고, 400 μL의 rhPTH (0.1 mg/kg) 또는 PTH/LysDOCA (rhPTH로서 0.1 mg/kg) 용액을 근위 공장(proximal jejunum)에 투여하였다. 상대적 생체이용률을 평가하기 위해 증류수에 녹인 150 μL의 rhPTH (0.02 mg/kg)를 별도로 피하주사 하였다. 약물 투여 후, 예정된 시간마다 혈액샘플 (200 μL)을 취하였고, 즉시 50 μL 시트르산나트륨(sodium citrate, 3.8%)용액과 혼합하였다. 이 샘플들은 2,500 x g, 4℃ 조건에서 15분 동안 원심분리 하였고 분리된 혈장샘플은 분석을 위해 70℃에 보관하였다. 혈장 중 rhPTH의 농도는 human PTH(1-34) ELISA kit (ALPCO Diagnostics, USA)를 사용해 620 nm 파장에서 측정하였다. 약동학적 파라미터들은 WinNonlin® Software(ver. 5.3; Pharsight Corporation, USA)을 사용하여 non-compartment method를 통해 추정하였다.Animal testing was approved by the Seoul National University IACUC and was performed according to the NIH guidelines for the Care and Use of Laboratory Animals and the IACUC guidelines. Sprague-Dawley rats (female, 200-250 g) were purchased from Orient Co., Ltd. (Korea). Rats were anesthetized with intraperitoneal injections of ketamine (45 mg / kg) and xylazine (5 mg / kg). Anesthetized rats were incised to excise the small intestine and 400 μL of rhPTH (0.1 mg / kg) or PTH / LysDOCA (0.1 mg / kg as rhPTH) solution was administered to the proximal jejunum. To evaluate relative bioavailability, 150 μL of rhPTH (0.02 mg / kg) dissolved in distilled water was separately injected subcutaneously. After drug administration, blood samples (200 μL) were taken at scheduled times and immediately mixed with 50 μL sodium citrate (3.8%) solution. The samples were centrifuged at 2,500 xg, 4 ° C for 15 minutes and the separated plasma samples were stored at 70 ° C for analysis. The concentration of rhPTH in plasma was measured at 620 nm wavelength using the human PTH (1-34) ELISA kit (ALPCO Diagnostics, USA). Pharmacokinetic parameters were estimated using a non-compartment method using WinNonlin ® Software (ver. 5.3; Pharsight Corporation, USA).
도 4는 랫드에서 상기 rhPTH(1-34)의 피하주사 (0.02 mg/kg)와 rhPTH(1-34)의 공장 내 투여 (0.1 mg/kg) 또는 상기 PTH/LysDOCA (1:10) 나노복합체 (rhPTH(1-34)로서 0.1 mg/kg)의 공장 내 투여 후 rhPTH(1-34)의 정맥 혈장 내 농도-시간 프로파일이다. 각 값은 평균±표준편차(각 그룹 당 n=4)를 의미한다. 도 4를 확인하면, 0.1 mg/kg rhPTH 공장 내 (intrajejunal) 투여 후 Cmax는 39.16±19.80 pg/mL였고 AUClast는 1,853.2±1,491.8 pg·min/mL이었으며 피하주사와 비교한 상대 생체이용률은 1.06%±0.85%였다. 반면, 표 2에서 보는 바와 같이, PTH/LysDOCA (1:10) 나노복합체 (rhPTH로서 0.1 mg/kg)를 공장 내 투여한 경우 PTH 단독으로 공장 내 투여한 경우와 비교했을 때 Cmax는 11.16배 (437.1±107.2 pg/mL), AUClast는 16.26배 (30,133,243pg·min/mL), 피하주사와 비교한 상대 생체이용률은 16.20배 (17.2%±6.41%) 증가하였다. 따라서 랫드에서 rhPTH의 장관 투과도와 흡수는 LysDOCA와의 복합체 형성을 통해 유의하게 증가되었다. 게다가 Tmax는 PTH 피하주사에서 15.0, PTH 공장 내 투여에서 30.0±0.0, PTH/LysDOCA의 공장 내 투여에서 35.0분이었으며 PTH/LysDOCA 공장 내 투여 후의 혈장농도는 120분에 이르기까지 정량을 위한 하한점(lower limit)보다 높게 나타났다.Figure 4 shows the subcutaneous injection of rhPTH (1-34) and rhPTH (1-34) in the rat (0.1 mg / kg) or the PTH / LysDOCA (1:10) nanocomposite in rats. Intravenous concentration-time profile of rhPTH (1-34) after in-plant administration of (0.1 mg / kg as rhPTH (1-34)). Each value represents the mean ± standard deviation (n = 4 for each group). 4, the C max was 39.16 ± 19.80 pg / mL after the 0.1 mg / kg rhPTH intrajejunal administration and the AUC last was 1,853.2 ± 1,491.8 pg · min / mL, and the relative bioavailability compared to subcutaneous injection was 1.06. % ± 0.85%. On the other hand, as shown in Table 2, when the PTH / LysDOCA (1:10) nanocomposite (0.1 mg / kg as rhPTH) was administered in-plant, C max was 11.16 times compared to the in-plant administration of PTH alone. (437.1 ± 107.2 pg / mL), AUC last was 16.26 times (30,133,243 pgmin / mL), and relative bioavailability was 16.20 times (17.2% ± 6.41%) compared with subcutaneous injection. Therefore, the intestinal permeability and absorption of rhPTH in rats were significantly increased by complex formation with LysDOCA. In addition, T max was 15.0 for PTH subcutaneous injection, 30.0 ± 0.0 for in-plant PTH administration, 35.0 minutes for in-plant administration of PTH / LysDOCA, and plasma concentrations after administration in PTH / LysDOCA plant reached 120 min. (lower limit).
| Test materialTest material | rhPTH (1-34)rhPTH (1-34) | rhPTH (1-34)rhPTH (1-34) | PTH/LysDOCA (1:10) nanocomplexPTH / LysDOCA (1:10) nanocomplex |
| AdministrationAdministration | SubcutaneousSubcutaneous | IntrajejunalIntrajejunal | IntrajejunalIntrajejunal |
| Dose of rhPTH (1-34) (mg/kg)Dose of rhPTH (1-34) (mg / kg) | 0.020.02 | 0.10.1 | 0.10.1 |
| Tmax a(min)T max a (min) | 15 ± 0.0015 ± 0.00 | 30 ± 0.0030 ± 0.00 | 35 ± 8.735 ± 8.7 |
| Cmax b(pg/mL)C max b (pg / mL) | 710.8 ± 97.75710.8 ± 97.75 | 39.16 ± 19.8039.16 ± 19.80 | 437.1 ± 107.2437.1 ± 107.2 |
| AUClast c(pgmin/mL)AUC last c (pgmin / mL) | 35,108 ± 9,077.735,108 ± 9,077.7 | 1,853.2 ± 1,491.81,853.2 ± 1,491.8 | 30,133 ± 11,24330,133 ± 11,243 |
| AUCinf d(pgmin/mL)AUC inf d (pgmin / mL) | 36,161 ± 9,843.936,161 ± 9,843.9 | 2,688.9 ± 1,299.62,688.9 ± 1,299.6 | 30,790 ± 11,21930,790 ± 11,219 |
| Bioavailabilitye(%)Bioavailability e (%) | 100100 | 1.06 ± 0.851.06 ± 0.85 | 17.2 ± 6.4117.2 ± 6.41 |
a; Tmax, Cmax에 도달한 시간,a; T max , C max time,
b; Cmax, 최대혈장농도,b; C max , maximum plasma concentration,
c; AUClast, 0에서 마지막 혈장농도 측정시간까지의 혈장농도-시간 곡선 아래 면적,c; AUC last , the area under the plasma concentration-time curve from 0 to the last measurement of plasma concentration,
d; AUCinf, 0에서 무한대까지의 혈장농도-시간 곡선 아래 면적,d; AUC inf , area under the plasma concentration-time curve from 0 to infinity,
e; 생체이용률, (AUClast,
intrajejunal/DoserhPTH
(1-34),
intrajejunal)/(AUClast
, subcutaneous/DoserhPTH (1-34), subcutaneous) ×100. 각 값은 평균±표준편차(n=4)e; Bioavailability, (AUC last , intrajejunal / Dose rhPTH (1-34), intrajejunal ) / (AUC last , subcutaneous / Dose rhPTH (1-34), subcutaneous ) × 100. Each value is mean ± standard deviation (n = 4)
실시예 5: PTH/LysDOCA의 장용성 미립자 특성평가Example 5: Enteric Particulate Characterization of PTH / LysDOCA
1. PTH/LysDOCA의 장용성 미립자 입자 크기, 표면 형태 및 분포 분석1. Analysis of Enteric Particle Particle Size, Surface Morphology and Distribution of PTH / LysDOCA
상기 제조된 PTH/LysDOCA의 장용성 미립자의 크기를 레이저 회절 입자 크기 분석기(laser diffraction particle size analyzer, Mastersizer X, Malvern Instruments Ltd., UK)를 사용해 측정하였다. 또한 상기 제조된 PTH/LysDOCA의 장용성 미립자의 표면 형태 및 분포를 주사전자현미경(SEM; Scanning Electron Microscopy, JSM-5300, JEOL, Japan) 및 공초점 레이저 주사 현미경(argon/HeNe laser and a Zeiss Axio Observer inverted microscope; JSM 710 NLO and LSM 780 NLO, Carl Zeiss Microscopy GmbH, Germany)을 사용해 측정하였다. FITC-dextran과 RITC의 공초점 형광 이미지는 20×대물렌즈와 각각 488 nm, 568 nm의 여기(excitation) 파장을 사용해 측정하였다.The size of the enteric particulates of the prepared PTH / LysDOCA was measured using a laser diffraction particle size analyzer (Mastersizer X, Malvern Instruments Ltd., UK). In addition, the surface morphology and distribution of the prepared enteric fine particles of PTH / LysDOCA were analyzed by scanning electron microscope (SEM; Scanning Electron Microscopy, JSM-5300, JEOL, Japan) and a confocal laser scanning microscope (argon / HeNe laser and a Zeiss Axio Observer). Inverted microscope; JSM 710 NLO and LSM 780 NLO, Carl Zeiss Microscopy GmbH, Germany). Confocal fluorescence images of FITC-dextran and RITC were measured using 20 × objective lenses and excitation wavelengths of 488 nm and 568 nm, respectively.
도 5는 PTH/LysDOCA (1:10) 나노복합체를 함유하는 장용성 미립자의 주사전자현미경(SEM)과 공초점 레이저 주사 현미경(CLSM; confocal laser scanning microscopy) 이미지이다. Eudragit L100-55 고분자 물질은 로다민아이소티오시아네이트(RITC; rhodamine isothiocyanate, 빨간색)와 혼합되었으며, PTH/LysDOCA (1:10) 나노복합체를 함유하는 중심(core)은 플로오레세인아이소티오시아네이트-표지 덱스트란(FITC; fluorescein isothiocyanate-labeled dextran, 녹색)과 혼합되었다.FIG. 5 is a scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) image of enteric particulates containing PTH / LysDOCA (1:10) nanocomposites. Eudragit L100-55 polymer was mixed with rhodamine isothiocyanate (RTC), the core containing the PTH / LysDOCA (1:10) nanocomposite is fluorescein isothiocyanate Mixed with labeled dextran (FITC) (green, fluorescein isothiocyanate-labeled dextran, green).
상기 rhPTH의 경구 투여용 제형을 위해 이중축 초음파 미립화를 이용해 상기 PTH/LysDOCA 나노복합체를 함유하는 장용성 미립자를 제조하였다. 장용성 미립자를 코어-쉘 구조를 디자인하기 위해 먼저 외측 유속(outer flow rate) (장용성 고분자 물질 용액)을 고정한 상태로 내측 유속(inner flow rate) (약물 용액)을 다양하게 변화시켜 보았다. 내측 유속이 0.5 mL/min보다 작았을 때 더 균질한 미세입자가 얻어졌다. 내측 유속이 0.6 mL/min을 초과할 때에는 장용성 미립자의 코어-쉘 구조를 형성하지 않았다. 뿐만 아니라, 교반 속도가 800 rpm 이상일 때 매끄러운 표면을 갖는 보다 균일한 형태의 미립자가 형성되었다. 도 5를 살펴보면, 최적의 조건 하에서 상기 PTH/LysDOCA의 장용성 미립자는 약 80%의 효율로 캡슐화 되었고 입자의 평균 지름은 60.2 μm로 나타났다. 공초점 이미지 역시 대부분의 PTH/LysDOCA가 립자의 중심(core)에 자리 잡고 있으며 장용성 고분자 물질의 쉘에 의해 균질하게 캡슐화 되었다는 것을 시사했다.Enteric fine particles containing the PTH / LysDOCA nanocomposites were prepared using dual axis ultrasonic atomization for oral administration of the rhPTH. In order to design the enteric particulate core-shell structure, the inner flow rate (drug solution) was variously changed with the outer flow rate (enteric polymer solution) fixed. More homogeneous microparticles were obtained when the inner flow rate was less than 0.5 mL / min. When the inner flow rate exceeded 0.6 mL / min, no core-shell structure of the enteric particulates was formed. In addition, more uniform forms of fine particles with a smooth surface were formed when the stirring speed was 800 rpm or higher. Referring to FIG. 5, under optimum conditions, the enteric fine particles of PTH / LysDOCA were encapsulated with an efficiency of about 80% and the average diameter of the particles was 60.2 μm. Confocal images also suggest that most PTH / LysDOCA are located at the core of the particles and are homogeneously encapsulated by a shell of enteric polymer.
2. PTH/LysDOCA의 장용성 미립자로부터 약물의 용출시험2. Dissolution of Drugs from Enteric Fine Particles of PTH / LysDOCA
상기 제조된 PTH/LysDOCA의 장용성 미립자로부터 약물의 용출시험은 0.1 N HCl (pH 1.2) 또는 phosphate buffer (pH 6.8)를 함유하는 500 mL의 배지를 가지고 37℃±0.2℃에서 수행하였고, 100 rpm의 USP type 1 (basket) 장치로 시험하였다. 상기 rhPTH 또는 PTH/LysDOCA를 함유하는 장용성 미립자는 경질 젤라틴 캡슐(hard gelatin capsule)로 캡슐화 하였다. 각각의 캡슐은 rhPTH로서 50 mg을 함유하도록 하였다. 용출 시험 시작 후 15, 30, 45, 60, 90, 120분째에 3 mL의 샘플을 취하였다. 필터과정을 거친 후, 용출시험액에 방출된 상기 rhPTH 또는 PTH/LysDOCA의 양은 HPLC-UV detector를 사용해 상기한 기술한 대로 측정하였다.The dissolution test of the drug from the enteric fine particles of the prepared PTH / LysDOCA was performed at 37 ℃ ± 0.2 ℃ with 500 mL of medium containing 0.1 N HCl (pH 1.2) or phosphate buffer (pH 6.8), 100 rpm Tested with USP type 1 (basket) device. The enteric microparticles containing rhPTH or PTH / LysDOCA were encapsulated in hard gelatin capsules. Each capsule was to contain 50 mg as rhPTH. 3 mL samples were taken 15, 30, 45, 60, 90, 120 minutes after the start of the dissolution test. After filtering, the amount of rhPTH or PTH / LysDOCA released in the elution test solution was measured using the HPLC-UV detector as described above.
도 6은 pH 1.2와 pH 6.8의 용출 시험액에서 상기 장용성 미립자로부터 rhPTH(1-34) 또는 PTH/LysDOCA (1:10) 나노복합체의 in vitro 누적 약물 방출률을 나타낸다. 각 값은 평균±표준편차(각 그룹 당 n=6)을 의미한다.FIG. 6 shows the in vitro cumulative drug release rate of rhPTH (1-34) or PTH / LysDOCA (1:10) nanocomposites from the enteric particulates in elution test solutions at pH 1.2 and pH 6.8. Each value represents the mean ± standard deviation (n = 6 in each group).
도 6을 살펴보면, pH 1.2의 용출시험액에서는 장용성 미립자는 120분 동안 붕해되거나 약물이 방출되지 않았다. 그러나 pH 6.8의 용출 시험액에서는 120분 동안 90% 이상의 rhPTH 또는 PTH/LysDOCA가 장용성 미립자로부터 완전히 방출되었다.6, in the elution test solution of pH 1.2, the enteric particulates did not disintegrate or release the drug for 120 minutes. However, in the dissolution test solution at pH 6.8, more than 90% of rhPTH or PTH / LysDOCA was completely released from the enteric particulates for 120 minutes.
실시예 6: 난소 적출에 의한 골다공증 유발 랫드 모델(OVX(ovariectomized) rat model)에서 경구 투여 후 미세-컴퓨터단층촬영(μ-CT scanning)과 정강이뼈(tibiae)의 조직학적 관찰Example 6 Histological Observation of Micro-CT Scanning and Tibiae After Oral Administration in Ovarian Extraction-Induced Osteoctomized Rat Model (OVX)
경구 투여된 상기 rhPTH와 PTH/LysDOCA의 골다공증 치료 효과는 60마리의 Sprague-Dawley 랫드 (암컷, 생후 8주 개체)에서 평가되었다. 골다공증 유발을 위해 50마리의 랫드의 양측 난소를 절제하였으며 난소적출 없는 플라세보 수술(SHAM)이 10마리의 랫드에서 수행되었다. 2주 후, 난소 절제한 랫드들은 무작위로 하기의 5개 그룹(각 8마리씩)으로 나누었다.Osteoporosis treatment effects of orally administered rhPTH and PTH / LysDOCA were evaluated in 60 Sprague-Dawley rats (female, 8 weeks old). Bilateral ovaries of 50 rats were excised to induce osteoporosis and placebo surgery without ovarian extraction (SHAM) was performed on 10 rats. Two weeks later, ovarian-dissected rats were randomly divided into the following five groups (eight of each).
① OVX (약물투여 안함)① OVX (Drug not administered)
② OVX-PTH-SC (1일 1회, 10 μg/kg rhPTH 피하주사)② OVX-PTH-SC (Once a day, 10 μg / kg rhPTH subcutaneous injection)
③ OVX-PTH-ORAL (1일 1회, 200 μL 3% Mg(OH)2 제산제 투여 후, 50 μg/kg rhPTH 경구투여)③ OVX-PTH-ORAL (Once administered 200 μL 3% Mg (OH) 2 antacid once a day, orally administered 50 μg / kg rhPTH)
④ OVX-PTH/LysDOCA-ORAL (1일 1회, 200 μL 3% Mg(OH)2 제산제 투여 후, rhPTH로서 50 μg/kg rhPTH/LysDOCA 경구투여)OVX-PTH / LysDOCA-ORAL (Once administered once daily, 200 μL 3% Mg (OH) 2 antacid, 50 μg / kg rhPTH / LysDOCA as rhPTH)
⑤ OVX-PTH/LysDOCA-MP-ORAL (1일 1회, rhPTH로서 50 μg/kg PTH/LysDOCA 함유 장용성 미립자 경구투여)⑤ OVX-PTH / LysDOCA-MP-ORAL (Orally administered enteric particulate containing 50 μg / kg PTH / LysDOCA as rhPTH once a day)
약물 투여기간 동안 약물의 용량은 매일 체중에 맞게 조절하였다. 투여가 시작된 지 12주 후 랫드에서 좌·우 정강이뼈(tibiae)를 분리하였고 neutral buffered 4% 포르말린(formalin)으로 고정하였다.During the drug administration, the dose of the drug was adjusted to the body weight daily. Twelve weeks after the start of administration, the left and right tibiae were isolated from rats and fixed with neutral buffered 4% formalin.
정강이뼈는 그것의 미세구조를 평가하고 구조적 파라미터들과 골밀도(BMD)를 측정하기 위해 In-
vivo μ-CT (Skyscan 1076, Bruker Corp., Germany)를 사용해 스캔하였다. X-ray source 세팅은 100kV, 100 μA에 1.0-mm-thick 알루미늄 필터를 통한 beam filtration이었다. 0°에서 360°까지 매 0.5°의 회전단계마다 35mm width ×17mm height 크기의 2D 영상(projection)이 기록되었다. 3D 미세구조의 이미지는 modified Feldkamp back-projection 알고리즘을 사용하여 재구성되었다. 재구성 후 3D 이미지는 무기질 상(mineral phase)을 추출하기 위해 low-pass 필터 (Gaussian filter)되었고 0.0에서 0.04 cross-section의 고정된 범위에서 global thresholding되었다. 해면골(trabecular bone)은 CT-Analyzer 1.13 (Bruker Corp., Germany)를 사용해 semiautomatic contouring함으로써 전체 이미지에서 추출하였다. 정강이뼈의 자세한 분석과 정량을 위해 정강이뼈의 원위부 골간단(distal metaphysis)을 VOI(volume of interest)로 정하였다. VOI는 정강이뼈에 있는 근위부 성장판(proximal growth plate)으로부터 0.54 mm 아래로 1.8 mm이다. 하기의 구조적 파라미터들이 계산되었다.Tibia was scanned using a In- vivo μ-CT (Skyscan 1076 , Bruker Corp., Germany) to assess its microstructure, and measuring the structural parameters and the bone mineral density (BMD). The X-ray source setting was beam filtration through a 1.0-mm-thick aluminum filter at 100 kV, 100 μA. A 2D projection of 35mm width x 17mm height was recorded at every 0.5 ° rotational step from 0 ° to 360 °. Images of 3D microstructures were reconstructed using the modified Feldkamp back-projection algorithm. After reconstruction, the 3D image was a low-pass filter (Gaussian filter) to extract the mineral phase and globally thresholded over a fixed range of 0.0 to 0.04 cross-section. Trabecular bone was extracted from the entire image by semiautomatic contouring using CT-Analyzer 1.13 (Bruker Corp., Germany). For the detailed analysis and quantification of the shin bone, the distal metaphysis of the shin bone was defined as the volume of interest (VOI). VOI is 1.8 mm below 0.54 mm from the proximal growth plate in the tibia. The following structural parameters were calculated.
① BMD① BMD
② BV/TV (bone volume fraction)② BV / TV (bone volume fraction)
③ BS/BV (bone surface/volume ratio)③ BS / BV (bone surface / volume ratio)
④ Tb.Th (trabecular thickness)④ Tb.Th (trabecular thickness)
⑤ Tb.Sp (trabecular separation)⑤ Tb.Sp (trabecular separation)
⑥ Tb.N (trabecular number)⑥ Tb.N (trabecular number)
⑦ Tb.Pf (trabecular pattern factor)⑦ Tb.Pf (trabecular pattern factor)
⑧ SMI (structural model index)⑧ SMI (structural model index)
정강이뼈의 골간(diaphysis)에 있는 골피질 또한 평가하였다. 골피질의 분석을 위한 VOI는 정강이뼈에 있는 해면골의 VOI가 끝나는 지점으로부터 0.54 mm 아래로 0.9 mm이다. 골피질에 대해서도 하기와 같은 구조적 파라미터들이 계산되었다:The cortex in the diaphysis of the tibia was also evaluated. The VOI for the analysis of the cortex is 0.9 mm below 0.54 mm from the end of the VOI of the spongy bone in the tibia. The following structural parameters were also calculated for the cortex:
① Ct.BMD (cortical bone mineral density)① Ct.BMD (cortical bone mineral density)
② Ct.BV (cortical bone volume)② Ct.BV (cortical bone volume)
③ Ct.Th (cortical thickness)③ Ct.Th (cortical thickness)
④ MMI(polar) (mean polar moment of inertia)④ MMI (polar) (mean polar moment of inertia)
조직학적 관찰을 위해, 포르말린으로 고정된 정강이뼈는 10% EDTA에서 탈칼슘화시켰고 단계적 에탄올(graded ethanol) 처리를 통해 탈수시켰다. 그 후 파라핀 왁스 블록(paraffin wax block)에 담아 5 μm 두께로 절단하였으며 헤마톡실린(hematoxylin)과 에오신(eosin) (H&E)으로 염색하였다. 조직학적 절편들은 광학현미경 (Bx41, Olympus, Japan)으로 관찰하였고 성장판의 중앙부분에 초점을 맞춰 사진 촬영하였다.For histological observation, formalin-fixed tibia was decalcified in 10% EDTA and dehydrated through graded ethanol treatment. After that, it was placed in a paraffin wax block, cut into 5 μm thickness, and stained with hematoxylin and eosin (H & E). Histological sections were observed under an optical microscope (Bx41, Olympus, Japan) and photographed with focus on the center of the growth plate.
폐경 후 골다공증 랫드 모델에서의 in vivo 치료적 효능을 평가하였다. 해면골 미세구조의 특성을 탐구하고 형태학적 파라미터들을 계산하기 위해서 in vivo μ-CT 측정을 수행했다. 도 7을 살펴보면, SHAM(플라세보 수술) 그룹과 비교해서, OVX 대조군은 유의한 수준으로In vivo therapeutic efficacy in postmenopausal osteoporotic rat models was evaluated. In vivo μ-CT measurements were performed to explore the properties of cavernous bone microstructure and to calculate morphological parameters. Looking at Figure 7, compared to SHAM (placebo surgery) group, OVX control group to a significant level
① BMD (0.1046±0.0391 vs 0.4885±0.0414 g/cm3)① BMD (0.1046 ± 0.0391 vs 0.4885 ± 0.0414 g / cm 3 )
② BV/TV (4.3508%±0.9652% vs 31.0642%±2.6533%)② BV / TV (4.3508% ± 0.9652% vs 31.0642% ± 2.6533%)
③ Tb.Th (0.0721±0.0047 vs 0.0814±0.0061 mm)③ Tb.Th (0.0721 ± 0.0047 vs 0.0814 ± 0.0061 mm)
④ Tb.N (0.5545±0.1530 vs 3.7966±0.3036 mm-1) 감소와④ Tb.N (0.5545 ± 0.1530 vs 3.7966 ± 0.3036 mm -1 ) decrease and
유의한 수준의Significant level
① BS/BV (52.3512±4.3507 vs 40.9926±4.0598 mm-1),① BS / BV (52.3512 ± 4.3507 vs 40.9926 ± 4.0598 mm −1 ),
② Tb.Sp (0.9234±0.1556 vs 0.1798±0.010 mm), ② Tb.Sp (0.9234 ± 0.1556 vs 0.1798 ± 0.010 mm),
③ Tb.Pf (17.2727±1.1483 vs 2.5354±2.7166 mm-1), ③ Tb.Pf (17.2727 ± 1.1483 vs 2.5354 ± 2.7166 mm −1 ),
④ SMI (2.3973±0.0899 vs 1.1430±0.2227) 증가를 보였다.④ SMI (2.3973 ± 0.0899 vs 1.1430 ± 0.2227) was increased.
그러나 OVX 랫드에서의 BMD, BV/TV, Tb.Th, Tb.N 감소는 OVX-PTH-SC 그룹과 OVX-PTH/LysDOCA-MP-ORAL 그룹에서 유의한 수준으로 억제되었다. 도 7의 A에서 볼 수 있듯이, 12주째에 OVX-PTH/LysDOCA-MP-ORAL 그룹 (rhPTH로서 50 μg/kg 투여)은 OVX 대조군보다 높은 BMD (0.1666±0.0293 vs 0.1046±0.0391 g/cm3) (159%)를 보였다. 반면, OVX-PTH-ORAL 그룹에선 OVX rat에서 관찰된 BMD의 감소가 유의한 수준으로 억제되지 않았다.However, BMD, BV / TV, Tb.Th and Tb.N reductions in OVX rats were significantly inhibited in OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups. As can be seen in FIG. 7A, at week 12 the OVX-PTH / LysDOCA-MP-ORAL group (administered 50 μg / kg as rhPTH) had higher BMD (0.1666 ± 0.0293 vs 0.1046 ± 0.0391 g / cm 3 ) than the OVX control. (159%). In contrast, in the OVX-PTH-ORAL group, the decrease in BMD observed in OVX rats was not significantly suppressed.
OVX-PTH/LysDOCA-MP-ORAL 그룹은 OVX-PTH-SC 그룹과 유사하게 해면골 미세구조 파라미터에서도 유의한 수준의 개선을 보였다: OVX 대조군에 비해 BV/TV에서 175% (7.6194%±1.4202%), Tb.N에서 174% (0.9673±0.1968 mm-1). 반면, OVX-PTH-ORAL 그룹에선 OVX 랫드에서 관찰된 BV/TV, Tb.N의 감소가 유의한 수준으로 억제되지 않았다. 게다가 OVX 랫드에서의 Tb.Sp, Tb.Pf, SMI 증가가 OVX-PTH-SC 그룹과 OVX-PTH/LysDOCA-MP-ORAL 그룹에선 유의한 수준으로 억제되었지만, OVX-PTH-ORAL 그룹에선 그렇지 않았다.The OVX-PTH / LysDOCA-MP-ORAL group showed significant improvement in the sponge bone microstructural parameters similar to the OVX-PTH-SC group: 175% (7.6194% ± 1.4202%) in BV / TV compared to the OVX control group. , 174% (0.9673 ± 0.1968 mm −1 ) at Tb.N. On the other hand, in the OVX-PTH-ORAL group, the decreases in BV / TV and Tb.N observed in OVX rats were not suppressed to a significant level. In addition, Tb.Sp, Tb.Pf, and SMI increases in OVX rats were significantly inhibited in OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups, but not in OVX-PTH-ORAL groups. .
정강이뼈의 중간축 부분(midshaft region)에 있는 골피질 평가에는 μ-CT가 사용되었고, 도 8에 결과를 나타내었다. 약물투여 12주 후 OVX 대조군의 모든 파라미터들은 SHAM 그룹에 비해 유의한 수준으로 감소하였다. 97% 낮은 Ct.BMD, 88% 낮은 Ct.BV, 96% 낮은 Ct.Th, 77% 낮은 MMI(polar), 반대로, OVX-PTH/LysDOCA-MP-ORAL 그룹에선 OVX control 그룹에 비해 각각 103%, 114%, 107%, 131% 높은 Ct.BMD, Ct.BV, Ct.Th, MMI(polar)를 보였다. OVX-PTH-ORAL 그룹과 비교했을 땐 OVX-PTH/LysDOCA-MP-ORAL 그룹에서 각각 103%, 115%, 109%, 135% 높은 Ct.BMD, Ct.BV, Ct.Th, MMI(polar)를 보였다. OVX-PTH-SC 그룹 역시 OVX 대조군에 비해 102% 높은 Ct.BMD, 118% 높은 Ct.BV, 110% 높은 Ct.Th, 132% 높은 MMI(polar)를 보였으며 모든 파라미터들의 값이 OVX-PTH/LysDOCA-MP-ORAL 그룹에서와 비슷하였다.Μ-CT was used to evaluate the cortex in the midshaft region of the tibia, and the results are shown in FIG. 8. After 12 weeks of drug administration, all the parameters of the OVX control group decreased to a significant level compared to the SHAM group. 97% lower Ct.BMD, 88% lower Ct.BV, 96% lower Ct.Th, 77% lower MMI (polar), in contrast, 103% in the OVX-PTH / LysDOCA-MP-ORAL group compared to the OVX control group. , Ct.BMD, Ct.BV, Ct.Th and MMI (polar) were high. Compared to OVX-PTH-ORAL group, 103%, 115%, 109%, 135% higher Ct.BMD, Ct.BV, Ct.Th, MMI (polar) in OVX-PTH / LysDOCA-MP-ORAL group, respectively Showed. The OVX-PTH-SC group also showed 102% higher Ct.BMD, 118% higher Ct.BV, 110% higher Ct.Th, 132% higher MMI (polar) than the OVX control group. Similar to / LysDOCA-MP-ORAL group.
대표적인 3D μ-CT 재구성과 근위부 정강이뼈(proximal tibia)를 가로지르는 종단 단면도(longitudinal cross-sections)는 도 9에 나타나있다. SHAM 그룹에서 관찰된 자연적인 뼈의 성장과 대조적으로, OVX 대조군에선 해면골 구조의 쇠퇴가 관찰되었다. 그러나 OVX-PTH-SC 그룹과 OVX-PTH/LysDOCA-MP-ORAL 그룹에선 OVX 대조군이나 OVX-PTH-ORAL 그룹에서보다 치밀한 근위부 정강이뼈의 해면골 미세구조가 나타났으며 이는 rhPTH의 피하주사나 PTH/LysDOCA 함유 장용성 미립자의 경구투여에 기인한 보다 큰 동화작용이 있음을 시사한다.Representative 3D μ-CT reconstructions and longitudinal cross-sections across the proximal tibia are shown in FIG. 9. In contrast to the natural bone growth observed in the SHAM group, the decline in spongy bone structure was observed in the OVX control group. However, in the OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups, there was a more fine spongy bone structure of the proximal tibia than in the OVX control group or the OVX-PTH-ORAL group. It suggests that there is a greater assimilation due to oral administration of LysDOCA containing enteric particulates.
도 10을 살펴보면, 동일한 경향이 H&E로 염색한 정강이뼈의 조직학적 절편(histological section)에서도 관찰되었다. SHAM 그룹은 붕괴된 형태의 해면골 연결성(connectivity)을 보인 OVX 대조군과 대조적으로, 치밀하고 균일한 형태의 해면골을 보였다. OVX-PTH-SC 그룹과 OVX-PTH/LysDOCA-MP-ORAL 그룹은 OVX 대조군과 OVX-PTH-ORAL 그룹에서보다 증가된 구조적 온전함과 보다 치밀한 해면골 미세구조를 보였다.Referring to Figure 10, the same trend was observed in the histological section of the shank bone stained with H & E. The SHAM group showed a dense and uniform form of cavernous bone, in contrast to the OVX control group, which showed collapsed cavernous connectivity. The OVX-PTH-SC and OVX-PTH / LysDOCA-MP-ORAL groups showed increased structural integrity and denser spongy bone microstructure than the OVX control and OVX-PTH-ORAL groups.
Claims (11)
- PTH(부갑상선 호르몬) 및 담즙산 유도체가 포함된 나노복합체를 장용성 물질로 코팅한 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물Formulation composition for oral administration of parathyroid hormone characterized in that the nanocomposite containing PTH (parathyroid hormone) and bile acid derivatives is coated with an enteric material
- 제 1항에 있어서, 상기 PTH는 PTH(1-28), PTH(1-31), PTH(1-34), PTH(1-38) 및 PTH(1-41)로 이루어진 군에서 어느 하나로 선택된 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물According to claim 1, wherein the PTH is selected from any one of the group consisting of PTH (1-28), PTH (1-31), PTH (1-34), PTH (1-38) and PTH (1-41) Formulation composition for oral administration of parathyroid hormone
- 제 2항에 있어서, 상기 PTH(1-34)는 rhPHT(1-34) 또는 hPTH(1-34)인 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물According to claim 2, wherein the PTH (1-34) is rhPHT (1-34) or hPTH (1-34) formulation composition for oral administration of parathyroid hormone, characterized in that
- 제 1항에 있어서, 상기 담즙산 유도체는 데옥시콜산 유도체인 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물The preparation composition for oral administration of parathyroid hormone according to claim 1, wherein the bile acid derivative is a deoxycholic acid derivative.
- 제 1항에 있어서, 상기 담즙산 유도체는 데옥시콜산에 라이신이 화학적으로 결합된 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물According to claim 1, wherein the bile acid derivative is a composition for oral administration of parathyroid hormone, characterized in that lysine is chemically bonded to deoxycholic acid.
- 제 1항에 있어서, 상기 PTH에 상기 담즙산 유도체가 1:1 내지 1:10의 몰비율로 결합된 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물The preparation composition for oral administration of parathyroid hormone according to claim 1, wherein the bile acid derivative is bound to the PTH in a molar ratio of 1: 1 to 1:10.
- 제 1항에 있어서, 상기 장용성 물질은 쉘락, 유드라짓(Eudragit : 메타크릴산-에틸 아크릴레이트 공중합체), 하이드록시프로필메칠셀룰로오스 프탈레이트, 아세틸호박산하이드록시프로필메칠셀룰로오스, 셀룰로오스 아세테이트 프탈레이트로 이루어진 군에서 선택된 하나 또는 둘 이상인 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물The method of claim 1, wherein the enteric material is a group consisting of shellac, Eudragit (Eudragit: methacrylate-ethyl acrylate copolymer), hydroxypropyl methyl cellulose phthalate, acetyl hydroxypropyl methyl cellulose, cellulose acetate phthalate Formulation composition for oral administration of parathyroid hormone, characterized in that one or more selected from
- 제 1항에 있어서, 상기 나노복합체는 입자크기가 10 nm 내지 50nm인 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물According to claim 1, wherein the nanocomposite formulation composition for oral administration of parathyroid hormone, characterized in that the particle size of 10 nm to 50 nm.
- 1) PTH와 데옥시콜산 유도체의 화학적 결합에 의한 나노복합체(PTH/데옥시콜산 유도체)를 형성하는 단계; 1) forming a nanocomposite (PTH / deoxycholic acid derivative) by chemical bonding of PTH and deoxycholic acid derivative;2) 상기 나노복합체를 물에 용해시킨 후 장용성 물질이 용해된 용액에 분산시켜 현탁액을 제조하는 단계; 2) preparing a suspension by dissolving the nanocomposite in water and then dispersing it in a solution in which the enteric material is dissolved;3) 상기 현탁액을 시린지 펌프로 이중축 초음파 분사 노즐의 내측 주입구로 이동시키는 단계; 3) moving the suspension to the inner inlet of the dual axis ultrasonic spray nozzle with a syringe pump;4) 이중측 초음파 분사 노즐의 외측 주입구로 장용성 물질 용액을 이동시키는 단계; 4) moving the enteric material solution to the outer inlet of the double side ultrasonic spray nozzle;5) 초음파 분사노즐의 진동분사에 의해 노즐 내측 약물 용액이 외측 장용성 물질 용액으로 둘러싸인 미세액적을 형성하는 단계; 5) forming microdroplets in which the drug solution inside the nozzle is surrounded by the external enteric material solution by vibration spraying of the ultrasonic jet nozzle;6) 미세액적이 유동 파라핀(liquid paraffin)에 분사 및 교반되어 미세액적의 껍질이 단단하게 굳어지는 단계;를 포함하는 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물의 제조방법6) a method of preparing a formulation composition for oral administration of parathyroid hormone, comprising: spraying and stirring the microdroplets into a liquid paraffin to harden the shell of the microdroplets.
- 제 9항에 있어서. 1) 단계에서 상기 PTH 및 상기 데옥시콜산 유도체가 1:1 내지 1:10의 몰비율로 혼합된 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 제제 조성물의 제조방법The method of claim 9. Method of preparing a formulation composition for oral administration of parathyroid hormone, characterized in that in step 1) the PTH and the deoxycholic acid derivatives are mixed in a molar ratio of 1: 1 to 1:10.
- 제 9항에 있어서, 상기 4)단계 이후에 진공필터를 통해 회수하고 헥산으로 세척 및 건조하는 단계를 더 포함하는 것을 특징으로 하는 부갑상선 호르몬의 경구 투여용 약학 조성물의 제조방법The method of claim 9, further comprising recovering through a vacuum filter after step 4), washing and drying with hexane, and preparing a pharmaceutical composition for oral administration of parathyroid hormone.
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| KR102216578B1 (en) * | 2019-09-05 | 2021-02-17 | 주식회사 아이큐어비앤피 | Oral pharmaceutical composition comprising teriparatide and method for preparing the same |
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| US20040186050A1 (en) * | 2001-06-01 | 2004-09-23 | Ault Joseph M | Method for orally administering parathyroid hormone (pth) |
| US20050032698A1 (en) * | 2003-07-14 | 2005-02-10 | Nps Allelix Corp. | Stabilized formulation of parathyroid hormone |
| WO2007044069A2 (en) * | 2005-10-06 | 2007-04-19 | Nastech Pharmaceutical Company Inc. | Pth formulations and methods of use |
| US20070155664A1 (en) * | 2003-07-04 | 2007-07-05 | Nycomed Danmark A/S | Parathyroid hormone (pth) containing pharmaceutical compositions for oral use |
| US20090264367A1 (en) * | 2001-08-17 | 2009-10-22 | Moise Azria | 5-cnac as oral delivery agent for parathyroid hormone fragments |
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| US20040186050A1 (en) * | 2001-06-01 | 2004-09-23 | Ault Joseph M | Method for orally administering parathyroid hormone (pth) |
| US20090264367A1 (en) * | 2001-08-17 | 2009-10-22 | Moise Azria | 5-cnac as oral delivery agent for parathyroid hormone fragments |
| US20070155664A1 (en) * | 2003-07-04 | 2007-07-05 | Nycomed Danmark A/S | Parathyroid hormone (pth) containing pharmaceutical compositions for oral use |
| US20050032698A1 (en) * | 2003-07-14 | 2005-02-10 | Nps Allelix Corp. | Stabilized formulation of parathyroid hormone |
| WO2007044069A2 (en) * | 2005-10-06 | 2007-04-19 | Nastech Pharmaceutical Company Inc. | Pth formulations and methods of use |
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