KR101915735B1 - Pharmaceutical composition for Prevention or treatment of bone diseases comprising postpartum tissue extracts and inhibitor of epidermal growth factor as active ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising, as an active ingredient, an extract from postnatal tissue selected from a combination of amniotic membrane extract, chorionic membrane extract, and amniotic membrane and chorion epithelial cell and an epidermal growth factor (EGF) inhibitor ≪ / RTI >

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises, as an active ingredient, a tissue-derived extract and an epithelial growth factor inhibitor after delivery,

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises an extract from postnatal tissue and an inhibitor of epidermal growth factors (EGF) as an active ingredient.

The bone not only supports the muscles or organs structurally, but also supports the body's soft tissues and body weight, surrounds the internal organs, protects the internal organs from external impacts, stores calcium or other essential minerals, such as phosphorus or magnesium, Is an important part of the human body. Adult bone in growth is balanced by continuous repetitive regeneration of production and absorption processes that remove old bone and replace it with new bone, which is called bone remodeling (Yamaguchi A. et al. Tanpakushitsu Kakusan Koso., 50 (6Suppl); 664-669, 2005). This circulation of the bones is essential for restoring the fine damage of the bones caused by growth and stress, and maintaining its function. In adults, about 10-30% of the bone is reformed annually through remodeling of bone resorption - bone formation.

Bone regeneration involves osteoblasts that produce bone and osteoclasts that destroy bone, and bone homeostasis is maintained in close association with each other. For example, osteoblast cells regulate the differentiation of osteoclasts responsible for bone resorption through secretion of substances such as RANKL (receptor activator of nuclear factor-κB ligand) and its inducible receptor, OPG (osteoprotegerin) Lt; / RTI > In the past, bone mineralization, that is, the metabolic abnormalities of calcium and phosphorus, has been studied mainly in the case where the homeostasis of the bone is not maintained by the physical effect, the hormonal system, I have not made any progress in the identification of the mechanism. In general, calcium-containing diets are recommended for the treatment and prevention of osteoporosis, and estrogen or vitamin D is recommended for postmenopausal women. In addition, a bisphosphonate family such as Fosamax (alendronate) and Actonel (ingredient name: risedronate) has been attracting attention as a new alternative therapeutic agent as a bone resorption inhibitor that inhibits and kills osteoclasts. However, calcium supplementation, which is widely used as a bone disease treatment agent, inhibits the secretion of parathyroid hormone and prevents bone loss due to bone resorption, but it is known that individual differences in bone mass maintenance are severe (Heandy RP, 1007-1017,1996). In addition, hormone therapy using estrogen or calcitonin has been reported to increase bone density and reduce the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction, and venous thrombosis have been reported (Nelson, HD et al., JAMA, 288: 872-881, 2002; Lemay, A., J. Obstet, Bynaecol. Can., 24: 711-7152-3). In the case of bisphosphonate preparations, cases of jawbone necrosis, severe atrial fibrillation, inactivation of bones or joints, or pain in the musculoskeletal system are increasing every year in patients taking a drug recently (Coleman RE., Br J Cancer, 98: 1736- 1740 (2008). Therefore, there is a need to develop a new therapeutic agent for bone diseases that can effectively inhibit bone resorption while exhibiting few side effects.

Amniotic membrane and amnion / chorion membrane are the innermost membranes of the placenta. They protect the fetus from external pathogens by surrounding amniotic fluid and embryo, And the fetus is able to grow and develop normally. The amniotic membrane / chorion is composed of collagen-composed extracellular matrix and epithelial cells. The epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-b), nerve growth factor (NGF) and hepatic growth factor (HGF), as well as anti-inflammation and anti-bacterial components And angiogenic modulatory properties (Hao Y et al., Cornea, 19 (3): 348-352, 2000).

Davis JW et al., Johns Hopkins Hosp Rep, 15; 307, 1910) has been widely used for wound healing after burn, wound and ophthalmic surgery (Faulk WP et al 39 (2): 349-353, 2013, Dua HS et al., Survey of Ophthalmology, 49 (1) : 51-77, 2004). In addition, Korea Patent No. 10-0756974 discloses a pharmaceutical composition for the treatment of allergic diseases and chronic inflammatory diseases containing histamine, immunoglobulin and placenta extract, and Korean Patent No. 10-0725133 discloses a composition comprising Discloses a method for culturing fibroblast, a cell that produces collagen using a placenta extract, and a composition for skin regeneration using the same.

The first problem to be solved by the present invention is to provide a pharmaceutical composition comprising, as an active ingredient, an after-delivery tissue-derived extract and an epithelial cell growth factor inhibitor showing the effect of preventing or treating bone diseases.

Another object of the present invention is to provide a method for producing the pharmaceutical composition.

Another object to be solved by the present invention is to provide a pharmaceutical composition comprising an amniotic membrane extract, a chorionic membrane extract, and a combination of amniotic membrane and chorion epithelium selected from a combination of an extract from postnatal tissue and an epithelial growth factor (EGF) inhibitor, To control the rate of bone formation, and to control the amount of bone formation.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises an extract from postnatal tissue and an inhibitor of epidermal growth factor (EGF) as an active ingredient.

According to one embodiment of the present invention, the postpartum tissue-derived extract may be any one selected from amniotic membrane extract, chorionic membrane extract, and complex extract of amnion membrane and chorion membrane.

According to another example implementation of the present invention, the postpartum tissue extracts amnion, chorion, and the tissue after delivery is selected from the amnion and chorion complex of water extract supernatant or 2-30% by volume of C _{1 - 4} alcohol aqueous extract It may be a supernatant.

The bone disease according to the present invention is a disease caused by bone loss or damage of bone tissue, and specifically includes bone defect, osteoporosis, fracture, osteoporotic fracture, diabetic fracture, nonunion fracture, osteoporosis, osteomalacia, , Osteoarthritis, degenerative bone disease, and malocclusion.

In addition,

(1) peeling the amniotic membrane and chorionic membrane from the placenta respectively, or peeling and washing the complex of the amniotic membrane and the hair thin membrane;

(2) pulverizing the complex of washed amniotic membrane, chorionic membrane or amniotic membrane and chorionic membrane to obtain a pulverized product;

Obtaining the extract by eluting the protein by introducing the aqueous alcohol solution _{4 -} (3) the pulverized C ₁ of water or 2-30% by volume;

(4) centrifuging the extract to collect a supernatant; And

(5) a step of adding an epithelial growth factor inhibitor to the supernatant, and (5) a method for preparing a pharmaceutical composition for preventing or treating bone diseases, which comprises an extract from postnatal tissue and an epithelial growth factor inhibitor as an active ingredient do.

In addition, the present invention shows the effect of treating or preventing bone diseases by increasing bone formation or increasing calcium deposition (calcification).

In addition, the present invention relates to a method for inhibiting osteogenesis by mixing an extract of postnatal tissue selected from an amniotic membrane extract, a chorionic membrane extract and a combination of amniotic membrane and chorionic villus with an epithelial growth factor (EGF) inhibitor, Thereby providing a method for controlling the promoting rate or the promoting rate. .

The postnatal tissue-derived extract according to the present invention; And an epithelial cell growth factor inhibitor as an active ingredient are excellent in the effect of promoting bone formation and inducing calcium deposition. Therefore, it is an object of the present invention to provide a method for treating osteoarthritis or osteoarthritis, which comprises administering a therapeutically effective amount of at least one compound selected from the group consisting of osteoprotegerin, And can be usefully used for preventing or treating bone diseases such as bone diseases and malocclusion. In addition, the pharmaceutical composition according to the present invention can be used alone or in combination with a pharmaceutical composition and an aggregate biomaterial support when used as a support for aggregate biosynthesis selected from? -Triticalcium phosphate (HTC), hydroxyapatite and mixtures thereof Compared to that of the control group.

FIG. 1A is an image of amnion membrane, chorion membrane, amnion / chorion membrane complex isolated from the placenta, and FIG. 1B is amniotic membrane extract (AME) and chorionic villus extract (CME).
FIG. 2 shows the results of analysis of bone regeneration-related growth factors contained in amniotic membrane extract (AME) and chorion membrane extract (CME).
FIG. 3 shows the results of ALP (Alkaline Phosphatase) activity test of the amniotic membrane extract.
FIG. 4 shows the result of measuring calcium deposition of the amniotic membrane extract.
FIG. 5 shows the results of measurement of calcium deposition of amniotic membrane extract treated with EGF inhibitor.
FIG. 6 shows the results of verifying the expression of the promoter of osteogenic differentiation in the amniotic membrane extract treated with the EGF inhibitor.
FIG. 7 shows the results of measurement of calcium deposition of EGF treated chorionic villi extracts.
8 shows the results of comparing bone morphogenetic formation effects of chorionic villus extract and BMP-2.
FIG. 9 is a table listing kinds of EGF inhibitors according to the mechanism.
FIG. 10 shows the results of measurement of calcium deposition of a pharmaceutical composition containing AG1478 as an EGF inhibitor instead of PD153035.
FIG. 11 shows the results of measurement of calcium deposition of a pharmaceutical composition containing Gefitinib as an EGF inhibitor instead of PD153035.
FIG. 12 shows the results of measurement of calcium deposition of a pharmaceutical composition containing AG1478 as an EGF inhibitor, according to the concentration of the amniotic membrane extract.
FIG. 13 shows the results of comparing the calcium deposition of the pharmaceutical composition according to the kind of the EGF inhibitor.
14 shows the results of measurement of calcium deposition of a pharmaceutical composition containing only PD153035.
FIG. 15 shows the results of measurement of calcium deposition of pharmaceutical compositions containing amniotic membrane extract, chorionic villus extract, EGF inhibitor and amniotic membrane extract.

The inventors of the present invention have found that biomass waste which is largely discarded after childbirth promotes osteogenesis and increases the degree of calcium deposition after efforts to find a biomaterial capable of preventing or treating bone diseases, thereby completing the present invention .

Hereinafter, the present invention will be described in more detail.

The present invention relates to an extract from postnatal tissue selected from amniotic membrane extract, chorionic membrane extract, and complex extracts of amnion membrane and chorion membrane; And Epidermal Growth Factors (EGF) inhibitors as active ingredients. The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis.

According to the present invention, the bone disease may be a disease caused by bone defect, bone loss or damage of bone tissue. Bone regeneration involves osteoblasts that produce bone and osteoclasts that destroy bone. Bone defect or bone loss is a disease caused by lack of bone tissue due to more bone loss than the bone formed. As a method of increasing bone tissue, there is a method of increasing osteoclast activity and increasing bone formation, and a method of reducing osteoclast loss by inhibiting osteoclast activity.

The postpartum tissue-derived extract according to the present invention; And an epithelial cell growth factor inhibitor as an active ingredient increase the amount of bone production by promoting osteoblast differentiation and osteogenesis and increasing calcium deposition (petrification). Accordingly, the composition according to the present invention can be applied as a pharmaceutical composition for preventing or treating bone diseases.

According to one embodiment of the present invention, amniotic membrane and chorionic villus contain various kinds of growth factors related to bone regeneration promotion. Specifically, fibroblast growth factor (FGF), transforming growth factor beta-1 Transforming Growth Factor beta-1, TGF beta-1, and epidermal growth factor (EGF). In particular, epithelial growth factor (EGF) is a growth factor contained in amniotic membrane only in postpartum tissues such as placenta and umbilical cord. The inventors of the present invention found that when the epithelial growth factor inhibitor was treated with the postpartum extract derived from amniotic membrane extract, chorionic villus extract, and complex extract of amniotic membrane and chorionic villus, the effect of promoting osteogenesis was significantly improved compared to that before epithelial growth factor inhibitor treatment .

According to the present invention, since the chorion epithelium has a low content of epithelial growth factor, a pharmaceutical composition containing chorionic villus extract without addition of an epithelial growth factor inhibitor may be expected to promote osteogenesis. However, It was confirmed that bone formation was further promoted by the addition of.

According to the invention, the tissue after the delivery of the water extract of C ₁ 2 to 30% by volume of the extract or supernatant of postpartum tissue (amniotic membrane, chorionic or amniotic and chorionic complex of) _{- 4} alcohol may be aqueous solution extracts supernatants.

The C _{1 - 4} alcohol can be selected from methanol, ethanol, propanol, butanol and isopropanol, and preferably is excellent in promotion of bone formation compared to the extract is extracted with ethanol in different alcohols.

The extract according to the present invention may be an extract extracted at 2 to 15 ° C, preferably 2 to 10 ° C, and the water extract is more excellent in bone formation promoting effect than the alcohol extract.

The epithelial growth factor inhibitor according to the present invention may be used in combination with tyrosine kinase inhibitor (TKI), monoclonal antibody inhibitor, mitogen activation inhibitor (EGFR), epidermal growth factor receptor May be any one selected from mitogen-activated protein kinase kinase inhibitors (MEK inhibitors) and phosophoinositide-3-kinase inhibitors (PI3K inhibitors), but epithelial cell growth Is not limited to those exemplified above as long as it can inhibit the factor receptor.

As described above, the epithelial growth factor inhibitors may be variously classified according to the mechanism for inhibiting epithelial growth factors. Among them, preferred epithelial growth factor inhibitors include monoclonal antibody inhibitors, , EGFR tyrosine kinase inhibitors, mitogen activated protein kinase enzyme inhibitors (MEK1 / 2 inhibitors), and PI3K inhibitors (PI3K inhibitors).

Wherein said monoclonal antibody inhibitor is selected from the group consisting of Matuzumab, Nimotuzumab, Zalutumumab, Cetuximab, and panitumumab, said monoclonal antibody inhibitors being selected from the group consisting of PD153035, Gefitinib, Erlotinib, AG494 and AG1478 , The mitogen-activated protein kinase enzyme inhibitor (MEK1 / 2 inhibitor) may be U0126, and the PI3K inhibitor may be Wortmannin.

The amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane complex of the present invention, without affecting signal transduction of growth factors other than EGF among the epithelial growth factor (EGF) inhibitors, Most preferred is a tyrosine kinase inhibitor such as PD 153035, Gefitinib, Erlotinib, AG494 and AG1478, which do not cause problems such as lowering the activity of the tyrosine kinase inhibitor, or the like.

In the case of other epithelial cell growth factor inhibitors other than the tyrosine kinase inhibitor, not only are there problems such as involvement in signal transduction of growth factors other than EGF, cytotoxicity when administered in vivo, side effects, and the like, It may cause problems such as aggregation when mixed with amniotic membrane extract, chorionic villus extract and complex extract of amniotic membrane and chorion, deterioration of effect of extract, decrease of activity, increase of cost, and the like.

In addition, the tyrosine kinase inhibitor is most preferably PD153035, and exhibited an inhibitory effect on EGF receptor 1 specifically when mixed with amniotic membrane extract, chorionic membrane extract, and combined amniotic membrane and chorionic membrane extract. Inhibitory effect of tyrosine kinase It was confirmed by cell experiments that the calcium deposition was improved by 2 to 2.5 times as compared with the case where the inhibitor was mixed. Also, it was confirmed that the group administered with PD153035 increased the expression of bone regeneration related gene by 2 to 10 times as compared with the control group without PD153035.

In addition, it was confirmed that when the above tyrosine kinase inhibitor PD153035 alone was treated without the combined extract of amniotic membrane extract, chorionic membrane extract and amniotic membrane and chorionic membrane, calcium deposition did not increase at all during induction of bone differentiation in cell experiments. That is, the tyrosine kinase inhibitor PD153035 itself has no effect of promoting bone regeneration, as can be seen from the following experimental examples.

The pharmaceutical composition contains the amniotic membrane extract, chorionic membrane extract, amniotic membrane, and chorion epithelium at a concentration of 50 to 1000 μg / ml and an Epidermal Growth Factor (EGF) inhibitor at a concentration of 10 to 40 μM . In the pharmaceutical composition, the epithelial growth factor (EGF) inhibitor is contained at a concentration of 10 to 20 μM when the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane combined extract is 100 to 400 μg / Is most preferable because it can be confirmed that the calcium deposition degree and the degree of expression of the bone regenerating factor increase by 2 to 10 times or more.

In the pharmaceutical composition, if the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorion epithelium are less than 100 μg / ml in the pharmaceutical composition and the epidermal growth factor (EGF) inhibitor is less than 10 μM, , And no significant effect was observed compared to the control group without any treatment.

In the pharmaceutical composition, the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane complex extract of the pharmaceutical composition may have a concentration exceeding 400 μg / ml, or the epidermal growth factor (EGF) inhibitor may be 20 μM It is understood that it is preferable for the cost reduction to include the concentration in the above range since the increase in the effect against the increase in concentration is insignificant. Furthermore, increasing the concentration of the drug present in the pharmaceutical composition means that the amount of drug capable of simultaneously contacting the cell is increased, and the strong action of the drug on the cell causes a secondary effect on tolerance or in vivo mechanism It is preferable to administer the drug at a minimum drug concentration which gives the maximum effect.

The pharmaceutical composition according to the present invention is useful for prevention of bone diseases such as calcium deposition, expression of bone differentiation promoting genes or the like, even when the amount of the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane combined is used alone, Since the therapeutic effect is maintained or improved, the cost saving effect can be obtained. Specifically, the amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorionic membrane complex extract according to the present invention; And epithelial growth factor (EGF) inhibitors, can be obtained by reducing the dose by 2 to 4 times or more, compared with the use of amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorion complex extract alone, And an increase in osteogenic differentiation promoting gene expression, it is possible to obtain 500 to 1600 times of extracts of the effect obtained by treatment with BMP-2 500 ng / mL per placenta with the extract obtained per placenta.

The pharmaceutical composition is preferably a combination of amniotic membrane extracts, amniotic membrane extracts, chorionic villi extracts, amniotic membrane extracts and amniotic membrane and chorion epithelium, which exhibit excellent effects when mixed with PD153035. In particular, when mixed with PD153035, It is most preferable that the amniotic membrane extract has a pharmacological effect in the skin.

The pharmaceutical composition according to the present invention can exhibit the effect of promoting bone formation by using the pharmaceutical composition alone. In addition, it can be used in combination with or mixed with an aggregate repellent support selected from? -Triticalcium phosphate (? TCP), hydroxyapatite, and a mixture thereof. There is no particular limitation on the method of using the pharmaceutical composition and the aggregate-producing support. For example, the pharmaceutical composition according to the present invention may be coated on the surface of the aggregate-producing support, or the aggregate- May be chemically bonded to each other.

According to one embodiment of the present invention, coating or chemically binding the pharmaceutical composition according to the present invention to the surface of an aggregate biomaterial support is preferable because quantitative bone regeneration efficiency can be quantitatively increased at a specific site for bone regeneration . According to one embodiment of the present invention, it was confirmed that the bone regeneration effect was remarkably improved as compared with the case where the pharmaceutical composition according to the present invention and the aggregate-producing support were used singly.

The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable carriers in addition to the postpartum tissue-derived extract and the epithelial growth factor inhibitor, and may be appropriately selected according to techniques known in the art.

In the present invention, a pharmaceutically acceptable carrier means a known pharmaceutical excipient that is useful when formulating the pharmaceutically active compound for administration and is substantially non-toxic and non-sensitive under the conditions of use. The exact ratio of such excipients is determined by standard pharmaceutical practice, as well as the solubility and chemical properties of the active compound, the route of administration chosen.

The pharmaceutical composition of the present invention may be formulated into a form suitable for a desired administration method using suitable and physiologically acceptable excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, .

The pharmaceutical composition according to the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, solutions or injections.

Formulations suitable for oral administration include solid preparations such as tablets, microparticles, capsules containing liquid or powder, pills, granules, powders, lozenges (including liquid-filled ones), chews, Gels, solid solutions, liposomes, films (including mucosal-tackiness), ovules, sprays and liquids. Liquid preparations include, for example, suspensions, solutions, syrups and elixirs.

The tablets generally contain a disintegrant in addition to the drug. Starch or modified starch such as sodium starch glycolate, corn starch, potato starch or pregelatinized starch as a disintegrant, clay such as bentonite, montmorillonite, veegum, microcrystalline cellulose, hydroxypropylcellulose or carboxy Cellulose such as methyl cellulose, alginate such as sodium alginate or alginic acid, cross-linked cellulose such as croscarmellose sodium, gums such as guar gum and xanthan gum, cross-linking such as crospovidone, A polymer, a boiling agent such as sodium bicarbonate, citric acid or the like may be mixed and used. In general, the disintegrant will include, but is not limited to, from about 1% to about 25% by weight of the dosage form, preferably from about 2% to about 10% by weight of the dosage form.

Binders are generally used to impart tack to the tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, starch, copovidone, highly disperse silica, mannitol, lactose , Hydroxypropylcellulose, and hydroxypropylmethylcellulose. Generally, the binder will comprise from about 1% to about 40% by weight of the dosage form, preferably from about 2% to about 25% by weight of the dosage form, but is not limited thereto. The tablets may be, for example, starch, microcrystalline cellulose, lactose, glucose, mannitol, alginate, alkaline earth metal salts, clay, polyethylene glycol and dicalcium phosphate as diluents. Generally, the diluent will comprise from about 1% to about 70% by weight of the dosage form, preferably from about 2% to about 50% by weight of the dosage form, but is not limited thereto.

Tablets may also optionally contain a surfactant, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. If present, the surfactant may comprise from about 0.2% to about 5% by weight of the tablet, and the glidant may comprise from about 0.2% to about 1% by weight of the tablet.

As lubricants there may also be mentioned lubricants such as talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, magnesium stearate, sodium lauryl sulfate, lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, glyceryl monostearate Polyethylene glycol 4000, and the like can be used. The lubricant generally comprises from about 0.25% to about 5%, preferably from about 0.5% to about 3% by weight of the tablet.

Other possible ingredients include antioxidants, colorants, flavors, preservatives and taste-masking agents.

Tablet formulations can be pressed directly or squeezed with a roller to form tablets. Alternatively, the tablet formulation or a portion of the formulation may be wet, dry, melt-granulated, melt congealed or extruded prior to tableting. The final formulation may comprise one or more layers, may be coated or uncoated, and may be encapsulated.

Solid preparations for oral administration can be formulated in immediate release and / or modified release form. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release types.

In one embodiment, the pharmaceutical composition of the present invention may be a tablet. Tablets may optionally be film coated. The total amount of the drug per unit dose may be an amount that provides a convenient size dosage form to the patient.

According to the present invention, the pharmaceutical composition can be administered orally or parenterally in any desired dosage form, depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, The effective amount can be adjusted depending on various factors including time, route of administration and minute of composition, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, doses of 0.0001 to 4000 mg / day may be administered at intervals of one time to several times a day at predetermined intervals according to the judgment of a doctor or a pharmacist.

The pharmaceutical composition for preventing or treating bone diseases according to the present invention can be prepared by carrying out the following steps.

(1) peeling the amniotic membrane and chorionic membrane from the placenta respectively, or peeling and washing the complex of the amniotic membrane and the hair thin membrane;

(2) pulverizing the complex of washed amniotic membrane, chorionic membrane or amniotic membrane and chorionic membrane to obtain a pulverized product;

Obtaining the extract by eluting the protein by introducing a _3-alcohol solution _- (3) the pulverized C ₁ of water or 2-30% by volume;

(4) centrifuging the extract to collect a supernatant; And

(5) adding an epithelial growth factor inhibitor to the supernatant.

In the above production method, peeling of the amniotic membrane, washing, crushing and protein elution in the amniotic membrane can be prepared by using a conventional method for producing a combined extract of amniotic membrane extract, chorionic membrane extract or amniotic membrane and chorionic membrane, no.

According to the present invention, it is possible to further include the step of treating with an ultrasonic pulverizer to facilitate protein elution before centrifugation in the step (4). The collected supernatant is preferably filtered and then frozen or stored at an extremely low temperature for use in the experiment. The extracted extract was used for the experiment after quantifying the protein concentration.

The present invention also relates to an extract from postnatal tissue selected from amniotic membrane extract, chorionic membrane extract, and complex extract of amniotic membrane and chorionic villus; And an Epidermal Growth Factors (EGF) inhibitor, in combination, and a method for promoting osteogenesis.

The epithelial growth factor inhibitor according to the present invention may be used in combination with tyrosine kinase inhibitor (TKI), monoclonal antibody inhibitor, mitogen activation inhibitor (EGFR), epidermal growth factor receptor May be any one selected from mitogen-activated protein kinase kinase inhibitors (MEK inhibitors) and phosophoinositide-3-kinase inhibitors (PI3K inhibitors), but epithelial cell growth Is not limited to those exemplified above as long as it can inhibit the factor receptor.

As described above, the epithelial growth factor inhibitor may be classified into various types according to the mechanism for inhibiting epithelial growth factor. Among them, preferred epithelial growth factor inhibitors include monoclonal antibody inhibitors, , EGFR tyrosine kinase inhibitors, mitogen activated protein kinase enzyme inhibitors (MEK1 / 2 inhibitors), and PI3K inhibitors (PI3K inhibitors).

The amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane complex of the present invention, without affecting signal transduction of growth factors other than EGF among the epithelial growth factor (EGF) inhibitors, Most preferably selected from the group consisting of PD153035, Gefitinib, Erlotinib, AG494, and AG1478, which are tyrosine kinase inhibitors, which do not cause problems such as deterioration of the activity or degradation of activity.

In the case of other epithelial cell growth factor inhibitors other than the tyrosine kinase inhibitor, not only are there problems such as involvement in signal transduction of growth factors other than EGF, cytotoxicity when administered in vivo, side effects, and the like, It may cause problems such as aggregation when mixed with amniotic membrane extract, chorionic villus extract and complex extract of amniotic membrane and chorion, deterioration of effect of extract, decrease of activity, increase of cost, and the like.

In addition, the tyrosine kinase inhibitor is most preferably PD153035, and exhibited an inhibitory effect on EGF receptor 1 specifically when mixed with amniotic membrane extract, chorionic membrane extract, and combined amniotic membrane and chorionic membrane extract. Inhibitory effect of tyrosine kinase The results of cell experiments confirmed that calcium deposition was improved by 2 ~ 2.5 times compared with that of inhibitor. Also, it was confirmed that the group administered with PD153035 increased the expression of bone regeneration related gene by 2 to 10 times as compared with the control group without PD153035.

In addition, it was confirmed that when the above tyrosine kinase inhibitor PD153035 alone was treated without the combined extract of amniotic membrane extract, chorionic membrane extract and amniotic membrane and chorionic membrane, calcium deposition did not increase at all during induction of bone differentiation in cell experiments. That is, the tyrosine kinase inhibitor PD153035 itself has no effect of promoting bone regeneration, as can be seen from the following experimental examples.

In step (5), the complex extract of the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane is added to the supernatant at a concentration of 50 to 1000 μg / ml, and the epidermal growth factor (EGF) lt; RTI ID = 0.0 > uM. < / RTI > In the pharmaceutical composition, the epithelial growth factor (EGF) inhibitor is contained at a concentration of 10 to 20 μM when the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane combined extract is 100 to 400 μg / Is most preferable because it can be confirmed that the calcium deposition degree and the degree of expression of the bone regenerating factor increase by 2 to 10 times or more.

In the pharmaceutical composition, if the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorion epithelium are less than 100 μg / ml in the pharmaceutical composition and the epidermal growth factor (EGF) inhibitor is less than 10 μM, , And no significant effect was observed compared to the control group without any treatment.

In the pharmaceutical composition, the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane complex extract of the pharmaceutical composition may have a concentration exceeding 400 μg / ml, or the epidermal growth factor (EGF) inhibitor may be 20 μM It is understood that it is preferable for the cost reduction to include the concentration in the above range since the increase in the effect against the increase in concentration is insignificant. Furthermore, increasing the concentration of the drug present in the pharmaceutical composition means that the amount of drug capable of simultaneously contacting the cell is increased, and the strong action of the drug on the cell causes a secondary effect on tolerance or in vivo mechanism It is preferable to administer the drug at a minimum drug concentration which gives the maximum effect.

The pharmaceutical composition according to the present invention is useful for prevention of bone diseases such as calcium deposition, expression of bone differentiation promoting genes or the like, even when the amount of the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane combined is used alone, Since the therapeutic effect is maintained or improved, the cost saving effect can be obtained. Specifically, the amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorionic membrane complex extract according to the present invention; And epithelial growth factor (EGF) inhibitors, can be obtained by reducing the dose by 2 to 4 times or more, compared with the use of amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorion complex extract alone, And an increase in osteogenic differentiation promoting gene expression, it is possible to obtain 500 to 1600 times of extracts of the effect obtained by treatment with BMP-2 500 ng / mL per placenta with the extract obtained per placenta.

In step (1), it is most preferable to use a complex of amniotic membrane or amniotic membrane and chorionic membrane because it shows excellent effect when mixed with PD153035. More preferably, it is most preferable to use amniotic membrane which has a pharmacological effect at a high concentration of 2 to 4 times or more when mixed with PD153035.

In addition, the present invention relates to a method for inhibiting osteogenesis by mixing an extract of postnatal tissue selected from an amniotic membrane extract, a chorionic membrane extract and a combination of amniotic membrane and chorionic villus with an epithelial growth factor (EGF) inhibitor, The acceleration or acceleration rate can be adjusted.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are for the purpose of describing the present invention more specifically and that the scope of the present invention is not limited thereby.

Example

Manufacturing example

A normal human placenta (= 37 weeks pregnancy) without any signs of internal, obstetric, or surgical complications and not infected with HBV, HCV and HIV virus through blood tests was obtained after cesarean section and received consent from all donors. The collection of samples and their use for experimental purposes were approved by IRB from Korea University Medical School.

Manufacturing example  One: Amniotic membrane extract  Produce

The amniotic membrane was carefully removed from the placenta with a sheath around the umbilical cord of the placenta. The amniotic membrane was collected and washed in PBS or saline containing 1% penicillin / streptomycin. After the amniotic fluid and blood of the amniotic membrane were removed, the mixture was pulverized using a homogenizer, and the buffer was added at a ratio of 1: 1 (mass) of the amniotic membrane (g) to PBS (mL) Respectively. Next, the protein was released using an ultrasonic disintegrator and centrifuged. The supernatant was collected and filtered through a syringe filter and stored at -80 ° C. Amniotic membrane extracts were prepared by measuring the protein concentration and using the required amount.

Manufacturing example  2: Production of amniotic membrane / chorion complex extract

The amniotic membrane and chorionic membrane complex were separated from the placenta, washed, and then ground to produce amniotic membrane / chorionic membrane complex in the same manner as the amniotic membrane extract.

Manufacturing example  3: Production of chorionic membrane extract

The chorion epithelium was removed from the amniotic membrane instead of the amniotic membrane.

Test Example

Growth factor analysis

Enzyme-linked immunosorbent assay (ELISA) was performed to identify bone growth-related growth factors contained in amniotic membrane extract (AME) and chorion membrane extract (CME) prepared in Preparation Examples 1 and 3 Respectively. The concentration used in the experiment is pg of growth factor / mg of protein in extraxt.

As shown in the following Table 1 and FIG. 2, Fibroblast Growth Factor (FGF) and Transforming Growth Factor beta-1 (TGFβ-1) , And TGFβ-1 contained in the amniotic membrane extract was found to contain about 40% more than chorionic villus extract.

On the other hand, both BMP and BMP-2 were not detected in amniotic membrane extract and chorionic villus extract, and epidermal growth factor (EGF) was detected only in amniotic membrane extract And chorionic villus extracts were found to be present in very small amounts.

division AME CME FGF 3.310 3.024 BMP-2 -1.846 -1.604 TGFβ-1 33.070 23.820 EGF 8.229 0.008

Amniotic membrane extract  ALP (Alkaline Phosphatase ) Activity and Calcium Measurement

The human osteosarcoma cell line MG-63 (Korean Cell Line Bank, KCLB), known as osteoblast-like cell line, was used for in vitro bone formation experiments, and 10% (v / v) FBS (Fetal Bone Serum) v) and incubated for penicillin / streptomycin DMEM (Dulbecco's modified Eagle's medium containing a) and 37 ℃, cells in 5% CO ₂ humidified environment.

① ALP (Alkaline Phosphatase ) Activity

An experiment was conducted to measure ALP enzyme, which is an index of bone formation at the initial stage of bone formation. To evaluate ALP activity of amniotic membrane extracts, ALP assay was compared with chorionic villus extract and amniotic membrane / chorion complex extract, which promoted osteogenesis and promoted ALP activation. Osteogenesis induction medium (OIM) (AME: amniotic membrane extract, ACME: amniotic membrane / chorionic villus complex, CME: chorionic villus extract) were administered to MG-63 cells and the ALP activity was measured with the control (OIM) Respectively.

3, and 7 days were taken, washed twice with cold PBS buffer, and lysed with cell lysis buffer containing 0.1% Triton X-10. The dissolved cells were centrifuged at 13000 rpm for 30 minutes at 4 DEG C, and the supernatant was recovered and quantitated by Bradford method. The same amount of protein was analyzed with ALP kit (AnaSPEC, Inc, Fermont, Canada) Nm was measured and shown in FIG.

On the 7th day after differentiation, chorionic villus extract showed the highest ALP activity, followed by amniotic membrane / chorion complex extract and amniotic membrane extract.

② Measurement of calcium deposition

Assessment of calcium deposition (petrification) is widely used as a mid and late indicator of osteoblast differentiation. To evaluate calcium deposition of amniotic membrane extracts, we performed this experiment in comparison with chorionic villus extract and amniotic membrane / chorion complex extract which promoted bone formation and promoted calcium deposition. In the same manner, MG-63 cells were differentiated in osteogenesis induction medium (OIM), treated with 100, 200, 400, and 800 ㎍ / mL of extracts, Was determined 7 days after differentiation. The content of calcium was measured according to the manufacturer's instructions using the QuantiChromTM Calcium Assay Kit (DICA-500), and the results are shown in FIG.

The calcium content of the amniotic membrane extracts was lower than that of the chorionic villus extract or amniotic membrane / chorion complex extracts (* P <0.05).

EGF  Inhibitory Amniotic membrane extract  Calcium deposition degree and Bone differentiation  Verification of promoted gene expression increase

The human osteosarcoma cell line MG-63 (Korean Cell Line Bank, KCLB), known as osteoblast-like cell line, was used for in vitro bone formation experiments, and 10% (v / v) FBS (Fetal Bone Serum) v) and incubated for penicillin / streptomycin DMEM (Dulbecco's modified Eagle's medium containing a) and 37 ℃, cells in 5% CO ₂ humidified environment.

① Calcium deposition rate measurement

In order to measure the degree of calcium deposition after treatment with EGF inhibitor in amniotic membrane extract, MG-63 cells were differentiated in the same osteogenesis induction medium (OIM), and PD153035 (Calbiochem, intracellular receptor delivery of EGF (A) were treated with 5, 10 and 20 μM of aminotransferase inhibitor, followed by treatment with 200 μg / mL of amniotic membrane extract for 1 hour. The calcium content was measured by inducing differentiation for 7 days (A) .

After 1 hour of treatment with 10 μM of PD153035 under the same differentiation conditions, the amniotic membrane extract was treated at a concentration of 100, 200 and 400 μg / mL, and then treated to measure the calcium content for 7 days (B). The calcium content was measured according to the manufacturer's instructions using the QuantiChromTM Calcium Assay Kit (DICA-500) and the results are shown in FIG.

5A and 5B, the calcium content of the pharmaceutical composition to which amniotic membrane extract (AME 200 μg / ml) and PD153035 10-20 μM were added was 1.5 to 2 times higher than that of amniotic membrane extract alone. This is 1.5 to 2 times more than the equivalent amount of chorionic villi extract or amniotic membrane / chorioallantoic extract. The content of amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorioallantoic extract (necessary dose), which is difficult to obtain, is 2 to 4 times or more It can be seen that the effect can be equal or improved than before the content is lowered.

However, when the amniotic membrane extract was in a concentration of 100 to 400 占 퐂 / ml and the PD153035 was less than 10 占 즉, that is, 5 占 퐉, calcium fixation, i.e., bone disease treatment effect was not observed.

That is, the pharmaceutical composition according to the present invention exhibits a better bone disease treatment effect than the prior art despite the fact that the concentration of the amniotic membrane extract, chorionic villus extract or amniotic membrane and chorioamnion complex extract is reduced by 2 to 4 times that of the conventional art, .

In both of FIGS. 5A and 5B, it was confirmed that calcium content was significantly higher in the group treated with the EGF inhibitor (PD153035), and that the calcium content was increased in a concentration-dependent manner by the EGF inhibitor.

The inventors of the present invention have found that a pharmaceutical composition comprising an epithelial growth factor inhibitor (preferably a tyrosine kinase inhibitor, PD153035) and amniotic membrane, chorionic membrane or amniotic membrane and chorioallantoic membrane extract is an epithelial growth factor inhibitor (preferably a tyrosine kinase inhibitor (Or inhibitory effect) of EGF, which is a growth factor contained in amniotic membrane extract, chorionic membrane extract or amniotic membrane and chorion complex, It was confirmed that it could be effective in promoting effective bone formation and treating bone diseases.

② Verification of increased gene expression promoting bone differentiation

MG-63 cells were differentiated from osteogenesis induction medium (OIM) in the same manner as PDGF3035 (Calbiochem, Inc.) to measure the degree of calcium deposition after treating the amniotic membrane extract with the EGF inhibitor (PD153035) After incubation at 10 μM for 1 h, amniotic membrane extracts were treated with 100 μg / ml of amniotic membrane extract to induce differentiation. On day 4, The increased expression of promoting bone differentiation was verified and shown in Fig.

To confirm gene changes during differentiation, total RNA was isolated from the cells at the differentiation stage using the triazole reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. ALP and IBSP (integrin-binding sialoprotein) were the index proteins of bone formation at the early stage of osteogenesis, and RUNX2 and OSTERIX were important gene transcription factors for bone differentiation. . The cultured cells were washed twice with cold PBS, and then lysed by adding 1 ml of Trizol reagent. After adding 200 μl of chloroform, the supernatant was separated by centrifugation at 4 ° C and 12,000 rpm for 20 minutes. 500 μl of isopropanol was added to the separated supernatant, followed by centrifugation again at 4 ° C and 12,000 rpm. The supernatant was discarded and the remaining pellet was washed three times with 70% ethanol and the RNA was isolated. For RT-PCR, RT-PCR amplification kit was added to the same amount of total RNA (5 μl) and incubated at 45 ° C for 60 minutes to prepare cDNA. cDNA was amplified by real-time PCR using primers specific for ALP, IBSP, RUNX2 and OSTERIX, respectively. Real-time PCR was performed using SYBR green reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. Specifically, 10 μl of 2 × SYBR green and 1 μl of each 0.5 pmol / μl primer were added to the same amount of cDNA, followed by 40 cycles of reaction at 95 ° C. for 30 seconds and 60 ° C. for 1 minute.

As shown in FIG. 6, RT-PCR showed that expression of osteogenic marker genes was increased only when amniotic membrane extract of the present invention and EGF inhibitor were treated at the same time. It can be seen that the pharmaceutical composition of the present invention has an increased expression of bone morphogenesis-promoting genes 2 to 10 times or more as compared with a control group in which amniotic membrane extract alone or nothing is treated.

EGF  Calcium deposition of processed chorionic villi extracts

To demonstrate inhibition of osteogenesis by EGF, experiments were performed by adding EGF to a chorioallantoic extract (400-800 [mu] g / ml) at a high concentration which exhibited a sufficiently good degree of calcium deposition under single extract conditions.

MG-63 cells were differentiated in the same osteogenesis induction medium (OIM) and EGF (100 ng / mL, PeproTECH, recombinant human) was added to chorionic villus extract at various concentrations (100, 200, 400, and 800 μg / mL) EGF) was treated or not treated to induce differentiation for 9 days, and the degree of calcium deposition was measured (A). As shown in FIG. 7A, when the chorionic villi extract alone was used, calcium deposition was significantly increased only at a high concentration of 400 to 800 μg / ml or more.

The concentration of EGF was increased to 50, 100 and 500 ng / mL, and the differentiation was induced for 9 days. The EGF concentration was then adjusted to 400 ㎍ / Changes were measured (B). As shown in FIG. 7B, the calcium deposition was significantly increased in the untreated group (0 ng / mL), but the addition of EGF (50, 100, 500 ng / .

Chorioallantoic extract and BMP-2 Osteogenesis  Comparative experiment

MG-63 cells were differentiated in the same osteogenesis induction medium (OIM) to confirm the effect of the extract of the present invention in comparison with BMP-2, a type 2 osteogenic protein, and BMP-2 and chorionic villus Extracts (CME) were treated by concentration to induce differentiation for 18 days.

As shown in FIG. 8, the results were better than those obtained by treatment with BMP-2 at 500 μg / mL at a concentration of 400 μg / mL of chorionic villus extract. From the results of Table 2, it was calculated that the chorioallantoic extract contained 1.209 pg of FGF and 9.258 pg of TGF-? 1 in 400 占 퐂 / mL.

Amniotic membrane / chorionic tissue was obtained 50-80 g per placenta. Amount of total protein per tissue and amount of total protein per extract of amniotic membrane / chorioallantoic membrane were determined and shown in Table 2 below.

division Total protein amount mg / tissue g Amniotic membrane extract 1.46 ㅁ 0.44 Chorionic villus extract 2.50 ㅁ 0.56

From the above results, it was confirmed that the amount of amniotic membrane extract protein of placenta was 36.5-58.4 mg, that of chorionic villus extract was 63-100 mg, and that of amniotic membrane / chorionic membrane complex was 99-158 mg. Thus, 250-400 batches of extracts obtained by treatment with BMP-2 at 500 ng / mL per placenta were obtained.

Furthermore, the amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorionic membrane complex extract according to the present invention; And epithelial growth factor (EGF) inhibitors, can be obtained by reducing the dose by 2 to 4 times or more, compared with the use of amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorion complex extract alone, And an increase in osteogenic differentiation promoting gene expression, it is possible to obtain 500 to 1600 times of extracts of the effect obtained by treatment with BMP-2 500 ng / mL per placenta with the extract obtained per placenta.

As evidenced by the above results, amniotic membrane extract, chorionic membrane extract or amniotic membrane / chorionic membrane complex extract; And an epithelial cell growth factor (EGF) inhibitor are useful for the treatment or prevention of osteopathy caused by bone loss or damage of bone tissue by promoting bone formation and improving calcium deposition. In addition, high added value can be created by using amniotic membrane / chorion.

PD153035 Of a pharmaceutical composition comprising an epithelial cell growth factor inhibitor other than Calcium saliva Complexity experiment

① Cell culture

The human osteosarcoma cell line MG-63 (Korean Cell Line Bank, KCLB), known as osteoblast-like cell line, was used for in vitro bone formation experiments, and 10% (v / v) FBS (Fetal Bone Serum) v) and incubated for penicillin / streptomycin DMEM (Dulbecco's modified Eagle's medium containing a) and 37 ℃, cells in 5% CO ₂ humidified environment.

② AG1478  or Gefitinib As an epithelial cell growth factor inhibitor. Calcium deposition degree  Measure

Assessment of calcium deposition (petrification) is widely used as a mid and late indicator of osteoblast differentiation. In order to confirm the effect of the pharmaceutical composition according to the present invention on the promotion of osteogenesis and prevention or treatment of osteoporosis according to the types of epithelial growth factor inhibitors, the calcium deposition degree was compared and the present experiment was conducted. At this time, the epithelial growth factor inhibitor was selected from the same tyrosine kinase inhibitors as PD153035. Of the known tyrosine kinase inhibitors, AG1478 (Tyrphostin AG1478) and Gefitinib (ZD1839), which have an effect similar to PD153035 (known to specifically inhibit EGF receptor 1 signaling), were used.

Similarly, MG-63 cells were differentiated in osteogenesis induction medium (OIM) and AG1478 (Calbiochem, a substance that inhibits tyrosine kinase, which is an intracellular receptor for EGF) or Gefitinib (Calbiochem, EGF 5, 10, and 20 μM), followed by treatment with amniotic membrane extract (200 μg / mL) for 1 hour, and induced differentiation for 7 days. And the content thereof was measured. The content of calcium was measured using a QuantiChromTM Calcium Assay Kit (DICA-500) according to the manufacturer's instructions, and the results are shown in FIGS. 10 and 11.

As shown in Figs. 10 and 11, the calcium content measured on the 7th day of differentiation showed that the pharmaceutical composition using AG1478 and Gefitinib instead of PD153035 showed a change in calcium deposition inducing effect compared to the control (OIM) (* P < 0.05).

③ Of amniotic membrane composition  Depending on concentration, AG1478 As an epithelial cell growth factor inhibitor. Calcium deposition degree  Measure

FIG. 12 shows that MG-63 cells were differentiated into osteogenesis induction medium (OIM), treated with AG1478 at a concentration of 10 μM, and then treated with amniotic membrane extract 100, 200, 400 (* P < 0.05). The results are shown in FIG. 2 (b). The results are shown in FIG. ).

As shown in Fig. 12, the pharmaceutical composition including AG1478 instead of PD153035 showed that although the concentration of the amniotic membrane extract having the bone formation promoting effect was increased, the increase in the calcium deposition effect was insignificant compared with the concentration increase have. In addition, the pharmaceutical composition comprising AG1478 showed little change in calcium deposition when compared to 400 μg / ml amniotic membrane extract alone (see FIG. 4).

④ According to the epithelial growth factor inhibitor, Calcium deposition degree  compare

FIG. 13 shows the results of treatment of PD-153035, AG1478 and Gefitinib with 5, 10 and 20 μM, respectively, on MG-63 cells differentiated from osteogenic induction medium (OIM) (* P < 0.05) by inducing differentiation for 7 days and measuring the calcium content according to the manufacturer's instructions using a QuantiChromTM Calcium Assay Kit (DICA-500).

As shown in Fig. 13, when the PD153035 was treated with a pharmaceutical composition containing 10-20 [mu] M of amniotic membrane extract and 200 [mu] g / ml of amniotic membrane extract, the degree of calcium deposition was 1.5 to 2 times higher than that of the control (OIM) Respectively.

 The pharmaceutical composition comprising PD153035 at 10-20 [mu] M and the amniotic membrane extract at 200 [mu] g / ml has the same or better calcium deposition than 800 [mu] g / ml amniotic membrane extract or 400 [ .

Thus, it was confirmed that the pharmaceutical composition containing PD153035 of 10-20 μM and amniotic membrane extract of 200 μg / ml can reduce the amount of amniotic membrane extract (amount of use) by 2 to 4 times or more.

PD153035 &Lt; / RTI &gt; Calcium deposition degree  Comparative experiment

① Cell culture

The human osteosarcoma cell line MG-63 (Korean Cell Line Bank, KCLB), known as osteoblast-like cell line, was used for in vitro bone formation experiments, and 10% (v / v) FBS (Fetal Bone Serum) v) and incubated for penicillin / streptomycin DMEM (Dulbecco's modified Eagle's medium containing a) and 37 ℃, cells in 5% CO ₂ humidified environment.

② Calcium deposition degree  Measure

Assessment of calcium deposition (petrification) is widely used as a mid and late indicator of osteoblast differentiation. When the epithelial cell growth factor inhibitor alone was included, the calcium deposition degree was compared to confirm the effect of promoting bone differentiation, prevention or treatment of osteoporosis of the pharmaceutical composition. At this time, PD153035, the most effective epithelial growth factor inhibitor, was used.

Similarly, MG-63 cells were differentiated in osteogenesis induction medium (OIM), and PD153035 (Calbiochem, a substance that inhibits tyrosine kinase, which is an intracellular receptor for EGF receptor) was cultured in 5, 10 and 20 μM And the calcium content was measured by inducing differentiation for 7 days. The content of calcium was measured according to the manufacturer's instructions using a QuantiChromTM Calcium Assay Kit (DICA-500). The results are shown in FIG.

As shown in Fig. 14, when the PD 153035 alone was used, there was almost no change in the calcium deposition inducing effect compared to the control group (OIM) in which nothing was treated, as can be seen from the calcium content measured on the 7th day of differentiation (* P < 0.05). That is, it can be seen that the epithelial cell growth factor inhibitor itself does not have the bone regeneration promoting effect.

Amniotic membrane extract , Chorionic villi extract, epithelial growth factor inhibitor Amniotic membrane extract  Of the pharmaceutical composition Calcium deposition degree  Comparative experiment

① Cell culture

The human osteosarcoma cell line MG-63 (Korean Cell Line Bank, KCLB), known as osteoblast-like cell line, was used for in vitro bone formation experiments, and 10% (v / v) FBS (Fetal Bone Serum) v) and incubated for penicillin / streptomycin DMEM (Dulbecco's modified Eagle's medium containing a) and 37 ℃, cells in 5% CO ₂ humidified environment.

② Calcium deposition degree  Measure

Assessment of calcium deposition (petrification) is widely used as a mid and late indicator of osteoblast differentiation. In order to confirm the effect of the amniotic membrane or chorion epithelium alone and the effect of the pharmaceutical composition (AME + PD) according to the present invention on the bone differentiation promotion and prevention or treatment of bone diseases, the calcium deposition degree was compared and the present experiment was conducted.

FIG. 15 shows the results obtained by differentiating MG-63 cells from osteogenesis induction medium (OIM), followed by treatment with 200 μg / ml of amniotic membrane extract (AME 200 μg / ml) (CME 200, 400 ㎍ / ml) and PD153035 (Calbiochem) were treated at a concentration of 10 and 20 μM, followed by treatment with 200 μg / ml of amniotic membrane extract AME 200 μg / ml + PD 10 (or 20) μM). (* P < 0.05) after inducing differentiation of each group for 7 days and measuring the content of calcium according to the manufacturer's instructions using a QuantiChromTM Calcium Assay Kit (DICA-500).

As shown in FIG. 15, there was no bone regeneration effect in the group treated with only AME 200 μg / ml and CME 200 μg / ml. However, when the EGF inhibitor PD153035 was treated, calcium deposition was significantly increased, Of CME at a concentration of 400 &lt; RTI ID = 0.0 &gt; pg / ml. &Lt; / RTI &gt;

Although not shown in the present experimental example, the pharmaceutical composition containing chorionic villus extract or amniotic membrane and chorion complex extract and PD153035 was lower than the pharmaceutical composition containing amniotic membrane extract and PD153035, but it was confirmed that the effect was increased compared with the control. Specifically, it was confirmed that the pharmaceutical composition containing chorionic membrane extract and PD153035 exhibited an effect that can be exhibited at a concentration of 1.1 to 1.5 times, and the pharmaceutical composition including the amniotic membrane and chorion complex extract and PD153035 The results of this study are as follows.

When low concentrations of amniotic membrane extract, chorionic membrane extract, or amniotic membrane and chorion epithelium were used with PD153035, it was found that equivalent or better efficacy of amniotic membrane extract, chorionic membrane extract or amniotic membrane and chorionic villus extract (increased calcium deposition, Promoting gene expression, promoting bone differentiation, and preventing or treating bone diseases).

Claims (21)

Extracts from postnatal tissue selected from amniotic membrane extracts and complex extracts of amnion membrane and chorion membrane; And Epidermal Growth Factors (EGF) inhibitors as active ingredients,
Wherein the epithelial growth factor inhibitor is PD153035. The method according to claim 1,
The postpartum tissue extracts amnion, chorion, and amniotic and chorionic of complex tissue after delivery is selected from the water extract supernatant or 2-30% by volume of C _{1 - 4} A pharmaceutical composition, characterized in that the alcohol aqueous extract supernatant. delete delete delete The method according to claim 1,
Wherein the pharmaceutical composition comprises the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane combined extract at a concentration of 100 to 400 μg / ml, and the epithelial growth factor inhibitor is contained at a concentration of 10 to 20 μM A pharmaceutical composition. delete The method according to claim 1,
The bone disease is selected from the group consisting of osteosarcoma, bone defect, osteoporosis, fracture, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalacia, osteomyelitis fracture, osteogenesis disorder, degenerative bone disease and malocclusion &Lt; / RTI &gt; The method according to claim 1,
Wherein said pharmaceutical composition is for use in combination with an aggregate biomedical support selected from? -Triticalcium phosphate (? TCP), hydroxyapatite, and mixtures thereof. The method according to claim 1,
Wherein said pharmaceutical composition is physically coated or chemically bound to the surface of an aggregate biomedical support selected from? -Triticalcium phosphate (? TTP), hydroxyapatite and mixtures thereof. . The method according to claim 1,
Wherein the composition is formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, solutions or injections. The method according to claim 1,
(Calcitization) of bone formation or promoting osteogenesis. (1) peeling and washing the complex of the amniotic membrane or amniotic membrane and the hair thin membrane from the placenta;
(2) pulverizing a washed amniotic membrane or a complex of amniotic membrane and chorionic membrane to obtain a pulverized product;
(3) introducing the pulverized material into water or an aqueous solution of C1-4 alcohol at 2-30 vol% to elute the protein to obtain an extract;
(4) centrifuging the extract to collect a supernatant; And
(5) a step of adding PD153035 as an epithelial growth factor inhibitor to the supernatant, and a method for producing a pharmaceutical composition for preventing or treating osteoporosis, which comprises the tissue-derived postpartum extract and the epithelial growth factor inhibitor as an active ingredient. delete delete delete 14. The method of claim 13,
In step (5), the supernatant contains the amniotic membrane extract, chorionic membrane extract, amniotic membrane and chorionic membrane complex extract at a concentration of 100 to 400 μg / ml, and the epithelial growth factor inhibitor is mixed at a concentration of 10 to 20 μM &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. delete 14. The method of claim 13,
The bone disease is characterized in that it is selected from osteosarcoma, bone defect, osteoporosis, fracture, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalacia, osteomyelitis fracture, osteogenic disorder, degenerative bone disease and malocclusion &Lt; / RTI &gt; Extracts from postnatal tissue selected from amniotic membrane extracts and complex extracts of amniotic membrane and chorionic villus; And PD153035, which is an inhibitor of epidermal growth factors (EGF), were mixed and administered to animals other than humans &Lt; / RTI &gt; (EGF) inhibitor PD153035, which is selected from a combination of amniotic membrane extract and amniotic membrane and chorionic villus complex, is administered to an animal other than human, and the amount of epithelial growth factor inhibitor A method for controlling the rate of formation or acceleration.

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