TW201014614A - Composition for preventing or treating chemotherapeutic agent-induced alopecia - Google Patents

Abstract

Provided is a topical pharmaceutical composition for preventing or treating chemotherapeutic agent-induced alopecia, wherein the topical pharmaceutical composition includes a nanoliposome made of a lecithin-containing liposome membrane, into which an epidermal growth factor is encapsulated. Provided is also a combination preparation which includes the topical pharmaceutical composition and a chemotherapeutic agent so that they can be separately administered.

Description

201014614 VI. Description of the Invention: [Technical Field] The present invention relates to a composition for preventing or treating baldness caused by a chemotherapeutic agent, and more particularly, the present invention relates to a preventive or therapeutic treatment by chemotherapy The composition of the agent-initiated composition comprising nanolipid particles, wherein the nanolipid particles are encapsulated as an epidermal growth factor. [Prior Art] Baldness is an abnormal hair reduction caused by a decrease in the number of hairs in the growth phase of the hair cycle and an increase in the number of hairs in the degenerative or quiescent period of the hair cycle. Although (4) does not explicitly reveal the machine of baldness, it can be caused by the following reasons: excessive sebum production caused by the endocrine system Wei (such as irritating imbalance), autonomic nerve green Wei Lai ring pure Wei (such as blood circulation disorder); Degeneration of scalp function caused by nutrient deficiencies, dandruff-causing fungi, and other fungi; allergies; genetic causes; or aging. Baldness is one of the most serious side effects of cancer treatment and is also caused by the administration of various chemotherapeutic agents. Because chemotherapeutic agents affect actively dividing cells, chemotherapeutic agents cause side effects in metabolically active organs such as bone marrow, hair, finger licks, toes, skin, or the intestines, and thus cause baldness (Cassagnol and Cohen, US Pharm, 33, 10-18, 2008). Although 8〇% or more than 8% of patients receiving chemotherapy are the most painful (Macquart-Moulin et al., Br j Cancer, %, 164〇_1645, 1997), they still have an effective rib or turn The method of chemotherapy-induced bald 32396pif 4 201014614. Baldness occurs about 2 to 4 weeks after chemotherapy and the hair does not grow until 3 to 6 months after the end of treatment. The degree of baldness varies with the amount of drug used, the amount of drug, and the time course of administration. Examples of highly bald drugs include cyclophosphamide, doxorubicin, cisplatin, cytosine arabinoside, and etoposide (Botchkarev, JID) ❷ Symposium Proceedings, 8, 72-75, 2003). These drugs can cause baldness even when administered locally. To date, since scalp biopsy samples from chemotherapeutic patients are ethically unacceptable, only some mechanisms of hair follicle cell death caused by chemotherapeutic agents have been identified. For example, chemotherapeutic agents can affect the hair follicles during active cell division, which can cause hair follicle cell death, or can promote the transition from autogenous to long-term (Cassagn〇i and Cohen, US Pharm, 33, 10-18, 2008). . In general, 2 to 4 weeks after the start of chemotherapy, most hair shafts are exfoliated after emergence (the growth period is bald). The chemotherapeutic agent interrupts the somatic cell division and metabolism of the actively growing hair follicles, and thus the hair shaft becomes thin and falls off. Subsequently, the hair follicles of the skin degenerate due to cell death and reach a stationary phase (B〇tchkarev, JID Symposium Proceedings, 8, 72-75, 2003) ° Epidermal growth factor (EGF) is effective in preventing or treating baldness. EGF is a polypeptide having 53 amino acids and three disulfide bonds and having a molecular weight of 6, 〇45 Daltons, also known as urinary gastrin (ur〇gastr〇ne), which effectively blocks gastric acid secretion. EGF promotes mitosis and cell growth of various cells (including epidermal and mesenchymal cells) and blocks gastric acid secretion, thus effectively treating skin 32396pif 5 201014614 skin, corneal wound or gastric ulcer (Carpenter, Exp. Cell Res) 164, 1-1〇, 1986). It is disclosed in various patent documents that EGF can effectively treat baldness. U.S. Patent Nos. 5,616,471 and 6,113,889 disclose a method for promoting the proliferation of hair follicle cells by adding EGF to a culture medium for growing hair follicle cells. European Patent No. 1655015 A composition for regenerating hair is disclosed, which comprises EGF, dexpanthenol, socji Umhyaluronate, etc. extracted from bovine amniotic fluid. Japanese Patent No. 2003-081866 discloses a composition for growing hair, It comprises a sustained release gelatin hydrogel preparation containing a cell growth factor. Japanese Patent Publication No. 2004-284968 discloses a preparation for preventing baldness, which comprises EGF and an anthraquinone derivative or a pharmaceutically acceptable salt thereof. It is known that EGF can be used alone or in combination with other active substances to treat Baldness and other types of baldness caused by learning therapy. However, the prevention and treatment of baldness caused by chemotherapeutic agents by EGF is unsatisfactory. At the same time, EGF is extremely unstable at room temperature (especially in the presence of humidity). Therefore, there is a need to develop a method for stabilizing the physicochemical properties of EGF. In addition, due to the medical effects of EGF, it is usually required to be injected into the skin by topical administration, and it is therefore necessary to develop a method for effectively delivering the drug through the skin. For this reason, the applicant of the present invention has developed a nanolipid granule containing esterified lecithin and encapsulating coenzyme Qi 〇 and/or egf, and has filed an application for this purpose (Korean Patent Application Publication No. 2007-0101426) Lecithin is commonly used in cosmetics and foods because of its high moisturizing effect and harm to the human body. However, because lecithin is highly hydrophobic, it needs 32396pif 6 201014614 to heat and disperse lecithin at 70 ° C. In order to form a nanolipid in the form of a lipoid, however, it has not been developed to encapsulate a substance such as EGF which is unstable under temperature. However, according to Korean Patent Application Publication No. 2007-0101426, the above problem can be solved by forming a vesicle using vinegar lecithin. However, Korean Patent No. 2007-010M26 is not disclosed. EGF-containing nanolipid granules treat the possibility of baldness caused by chemotherapeutic agents. 0 The inventors of the present invention have devised to develop a medical substance effective for preventing and treating baldness caused by chemotherapeutic agents, and found to contain When the lecithin of the lecithin liposome membrane encapsulates EGF, it is more effective in preventing and treating baldness caused by the chemotherapeutic agent than the treatment of any other kind of baldness, and the present invention has been completed. SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition for effectively preventing and treating baldness caused by a chemotherapeutic agent. It is also an object of the present invention to provide a combined preparation for the independent administration of a chemotherapeutic agent and for the effective prevention and treatment of a medicated composition-initiated pharmaceutical composition. According to one aspect of the present invention, there is provided a topical pharmaceutical composition for preventing or treating baldness caused by a chemotherapeutic agent, wherein the topical pharmaceutical composition comprises a capsule made of a membrane containing ovoid vesicles Nano-lipids of epidermal growth factor. According to an aspect of the present invention, there is also provided a combined preparation comprising the topical pharmaceutical composition and a chemotherapeutic agent such that the ingredient can be administered independently of 32396pif 7 201014614. The above and other objects, features and advantages of the present invention will become more <RTIgt; [Embodiment] Hereinafter, the present invention will be described in detail. In the present specification, the term "encapsulation" means the inclusion of a hydrophilic substance in the hydrophilic region of the center of the vesicles, or the retention of hydrophobic substances in the lipid bilayer of the vesicles. The term "nanolipids" refers to vesicles having a diameter of from about 1 to about 2 nanometers, typically dispersed by a pressure of about 1000 pounds per square inch (psi) or higher. Made from micron vesicles. The composition of the present invention comprises: a nanolipid granule which is encapsulated with epidermal growth factor (EGF) made of a lecithin-containing liposome membrane. Since the nanolipid granules made of the lecithin-containing micro-salt granules are encapsulated with Egf, the baldness caused by the chemotherapeutic agent may be more effectively prevented or treated than any other type of baldness. Further, since EGF is encapsulated by a nanolipid in the composition of the present invention, it is ensured that the stability is extremely unstable at room temperature and at the time of reduction. The egg fat used for preparing the nanolipid can be hydrogenated egg yolk. When the (4) lipid is formed into a nano-lipid, the S-seed egg fat can provide a higher moisturizing effect than the hydrogenated egg fat when it is administered locally. Thus, the use of the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The amount of baldness elicited by the chemotherapeutic agent is used, and the therapeutically effective amount may vary depending on the degree of baldness caused by the chemotherapeutic agent and the age, sex, and sensitivity of the patient. With respect to the composition of the present invention, the amount of EGF may range from 0 0001 to 0.1 weight A (1 to 1000 μg/ml) based on the total weight of the nanolipid particles (g/v; usually g/ml). For example, in the range of up to 0.01% by weight (1 Torr to 100 μg/ml), and the 0 dose of EGF can be administered to the affected area as a single dose or divided into several portions. The encapsulated EGF-containing nanolipid granules made of a lecithin-containing liposome membrane can be prepared according to the disclosure of Korean Patent No. 2007-0101426, which is filed by the applicant of the present application and is incorporated by reference in its entirety. The manner is incorporated herein. The liposome membrane comprises egg wall fat. For example, the egg yolk may comprise hydrogenated egg yolk fat, esterified egg can or a combination thereof. Further, if necessary, the liposome membrane may further comprise any of the vesicle membrane components (i.e., lipidoids) which are usually used to form vesicles. The lipidoid may comprise phosphoglyceride or sphingolipid, such as phospohatidylcholine (ie lecithin), hydrogenated lecithin, phosphatidylethanolamine, squamous muscle Phosphadydylinisitol, ceramide, cereborside (ie galactosyl ceramide), sphingomyelin, gangloside, and combinations thereof. Lipids such as esterified lecithin provide moisturization. The liposome membrane may comprise hydrogenated lecithin and/or ceramide in the lipidoid. Hydrogenated egg phosphorus 32396pif 9 201014614 曰疋 = all unsaturated hydrocarbons are reduced to form a saturated hydrocarbon (IV) lipid. The esterified egg-breaking fat contained in the microlipid film of the diced rice gluten has high dispersibility with respect to water or alcohol and has both hydrophilicity and hydrophilicity. Therefore, since the esterified lecithin has a polarity suitable for dispersion in a hydrophilic solvent but is not suitable for complete dissolution in a hydrophilic solvent, when the esterified egg wall is used to form a liposome film, the obtained liposome solution is 2 〇 至 to 6〇. 〇 It is stable at low temperatures. S-specific lecithin can be prepared by reacting lecithin or hydrogenated lecithin with an organic acid. The esterification reaction is a reaction in which an alcohol group of lecithin is reacted with an organic acid to remove water molecules. The esterified lecithin reacts with water in an aqueous solution to dissociate into lecithin (alcohol group) and an organic acid. Therefore, esterified lecithin, lecithin (or hydrogenated lecithin), and an organic acid may coexist when an appropriate equilibrium state is maintained in the following dissociation reaction depending on the acidity. KCOOR' (esterified lecithin) public RCOOH (organic acid) + R, 〇 H (egg fat or hydrogenated egg fat) The organic acid used to form the esterified lecithin can be the organic acid commonly used in cosmetics or foods. For example, an anhydrous organic acid, and may be acetic acid, malic acid, lactic acid, glycolic acid, citric acid or oxalic acid, for example. Anhydrous malic acid or anhydrous acetic acid. When anhydrous acetic acid is used, the esterified lecithin has a stronger hydrophilic property than when anhydrous malic acid is used. The organic acid can be appropriately selected depending on the amount of EGF encapsulated by the nanolipid. When applied to the skin, the above organic acids may cause the action of exfoliating or softening the skin. 32396pif 10 201014614 Esterified lecithin can be used in an amount capable of forming vesicles. For example, the amount of esterified egg fat can range from about j to 5 parts by weight based on 1 part by weight of EGF. The nanolipid may further comprise at least one selected from the group consisting of triglyceride-based organic compounds (for example, caprylic/capric triglyceride) as a dispersing agent. Anionic surfactant (such as diethylamine cetylphosphate, phosphatidylcholine sodium salt or triethylaminecoconyl glutamine sodium) Softener (such as butylated hydroxy toluene), amphoteric surfactant based on beet test (eg propylbetainelolylamine propylbetaine, laurel beet test (lolylbetaine) Or cocoamphopropyl propyl betaine; and a chelating agent (eg, ethylenediamine tetraacetate sodium salt). The nano vesicles can be prepared according to the method disclosed in Korean Patent No. 2007-0101426. For example, a method of preparing a nanoliposome can comprise reacting lecithin with an organic acid to form an esterified lecithin (step 1); dissolving the acetified lecithin and EGF in a solvent (step 2); and dispersing The resulting solution was obtained to obtain nanolipids (step 3). In step 1, an esterified egg structure is formed by condensing lecithin with an organic acid. For example, the organic acid may be selected from the group consisting of acetic acid, malic acid, lactic acid, glycolic acid, citric acid, and oxalic acid. 32396pif 11 201014614 Oil phase::=::::= Oil phase solution and aqueous phase solution. Prepare the aqueous phase solution, and mix the alcohol used to prepare the oil phase solution. When the oil phase solution is made, it is not limited to) a hydrophilic organic compound such as caprylic acid/capric acid triacetate (all amines, sulphur-based vinegar, anti-bad money = such as -ethylamine cocoyl bran Amino acid sputum. Phosphorus choline or triethylbenzene). Amino acid), or a softening agent (such as a butylated permeation group f to dissolve H^=, T_EGF roll aqueous active ingredient such as diethylamine ten!" =, an anionic surfactant can be added (the amines ten, An aqueous phase solution is prepared by calcining vinegar, sodium ascorbate, sodium sulphate, or a chelating agent such as sodium edetate. At the temperature of 〇C, the dissolution temperature of the acetated printing grease and EGF is lower than 2〇rc', although the acetification _lipid and egf 'W will be too long and the solution may be unstable. The temperature is higher than 6 〇. (:, EGF may be unstable. S / Mi 〇 oil phase / gluten solution and aqueous phase solution, use a uniform mixing type mixer (h0m 〇 miXerstirrer) to evenly stir the mixture. In the liposome solution, the microlipids have a micron diameter. In step 3, 'the uniformly mixed solution prepared in step 2 is passed through the microfluidizer at a pressure higher than about ten square feet. 32396pif 12 201014614 (microfluidizer, M/F) at least once to disperse the liposome to the nanometer level, from A nanolipid solution is produced. If the pressure is less than about 1 〇〇〇 psi, the nano vesicles may not be obtained. The uniformly stirred mixed solution may be passed through the M/F twice or more. In the nanolipid solution prepared as described above, the nanolipid particles form an oil phase-containing aqueous phase type liposome in the aqueous solution, and thus, can be located at the top of the nanolipid The encapsulated EGF in the aqueous phase is stable. φ The composition of the present invention can be formulated into a dosage form suitable for topical administration. For example, the composition of the present invention can be formulated into a solution, an ointment, an emulsion or a cream for local application. Dosing. If necessary, 'mix the composition with at least one pharmaceutically acceptable carrier or additive selected from the group consisting of: stabilizers such as amino acid, sodium bisulfite (sodium bisulfite), sodium metabisulfite, sodium sulphite, disodium ethylenediaminetetraacetate, sodiumbisulphite, sodium formaldehyde sulfoxylate (so Dylformaldehyde ❹ sulfoxylate ), thiourea or acetone sodium bisulfate; humectants such as ceramide, glycerine, propyleneglycol, ammonium alginate, Cyclomethicone, dimethicone, polydextrose, sodium hyalurondate, sodium lactate, sorbitol, triacetine , triethanolamine or xylitol; an emulsifier such as poly 32396pif 13 201014614 polyoxyethylene stearate; and sodium benzoate, bismuth benzoate Methyl (paraffoxybenzoate), isopropyl paraoxybenzoate, isobutyl paraoxybenzoate, sorbic acid, sorbic acid Sorate ), sodium sorbate, dihydro acetic acid, sodium dihyd Roacetate ), benzalkonium chloride, benzethonium chloride, phenol, crest (cresro), phenol (chi〇rocres〇i), and benzene Methylalcohol. The above pharmaceutically acceptable carriers or additives are described in detail in Remington&apos;s Pharmaceutical Sciences (19th Ed., 1995). The composition for preventing or treating the baldness caused by the chemotherapeutic agent of the present invention can be separately formulated and sold to the relevant market. However, the composition may also be packaged and distributed with a chemotherapeutic agent to prevent or treat the priming caused by the priming chemotherapeutic agent. The combination preparation according to the embodiment comprises a composition and a chemotherapeutic agent which are administered topically for preventing or treating baldness caused by a chemotherapeutic agent, each of which is separately administered. When a combination preparation is used, the composition of the chemotherapeutic agent can be simply administered to the patient before the administration of the chemical agent for inducing baldness, in order to prevent the development of the head, or, in the case of difficult scales. Thereafter, (d) § is administered to the patient to treat the developed bald head. 32396pif 201014614 The chemotherapeutic agent included in the combination preparation may be any chemotherapeutic agent that may cause baldness. Examples of chemotherapeutic agents include antimetabolites, alkylating agents, topoisomerase inhibitors, anti-microtubule agents, antitumor antibiotics, and plant alkaloids. In particular, the chemotherapeutic agent can be, for example, but not limited to, doxorubicin, cyclodecalamine, cisplatin, cytarabine, etoposide, dactinomycin, daunorubicin ), docetaxel, idarubicin, ifosfamide, irinotecan, paclitaxel, topotecan, bole Bleomycin, mechlorethamine, methotrexate, epirubicin, ifosphamide, Vindesine, vinorelbine , amsacrine, melphalan, vincristine, vinblastine, gemcitabine, 5-fluorouracil, busulphan, Lumustine or thi〇tepa. ® The present invention will be described in more detail with reference to the following examples. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: Preparation of encapsulated EGF-negative nanolipids using argonized lecithin (1) Preparation of an oil phase solution To 200 g of ethanol, 40.0 g of hydrogenated lecithin, 200 g of caprylic/capric triglyceride, 6.0 was added.克克 diethylamine cetyl phosphate and 10.0 g of butylated hydroxytoluene, followed by stirring at about 6 (TC heating 32396 pif 15 201014614) to the room temperature to dissolve the mixture. 'Therefore, an oil phase solution is produced. (2) Preparation of an aqueous phase solution Adding μg (10) gamma A, i 〇 and W g _ to 1541.5 g of pure water, and at room temperature _ to completely dissolve the corpse Thereby producing an aqueous phase solution. (3) Preparation of nano vesicles egf

= phase towel was added with an aqueous phase solution, and the mixture was thoroughly mixed with a (iv) homomixer for 5 minutes or longer until a homogeneous solution was obtained. The solution is passed through micro at a pressure of the square of the cake or higher. The dispersion is at least _ times to disperse the microbes to the nanometer level to produce a non-deficient EGF solution. When the mixed solution passes through the microfluidizer, the temperature is adjusted to be equal to or lower than room temperature using a coolant. Poor 2. Preparation of encapsulated EGF nano-deer using 磨化_碟廉 (ΌPreparation of esterified egg fat

31 g of hydrogenated saturated hydrocarbon egg yolk was dissolved in 200 g of tetrahydrofuran solution by heating at a temperature of 70 °C. When hydrogenated saturated hydrocarbon egg phosphorus was added, 3 g of anhydrous malic acid or 2 5 g of dihydrate acetic acid was added thereto and dissolved. When it is sufficiently dissolved, 〇5 Δg of an alkylamine or an alcoholamine as a catalyst is added thereto, followed by circulation/reflow cooling for 3 hours or longer. When the reaction is terminated, the reaction product is dried under vacuum at a temperature of S 45 〇C, and (iv) is produced as a white powder in the form of a vinegar. 32396pif 16 201014614 (2) Injury oil phase solution 30 grams of esterified lecithin, 200 grams of grams of diethylamine cetyl phosphate was then 'heated by heating at about 40 ° C. Subsequently, the obtained product was cooled to room temperature, and octanoic acid/capric acid triglyceride, 1 Torr, and 0.5 gram of butylated hydroxytoluene were added to U gram of ethanol to dissolve the mixture to produce an oil phase. Solution. (3) Preparation of aqueous solution

Under stirring, the aqueous solution was prepared by completely dissolving 650 g of pure water in a solution of 5 g of Na-EDTA, 1.0 g., and 1 g of EGF at room temperature. (4) Prepare a nano vesicle egf secret (4) 1 Γΐ + aqueous phase solution, and allow the homomixer to fully mix the H material for 5 minutes or more until a homogeneous solution is obtained. The hybrid test was passed through a microfluidic homogenizer under a pressure of a height of ten square feet to disperse the microfilaments to the natrix, thereby producing a nanolipid EGF solution. When the aged solution passes through the microfluidizer, the coolant is used to adjust the temperature to be equal to or lower than room temperature. Experimental Example 1: Stability Evaluation of Formulations Regarding the nanoparticle vesicles EGF solution prepared according to Example 1 (when hydrogenated egg yolk fat was used) and Example 2 (when esterified lecithin was used), the average particle diameter was compared and The gelation phenomenon occurs when the nano-lipid EGF solution is placed at room temperature for a long time. The results are shown in Table 1. In addition, the Ostwald ripening phenomenon (Oswald W, Z. Phys. Chem., 34} 32396pif 17 201014614 4 grains = 1 ΓΓ 四 (4) 瓦 咖 化Small particles are deposited on the larger particles due to the solution, and thus the particles are gelled, and the gel is dispersed again. The time elapsed when the gel dissolves is defined as the gelation time. Again [Table 1]

As illustrated in Table 1, the nanolipid EGF solution prepared according to Examples i and 2 had similar stability, and Ostwald ripening did not occur even when left for 8 weeks. Therefore, the nanolipids prepared according to the examples have an excellent stability. Experimental Example 2: Effect of EGF on the growth of rat vibrissa follicles Hair follicles were isolated and cultured from a 3 week old Wistar male rat and used in this experiment. A 24-well plate was prepared. 500 microliters of William E medium (William E medium X Gibco Inc., NY, USA) was placed in each well. It contained 2 millimoles per liter of L-glutamine (Gibco Inc. , NY, USA), 10 μg/ml insulin (Sigma, MO, USA), 50 mil/liter hydrocortisone (Sigma, MO, USA), 100 units/ml penicillin (enicillin) And 100 μg/ml streptomycin (Gibco Inc., NY, USA), and add a single hair follicle to each well and in a 32396 pif 201014614 incubator at 37 ° C, 5% C 〇 2 Cultivate. In this experiment, the concentration of EGF was G.Bu G.3, 1 and 1 G Ng/ml, and the concentration of 1 μmol/L (μΜ) of Mindidine Sulfate was added (min〇xidil made him) (Sigma, USA) was used as a positive control substance and the medium was changed every three days during the incubation period. On the first day and the 17th day after the incubation, the length of the hair follicle was measured using an image analyzer (Olympus Japan) to identify the growth rate of the hair follicle. The results are shown in Table 2 below. Drug group f-subgroup EGF (Nike/ml) 1 micromol/liter literidine 0.1 0.3 1 10 Day 1 (micron) 275 251 280 295 261 298 'Phase π (micron) 308 315 334 373 301 4Ϊ4 As shown in Table 2, when the EGF concentration was 1 Ng/ml, the growth of hair follicles was increased by 38%, which was significantly increased compared with the control group. Experimental Example 3: The effect of EGF on the proliferation of rat dermal papilla cells in the tentacles Q in the incubator, at a temperature of 37 ° C, 5% C02, using 100 units / ml penicillin, 1 〇〇 microgram / ml of Streptomyces Rat whisker dermal papilla cells were cultured in DMEM (Hyclone Inc, USA) medium of 10% FBS (Gibco Inc" NY, USA). Subculture was performed every three days. Dermal papilla cells were placed in 96-well plates. Incubation for 24 hours. Subsequently, the medium used was replaced with serum-free DMEM medium, followed by incubation for 24 hours, and the medium was treated with the sample. Subsequently, incubation was carried out for 4 days and the dermal papilla cells were evaluated by MTT assay (Sigma, MO, USA). Proliferation. 32396pif 19 201014614 In this experiment, the concentration of EGF was 0.1, 0·3, 1 and l〇N/ml, and the concentration of minoxididine sulfate (Sigma, USA) used as a positive control substance was 1 micro. Moere/liter. The results are shown in Table 3. [Table 3] Drug group control group EGF (Nike/ml) Ear/Male 0.1 0.3 1 10 Cell growth (%) 100 120.5 126.0 125.9 118.4 132.2 As shown in Table 3 As shown, when the EGF concentration is 〇.3 and 1 At gram/ml, the proliferative effect of dermal papilla cells increased by 26%, which was significantly increased compared with the control group. Experimental Example 4: Prevention of baldness in a bald model of a rat with a chemotherapeutic-inducing baldness In this experiment The 1-day-old SD rats were used to evaluate the preventive effect on the baldness induced by the chemotherapeutic agent. The body weight of the SD rats was measured and the average body weight was calculated, and each group contained UISD rats. After 1 to 5 days and 8 to 38 days, the drug was administered to the patient twice or six times a day, and the area of the bald head was evaluated. In this experiment, the intraperitoneal injection dose of 4 mg was given to the 8-day-old rat. / kg of the chemotherapeutic agent Doytomycin (Boryung Co., Ltd) to initiate the filling. In addition, the immunohistochemical staining & during the administration to assess the changes in the long finger receptor (EGFR) Effect on cell proliferation. In this experiment, the drug-injected tissue was sectioned at appropriate time points and used as a test sample. Using 32396pif 20 201014614 for Ki-67 (a cell proliferation marker molecule) mouse Derived anti-Ki-67 Mouse antibody (Dako, Denmark) was used to measure the ratio of cells growing in the hair follicle base. In addition, anti-EGFR rat antibody (Cell Signaling Technology, USA) was used to analyze EGFR. In this experiment, by using an image analyzer (Version 4.01 image measurement standard v4.01, Bersoft, Dominica) was measured to quantify all data covering the bald area. The drug was EGF or Nanomicro φ lipid granules (nano EGF) prepared according to Example 2. The evaluation results of the bald area are shown in Table 4 and Figure i. The area of the bald area was visually observed and the image thereof is shown in Fig. 2, which is a graph showing changes in EGFR, and Fig. 4 is a graph showing the results of analysis of cell proliferation. [Table 4] EGF against baldness induced by doxorubicin in baldness model (balt area (cm 2 )) After birth (days) 13 14 19 22 26 29 34 37 Unmedicated group 0.00 0.72 1.35 1.68 2.0 1.22 0.51 0.25 Negative control group 0.00 0.34 1.47 1.71 1.51 1.00 0.26 0.04 Experimental group 1 0.00 0.66 1.42 1.39 1.25 0 46 0.15 0.03 妙絚2 0.00 0.52 1.03 U3 1.05 0.29 0.08 0.01 Experimental group 3 0.00 0.53 0.97 1.10 1.27 0.55 0.02 0.00 Experimental group 4 0.00 0.05 0.35 0.50 0.33 0.00 0.00 0.00 Experimental group 5 0.00 0.09 0.27 0.61 0.57 0.00 0.00 0.00 Test group 6 0.00 0.00 0.04 0.14 0.21 0.00 0.00 0.00 (Untreated group (-X-); negative control group (_·), administration medium Group 1 (-△_), EGF (10 μg/ml) twice daily; Group 2 (-▲〇, EGF (50 μg/ml) twice daily; Group 3 ( _〇·), 32396pif 21 201014614 Daily administration of nano EGF (.10 μg/ml) twice; group 4 (_·_), daily administration of nano EGF (50 μg/ml) twice; group 5 (_◊_), Inject Nai # EGF (1G microgram/ml) six times a day; and group 6 (·♦ small each Tiantou and nano EGF (50 μg/ml) six times) As shown in Table 4, Figure 1 and Figure 2, nano EGF showed extremely high bald prevention. When EGF concentration was high (5G μg/ml) 1 〇 microgram / ml) and the number of local administration of EGF is also high (six times > twice), showing better prevention of baldness (Table 4 and Figure 另外. In addition, the prevention effect of nano egf is not changed The Egf of the nanolipids was high. In addition, as shown in the results of cell proliferation and EGFR analysis in Fig. 3 and Fig. 4, only in the group administered with EGF, the cell proliferation was improved & the Egfr expression was increased. Therefore, it is determined that the prevention of baldness is associated with the promotion of cell proliferation in the hair follicle base and the increase in EGFR expression. Therefore, EGF prevents baldness caused by chemotherapeutic agents by increasing the expression of EGFR and promoting the proliferation of hair follicle basal cells. Example 5: Evaluation of Stability of EGF To evaluate the stability of EGF encapsulated by nanolipids, the EGF solution prepared according to Example 2 was stored at 75% RH under severe conditions and at a temperature of 40 °C (1〇 Millol/L of phosphate buffered saline, pH 7 4 And the nano EGF solution' and compared their relative stability for 3 months. The amount of residue was measured using an ELISA method (Human EGF Quantikine ELISA kit [Cat. No. DEG00], R&amp;d Systems). The results are shown in Table 5 below. ' [Table 5] 32396pif 22 201014614

Nano EGF stability test results (n = 8)

4 weeks

In addition to the 'encapsulation of β by nanolipid granules, the stability of nano EGF was measured in the same condition as described above under refrigeration conditions (4. underarm for 48 weeks. The results are shown in Figure 5. As shown in Figure 5, the initial amount of 95% of nano EGF was maintained for 48 weeks. ❷ As described above, 'EGF is encapsulated by nanolipids made of lecithin-containing vesicles, so EGF The effect on baldness, especially EGF, on the prevention and treatment of baldness caused by chemotherapeutic agents is significantly increased to the extent that is unpredictable to those skilled in the art. Therefore, the composition of the present invention comprises microphospholipid-containing A composition of encapsulated EGF nanolipids made of lipid granules, which is effective in preventing or treating a representative side effect of a chemotherapeutic agent, namely baldness. Although the invention has been particularly shown and described with reference to exemplary embodiments of the invention It will be appreciated by those skilled in the art that various changes can be made in the form and details of the present invention, without departing from the spirit and scope of the invention as defined by the appended claims. Figure 1 shows a graph of the effect of nano-lipid EGF (negative EGF) on the prophylactic effect of the SD rat model induced by the chemotherapeutic drug doxorubicin. The evaluation was compared with the untreated group, the negative group, and the EGF solution group: untreated group (-X-), negative control group (__), administration medium; group 丨 (_△_), daily dose EGF (1 μg/ml) twice; Group 2 (_▲_), EGF (50 μg/μl) twice daily; Group 3 (_〇_), daily administered with EGF (l 〇 micrograms / ml) twice; group 4 (_·_), daily nano-egf (50 μg / ml) twice; group 5 (_ 〇 _), daily injection of nano egf (i 〇 microgram / ML) six times; and group 6 (-♦-), administered daily with nano EGF (50 μg/ml) six times. Figure 2 shows the macroscopic image 'The image shows nano EGF versus the chemotherapeutic agent doxorubicin The prevention of baldness in the SD rat model of baldness was evaluated by comparison with the effects of the unadministered group, the negative control group, and the Egf solution group. Fig. 3 is about the chemotherapeutic agent A graph of the distribution of epidermal growth factor receptor (egfr) administered by a drug in a SD-induced SD rat model compared to the EGFR distribution in the unadministered, negative, and EGF solutions To evaluate: untreated group (-〇-); negative control group (-▲-), administered medium; group i (__), administered EGF (10 μg/ml) six times a day; group 2 (_· _), daily administration of nano EGF (50 μg / ml) six times; and normal animal group 32396pif 24 201014614 Figure 4 is about cell proliferation of drug-treated SD rat model induced by chemotherapeutic drug doxorubicin-induced baldness (Ki_67 labeled molecule), the cell proliferation was evaluated by comparison with cell proliferation in the unadministered group, the negative control group, and the EGF solution group. · Unadministered group (-〇-), negative control group (-▲-), administration medium; group 丨 (__), administered EGF (10 μg / ml) six times a day; group 2 (_··), daily administration of nano EGF (50 μg / ml) six Times; and normal animal groups. Fig. 5 is a graph showing the evaluation results of the stability of nano EGF under cold storage conditions. β [Main component symbol description] None 32396pif 25

Claims (1)

201014614 VII. Patent application scope: _ i ^ Topical pharmaceutical composition 'Used for lion or treatment of (3) head caused by chemotherapeutic agent, the topical pharmaceutical composition includes: Nai made of egg fat-containing lipid film The liposome is encapsulated with epidermal growth factor. 2: The topical pharmaceutical composition according to item yi of the patent application, wherein the (IV) lipid contained in the micro-secret film is hydrogenated-lipid, _ oxidized egg squama or a combination thereof.局部 3: The topical pharmaceutical composition according to claim 2, wherein the microlipid film containing the brewing (tetra) lipid further comprises a gasified lipid, a neuronamine or a combination thereof. 4. The topical pharmaceutical composition according to claim 2, wherein the vinegar (tetra) lipid comprises a reaction product of a lipid or a hydrogenated (tetra) lipid and an organic acid. 5. The topical pharmaceutical composition according to claim 4, wherein the organic acid is acetic acid, anaphyllin, lactic acid, glycolic acid, or oxalic acid. 6. The topical pharmaceutical composition according to claim 2, wherein the ester-containing lecithin-containing nanolipid granule further comprises at least one selected from the group consisting of an anionic surfactant, An organic compound, a softener, a chelating agent, and an amphoteric surfactant mainly composed of triglyceride. / 7. The chemotherapeutic agent described in the section referred to in the scope of the patent application is an antimetabolite, a burning agent, a topoisomerase inhibitor, 32396pif 26 201014614, an anti-microtubule agent, an antitumor antibiotic or a plant alkaloid. . 8: The topical pharmaceutical composition according to claim 7, wherein the chemotherapeutic agent is doxorubicin, cyclomethamine, occupation, arsenic, etoposide, actinomycin D, ordosin , Docetaxel, Yirm Kang, Pacific Paclitaxel, Topotecan, Bole A (four) 呤, Epirubicin, Isocyclic Phosphate, Changchun Cellulite ί' π Acridine, Melphalan, Changchun New Alkali, vinblastine, garcinoxin, 5-fluorouracil, busulfan, lomustine or thiotepa. 9. A combination preparation comprising a topical pharmaceutical composition and a chemotherapeutic agent as described in paragraphs 1 to 8 of the Wei Wei, such that the ingredients can be administered independently. 10. The combination preparation according to claim 9 of the patent scope, wherein the treatment is Ahusu, cyclodamine, lysine, glucosinolate, lysine D, daunorubicin, docetaxel, Idarubicin, sedative rituximab, paclitaxel, topotecan, bolapiamide, epirubicin, vindesine, vinorelbine, Hi, vinca new test, periwinkle test, gemcitabine , 5_ fluorourine, busulfan, lomose juice or sputum. 32396pif 27