WO2010082789A2 - Compositions anticancéreuses comprenant du cédrol nanoparticulaire - Google Patents

Compositions anticancéreuses comprenant du cédrol nanoparticulaire Download PDF

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WO2010082789A2
WO2010082789A2 PCT/KR2010/000276 KR2010000276W WO2010082789A2 WO 2010082789 A2 WO2010082789 A2 WO 2010082789A2 KR 2010000276 W KR2010000276 W KR 2010000276W WO 2010082789 A2 WO2010082789 A2 WO 2010082789A2
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cedrol
cancer
acid
pharmaceutically acceptable
acceptable salt
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PCT/KR2010/000276
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WO2010082789A3 (fr
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Byoung Woo Kim
Hyun Ju Kwon
Eun Jin Seo
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Dong-Eui Educational, Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a method for preparing cedrol or a pharmaceutically acceptable salt thereof in a nanoparticle form, and a composition for the treatment and prevention of cancer, comprising the nanoparticles prepared by the same.
  • Nanobiotechnology initially got started with the manufacture of nano-scale structures and has become a growing field of interest worldwide. Most of the methods can be used in the preparation of drug particles as well as other various organic particles, and also for nano encapsulation (see, Zhiping Zhang and Si-Shen Feng, The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)tocopheryl polyethylene glycol succinate nanoparticles, Biomaterials, 27, 4025-4033. 2006).
  • Cedrol as used herein belongs to this set of poorly soluble drugs.
  • Juniperus chinensis an evergreen coniferous tree native to Korea, is widely distributed. It has the efficacies of relieving rheumatism, dispelling cold, extinguishing blood stasis, and detoxification, and has been used in traditional medicines for the treatment of cold, flu, joint pain caused by rheumatism, measles, induration, and the early stage of tumor. Recently, several chemical ingredients have been isolated from Juniperus chinensis, and their structures have been also clarified.
  • norpimarane and norabietanes were isolated from the leaf of Juniperus chinensis, and many chemical ingredients ( ⁇ -cuparenol, ⁇ -acorenol, thujopsen-12-ol, isoleptographiol, caryophylleneoxide, 6,7-epoxycaryophyll-3-en-14-ol, cedrol, cedran-3a-ol, cedrane-3/3,12-diol, ferruginol, hinokiol, dehydroabietinol, abieta-8, 11,13-trien-7-one, sugiol, totarol, sandaracopimaric, trans -communic acid, cis -communic acid, agathic acid, isocupressic acid, acety lisocupressic acid, 15-hydroxylabd-8-en-19-oic acid, 15,16-bisnor-13-oxolabda
  • cedrol or the pharmaceutically acceptable salt thereof exhibits excellent therapeutic and prophylactic effects on cancer.
  • cedrol is a poorly soluble compound, it has been difficult to formulate it into oral solid dosage forms by the processes of granulation, mixing, and tabletting. Accordingly, the present inventors considered many methods for introducing cedrol into the human body. They found that a nanoparticle form of cedrol prepared by the present method is easily introduced into a human body and exhibits an excellent sustained release effect in the body, and thus can be effectively used for the treatment and prevention of cancer, thereby completing the present invention.
  • Nano drug delivery systems are advantageous in that selective, topical treatment can be achieved. Therefore, much focus has been placed on nano drug delivery systems.
  • the nanoparticle form (NM) of a drug or poorly soluble molecule could reduce side effects of the drug and maximize its efficacy.
  • the composition prepared by the present invention is very useful for the treatment and prevention of cancer.
  • FIG. 1 is a diagram showing the process of preparing the nanoparticles comprising the cedrol or pharmaceutically acceptable salt thereof of the present invention
  • FIGs. 2 and 3 are diagrams showing the size of the cedrol nanoparticles prepared by the method of the present invention, depending on PVA concentration and sonication time;
  • FIGs. 4 to 9 are diagrams showing FE-SEM of the cedrol nanoparticles prepared by the method of the present invention, depending on PVA concentration and sonication time;
  • FIG. 10 is the result of DSC thermogram showing physical properties of the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 11 is a graph showing the cedrol release of the cedrol nanoparticles prepared by the method of the present invention, namely, the cedrol release of NM depending on PVA concentrations and time;
  • FIGs. 12 to 16 are the results showing cytotoxicity depending on PVA concentrations and sonication time, when HT29 cells were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 17 is the result of Western blot showing inhibition of MCM protein expression, when HT29 cells were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 18 is a graph showing changes in body weight, when cancer animal models were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 19 is a graph showing the tumor size over time, when cancer animal models were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 20 is a graph showing the weight of tumor, resected from cancer animal models that were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • FIG. 21 is a diagram showing the reduced volume of malignant tissue, when cancer animal models were treated with the cedrol nanoparticles prepared by the method of the present invention.
  • the present invention relates to a method for preparing cedrol or a pharmaceutically acceptable salt thereof in a nanoparticle form, comprising the steps of (a) dissolving the cedrol or the pharmaceutically acceptable salt thereof in an organic solvent, mixing the solution with an amphiphilic biopolymer and then mixing it with a surfactant at a concentration of 3 to 7%; (b) sonicating the mixture of (a) to form an emulsion; and (c) centrifuging and washing the emulsion of (b) 4 to 6 times and then drying it.
  • cedrol or pharmaceutically acceptable salt thereof according to the present invention is described in Korean Patent No. 673574, and the disclosure is incorporated herein in its entirety, as long as it is within the scope of this invention.
  • the cedrol of the present invention refers to a compound that induces apoptosis of cancer cells and has an inhibitory effect on cancer cell growth. More particularly, the cedrol of the present invention induces cell death of cancer cells by apoptosis through nuclear morphological change, release of mitochondrial cytochrome c, activation of caspase-8, caspase-9, and caspase-3, and cleavage of poly (ADP-ribose) polymerase, and has an inhibitory effect on the expression of a replication initiation factor, MCM protein.
  • MCM protein replication initiation factor
  • cedrol can be prepared by chemical synthesis methods well-known to those in the art, and preferably obtained by isolation from Juniperus chinensis, which is an evergreen coniferous tree belonging to the family Cupressaceae.
  • Cedrol as used herein embraces a pharmaceutically acceptable salt thereof, optionally.
  • the pharmaceutically acceptable salt of the cedrol of the present invention comprise all the acidic or basic salts which may be present in the compound of the present invention, as long as particular mention is not made.
  • the pharmaceutically acceptable salt includes sodium salt, calcium salt, and potassium salt of hydroxy group
  • other pharmaceutically acceptable salts of the amino group includes hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate).
  • the salts can be produced by a preparation method or preparation process thereof known in the related art.
  • acid addition salts prepared with free acids are preferred.
  • the acid addition salts are prepared by the conventional method, for example, a method comprising the steps of dissolving a compound in an excessive amount of acid aqueous solution, and precipitating the salt using water-miscible organic solvents such as methanol, ethanol, acetone or acetonitrile.
  • Acid or alcohol e.g., glycol monomethylether
  • the mixture is dried by evaporation or the precipitated salt can be suction-filtered.
  • organic acids and inorganic acids may be used.
  • the inorganic acids include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid
  • examples of the organic acids include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, and hydroiodic acid.
  • the pharmaceutically acceptable metal salt form may be prepared by using a base.
  • the alkali metal or alkali-earth metal salt thereof can be prepared by a conventional method, for example, after dissolving the compound in an excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof.
  • sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt (e.g., silver nitrate).
  • the first step is a step of dissolving the cedrol or the pharmaceutically acceptable salt thereof in an organic solvent, and mixing the solution with an amphiphilic biopolymer to mix it with a surfactant at a concentration of 3 to 7%.
  • Cedrol is a poorly soluble compound, which has the property of being poorly soluble in water. Because of this property, there have been many difficulties in the preparation of a therapeutic or prophylactic drug for cancer comprising cedrol. Therefore, in order to solve the problem, cedrol is first dissolved in an organic solvent in the present invention. Any organic solvent may be used, and preferably methylene chloride, acetone, dichloromethane, dimethylformamide, dimethylsulfoxide, acetonitrile, tetrahydrofuran, or methanol. If necessary, the mixtures thereof may be used. Further, if necessary, they may be mixed with a small amount of water.
  • the cedrol or pharmaceutically acceptable salt thereof dissolved in the organic solvent is mixed with an amphiphilic biopolymer.
  • the amphiphilic biopolymer in any organic solvent may be used, preferably poly(lactic acid) or poly(lactic acid-co-glycolic acid).
  • the cedrol or pharmaceutically acceptable salt thereof is preferably mixed with the amphiphilic biopolymer in a ratio of 1: 10, and more preferably in a ratio of 1: 5.
  • a surfactant is dissolved in the solution at a concentration of 3 to 7%.
  • the surfactant is dissolved in the solution at a concentration of 4 to 6%. If the concentration of the surfactant is more than 7%, there are disadvantages that the size of the final nanoparticle becomes larger, encapsulation efficiency becomes low, and sustained release of cedrol or pharmaceutically acceptable salt thereof increases. Therefore, when the concentration of the surfactant is within the proper range from 3 to 7%, preferably 4 to 6%, such disadvantages can be overcome. If the concentration of the surfactant is less than 0.5%, it may cause problems in the formation of nanoparticles.
  • any surfactant may be used, preferably polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid or derivatives thereof, cremophor or Tweens.
  • the second step is a step of forming an emulsion by homogenization of the mixture.
  • the homogenization may be performed by any known method in the art, preferably by sonication.
  • the sonication time can be readily determined by those skilled in the art, depending on the sonication method and formed emulsion. As one specific example thereof, the sonication time is 3 min to 12 min, preferably 4 min to 10 min. If the sonication time is more than 12 min, there is a problem in that the prepared nanoparticles display cytotoxic effects. If the sonication time is less than 3 min, there is a problem in that the emulsion is not sufficiently formed.
  • the third step is a step of centrifuging and washing the emulsion 4 to 6 times and then drying it.
  • the centrifugation can be performed to fully separate the emulsion layer, and at the speed and for the time known to those skilled in the art.
  • the washing is a process for removing impurities that may be included in the prepared nanoparticles.
  • the centrifugation and washing are performed for the preparation of pure nanoparticles, and they can be performed 4 to 6 times, and preferably 4 to 5 times.
  • the drying step is performed to obtain nanoparticle powder.
  • the drying step is not essential, but can be selectively performed according to the final formulation of the prepared nanoparticles.
  • the drying step is to prepare a particle form by removing any water or other compounds that are used during the process.
  • the drying step may be performed by the drying methods known in the art, including freeze-drying, spray granulation, and spray drying, preferably freeze-drying. These drying methods are well known in the art.
  • the nanoparticles of the cedrol or pharmaceutically acceptable salt thereof that are prepared by the method have a size of 100 to 500 nm, preferably 200 to 500 nm, and more preferably 200 to 400 nm.
  • the nanoparticle forms of the cedrol or pharmaceutically acceptable salt thereof that are prepared by the method have the advantages of excellent encapsulation efficiency and sustained release effect of the compounds, as compared to the native form of the cedrol or pharmaceutically acceptable salt thereof.
  • the nanoparticle forms also have an inhibitory effect on overexpression of MCM protein, equal to that of the native form.
  • the present invention provides a pharmaceutical composition for the treatment and prevention of cancer, comprising the nanoparticles of cedrol or a pharmaceutically acceptable salt thereof that are prepared by the method.
  • the cancer disease includes general cancer diseases, including stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or ocular melanoma, uterine cancer, ovarian cancer, large intestine cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin s disease, esophageal cancer, small intestine cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal tumor, brain stem glioma, and pitu
  • the composition for the treatment and prevention of cancer of the present invention comprises the nanoparticle in an amount of 0.1 to 50% by weight, based on the total weight of the composition.
  • the nanoparticle contains a smaller amount of the cedrol compound, it has excellent sustained release effect to maintain its efficacy for a long period of time.
  • the composition of the present invention may comprise any pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier used in the composition of the present invention may be at least one selected from an excipient, a disintegrating agent, a binder, and a lubricant.
  • an excipient such as microcrystalline cellulose, lactose, low-substituted hydroxycellulose
  • a disintegrating agent such as sodium starch glycolate, anhydrous calcium hydrogen phosphate
  • a binder such as polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, or hydroxypropylcellulose
  • a glidant such as magnesium stearate, silicon dioxide, or talc.
  • an additive imparting gloss to a tablet such as anhydrous dibasic calcium phosphate
  • a tablet may also be coated with a water-insoluble material to prevent the infiltration of moisture in air into the tablet.
  • the coating material should have a compact molecular structure and may not be easily dissolved in an aqueous solution.
  • the coating material may be a polymer material such as a methacrylic acid copolymer, hydroxypropylmethylcellulose phthalate, celluloseacetate phthalate, hydroxypropylmethylcellulose acetatesuccinate, and polyvinylalcohol. These polymer materials may be used alone or in combination.
  • a coating layer coated on a tablet may include an additive for a coating material commonly known in the art, e.g., a plasticizer, a preservative, a pigment, or a light shielding agent.
  • composition of the present invention may be presented in the form of solutions such as sterile aqueous solution, or injectables.
  • solutions such as sterile aqueous solution, or injectables.
  • Such solution contains, if necessary, from 10 to 40% of propylene glycol and sodium chloride sufficient to avoid hemolysis (e.g. about 1%).
  • the composition of the present invention may be formulated into pharmaceutical preparations common in the pharmaceutical field.
  • the formulations include injectable formulations, tablets, capsules, powders, granules, suspensions, emulsions, syrups, emulsions in water, plasters, ointments, aerosols, oils, gels, spirits, tinctures, baths, liniments, lotions, patches, pads and creams, preferably injectable formulations, tablets, capsules, powders, aerosols, or patches, and more preferably tablets or capsules for oral administration, aerosols inhalable via the respiratory tract, or patches applicable to a desired area of the skin, and injectable formulations for parenteral administration.
  • a propellant for spraying a water-dispersed concentrate or wetting powder may be used in combination with an additive.
  • a permeation stimulator may be used to increase the permeation of compounds through the skin.
  • the present invention relates to a method for treating and/or preventing cancer by administration of the pharmaceutical composition into an individual.
  • the term "individual” refers to any mammal in need of treatment for cancer, including human and non-human primates, domestic animals and livestock, pet or sports animals, for example, dogs, horses, cats, sheep, pigs, and cows.
  • the individual in need of treatment of cancer is preferably human.
  • composition of the present invention may be administered via various routes such as oral, parenteral, topical, rectal, and intranasal routes.
  • Parenteral administrations include indirect injections to generate a systemic effect or direct injections to the afflicted area. Examples of parenteral administrations are subcutaneous, intravenous, intramuscular, intradermal, intrathecal, intraocular injections or infusions techniques.
  • Topical administrations include the treatment of infectious areas or organs readily accessible by local application, for example, eyes, ears including external and middle ear infections, vaginal, open and sutured or closed wounds and skin. It also includes transdermal delivery to generate a systemic effect.
  • Intranasal administration includes nasal aerosol or inhalation applications
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient to exert the effects of the composition, and further an amount not to cause an adverse reaction, or serious or excessive immunity response.
  • An exact effective dose level may be readily determined by those skilled in the art, depending on the factors well known in the medical field such as the patient s age, weight, health condition, and sex, drug sensitivity, the route of administration, and administration method.
  • the composition of the present invention can be administered once to several times. For example, according to the severity of the disorder, it may be administered once daily or every 4 to 6 hours. To treat an acute case, the composition of the present invention is preferably administered every 4 to 6 hours. For the purpose of prevention (or maintenance), it is preferably administered once or twice daily.
  • the composition for the treatment and/or prevention of cancer according to the present invention may be administered two or more times daily, depending on the characteristics of disease.
  • Methylene chloride (Dichloromethane, DCM, analytical grade) was purchased from Merck (Germany), and acetonitrile (HPLC grade) was purchased from Malinckrodt Baker, Inc.
  • cedrol nanoparticles (Nanomedicin, NM), poly(DL-lactic-co-glycolic acid) (PLGA) was used as an amphiphilic biopolymer, and polyvinyl alcohol (PVA) was used as a surfactant.
  • PLGA poly(DL-lactic-co-glycolic acid)
  • PVA polyvinyl alcohol
  • the procedures are as follows. 40 mg of cedrol was fully dissolved in 16 ml of methylene chloride (DCM), and 200 mg of PLGA was mixed therewith. 100 ml of PVA solution was added to the mixed solution at the total concentrations of 0.5%, 5% and 10%, and sonication (60 W) was performed for 5 or 10 min to form an emulsion.
  • the formed emulsion was transferred to a 50 ml centrifuge tube, and centrifuged at 12,000 rpm for 15 min. The resultant was washed with 10 ml of triple deionized water, and then centrifuged at 12,000 rpm for 15 min. This procedure was repeated five times, and PPT was suspended in 10 ml of distilled water, and frozen at -80°C, and freeze-dried for 48 hours to prepare NM particles. The procedures are shown in FIG. 1.
  • the mean diameter of NM was 50 to 800nm, and poly-dispersity was 0.243 to 0.373.
  • the size varied depending on PVA concentration and sonication time. As the PVA concentration increased, the size increased. However, as the sonication time increased, the size decreased.
  • NP1 and NP3 showed no significant difference in their size (200 to 500 nm), but NP5 showed a great size difference of 600 to 900 nm.
  • NP4 had the smallest particle size, and subsequently, NP2 was the second smallest, and well-dispersed. There was no significant difference between NP5 and NP6 of FIG. 2.
  • the surface morphology of NM was measured by field emission scanning electron microscopy (FE-SEM, Quanta 200 FEG ESEM, Czech). The results are shown in FIGs. 4 to 9.
  • the measurement was performed under a nitrogen environment, and about 5 mg of blend sample was used to maintain the constant mass of polymeric components in the blend.
  • FIG. 10 is the results showing the physical properties of NM, in which each represents gelation temperature, melting temperature, and crystallization temperature, and showing crystallization during the first and second heating cycles, in which Tg, Tm, and Tc of the first cedrol(CR) were near 55°C, 90°C, and 50°C, respectively.
  • the biodegradable polymer, PLGA was melted at 57°C, and NM had both of the melting peaks appeared in CR and PLGA, indicating that NM is a nanoparticle to form a micelle.
  • Encapsulation efficiency of NM was analyzed by HPLC (Waters alliance 2690). Reverse phase Inertsil XTrra TM RP18 (4.6' 150mm i.d., pore size 5 ⁇ m, Waters, Tokyo, Japan) was used as a column, and acetonitrile-water (60:40) was used as a developing solvent for elution. The measurement was performed at 195 nm. 6 mg of NM 3 prepared in Example 1 was dissolved in 1 ml of DCM. After evaporating DCM completely, it was dissolved in 1.5 ml of acetonitrile-water (60:40), and the amount of eluted drug was measured by HPLC. The encapsulation efficiency was calculated by the following Equation 1.
  • Encapsulation efficiency (EE) was in the wide range from 48 to 90%, and within 10% in the correction experiment.
  • the correction values were shown in Table 1.
  • Table 1 As shown in Table 1, when the concentration of PVA was high, the encapsulation efficiency was considerably low. As the concentration of PVA decreased, the encapsulation efficiency increased. Therefore, it can be seen that when the concentration of PVA is within 5%, the encapsulation efficiency has the optimal value.
  • Example 1 8 mg of NM prepared in Example 1 was suspended in 10 ml of PBS (pH 7.4). While shaking the solution in a 37°C shaking water bath for 120 min at 100 rpm, the amount of drug release was measured by HPLC at suitable time intervals to evaluate sustained-release efficiency of the drug. The results are shown in FIG. 11.
  • Non PVA (emulsifier)-treated group had 50% of drug release, and the cedrol release increased at a low concentration of PVA. As the PVA concentration increased, the cedrol release remarkably decreased. Taken together, the sustained release of drug increased at the PVA concentration within 5% and higher sonication time, namely at small size and high dispersity.
  • MTT 3-(4,-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide
  • cell viability of the NM (15 to 30 ⁇ g/ml)-treated HT29 cells decreased in a concentration-dependent manner.
  • the highest cytotoxic activity against HT-29 cells was observed at 5% PVA and sonication time of 10 min.
  • Example 4 MCM protein expression of NM-treated HT29 cell
  • MCM protein is an essential eukaryotic DNA replication factor having a helicase activity, and functions to control DNA replication by phosphorylation and dephosphorylation in normal cells.
  • MCM protein expression is not tightly regulated, cells undergo unlimited proliferation and transformation into cancer cells. It was reported that the MCM protein expression is much higher in cancer cells than normal cells(Ishimi Y, et al., Eur J Biochem, 270(6), 1089-101, 2003).
  • NM prepared in Example 1 was dissolved in PBS, which was applied to HT29 cells at various concentrations, and then the sample-treated cancer cells were suspended in a CSK buffer (10 mM Pipes, pH 6.8, 100 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA ,1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride) supplemented with 0.1% Triton X-100, 1 mM ATP and Protease inhibitor (Pharminogen A).
  • CSK buffer 10 mM Pipes, pH 6.8, 100 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA ,1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride
  • the cell suspension was sonicated to obtain MCM-2, -3, -5, -6, and -7, PCNA, and actin proteins.
  • the sonicated cells were centrifuged at 20,000 x g for 30 min to obtain the supernatant, and the protein concentration of the supernatant was determined using a BCA protein assay kit (Bio-Rad). An equal amount of protein (40) was used, and actin was used as a control group to perform SDS-PAGE. 15% and 10% separation gels were used for actin and MCM-2, -3, -5, -6, and -7, respectively.
  • the proteins in the gels were transferred to a PVDF membrane, and blocking was performed using a blocking solution (Blockace TM , Dai-Nippon) at room temperature for 1 hr.
  • the PVDF membrane was reacted with primary antibodies at 37°C for 1 hr, and washed with TBS (50 mM Tris/HCl, pH 7.5, 0.15 M NaCl) containing 0.1% Triton X-100.
  • the PVDF membrane was reacted with peroxidase-conjugated secondary antibodies (Pierce) at 37 C for 1 hr, and detected by chemiluminescence system (SuperSignal West Femto Maximum sensitivity Substrate, Pierce). The reactivity was quantified using a Fluorchem TM 5500 (Alpha Innotech). The results are shown in FIG. 17.
  • MCM 2 to 7 proteins As shown in FIG. 17, reduced expression of MCM 2 to 7 proteins was observed in the NM-treated HT29 cells, indicating inhibition of the overexpression of MCM protein, which is an essential DNA replication factor having a helicase activity in cancer cells.
  • Example 5 Inhibitory effect of NM on malignant tissue
  • mice 4 to 5 week-old male C57bl/6 mice (weight of 15-20 g) were divided into four groups of 6 animals each. All mice were acclimated for 1 week, and subcultured B16/F10 melanoma cells of 5 x 10 6 cells/ml were injected into the right armpit region to induce cancer.
  • NM suspended in PBS was administered by intravenous, intraperitoneal, and oral injection. After 18 days, the mice were sacrificed to resect the malignant tissues.
  • Table 2 and FIG. 18 show changes in body weight of cancer mouse model after NM treatment.
  • mice treated with intraperitoneal injection were 2.24 g, whereas that of the control group was 5.1 g. That is, NM reduced the tumor weight by 50% or more.
  • the tumor weights of the mice treated with intravenous and oral injection were 3.61 g and 3.76 g, showing that the tumor weight reduced to 30%, compared to the control group (see Table 3 and FIGs. 19 to 21).
  • Tumor primary site (a x b)/2

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Abstract

L'invention concerne une méthode de préparation de cédrol ou de l'un de ses sels pharmacocompatible sous forme de nanoparticules, et sur une composition de traitement et de prévention du cancer comprenant les nanoparticules ainsi préparées.
PCT/KR2010/000276 2009-01-16 2010-01-15 Compositions anticancéreuses comprenant du cédrol nanoparticulaire WO2010082789A2 (fr)

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KR1020090003868A KR20100084399A (ko) 2009-01-16 2009-01-16 나노입자 형태로 세드롤을 포함하는 항암 조성물
KR10-2009-0003868 2009-01-16

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WO2010082789A2 true WO2010082789A2 (fr) 2010-07-22
WO2010082789A3 WO2010082789A3 (fr) 2010-11-18

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CN112870161B (zh) * 2021-02-02 2023-03-21 贵州大学 一种雪松醇纳米乳及其优化制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951836B2 (en) * 2000-01-05 2005-10-04 Basf Aktiengesellschaft Microcapsule preparations and detergents and cleaning agents containing microcapsules
KR100673574B1 (ko) * 2005-12-19 2007-01-24 학교법인 동의학원 향나무로부터 분리된 세드롤을 함유하는 암 예방 및치료용 조성물
WO2008054042A1 (fr) * 2006-11-01 2008-05-08 Jae Woon Nah Nanoparticules biodégradables de poly(dl-lactide-co-glycolide) encapsulant le chlorhydrate de ciprofloxacine ayant une propriété de libération prolongée et son procédé de fabrication
US20080213198A1 (en) * 2004-04-26 2008-09-04 Sederma Sas Cosmetic or Dermopharmaceutical Composition Comprising at Least one Udp Glucuronosyl Transferase (Ugt) Enzymes Inducer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951836B2 (en) * 2000-01-05 2005-10-04 Basf Aktiengesellschaft Microcapsule preparations and detergents and cleaning agents containing microcapsules
US20080213198A1 (en) * 2004-04-26 2008-09-04 Sederma Sas Cosmetic or Dermopharmaceutical Composition Comprising at Least one Udp Glucuronosyl Transferase (Ugt) Enzymes Inducer
KR100673574B1 (ko) * 2005-12-19 2007-01-24 학교법인 동의학원 향나무로부터 분리된 세드롤을 함유하는 암 예방 및치료용 조성물
WO2008054042A1 (fr) * 2006-11-01 2008-05-08 Jae Woon Nah Nanoparticules biodégradables de poly(dl-lactide-co-glycolide) encapsulant le chlorhydrate de ciprofloxacine ayant une propriété de libération prolongée et son procédé de fabrication

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KR20100084399A (ko) 2010-07-26

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