KR101048594B1 - Pharmaceutical composition containing cannabinoid derivatives that inhibit angiogenesis and cancer growth - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of diseases caused by angiogenesis and cancer diseases containing the cannabinoid derivative represented by Formula 1 as an active ingredient, wherein the cannabinoid derivative represented by Formula 1 is a neovascular vessel It is excellent in inhibiting formation and inhibiting cancer cell growth, and can be used as a pharmaceutical composition for preventing and treating diseases caused by cancer diseases and angiogenesis.

Cannabinoid derivatives, angiogenesis, cancer disease

Description

Pharmaceutical composition comprising cannabinoids having anti-angiogenic activity and cancer growth inhibitory activity}

The present invention relates to a pharmaceutical composition containing a cannabinoid derivative having an angiogenesis inhibitory activity and a growth inhibitory activity of cancer as an active ingredient.

Angiogenesis is the process by which new capillaries are formed from existing microvessels. Angiogenesis normally occurs during embryonic development, tissue regeneration and wound healing, and the development of the corpus luteum, a change in the genital system of women periodically, and in these cases is strictly controlled (Folkman J et al., Int Rev. Exp.Pathol . , 16, pp 207-248, 1976).

In adults, vascular endothelial cells grow very slowly and do not divide relatively well compared to other types of cells. Angiogenesis process is generally caused by the stimulation of angiogenesis factors, blood vessels are reconstructed by the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated.

However, there are diseases caused by angiogenesis that is not controlled autonomously and grows pathologically. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as atherosclerosis, angiogenesis, and edema sclerosis. Eye diseases caused by angiogenesis include corneal graft angiogenesis and angiogenesis. Glaucoma, diabetic retinopathy, corneal disease caused by neovascularization, degeneration of spots, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis and the like. Chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, dermatological diseases such as acne, Alzheimer's and obesity are also associated with angiogenesis, and cancer growth and metastasis necessarily depends on angiogenesis (D'Amato RJ et al., Ophthalmology, 102 (9), pp1261-1262, 1995; Arbiser JL, J. Am. Acad. Dermatol., 34 (3), pp486-497, 1996; O'Brien KD et al. Circulation, 93 (4), pp 672-682, 1996; Hanahan D et al., Cell, 86 , pp 353-364, 1996).

Angiogenesis, in particular, plays an important role in the growth and metastasis of cancer cells. Tumors are supplied with nutrients and oxygen for growth and proliferation through neovascularization, and new blood vessels that penetrate the tumor give metastasis to metastatic cancer cells by allowing them to enter the blood circulation (Folkman and Tyler, 1999). Cancer Invasion and metastasis , Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, pp 94-103, 1977; Polverini PJ, Crit. Rev. Oral. Biol. Med. , 6 (3), pp230-247, 1995). The main cause of death of cancer patients is metastasis, and cancer metastasis does not contribute to the survival rate of chemotherapy or immunotherapy currently used in the clinic.

Arthritis, a representative disease of inflammatory diseases, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis and destroys cartilage. In other words, synovial cells and vascular endothelial cells proliferate in the synovial cavity with the help of inflammation-inducing cytokines, forming joint pannus, a layer of connective tissue that develops in the cartilage as the angiogenesis progresses, destroying cartilage that acts as a cushion. (Koch AE et al., Arthritis. Rheum., 29, pp471-479, 1986; Stupack DG et al., Braz J. Med. Biol. Rcs., 32 (5), pp578-581, 1999; Koch AE , Atrhritis.Rheum ., 41 (6), pp951-962, 1998).

Many ocular diseases, which cause millions of blindness worldwide each year, are also caused by angiogenesis (Jeffrey MI et al., J. Clin. Invest., 103, pp1231-1236, 1999). For example, diseases such as macular degeneration, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma and corneal diseases caused by neovascularization are the diseases caused by neovascularization (Adamis AP). et al., Angiogenesis, 3, pp 9-14, 1999). Among them, diabetic retinopathy is a complication of diabetes, in which capillaries in the retina invade the vitreous body and eventually become blind.

Psoriasis, which is characterized by red spots and skin, is also a chronic proliferative disease of the skin. It does not heal and involves pain and malformations. In normal cases, keratinocytes proliferate once a month, whereas psoriasis patients proliferate at least once a week. This rapid proliferation requires a large supply of blood and angiogenesis is bound to occur (Folkman J, J. Invest. Dermatol., 59, pp 40-48, 1972).

Since angiogenesis inhibitors can be applied as a therapeutic agent for such various angiogenesis-related diseases, studies are being actively conducted to treat the above diseases by inhibiting angiogenesis. Such angiogenesis inhibitors usually require long-term administration to patients, so they should be of low toxicity and be orally available for use as the ideal therapeutic. Therefore, there is a demand for the development of a drug having low toxicity as an angiogenesis inhibitor.

Thus, the present inventors completed the present invention by confirming that the cannabinoid derivative of the present invention has excellent antiangiogenic and cancer cell growth inhibitory effects.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of diseases and cancer diseases caused by angiogenesis, containing a cannabinoid derivative as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by angiogenesis, containing a cannabinoid derivative represented by the following formula (1) and a pharmaceutically acceptable carrier:

[Formula 1]

In Formula 1, R ₁ and R ₂ are each selected from hydrogen, C 1 -C 10 alkylcarbonyl or C 1 -C 10 alkyl.

More preferably, the cannabinoid derivative is a compound represented by the following formula (2), that is, 3,6,6,9-tetramethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] Chromen-1-ol (3,6,6,9-tetramethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-1-ol) or a compound represented by formula (3) That is, (1-hydroxy-6,6,9-trimethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-2-yl) ethanone [(1- hydroxy-6,6,9-trimethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-2-yl) ethanone]:

[Formula 2]

(3)

The angiogenesis-related diseases include rheumatoid arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea, shogren Syndrome, myopia ophthalmic tumor, corneal transplant rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, Inflammatory bowel disease, periodontal membrane disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging and cancer infiltration and metastasis of any one selected from the group.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing a cannabinoid derivative represented by the formula (1) and a pharmaceutically acceptable carrier.

More preferably, the cannabinoid derivative is a compound represented by Formula 2, that is, 3,6,6,9-tetramethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] Men-1-ol (3,6,6,9-tetramethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-1-ol) or a compound represented by formula (3) , (1-hydroxy-6,6,9-trimethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-2-yl) ethanone [(1-hydroxy -6,6,9-trimethyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-2-yl) ethanone].

The cancer diseases include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, and fallopian tube carcinoma , Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer , Chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma It is any one selected from the group consisting of.

The cannabinoid derivatives of the present invention inhibited angiogenesis by treatment with neovascularization inducers such as vascular endothelial growth factor in chicken chorioangular model, and also inhibited neovascularization activated by transplantation of cancer cells into choriourema. It can be usefully used as a composition for the prevention and treatment of diseases caused by angiogenesis.

In addition, the cannabinoid derivative of the present invention can be usefully used as a composition for the prevention and treatment of cancer diseases by inducing apoptosis of human colon cancer cells through NAG-1 induction.

The amount and method of application of the pharmaceutical composition comprising the cannabinoid derivative of the present invention may vary depending on the formulation and the purpose of use.

The pharmaceutical composition comprising the cannabinoid derivative of the present invention comprises 0.1 to 50% by weight of the cannabinoid derivative based on the total weight of the composition.

In addition, the pharmaceutical composition comprising the cannabinoid derivative of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of the pharmaceutical composition.

Such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undetermined. Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition comprising the cannabinoid derivative of the present invention is in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the cannabinoid derivative of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times daily in an amount of 0.1 to 100 mg / kg. The dosage of the compound can also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The cannabinoid derivative according to the present invention inhibits angiogenesis by treatment with neovascularization inducers such as vascular endothelial growth factor in chicken choriourema model, and also inhibits neovascularization activated by transplantation of cancer cells into choriourema. Therefore, it can be usefully used as a composition for the prevention and treatment of diseases caused by angiogenesis.

In addition, the cannabinoid derivative of the present invention can be usefully used as a composition for the prevention and treatment of cancer diseases by inducing apoptosis of human colon cancer cells through NAG-1 induction.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Preparation of Compound (HHC-7)

Orcinol (124 mg, 1 mmol) and citronellal (308 mg, 2 mmol) as aldehyde, 24 hours xyl, using EDDA (36 mg, 0.2 mmol) / TEA (2 mL) mixed catalyst as catalyst The reaction was carried out under reflux conditions to prepare HHC-7 (182 mg, 70%).

¹ H NMR (CDCl ₃ , 300 MHz) δ 6.26 (1H, s), 6.08 (1H, s), 5.67 (1H, s), 3.09 (1H, br d, J = 12.6 Hz), 2.51-2.43 (1H , m), 2.16 (3H, s), 1.86-1.83 (2H, m), 1.73-1.59 (3H, m), 1.49-1.42 (1H, m), 1.38 (3H, s), 1.14-1.10 (1H m), 1.07 (3H, s), 0.94 (3H, d, J = 6.6 Hz);

¹³ C NMR (CDCl ₃ , 75 MHz) δ 154.9, 154.7, 137.2, 110.5, 110.2, 108.5, 77.1, 49.1, 38.8, 35.4, 35.3, 32.7, 27.9, 27.4, 22.5, 20.9, 18.7;

IR (neat) 3387, 2922, 1624, 1580, 1510, 1454, 1332, 1267, 1188, 1138, 1115, 1086, 1057, 1001, 821, 740 cm ^-1 .

Example 2 Preparation of Compound (HHC-8) of Formula 2

2,4-dihydroxyacetophenone (152 mg, 1 mmol) and citronellal (308 mg, 2 mmol) as aldehydes are used, and EDDA (36 mg, 0.2 mmol) / TEA (2 mL) mixed catalyst is used as a catalyst. The reaction was carried out under reflux conditions for 16 hours to prepare HHC-8 (219 mg, 76%).

¹ H NMR (CDCl ₃ , 300 MHz) δ 13.5 (1H, s), 7.43 (1H, d, J = 8.9 Hz), 6.27 (1H, d, J = 8.9 Hz), 3.17 (1H, br d, J = 12.6 Hz), 2.57-2.48 (1H, m), 2.49 (3H, s), 1.85-1.78 (2H, m), 1.65-1.57 (3H, m), 1.49-1.42 (1H, m), 1.37 (3H, s), 1.14-1.04 (1H, m), 1.04 (3H, s), 0.91 (3H, d, J = 6.6 Hz);

¹³ C NMR (CDCl ₃ , 75 MHz) δ 202.6, 164.2, 160.9, 129.8, 112.9, 112.8, 109.4, 78.6, 48.6, 38.1, 35.4, 34.9, 32.6, 27.8, 27.4, 26.1, 22.4, 19.1;

IR (neat) 2922, 1620, 1487, 1414, 1372, 1331, 1260, 1211, 1144, 1067, 912, 882, 847, 802 cm ^-1 .

Reference Example 1 Experiment Preparation and Cell Culture

1. Reagent and Instrument Preparation

Yoo Jung-ran was purchased from Yeungnam University ranch (Gyeongsan, Korea). HCT116 cells, HT-29 cells, and MCF-7 cells, human breast cancer cells, were purchased from ATCC (Rockville, MD, USA). .

2-pyridinecarboxaldehyde, 2-acetylfuran, and cortisone acetate were purchased from Aldrich Chemical Co., St. Louis, Mo., USA. Fibroblast growth factor (FGF) was purchased from Invitrogen, USA, and vascular endothelial growth factor (VEGF) was purchased from R & D systems, Minneapolis, MN, USA. Tetrac and XT199 were obtained from Albany College of Pharmacy, and whatman filter discs were purchased from Whatman, UK.

For structural characterization of the synthesized material, 1H-NMR spectra were obtained using the Bruker AMX 250 MHz model, and liquid chromatography / mass spectrometry was performed by Fininigan LCQ Advantage LC /. MS / MS spectrometry was analyzed using the Xcalibur program. Thin-layer chromatography and column chromatography used Merck's Silicagel Kieselgel 60 F254 (230-240 mesh).

2. Cell Culture

Human colon cancer cells, HCT116 cells, HT-29 cells, and human breast cancer cells, MCF-7 cells, were purchased from the American Type Culture Collection (ATCC, Rockville, Mass.). The cells were cultured in a humidified incubator at RPMI 1640 (Sigma Chemical CO.) Medium containing 10% fetal bovine serum (GIBCO), 37 ° C., 5% CO ₂ /95% air. At this time, the culture medium was replaced once every other day. After growing confluent, it was subcultured with trypsin treatment.

Experimental Example 1 Cytotoxicity Experiment

In order to measure cell viability, human colon cancer cells, HCT116 cells and HT-29 cells were treated with RPMI 1640 (Sigma Chemical CO.) Medium containing 10% fetal bovine serum (GIBCO), a temperature of 37 ° C, Incubated in a humidified incubator with 5% CO ₂ /95% air conditions.

At this time, the culture medium was replaced once every other day. After growing confluent, it was subcultured with trypsin treatment with 0.25% trypsin-EDTA solution.

After 4 to 5 days of incubation as described above, a 24 well plate was dispensed at a density of ⁵ × 10 ⁴ cells / well, and the media volume of each well was adjusted to 1 ml. Thereafter, the compounds of Examples 1 and 2 were treated with 0.1, 1, 5, 10, 20, 50, and 100 μM concentrations for 24 hours, and then 100 μl of MTT [3- (4,5-dimethylthiazol -2-yl) -2,5-diphenyl tetrazolium bromide; 5 g MTT / Lin H ₂ O] were added and the cells were further incubated for 4 hours.

Then, 200 μl of dimethylsulfoxide (DMSO) was added to the wells containing each cell and mixed by pipette to dissolve the reduced MTT crystals. Relative cell viability was determined by scanning with a microplate reader (Molecular Devices, Menlo Park, CA) with a 540 nm filter.

As a result, as shown in Figure 1, the cytotoxicity was not large.

Experimental Example 2 Induction of Apoptosis (Flow Cytometry)

HCT116 cells and HT-29 cells, which are human colorectal cancer cells, were inoculated in 6 well plates at a density of ⁵ × 10 ⁴ cells / well for 24 hours, followed by pretreatment of the compound of Example 2 (10 μM) in each well over time. Cells were collected using 0.25% trypsin-EDTA solution, fixed in 70% ethanol, and overnight at 4 ° C. The fixed cells were collected, washed once with PBS, and then reacted with 100 μg / ml of RNaes A 250 μl at 37 ° C. for 30 minutes. After the reaction, 250 μl of Propidium Iodide (1 g / l) was added and the reaction was filtered at 37 ° C. for 30 minutes, followed by filtration of the fluorescence emitted from the PI-DNA complex using a flow cytometer (FACScan cytometer; Becton Dickinson, USA). Was measured.

As a result, the compound of Example 2 induced apoptosis as shown in Figures 2a to 2c.

Experimental Example 3 Induction of p53 and NAG-1 Expression

HCT116 cells and HT-29 cells, which are human colon cancer cells, were inoculated at a concentration of ⁵ × 10 ⁴ cells / well in a 100 mm dish and incubated for 24 hours, followed by compound of Example 2 (10 μM) and sulindac sulfide (SS). After treatment, cells were harvested using 0.25% trypsin-EDTA solution and analyzed for protein expression of p53 and NAG-1 via Western blot.

The total protein extract was lysis buffer (10 mM Tris pH 7.6, 10 mM EDTA, 150 mM NaCl, 0.1% NP-40, 2 mM DTT, 1 mM PMSF, 0.7 μg / ml pepstatin A, 10 μg / ml Leupeptin, 1 μg / After reacting for 40 minutes by adding ml aprotinin), the supernatant was obtained by centrifugation at 12,000 rpm for 10 minutes.

Protein extract in the nucleus was extracted using NE-PER kit (Pierce, Rockford, IL, USA) and centrifuged at 12,000 rpm for 10 minutes to obtain a supernatant. Protein content of each extract was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA), bovine serum albumin (BSA) was used as a standard protein.

The above protein extract was subjected to electrophoresis using 12% SDS-PAGE. Transfer the electrophoresis gel to Hybond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and then membrane is blocked for 1 hour with 5% skim milk in Tris-buffered saline (TBS) -Tween-20 (TBST). The specific primary antibody was diluted in TBS-5% skim milk and overnight at 4 ° C.

After washing for 5 minutes with TBST for 5 minutes, the secondary antibody was diluted in TBS-5% skim milk and reacted for 1 hour at room temperature. After washing for 3 minutes with TBST for 10 minutes, the protein was purified using ECL kit (Thermao scientific, IL, USA). Confirmed.

As a result, the compound of Example 2 induced protein expression of p53 and NAG-1 in a concentration-dependent manner, as shown in Figures 3a and 3b.

In addition, expression of NAG-1 mRNA was analyzed by RT-PCR. First, TNF-α (10 ng / ml) stimulation was applied to HCT116 cells and HT-29 cells pretreated with the compound of Example 2, and then HCT116 cells and HT-29 cells were treated with trizol solution (Invitrogen, Carlsbad, CA). Messenger RNA was extracted.

mRNA concentrations were determined using a UV-Visible spectrophotometer instrument (Shimadzu, Japan) and reverse transcribed at 37 ° C. for 1 hour using a Ready-To-Go T-Primed First Strand Kit (Amersham Biosciences, USA).

RT-PCR was performed using primer sets specific for human MCP-1 and IL-8 in the presence of 0.5 U Taq DNA polymerase (TaKaRa, Japan). MRNA levels of all genes were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (Qiagen).

At this time, RT-PCR for mRNA expression of NAG-1 was for 4 minutes at 94 ℃ (one cycle); 60 seconds at 94 ° C., 60 seconds at 58 ° C., 60 seconds at 72 ° C. (35 cycles); And finally at 72 ° C. for 10 minutes, and the primers used are as shown in SEQ ID NOs: 1 and 2, respectively.

In addition, RT-PCR for the expression of the GAPDH gene was for 4 minutes at 94 ° C. (one cycle); 45 seconds at 94 ° C., 45 seconds at 58 ° C., 45 seconds at 72 ° C. (30 cycles); And finally at 72 ° C. for 10 minutes, and the primers used are as shown in SEQ ID NOs: 3 and 4, respectively.

The resulting PCR product was isolated on 2% agarose gel electrophoresis, stained with 0.5 μg / ml ethidium bromide and visualized, taken under UV light using a gel documentation system (UVP, Cambridge, UK) It became. As a result, NAG-1 mRNA expression analyzed by RT-PCR as shown in Figure 3c also induced the concentration of the compound of Example 2.

Experimental Example 4 Induction of p53 and NAG-1 Expression

Specific primers used to examine p53 and NAG-1 expression induction were human NAG-1 (SEQ ID NO 1: sense 5'-ACT CCG AAG ACT CCA GAT TCC GA-3 '; SEQ ID NO: 2: antisense 5'-ATG CAC ATG GTC ACT TGC ACC T-3 ') and human GAPDH (SEQ ID NO: 3: sense 5'-GGT GAA GGT CGG AGT CAA CG-3'; SEQ ID NO: 4: antisense 5'-CAA AGT TGT CAT GGA TGA CC-3 ' Was.

In addition, the antisense oligodeoxynucleotide was synthesized from Bioneer Co. (Korea), p53 antisense oligonucleotide (SEQ ID NO: 5: 5'-CGGCTCCTCCATGGCAGT-3 ') and random oligonucleotide (SEQ ID NO: 6 5'-CCGGTGAACGAGCGAGCACA- 3 ') was used.

The oligonucleotides above were used for transfection of human colon cancer cells, HCT116 cells and HT-29 cells. That is, the solution obtained by mixing oligonucleotide and Genejammer transfection reagent at a ratio of 1: 2.5 was added to cells sufficiently grown at a final concentration of 400 nM and reacted for 5 hours.

Thereafter, the transfection mixture was transfected by adding a medium containing 20% FBS for 24 hours without removing the transfection mixture.

Compounds of Example 2 (10 μM) were treated for 48 hours in wells transfected with p53 antisense oligodeoxynucleotides (p53 AS oligo) or random oligodeoxynucleotides (RD oligo). p53 and NAG-1 protein expression was analyzed via Western blot and cell viability was examined via MTT assay.

As a result, as shown in Figures 4a and 4b p53 inhibition induced cell death through downregulation of NAG-1 in HCT116 cells and HT-29 cells.

Experimental Example 5 Examination of Angiogenesis Inhibitory Effect by CAM Analysis

In order to confirm the anti-angiogenic effect in vivo , chorioallantoic membrane (CAM) analysis was performed (Nguyen M et al., Microvascular Res. , 47, pp31-40, 1994). .

Incubate the fertilized eggs of chickens while maintaining the temperature at 37 ℃ and relative humidity of 55%. On the 10th day, use a hypodermic needle (hypodermic needle, Green Cross Medical Industry, Korea) to drill the first small hole in the air sac. A second hole was drilled in the flat part of the fertilized egg for the window.

By venting air through the first hole in the air sac area, the villus urea separates from the shell of the fertilized egg, which is then cut with a grinding wheel (Multipro 395JA, Dremel, Mexico). made.

Next, the vascular endothelial growth factor (VEGF) concentration was 20 ng / CAM after treatment with 3 mg / ml of cortisone acetate in whatman filter disk # 1, whatman, USA. Soaked in.

The filter disc was placed on the blood vessel through the created window, and the compounds of Examples 1 and 2 were dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate buffer solution (PBS) for each concentration (0.5, 1, 5 µg / CAM).

After 3 days of drug treatment, the CAM part on the filter disc was removed and washed with phosphate buffer solution, followed by a stereo microscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA) was used to measure the number of vascular branches and analyze data.

As a result, as shown in FIGS. 5A and 5B, the increase in VEGF-induced angiogenesis was reduced concentration-dependently due to the compound treatment according to the invention.

Experimental Example 6 Examination of Inhibitory Effect of VEGF-Induced Proliferation on HUVEC Cells

HUVEC cells were cultured in a 0.2% gelatin coated flask. The HUVEC cells were cultured in EBM-2 (Endothelial Cell Basal Medium-2, Clonetics, San Diego, CA) medium.

The EBM-2 medium is fetal bovine serum (FBS), hydrocortisone (hydrocortisone), human basic fibroblast growth factor (hFGF-B), vascular endothelial growth factor (VEGF), R3-IGF-1 (human) recombinant insulin-like growth factor, ascorbic acid, human epidermal growth factor (hEGF), GA-1000 and heparin.

HUVEC cells were cultured for 4 to 5 days and aliquoted into 24 well plates at a density of 5 × 10 ⁴ cells / well, and the media volume of each well was adjusted to 1 ml. Thereafter, the compounds of Examples 1 and 2 were treated at 0.1, 1, 5, and 10 μM concentrations for 48 hours, and then 100 μl of MTT (3- (4,5-dimethylthiazol-2-yl) After adding -2,5-diphenyl tetrazolium bromide; 5 g MTT / Lin H ₂ O), the cells were further incubated for 4 hours.

Thereafter, 200 μl of dimethyl sulfoxide (DMSO) was added to the wells containing the respective cells and mixed by pipette to dissolve the reduced MTT crystals. Relative cell viability was measured by scanning with a microplate reader (Molecular Devices, Menlo Park, CA) with a 540 nm filter.

As shown in FIG. 6A, the compounds of the present invention reduced VEGF-induced cell proliferation in a concentration dependent manner.

<Experiment 7> Examination of chemotactic migration inhibitory effect in HUVEC cells

In order to determine the chemostatic migration inhibitory effect of the compounds of the present invention, experiments were carried out using HUVEC cells in vitro (Mi-Sung Kim et al ., Cancer Research , 63, 5454-5461, 2003 Sang- Oh Yoon et al ., The journal of Biological chemistry , 276, 20085-20092, 2001; Sonia Zorzet et al., The journal of pharmacology and experimental therapeutics , 295, 927-933, 2000). The culture plate used herein used a 24-well plate (Corning Costar, Cambridge, Mass.) Containing a polycarbonate filter with multiple 8 mm size pores.

The lower surface of the polycarbonate filter was coated with 20 l of collagen type 1 (type I collagen) at a concentration of 0.5 mg / ml, and the upper surface was coated with 20 l of Matrigel (BD biosciences, Bedford, MA) at a concentration of 1.5 mg / ml.

Here, the lower region of the polycarbonate filter was filled with medium containing 10% FBS, and HUVEC cells were seeded on top of the polycarbonate filter. At this time, the compounds of Examples 1 and 2 were dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate buffer solution (PBS) and treated for 4 hours at each concentration (1, 10 μM).

The inoculated HUVEC cells were incubated at 37 ° C. for 18 hours, and then the cells infiltrated on the lower surface of the polycarbonate filter were fixed with methanol and stained with hematoxylin and eosin. .

As a result, as shown in FIG. 6B, the compound of the present invention reduced VEGF-induced chemotactic migration in a concentration-dependent manner.

Experimental Example 8 Inhibition of Angiogenesis In HUVEC Cells

HUVEC cells were cultured in 48-well plates to determine the effect of the compounds of the present invention on angiogenesis in HUVEC cells. 40 μl of Matrigel (BD biosciences, Bedford, Mass.) Was coated on each well of a 48-well plate, and then used after storing the 96-well plate at 37 ° C. for 30 minutes.

HUVEC cells were suspended in EBM-2 (Endothelial Cell Basal Medium-2, Clonetics, San Diego, Calif.) Medium containing 2% fetal bovine serum. These HUVEC cells were allowed to inject 1 × 10 ⁴ cells into each well coated with the matrigel.

Next, different concentrations of the compound of the invention were added to each well and maintained for 14 hours. The HUVEC cells treated in this way were photographed with a digital camera connected with an inverted microscope, and as a result, as shown in FIG. 6C, the blood vessels formed in the control group decreased as the compound concentration of the present invention was increased.

Experimental Example 9 Examination of VEGF Expression Inhibitory Effect in MCF-7 Cells

After MCF-7 cells were treated with compounds of Examples 1 and 2 for 18 hours at different concentrations, cellular VEGF expression levels in total cell eluate were analyzed by Western blot.

As a result, as shown in FIG. 7a, cellular VEGF expression was inhibited in a concentration-dependent manner.

In addition, VEGF secretion levels in the cell supernatants were analyzed by ELISA. As a result, as shown in FIG. 7B, the secretion-dependent secretion of VEGF levels was suppressed.

In addition, CAMs were loaded with 2 × 10 ⁶ MCF-7 cells / CAM for tumor transplantation and inoculated with the compounds of Examples 1 and 2 only once at the same time as tumor transplantation. As a result, growth of tumor size was inhibited in a concentration-dependent manner as shown in FIG.

Experimental Example 10 Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Molugin and molugin synthetic derivatives prepared in Preparation Example were suspended in 0.5% methylcellulose solution using 5 animals per group and administered once orally at doses of 1 g / kg, 5 g / kg and 10 g / kg.

After the administration, the mortality, clinical symptoms, and weight changes of the animals were observed. Hematological and hematological examinations were performed. Necropsy was performed to visually observe the abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with HHC-8, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

As a result, the HHC-8 of the present invention does not show a change in toxicity in rats up to 10 g / kg, therefore, the oral administration intermediate dose (LD50) was determined to be a safe substance of more than 10 g / kg.

Hereinafter, the preparation examples of the pharmaceutical composition containing the cannabinoid derivative of the present invention, but the present invention is not intended to limit it, only intended to explain in detail.

Preparation Example 1 Preparation of Powder

Powder was prepared by mixing 500 mg of HHC-8, 100 mg of lactose and 10 mg of talc and filling into an airtight bag.

&Lt; Formulation Example 2 > Preparation of tablet

Tablets were prepared by mixing 500 mg of HHC-8, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, followed by compression according to a conventional method for preparing tablets.

Preparation Example 3 Preparation of Capsule

According to a conventional capsule preparation method, 500 mg of HHC-8, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and filled into gelatin capsules to prepare a capsule.

Preparation Example 4 Preparation of Injection

According to the conventional method for preparing an injection, it was prepared by containing 50 mg of HHC-8 per ampule (2 mL), a sterile distilled water dose for injection, and a pH adjuster dose.

Preparation Example 5 Preparation of Liquid

According to the conventional method of preparing a liquid solution, 100 mg of HHC-8, 10 g of isomerized sugar, and 5 g of mannitol are dissolved in an appropriate amount of purified water, and then mixed with an appropriate amount of lemon flavor. The bottle was filled and sterilized to prepare a liquid.

Preparation Example 6 Preparation of Drinks

With the composition and content of 0.5 mg HHC-8, 522 mg honey, 5 mg thioctoamide, 10 mg nicotinic acid amide, 3 mg riboflavin sodium hydrochloride, 2 mg pyridoxine hydrochloride, 30 mg inositol, 50 mg orthoic acid and 200 ml water To prepare a beverage using conventional methods.

1 is to examine the cytotoxicity of the cannabinoid derivatives according to the present invention,

Figure 2a and 2b is to examine the apoptosis induction effect of the cannabinoid derivatives according to the present invention by PI and DAPI staining, respectively

Figure 2c shows the morphological changes after the cannabinoid derivative treatment according to the present invention,

Figure 3a and 3b is a Western blot analysis of p53 and NAG-1 protein expression of the cannabinoid derivatives according to the present invention, respectively

Figure 3c is the analysis of the NAG-1 mRNA expression level by the cannabinoid derivatives according to the present invention by RT-PCR,

Figure 4a shows the apoptosis reduction effect of the cannabinoid derivative treatment according to the invention in cells transfected with p53 antisense,

Figure 4b shows the cell survival rate following the cannabinoid derivative treatment according to the invention in cells transfected with p53 antisense,

5a and 5b show the effect of inhibiting VEGF-induced angiogenesis by the cannabinoid derivatives according to the present invention, respectively

Figure 6a shows the effect of inhibiting cell proliferation by the cannabinoid derivatives according to the present invention,

Figure 6b shows the effect of inhibiting VEGF-induced chemotactic migration by the cannabinoid derivative according to the present invention,

Figure 6c shows the effect of inhibiting angiogenesis in HUVEC cells by the cannabinoid derivatives according to the present invention,

7a and 7b show the effect of VEGF expression by the cannabinoid derivative according to the present invention,

Figure 7c shows the growth inhibitory effect of tumor size by the cannabinoid derivatives according to the present invention.

<110> Yeungnam University Industry-Academic Cooperation Foundation <120> Pharmaceutical composition comprising cannabinoids having          anti-angiogenic activity and cancer growth inhibitory activity <130> dp-2009-0157 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer for human NAG-1 <400> 1 actccgaaga ctccagattc cga 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for human NAG-1 <400> 2 atgcacatgg tcacttgcac ct 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for human GAPDH <400> 3 ggtgaaggtc ggagtcaacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for human GAPDH <400> 4 caaagttgtc atggatgacc 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> P223 antisense oligonucleotide <400> 5 cggctcctcc atggcagt 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> random oligonucleotide <400> 6 ccggtgaacg agcgagcaca 20  

Claims (6)

delete A pharmaceutical composition for the prevention or treatment of diseases caused by angiogenesis, comprising the compound represented by Formula 3 as an active ingredient, wherein the disease caused by angiogenesis is rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, aging Macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, cone cornea, Sjogren's syndrome, myopic ocular tumor, corneal transplant rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm , Degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomeruli, immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin Angiogenesis, characterized in that any one selected from the group consisting of aging and cancer infiltration and metastasis A pharmaceutical composition for preventing or treating a disease: (3)

delete delete A pharmaceutical composition for the prevention and treatment of cancer diseases comprising the compound represented by the following formula (3) as an active ingredient, wherein the cancer diseases are lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or Intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine Cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS; central nervous system) prevention of cancer disease, characterized in that any one selected from the group consisting of tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma Therapeutic Pharmaceutical Compositions: (3)

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