KR20140038612A - Medicament for treating and preventing bone diseases containing costunolide - Google Patents

Abstract

The present invention relates to a medicament comprising costunolide for treating and preventing bone diseases. More specifically, the present invention relates to costunolide which is a sesquiterpene lactone compound extracted from roots of Saussurea lappa of Asteraceae, wherein the costunolide can be used as a medicament for treating and preventing osteoporosis.

Description

Medicine for Treating and Preventing Bone Diseases Containing Costunolide}

The present invention relates to agents for treating and preventing bone diseases containing costunolides. Specifically, the present invention relates to costunolide which can be used as a sesquiterpene lactone compound extracted from the root of Saussurea lappa , which can be used as an agent for treating and preventing osteoporosis disease.

Bone is produced and formed repeatedly from the prenatal period, and quality bone is maintained. In particular, the bone-forming osteoblasts (osteoblast) and bone-absorbing osteoclasts (osteoclast) is balanced in the activity of high-quality bones are maintained. However, in postmenopausal women and chronic inflammatory patients, such as periodontitis and rheumatoid arthritis, the balance between osteoblasts and osteoclasts is broken, leading to bone diseases such as osteoporosis. In particular, the female hormone estrogen is a hormone that strongly inhibits the formation and activity of osteoclasts, causing osteoporosis in postmenopausal women lacking estrogen.

The osteoclasts that cause osteoporosis are the differentiation of monocytes / macrophages derived from hematopoietic stem cells into multinuclear osteoclasts. Monocytes / macrophages, progenitor cells of osteoclasts, are differentiated into osteoclasts by stimulation of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-B (RANK) ligand (RANKL). RANKL (or ODF, OPGL, or TRANCE) is a cytokine of tumor necrosis factor (TNF) family that is expressed in osteoblasts and promotes differentiation into osteoclasts by activating the receptor RANK, which is expressed in monocytes / macrophages. When RANK of the TNF receptor family binds to RANKL, it becomes trimerization and binds to the TNF-associated factor (TRAF) family protein at the cytoplasmic end region. In particular, TRAF6 binds to the cytoplasmic RANK and activates various signaling systems, including NF-B, c-Jun N-terminal protein kinase (JNK), p38, ERK, and Akt. The activity of various signal transduction systems promotes the expression of the transcription factor c-Fos, which is essential for osteoclast differentiation. It also activates the immunoreceptor tyrosine-based activation motif (ITAM) at the cytoplasmic ends of DNAX-activating protein (DAP) 12 and Fc receptor common chain (FcR). The activity of ITAM increases Ca ²⁺ in cells and promotes the expression of the nuclear factor of activated T cells (NFAT) c1 with c-Fos (7,8). NFATc1, along with c-Fos, is an essential transcription factor for osteoclast differentiation and activity and induces the expression of osteoclast markers such as tartrate resistant-acid phosphatase (TRAP), calcitonin receptor, and osteoclast-associated receptor (OSCAR).

Recently, with the development of medicine and the development of new drugs, the elderly population is increasing, but osteoporosis patients and bone diseases such as arthritis are prominent in the elderly population. However, bisphosphonate, the most widely used drug for the treatment of osteoporosis, has a good effect, but has side effects such as esophageal and gastrointestinal ulcers and jaw necrosis. Do. Therefore, the discovery of natural substances that can be easily obtained without side effects can be said to be an important research for the development of bone disease treatments.

Costunolide is a sesquiterpene lactone compound extracted from the root of Saussurea lappa and has various activities such as anti-inflammatory, antifungal and anticancer effects. The present inventors have tried to find a drug that can suppress osteoporosis without side effects. The present inventors verified the effect of costunolide on osteoclast differentiation using a variety of experimental methods and examined the effect of costunolide on osteoclast differentiation by verifying the effect of costunolide on the expression of various genes and the activity of signal transduction proteins.

It is an object of the present invention to provide a medicament capable of treating and preventing bone diseases such as osteoporosis without side effects.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of bone diseases, including costunolide, and the treatment and prevention of bone disease, including costunolide and a pharmaceutically acceptable carrier. .

The costunolide is characterized in that it is extracted from the root of the mussel (Saussurea lappa). The costunolide is characterized in that it inhibits osteoclast differentiation. The bone disease is characterized in that osteoporosis.

In addition, the medicaments and compositions of the present invention may further comprise bisphosphonates or paratyroids.

By using a pharmaceutical composition for treating and preventing bone diseases containing costunolide according to the present invention, or for treating and preventing bone diseases including costunolide and a pharmaceutically acceptable carrier, such as osteoporosis without side effects, Can treat and prevent bone diseases.

1 shows that costunolide inhibits the differentiation of osteoclasts without cytotoxicity.
2 shows that costunolide inhibits NFATc1, TRAP and OSCAR expression.
3 shows that costunolide inhibits the expression of NFATc1.
4 shows that costunolide does not affect the RANKL-induced signaling pathway.
5 shows that RANKL inhibits the expression of HO-1 in BMM.
6 shows that HO-1 significantly inhibits the differentiation of osteoclasts.

Hereinafter, the present invention will be described in detail.

1. Samples and Mice

Costunolide was obtained from Prof. Yoon-Chul Kim (College of Pharmacy Wonkwang University), TRAF6, c-Fos, NFATc1, HO-1 antibody was used by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Actin antibodies were purchased from Sigma Aldrich (St. Louis, Mo., USA). Phospho (p) -ERK, ERK, p-JNK, JNK, p-p38, p38, I-B antibodies were manufactured from Cell Signaling Technology (Beverly, MA, USA). Mice used in this study were ICR mice.

2. Mouse bone marrow cell isolation and progenitor cell acquisition

5-6 weeks old mice of ICR species were sacrificed by cervical dislocation and the femur and tibia were separated. Bone marrow cells were obtained from the isolated femoral and tibial spaces by washing with a 1cc syringe using -minimum essential medium (-MEM) medium containing antibiotics. Erythrocytes contained in bone marrow cells were removed with erythrocyte decomposing solution and incubated for 3 days with medium added with 30 ng / ml M-CSF. After 3 days, the cultured cells were named as bone marrow-derived macrophage (BMM) and used as progenitor cells.

3. Influence of costunolide on osteoclast differentiation

To induce differentiation into osteoclasts, macrophages were added to a 48-well plate at a number of 3.5 X 10 ⁴ / well, treated with 30 ng / ml M-CSF and 100 ng / ml RANKL and constunolide by concentration. After 3 days, the cells were replaced with fresh medium containing M-CSF and RANKL, and the cells were cultured until day 4.

4. TRAP staining

The cultured cells were fixed with 3.7% formalin for 5 minutes and treated with 0.1% Triton X-100 for 1 minute to permeate the cells. TRAP solution (Sigma Aldrich, USA) was prepared according to the manufacturer's method and added to the cells and stained. After staining, the cells stained in red were regarded as osteoclasts.

5. Experimental impact of costunolide on gene expression

The cells used in the experiment were isolated from RNA using Qiazol reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's method. RNA 1 synthesized cDNA using oligo dT primer, dNTP, buffer, dithiothreitol, RNase inhibitor and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). cDNA 1 l was performed by PCR using each primer as follows. c-Fos sense, 5'-CTGGTGCAGCCCACTCTGGTC-3 '; c-Fos antisense, 5'-CTTTCAGCAGATTGGCAATCTC-3 '; NFATc1 sense, 5'- CAACGCCCTGACCACCGATAG-3 '; NFATc1 antisense, 5'-GGCTGCCTTCCGTCTCATAGT-3 '; TRAP sense, 5'-ACTTCCCCAGCCCTTACTAC-3 '; TRAP antisense, 5'-TCAGCACATAGCCCACACCG-3 '; OSCAR sense, 5; -ATCACTGGTGGCACTGCCTG-3 '; OSCAR antisense, 5'-GGCAGGAGACCATCAAAGGC-3 '; DAP12 sense, 5'-TTCCTTCCTGTCCTCCTGACTGTG-3 '; DAP12 antisense, 5'-TGCCTCTGTGTGTTGAGGTCACTG-3; FcR sense, 5'-ATCTCAGCCGTGATCTTGTTCTTG-3 '; FcR antisense, 5'-TCTCATGCTTCAGAGTCTCATATG-3 '; GAPDH sense, 5'-ACCACAGTCCATGCCATCAC-3 '; GAPDH antisense, 5'-TCCACCACCCTGTTGCTGTA-3 '. PCR products were observed at ultraviolet wavelength (280 nm) after electrophoresis on 1% agarose gel with Et-Br.

6. Experimental Effects of Costunolide on Protein Activity and Expression

The cells used in the experiment were lysed using lysis buffer (50 mM tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, PMSF). Centrifugation was performed for 20 minutes to obtain pure protein and the supernatant was used as protein. Proteins were quantified and the same amount of protein was electrophoresed using 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel. Electrophoresis gel was transferred to PVDF membrane (Amershan biosciences) and the protein expression was observed using the antibodies required for the experiment.

7. HO-1 Gene Cloning

The mouse HO-1 gene was synthesized with cDNA from macrophage RNA and cloned using primers with restriction enzymes EcoRI and XhoI at the ends of the gene. The primers used are as follows. DNA obtained using the above primer was about 1.5kb and cloned into TA vector. The cloned HO-1 gene was cloned back into the pMX-IRES-EGFP vector, a retrovirus vector.

8. Retrovirus Isolation and Intracellular Infection Experiment

To isolate the HO-1 retrovirus, Plat E cells were infected with pMX-HO-1 gene using Fugene 6. Infected Plat E cells were cultured for 2 days and the culture was used as HO-1 retrovirus. To infect macrophages with HO-1 retrovirus, polybrene was added to HO-1 retrovirus at 10 g / ml and macrophages were infected for 6 hours. The presence of HO-1 retrovirus was confirmed by observing GFP on a fluorescence microscope, and more than 90% of the cells were infected.

9. Statistical Analysis

The quantitative results obtained in this study were analyzed statistically using Student's t-test and considered statistically significant when p value was less than 0.05.

The results obtained from the examples of the present invention are as follows.

1. Effect of costunolide on osteoclast differentiation

To verify the effect of costunolide on the differentiation of osteoclasts, macrophages derived from bone marrow cells were treated with M-CSF and RANKL, and the experimental groups were treated with costunolide by concentration. The control group treated with M-CSF and RANKL only differentiated into multinucleated TRAP-positive cells, a typical form of osteoclasts, but the costunolide-treated group inhibited osteoclast formation with increasing concentration (Fig. 1A). In particular, it was confirmed that osteoclast differentiation was rapidly suppressed from the costunolide 5 M concentration (Fig. 1B). Next, to determine whether the effect of costunolide on the differentiation of osteoclasts was related to cytotoxicity, we examined the cytotoxicity by treating costunolide on macrophages. Cytotoxicity experiments showed no toxicity at costunolide 10 M concentration (Fig. 1C). As a result, the effect of costunolide on osteoclast differentiation is a specific effect of the drug.

2. Effect of costunolide on gene expression induced by RANKL

RANKL induces the expression of various transcription factors and promotes the expression of several genes important for osteoclast differentiation (7). Therefore, the effects of costunolide on the expression of TRAP and OSCAR, which are markers of osteoclasts, and major transcription factors were examined. The expression of osteoclast markers, TRAP and OSCAR, increased with time, but significantly decreased in the costunolide-treated control group (Fig. 2). As a result, it can be seen that costunolide inhibits the expression of TRAP and OSCAR, thereby suppressing TRAP positive cells. Among the various transcription factors, important transcription factors for osteoclast differentiation are c-Fos and NFATc1, which are known as important transcription factors for expression of TRAP and OSCAR. Therefore, it was confirmed whether the inhibition of TRAP and OSCAR was caused by the expression changes of c-Fos and NFATC1. Fig. As shown in Figure 2, it can be seen that the expression of NFATc1 is suppressed in the experimental group treated with costunolide.

3. Influence of costunolide on major transcription factor protein expression

The transcription factor c-Fos is a gene that is rapidly increased by stimulation of RANKL and is known as an important transcription factor that promotes the expression of NFATc1. In this study, costunolide did not affect the expression of c-Fos but strongly inhibited NFATc1. Therefore, the effect of costunolide on the transcription factor protein expression induced by RANKL was verified. The expression of c-Fos protein was the same as that of mRNA, and the c-Fos protein increased by RANKL was not inhibited by costunolide and did not affect the expression of TRAF6, an important protein in RANKL signaling system (Fig. 3). ). However, NFATc1 protein expression was suppressed in the experimental group treated with costunolide (Fig. 3). These results suggest that the effect of costunolide on osteoclast differentiation may be the result of inhibition of NFATc1 expression.

4. Influence of costunolide on RANKL signaling

RANKL activates a variety of signal transduction proteins through TRAF6. In particular, the transcription factor NF-B is also known as an important transcription factor for osteoclast differentiation (17). JNK and p38 are known as mediators that promote the expression of various transcription factors (18). Fig. As shown in Figure 4, it was confirmed that there is no effect of costunolide on the NF-B activity by RANKL and the activities of ERK, JNK, and p38. As a result, costunolide is expected to have no effect on RANKL early stage of honeymoon delivery and specifically inhibit NFATC1 expression by c-Fos.

5. Effect of costunolide on HO-1 expression

Recently, the expression of HO-1 was reduced during osteoclast differentiation, and cells overexpressing HO-1 were reported to be inhibited in osteoclast differentiation (19). Therefore, to verify this fact, the influence of HO-1 during osteoclast differentiation was confirmed. The expression of c-Fos and NFATc1 confirmed good osteoclast differentiation. However, HO-1 decreased rapidly after 12 hours (Fig. 5A). Next, it was confirmed whether costunolide can induce HO-1 expression. Fig. As shown in 5B, HO-1 expression was increased by increasing or decreasing the concentration of costunolide. Finally, the effect of costunolide on HO-1 expression during osteoclast differentiation was confirmed. In the control group not treated with costunolide, HO-1 was rapidly decreased, but in the experimental group treated with costunolide, HO-1 expression was maintained for up to 24 hours without affecting TRAF6 and c-Fos expression (Fig. 5C). . These results suggest that costunolide's mechanism of inhibiting osteoclast differentiation is related to HO-1.

6. Effect of HO-1 on Osteoclast Differentiation

To verify the effect of HO-1 on osteoclast differentiation, HO-1 retrovirus was acquired and infected with macrophages as a method for overexpressing HO-1 in macrophages. Macrophages infected with control retrovirus and HO-1 retrovirus were treated with M-CSF and RANKL to differentiate into osteoclasts. Fig. As shown in Fig. 6, cells overexpressing HO-1 were inhibited into osteoclasts (Fig. 6). As a result, costunolide induced HO-1 and inhibited osteoclast differentiation.

According to the present invention,

Bone homeostasis is regulated by the balance between osteoblasts that form bone and osteoclasts that absorb bone. Increasing the activity and number of osteoblasts increases bone density, but osteoporosis occurs in which the bone density decreases when the activity and number of osteoclasts increase. Osteoporosis is a metabolic disorder in which bone density continues to decrease due to excessive formation of osteoclasts and increased activity. Osteoblasts and osteoclasts are tightly regulated by various hormones and cytokines (20).

Bisphosphonate, the most widely used therapeutic agent for osteoporosis, is effective, but recently, it has been reported that bisphosphonate preparations cause fatal side effects such as esophageal and gastrointestinal ulcers and mandibular necrosis. In addition, estrogen, selective estrogen receptor modulator (SERM), and calcitonin preparations are used for the treatment of bone diseases such as osteoporosis. It is reported (20, 21). The purpose of this study was a discovery of osteoporosis medicine using a natural substance isolated from edible and medicinal plants author that selective several natural products to a single material costunolide extracted from the roots of Saussurea lappa (Asteraceae) inhibited the differentiation of osteoclasts Confirmed. The inhibitory effect of costunolide on osteoclast differentiation was not associated with cytotoxicity.

RANKL stimulates the expression of transcription factors important for the differentiation of osteoclasts by inducing the activation of various signaling proteins. In particular, c-Fos was recognized as a transcription factor induced by RANKL stimulation within 1 hour and was essential for the formation of osteoclasts through the study of c-Fos deficient mice (22). Recently, takayanagi et al. Reported that the transcription factor NFATc1 plays an essential role in osteoclast differentiation in NFATc1 deficient mice and that c-Fos plays an important role in the expression of NFATc1 (16). In this study, the authors confirmed the effects of costunolide on osteoclast differentiation and confirmed the expression of the transcription factors c-Fos and NFATc1, which are important for osteoclast differentiation. From 12 hours after RANKL treatment, the expression of c-Fos mRNA was increased and decreased after 48 hours. When RANKL was treated in the experimental group treated with costunolide, the expression of c-Fos mRNA was increased from 12 hours and decreased after 48 hours. However, NFATc1 mRNA induced by RANKL was significantly inhibited by costunolide. In addition, the effect of costunolide on c-Fos and NFATc1 protein expression was the same as that of mRNA expression. As a result, it can be said that costunolide specifically inhibits the expression process of NFATc1 induced by c-Fos.

HO-1 is an enzyme that promotes the breakdown of heme, producing biliverdin, iron, and carbon monoxide (23). HO-1 is also known as a gene that inhibits inflammation. Recently, HO-1 has been reported to be associated with osteoclast differentiation (24), Sakai et al. Reported that HO-1 expression is decreased during osteoclast differentiation, and that HO-1 overexpressing cells are inhibited into osteoclasts. 19). In addition, a study recently published that costunolide promotes the expression of HO-1. Therefore, the author identified whether osteoclast differentiation inhibitory mechanism is related to HO-1. Costunolide promoted HO-1 expression in macrophages, which are precursors of osteoclasts, and the costunolide-induced HO-1 expression was the same as inhibiting osteoclast differentiation. Costunolide also maintained HO-1 expression for up to 24 hours during osteoclast differentiation. Next, we confirmed whether HO-1 is related to osteoclast differentiation and the effect of HO-1 on osteoclast differentiation under experimental conditions used by the authors. HO-1 overexpressed macrophages inhibited osteoclast differentiation. The mechanism of inhibition of osteoclast differentiation by costunolide may be related to the induction of HO-1 expression.

In conclusion, we confirmed that costunolide suppressed osteoclast differentiation in a concentration-dependent manner without cytotoxicity and inhibited the expression of osteoclast markers, TRAP and OSCAR. Costunolide's mechanism of inhibition of osteoclast differentiation has been shown to inhibit osteoclast differentiation by inhibiting mRNA and protein expression of transcription factor NFATc1 and promoting HO-1 expression.

Claims (10)

Treatment and prevention of bone diseases containing costunolides. 2. The agent for treating and preventing bone diseases according to claim 1, wherein the costunolide is extracted from the root of Saussurea lappa. [Claim 2] The agent for treating and preventing bone diseases of claim 1, wherein the costunolide inhibits osteoclast differentiation. The agent for treating and preventing bone disease according to claim 1, wherein the bone disease is osteoporosis. The agent for treating and preventing bone disease according to claim 1, further comprising bisphosphonates or paratyroids. A pharmaceutical composition for preventing and preventing bone diseases, comprising costunolide and a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing and preventing bone disease of claim 1, wherein the costunolide is extracted from the root of Saussurea lappa. [Claim 2] The pharmaceutical composition of claim 1, wherein the costunolide inhibits osteoclast differentiation. According to claim 1, wherein the bone disease is a bone disease and preventive pharmaceutical composition, characterized in that osteoporosis. The pharmaceutical composition for preventing and preventing bone disease of claim 1, further comprising bisphosphonates or paratyroids.

Images