Patent Application
20160220582
1. Technical Field of the Invention
The present invention relates to a synergistic combination of lupeol acetate (LA) with curcumin and its use in the treatment or prevention of activated osteoclast precursor associated disorders. Especially, the present invention relates to a composition of lupeol acetate and curcumin at low dosage, used for regulating immune functions and inhibiting rheumatoid arthritis (RA) or osteoclastogenesis-related diseases.
2. Background
Reumatoid arthritis (RA) is a chronic autoimmune disorder that is closely correlated with the excessive activation of macrophages. The activation of macrophages in the joint will release proinflammatory cytokines, and attract more immune cells to infiltrate, result in more severe inflammatory response, and causes the disintegration of articular cartilage and bone injuries, which can lead to joint deformity at late stage and substantial loss of function and mobility. Macrophages may further differentiate into osteoclasts and lead to bone erosion in the joint cavity, which is the main reason for the progression of RA.
The major pharmaceuticals used in clinical treatment of RA are steroids, non-steroid anti-inflammation drugs, and certain biological agents against cytokine, such as TNF-α blockers, anti-IL-1β, anti-IL-6 antibodies, and the like (Breedveld F C. Arthritis Res 2002, 4(2):27). Such therapeutic agents are not only expensive, but also possess certain degree of side effects.
Lupeol acetate (LA), a type of triterpene, is an ingredient in the extraction of Shea nut, and exists in the mango, cabbage and green pepper. Lupeol acetate has a chemical structure similar to sterols, and has been known with capability of anti-inflammation, anti-oxidation, anticancer and immunomodulation (see, Akihisa T et al., J Oleo Sci 2010, 59(6):273-280; Saleem M. Cancer Lett 2009, 285(2):109-115; Siddique H R, Saleem M. Life Sci 2011, 88(7-8):285-293). It is also demonstrated that LA can effectively mitigate the inflammatory condition induced by carrageenan in mice (Lucetti D L et al. J Inflamm 2010, 7(60)).
In addition, US Patent Application no. 20120177754 has disclosed extraction of lupeol acetate from Boswellia frereana, and the significant therapeutic effect of lupeol acetate in inhibiting inflammation and treatment of rheumatoid arthritis. However, the animal model experiments show that long-term use of high doses (100 mg/kg, 12 days) is necessary for rheumatoid arthritis treatment in mice even a highly pure extract of natural lupeol acetate (95%) is used in the therapy.
Curcumin is a principal curcuminoid extracted from Curcuma Tonga (also known as Turmeric), which is a member of the ginger family (Zingiberaceae), and has been used in curry powder as a common and cheap spice component. Curcumin has been reported with effects of immune modulation and anticancer, and has been shown to have antioxidant, anti-inflammatory and anti-atherosclerosis effects in several animal experiments. It is also known to inhibit the occurrence of inflammation and progression of arthritis in mice. However, the clinical application of curcumin is limited by its poor bioavailability.
Therefore, the present invention contemplates to combine lupeol acetate with curcumin for significantly reducing the cost of drug production, and achieving synergistic effects in the treatment or prevention of activated osteoclast precursor associated disorders to benefit more kinds of patients in clinical use.
Based on the purpose described above, the present invention finds that the combination of lupeol acetate (LA) with curcumin at low doses significantly reduced the activation of macrophages and osteoclastogenesis. The composition of lupeol acetate and curcumin will not only create synergistic effects for inhibiting inflammation and alleviating bone loss at a reduced dosage of lupeol acetate, but also improve the bioavailability of curcumin for clinical application.
Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for treating or preventing osteoclastogenesis associated disorders, comprising lupeol acetate and curcumin combined at certain content or proportion of the composition.
In one preferable embodiment of the present invention, the composition comprises 25-50 mg/kg of lupeol acetate and 40-50 mg/kg of curcumin, and pharmaceutically acceptable carrier, diluent or excipient. In some embodiments of the present invention, the lupeol acetate and the curcumin are combined at a ratio of 0.5:1 to 1:2, and preferably at a ratio of 1:1 to 1:2.
In certain embodiments of present invention, the osteoclastogenesis associated condition includes rheumatoid arthritis (RA). In other embodiments of present invention, the osteoclastogenesis associated condition includes osteoporosis. In a further embodiment of present invention, the osteoporosis is a sterol anti-inflammatory agent triggered osteoporosis.
In another aspect, the present invention relates to a method treating or preventing an osteoclastogenesis associated condition by administering the pharmaceutical composition comprising lupeol acetate and curcumin, combined at a ratio of 0.5:1 to 1:2.
In certain embodiments, the osteoclastogenesis is suppressed by inhibiting a differentiation of osteoclast from macrophage.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
As used herein, term “activated osteoclast precursor associated disorders” refers to the disorders caused by the over-activation of osteoclast precursor. The “osteoclast precursor” is refers to macrophage in general. As shown in
Once macrophage is activated, it would secret TNF-α, IL-1β, and cause osteoblast to reduce the secretion of OPG and increase the secretion of RANKL (receptor activator of nuclear factor kappa-B ligand). The binding of OPG and RANKL can reduce the formation of osteoclasts. Macrophage acts as a precursor in osteoclast. There are RANKL receptors on its cell membrane, once binding with RANKL would trigger osteoclastogenesis to differentiate into osteoclasts, and further result in bone erosion and osteoporosis. Thus, in certain embodiments of the present invention, the activated osteoclast precursor associated disorders include rheumatoid arthritis and osteoporosis.
The other characteristics and advantages of the present invention will be further illustrated and described in the following examples. The examples described herein are using for illustrations, not for limitations of the invention.
RAW 264.7 cells (a mouse macrophage cell line) were treated with different concentrations of lupeol acetate (10, 20, 40, 80 μM), curcumin (2.5, 5, 7.5, 10 μM) and combination (10 μM Cur+10 μM LA, 10 μM Cur+20 μM LA, 10 μM Cur+40 μM LA, 10 μM Cur+80 μM LA) for 24 h. Cell viability was evaluated with MTT assay, and compared to the results of the control group.
5×104 RAW264.7 cells/well were seeded in 96-well culture dishes, and treated with directed concentration of lupeol acetate (LA), curcumin (Cur) and combination for 24 hours after the 24-hr attachment. After removing the culture medium, 100 μl of 0.5 mg/ml MTT solution was added, and the cells were incubated at 37° C. for 4 hours. The mitochondrial enzyme (succinate dehydrogenase, SDH) in living cells will react with tetrazolium bromide in the MTT solution and form formazan blue-purple crystals. The MTT solution was removed and 200 μl DMSO was added to dissolve the blue-purple crystal, and then the O.D. (optical density) value was read under wavelength of 570 nm with an ELISA reader (TECAN Sunrise, USA). The relative cell viability is calculated by comparing the absorbance value of drug treated group with that of the control group (setting the value of control group as 100%).
From the analytic results shown in
In this example, the expressions of TNF-α, IL-6 and IL-1β, all are pro-inflammatory cytokines, assayed by ELISA are used to determined the optimal combination of LA and curcumin. RAW264.7 macrophages were pretreated with different concentrations of LA, curcumin and combination for an hour prior to the addition of 1 μg/ml LPS, then incubated for another 24 h at 37° C., the supernatants from each group were collected, and the expression levels of TNF-α, IL-1β and IL-6 were detected by ELISA.
As shown in
In addition, under LPS stimulation, CD86 and CD80, the two co-stimulation factors expressed on the surface of macrophage, were decreased after the combination treatment of 40 μM LA+10 μM Cur (
Macrophages are usually immobile but become actively mobile when stimulated by inflammation, immune cytokines and microbial products. To examine whether LA+Cur could decrease the migratory ability of macrophage, LPS was used to activate RAW264.7 macrophage cells. Transwell assay was used to determine the migration ability of macrophage cells which could be changed by combination treatment.
1×105 RAW264.7 macrophages cells were seeded in Transwell upper chamber (5.0 μm polycarbonate membrane, 6.5 mm insert, 24-well plate, Corning, USA). LA, Cur and combination (40 μM LA+10 μM Cur) are added into the upper chamber an hour prior to the addition of 1 μg/ml LPS to the lower chamber. After incubating for 24 hr at 37° C., 5% CO2, the medium was removed from the Transwell upper chamber and the membrane was washed twice with PBS. The migrated cells on the membrane were fixed with the fixative (methanol:glacial acetic acid=3:1) for 15 min and stained with hematoxylin for 10 min. Hematoxylin positive cells in each group were counted for five views under the microscope (100×) and were quantified.
As shown in
The protein levels of COX-2 and CCL-2 were found to modulate cell migration under LPS stimulation. Therefore, the expressions of COX-2 and MCP-1 were further confirmed by Western blotting. Results have shown that LPS stimulation would cause large amount of cells to migrate. However, cells harvested from LPS-stimulated RAW264.7 cells treated with 40 μM LA+10 μM Cur, and the protein levels were assayed with Western blotting. As shown in
The generation and metabolism of bones are kept in a state of dynamic equilibrium, and the destruction of this equilibrium will cause the damage of bones. In rheumatoid arthritis, there is excessive osteoclast formation occurred and resulting in the erosion of bone.
To evaluate the effects of the combination of present invention on the suppression of rheumatoid arthritis, we firstly analyze the differentiation of osteoclasts from certain cells using the tartrate-resistant acid phosphatase (TRAP) staining method. The TRAP staining is used to detect the internal acid phosphatase activity of leukocytes in a blood, bone or tissue sample. Because osteoclasts contain acid phosphatase, we can use this staining method to determine the formation of osteoclast cells.
1×104 RAW 264.7 cells were seeded in 96-well plates and treated with LA (40 μM LA, 80 μM LA), Cur (10 μM curcumin) and combination (40 μM LA+10 μM curcumin) co-incubated with 100 nM RANKL in α-MEM medium supplemented with 10% bovine calf serum (BCS, Sigma, USA), 1% L-glutamine (Gibco-BRL, CA, USA) and 1% penicillin-streptomycin (Gibco-BRL, CA, USA). On Day 5, cells were stained for tartrate-resistant acid phosphatase (TRAP). After removing the supernatant of culture, the cells are rinsed twice with PBS, and then fixed with 3.7% paraformaldehyde for 1 hour. The fixed cells are washed with PBS. The Acid Phophatase Leukocyte kit (TRAP stain, Cat. 387-A, Sigma-Aldrich, USA) is used in the TRAP staining and the method is briefly described as follows. Before staining, the temperature of ddH2O used for adjuvant preparation is confirmed to be 37° C. The Fast Garnet GBC base and sodium nitrite solution at equal volumes are uniformly mixed for 30 seconds and incubated at room temperature for at least two minutes.
Then following the procedures described in the instruction manual, a staining agent is prepared by adding a well-mixed solution of 1 ml Fast Garent GBC base, 0.5 ml Naphthol AS-BI phosphate solution, 2 ml acetate solution and 1 ml tartrate solution to 45 ml of 37° C. ddH2O. The staining agent is uniformly mixed and added to each well of 96-well plate at 100 μl aliquot, and then placed in a 37° C. dark incubator for one hour. After the reaction, the 96-well plate is wetted by ddH2O, and stained with a hematoxylin solution included in the kit for ten minutes, then rinsed with tap water and air dried. Finally, the osteoclast differentiation is observed under a microscope, those contain 3 or more nuclei will be identified as osteoclast.
As shown in
Next, the real time Q-PCR is used for analyzing the change of NFATc1 (nuclear factor of activated T cell, the major factor of osteoclast proliferation and known as cytoplasmic 1) under the RANKL induction and drug treatments. As shown in
The animal model used in present studies of rheumatoid arthritis is the collagen-induced arthritis animal model; the progression of rheumatoid arthritis in this animal model is similar to that in human. In this embodiment, bovine type II collagen combined with complete Freund's adjuvant (CFA) is used to induce rheumatoid arthritis in DBA/1J mice, also known as collagen-induced arthritis (CIA) mice.
The type II collagen is a major component of cartilage, and the use of heterologous (bovine) collagen will induce the production of anti-CII antibody in mice, resulting in the self-immune response to attack its own joint cartilage. In the early stage, the complement system is initially activated to attract neutrophils and macrophages, and stimulate the release of inflammatory cytokines from the activated cells. The inflammatory mediators will further affect T cells, B cells and macrophages to produce a more severe inflammation and further attack joints to progress into rheumatoid arthritis.
Eight-week-old DBA/1J mice (purchased from Jackson lab, ME, USA and housed in the Animal center of National Yang Ming University under pathogen-free conditions according to the Institutional Animal Care and Committee guidelines) were used. 100 μl of arthritis-inducing adjuvant (prepared by mixing Complete Freund's Adjuvant (5 mg/ml heat-killed M. tuberculosis in incomplete Freund's Adjuvant) (Chondrex, WA, USA) with equal volume of bovine type II collagen (2 mg/ml solution in 0.05 M acetic acid) (Chondrex, WA, USA)) is injected into the dermis of tail (intra-dermal, i.d.) using a 30 G syringe; and a 50 μl second dose of same ingredients is injected in the same way at an interval of 21 days. The symptoms are produced at about six days after the second dose injection, with an induction rate of 100%. The CIA mice were established and treated with LA 25 mg/kg alone, LA 50 mg/kg alone, Cur 50 mg/kg alone and LA 25 mg/kg+Cur 50 mg/kg after second immunization once per day. Using animals treated with deionized distilled water (ddH2O) containing 0.1% dimethyl sulfoxide (DMSO, Sigma, USA) as the normal mice group. Signs of arthritis were monitored until Day 43.
Clinical score is used to assess the redness and swelling of limbs of each mouse. Arthritis score: 0=normal, 1=slight swelling and/or erythema, 2=extensive swelling and/or erythema, 3=joint distortion and/or rigidity, 4=very inflamed and swollen paw or ankylosed paw. Each limb will be measured, the scores of four limbs will be summed up, and the maximum score of each mouse will be 16.
The body weight tracking shows that changes were with ±10%, that means no general toxicity was found throughout the experimental period (
Apart from blood analysis, current clinical diagnosis will also use X-ray as an evaluation basis. Since X-ray image can only diagnose rheumatoid arthritis when the bone has been damaged severely at late stage, it could not be an effective evaluation tool. 18F-FDG can be used as a tracer to observe and evaluate its accumulation during rheumatoid arthritis. Therefore, in further experiments, we choose 18F-FDG to tract the inflammation in the joint of mice, and use micro-CT to prove the treatment group could diminish the bone loss.
Repeated 18F-FDG PET in vivo determination of disease severity in knee joints and paws on 20, 25, 32, 39 and 43 days after arthritis induction. Mice were anesthetized using 1-3% isoflurane and were intravenous injected with 0.5 mCi/100 μl (18.5 MBq) of 18F-FDG 40 min before images acquisition. The emission scans were performed using an animal micro/PET scan (Tri-Modality FLEX Triumph™ Pre-Clinical Imaging System). All images were acquired for 40 min and reconstructed by software supplied by the manufacturer. The ROIs were selected, and the accumulation of 18F-FDG of each ROI was further analyzed by Amide software.
The results are shown in
Micro-CT images were acquired from the Tri-Modality FLEX Triumph™ Pre-Clinical Imaging System (Gamma MedicaIdeas; Northridge, Calif.). The ROI was analyzed visually in three different slices: transverse, coronal, and sagittal. Individual ROI was used to create 3D representations of the bones of interest. For each specific bone ROI, the total number of pixels and mean pixel intensity across all slices were used to measure bone volume and density. As shown in
The mice were sacrificed by cervical dislocation on the 32nd day of the animal experiment. The whole leg of mouse was removed, and the leg tissue was ground by adding an appropriate amount of lysis buffer (tissue protein extraction reagent, T-PER, Pierce Protein Biology Products, IL, USA), centrifuged at 15,000 rpm for 20 minutes, and the supernatant is taken as the sample for each group. The protein levels of angiogenesis, cell migration, bone erosion and immunosuppressor factor, including VEGF, COX-2, MCP-1, TGF-β, IL-10, Granzyme B, MMP-9, OPG and RANKL, were confirmed with ex vivo Western blotting.
As shown in
The LightShift Chemiluminescent EMSA kit (Pierce, Rockford, Ill., USA) was used in the analysis for NF-κB/DNA binding activity. Nuclear extracts were incubated with the biotin labeled DNA probes at room temperature for 20 minutes. The separated DNA/protein complexes from the free oligonucleotide on 10% polyacrylamide gel were transferred to a nylon membrane. The nylon membrane was immersed in ECL (Pierce, Rockford, Ill., USA) and reacted to emit cold light (luminescence), and then exposed to the film for the observation of NF-κB activity. Using IMAGE J software (National Institutes of Health), the obtained images were quantified to blackening degree; the blackening degree of the protein to be observed is divided by the value obtained in the control group to compare the differences of each group in the expression level of nuclear proteins. The result of ex vivo electrophoretic mobility shift assay (EMSA) shown in
Treg cell is an immunosuppression-related T cell, and usually less differentiated in autoimmune diseases. Some literatures have indicated that injection of Treg cells to the back of mice will effectively reduce the incidence of rheumatoid arthritis. Therefore, we implicate that the increase in the number of Treg cells induced by the treatment of the LA+Cur can reduce the occurrence of rheumatoid arthritis.
The mice were sacrificed at the peak of the incidence, that is the 32nd day of the experiment. The spleen and drained lymph nodes (DLNs) were harvested and labeled with anti-FoxP3-Alexa Fluor 488/CD4-APC/CD25-PE Abs according to manufacturer's protocol of Mouse Treg Flow Kit (Biolegend, San Diego Calif., USA). The percentage of positive stained cells was analyzed by FACS instrument (BD Biosciences, San Jose, Calif., USA).
The results showed that the percentage of Treg cells in the combination group had no significant difference with that of the LA50 mg/kg group. Also, the combination group had a significantly higher percentage of Treg cells as compared to those of the other groups (
Celecoxib (Celebrex® capsule) is a known COX-2 inhibitor, a kind of non-steroidal anti-inflammatory analgesics (referred to as NSAIDs), and currently used in the clinical treatment of menstrual pain or chronic pain caused by rheumatoid arthritis, degenerative arthritis. In this embodiment, the inhibitory effects of combination (lupeol acetate plus curcumin) on the expressions of arthritis-related proteins and osteoclastogenesis inducing factor RANKL are compared with the inhibition by Celecoxib. Furthermore, immunohistochemical staining was used to evaluate the inhibition of joint inflammation by these drugs.
Mice were sacrificed at the peak of arthritis (on day 32), the protein extraction was isolated from legs of each group. The protein levels of angiogenesis, cell migration, bone erosion and immunosuppressor factor were confirmed with ex vivo Western blotting. As shown in
For the analysis of IL-6 expression, mouse cheek blood was sampled on day 20, 32 and 43 after the first immunization, and serum was isolated and subjected to ELISA IL-6 (Cat. No. 88-7064, eBioscience, CA, USA) according to the manufacturer's instruction manual. As shown in
To investigate the therapeutic efficacy of LA combined with curcumin on the histological damage in CIA mice, the mice were scarified on day 43 after arthritis induction. The joints of four limb of each mouse were excised and fixed with paraformaldehyde then embedded by paraffin and sectioned. Slides were stained with haematoxylin and eosin (H & E), and were observed under microscope.
As shown in
In summary of the experimental results described above, it has proven that combination of lupeol acetate with curcumin at half dosage can synergistically alleviate the inflammatory response by inhibiting the release of cytokines, such as COX-2, MCP-1, TNF-α, IL-1β and the like by macrophages, and reduce the expression of osteoclastogenesis-related proteins, such as MCP-1, COX-2, granzyme B, MMP-9, TGF-β, IL-1β, OPG and RANKL by regulating the levels of NF-κB and NFATc1. Moreover, it has demonstrated in the in vivo CIA animal experiments that combination of lupeol acetate plus curcumin of the present invention can effectively alleviate the joint inflammation, swelling, bone erosion and the incidence of rheumatoid arthritis in mice.
According to the present invention, the development of autoimmune arthritis is suppressed by the combination of lupeol acetate (LA) and curcumin (Cur) via blockading the release of pro-inflammatory cytokines and decreasing the formation of osteoclasts in vitro and in vivo. Also, the golden section of combination of LA+Cur (lupeol acetate 25 mg/kg plus curcumin 50 mg/kg) not only reduces the cost but also a potential treatment in patient with RA and osteoporosis. Accordingly, the present invention has reached the purpose in reducing the clinical dosage of lupeol acetate by combination with curcumin for the treatment of osteoclast precursor associated disorders, including rheumatoid arthritis (RA) or osteoclastogenesis-related diseases. The composition of lupeol acetate and curcumin at low dosage will be useful in inhibiting inflammation, improving the severity of bone erosion and joint swelling, and alleviating bone loss in arthritis patients.
In addition, the current therapy of rheumatoid arthritis is often in combination with steroid drugs, which will increase the chance of occurring side effects such as osteoporosis. Therefore, the present invention further provides a composition comprising lupeol acetate and curcumin combined with conventional steroids for the treatment of rheumatoid arthritis, which is beneficial to reduce the probability and severity of osteoporosis in the patients with rheumatoid arthritis by the effects on inhibiting osteoclastogenesis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 104103096 | Jan 2015 | TW | national |
