PROCESS FOR EXTRACTING THE ALKALOID FRACTION OF RHODOPHIALA BIFIDA (HERB.) TRAUB AND USES THEREOF

Abstract

The present invention describes a method for completely extracting the alkaloid fraction (montanine) of Rhodophiala bifida (Herb.) Traub from bulbs of Rhodophiala bifida. The present invention further describes a method for treating inflammation using pharmaceutical compositions containing the alkaloid fraction of Rhodophiala bifida as the active ingredient. The present invention therefore comprises an extraction method that is faster than other extraction methods described in the literature for the alkaloid fraction of Rhodophiala bifida, dispensing with numerous changes of solvent in order to strain the plant, and the use thereof as an anti-inflammatory. The present invention is further characterised by the development of an anti-inflammatory medicinal drug for treating and preventing diseases involving inflammation and/or the local increase in the number of fibroblasts as its etiopathogenesis, such as: rheumatoid arthritis, ulcerative colitis, sepsis, acute pulmonary disease, inflammatory infections, in particular inflammatory and fibrosing diseases related to the lungs and kidneys, osteoporosis, Castleman disease, psoriatic arthritis, juvenile chronic arthritis and other non-specific inflammatory joint diseases.

Claims

1-4. (canceled)

5. A method of treating a disease having inflammation and/or localized increase of fibroblasts activity as etiopathogenesis, comprising administering an effective amount of montanine to an individual in need thereof.

6. The method of claim 5, wherein the disease is chosen from Rheumatoid Arthritis, juvenile rheumatoid arthritis, and osteoporosis.

7. The method of claim 5, wherein the disease is chosen from Castleman's disease, psoriatic arthritis, and non-specific acute and chronic arthritis.

8. The method of claim 5, wherein the disease is chosen from fibrotic diseases related to the lungs and kidneys.

9. The method of claim 5, wherein the administering does not impact or depress the individual's immune system.

10. The method of claim 5, wherein the treating is for a disease having migration-invasion, proliferation or activation of fibroblasts as etiopathogenesis.

11. The method of claim 10, wherein the method of treating is for treating rheumatoid arthritis, juvenile rheumatoid arthritis, and osteoporosis.

12. The method of claim 10, wherein the administering does not impact or depress the individual's immune system.

13. The method of claim 5, wherein the treating is for a disease whose etiopathogenesis derives from the inflammatory process, lymphocyte migration, and/or proliferation.

14. The method of claim 13, wherein the method of treating is for treating Castleman's disease, psoriatic arthritis and non-specific acute and chronic arthritis.

15. The method of claim 13, wherein the administering does not impact or depress the individual's immune system.

16. The method of claim 5, wherein the disease is for treating fibrotic disease of the osteo-articular system.

17. The method of claim 16, wherein the method of treating is for treating fibrotic lung forms of rheumatoid arthritis, idiopathic pulmonary fibrosis, renal fibrosis, or retroperitoneal fibrosis.

18. The method of claim 16, wherein the administering does not impact or depress the individual's immune system.

19. The method of claim 5, wherein the administering is systemic.

20. The method of claim 19, wherein the disease is rheumatoid arthritis.

21. The method of claim 20, wherein the administering does not impact or depress the individual's immune system.

22. The method of claim 5, wherein the inflammation is caused by the individual's reaction to infection, ischemia, toxic agents, autoimmunity, or tissue injury.

23. The method of claim 5, wherein the inflammation is caused by the individual's reaction to autoimmunity.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 shows the chemical structure of the alkaloid montanine.

[0050] FIG. 2 demonstrates the chromatogram with mass spectrometry detection of isolated montanine.

[0051] FIG. 3 demonstrates the chromatogram with detection in the UV region of isolated montanine.

[0052] FIG. 4 shows the mass spectrum of montanine.

[0053] FIGS. 5A-B show the effect of montanine on leukocyte migration to the femorotibial articulation and joint hypernociception in a model of mBSA-induced monoarthritis. *P<0.05. FIG. 5A demonstrates leukocyte migration. FIG. 5B shows hypernociception.

[0054] FIG. 6 shows the effect of montanine on lymphocyte viability for 24 h. *P <0.05.

[0055] FIGS. 7A-B show the effect of montanine on the LPS- or ConA-induced lymphocyte proliferation. *P<0.05. FIG. 7A shows the LPS-induced lymphoproliferation. FIG. 7B shows the ConA-induced lymphoproliferation.

[0056] FIG. 8 shows the effect of montanine on synovial fibroblast invasion. *P<0.05.

[0057] FIGS. 9A-B show the cell blood count, immunosuppression test of montanine, at time zero, after 3 days of administration and after 3 more days with no administration given.

[0058] FIG. 10 shows the cell viability in 24 hours with different concentrations of montanine.

[0059] FIG. 11A shows lymphoproliferation with lipopolysaccharide (LPS).

[0060] FIG. 11B shows lymphoproliferation with conconavalin A (ConA).

[0061] FIG. 12A shows the invasion of fibroblasts with and without 1 M montanine.

[0062] FIG. 12B shows the invasion of fibroblasts with and without 1 M montanine.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The general methods for the extraction of alkaloids are based on solubility in water-immiscible organic solvents (ether, ethyl acetate, benzene, etc.) and water insolubility. Alkaloid salts exhibit inverse properties.

[0064] In the present invention there is described an effective method for the extraction of the alkaloid fraction of Rhodophiala bifida.

[0065] The bulbs of Rhodophiala bifida are first washed with tap water, cut into chips and dried in a stove until complete removal of water. Thereafter, the dry bulbs are milled in a knife mill.

[0066] For extraction of chemical substances, subjecting the dried and milled bulbs to a liquid medium with sulfuric acid at a concentration of 0.5 to 50%, at a ratio of 0.5 to 5 grams of bulbs to 5 to 50 mL of the acidic solution and are placed in an ultra-sound bath for a time that ranges from 1 to 48 hours at a temperature ranging from ambient to 100 C. Preferably the following conditions are used: sulfuric acid 2% (or hydrochloric acid 50%) at the ratio of 1 g of bulb to 10 mL of acidic solution for 4 hours in the ultra-sound bath at room temperature;

[0067] Thereafter, the mixture is centrifuged and the supernatant is basified. The basified solution is extracted with ethyl acetate (or ethyl ether, petroleum ether, chloroform, benzene, dichloromethane). Preferably, from among the nonpolar solvents ethyl acetate is used, which is then evaporated.

[0068] The residue of the organic phase is subjected to vacuum liquid chromatography, first using hexane as the mobile phase to remove possible impurities, and then the step of montanine isolation with solvents is performed using a polar solvent, preferably C1 to C4 alcohols, more preferably using methanol. To each 10 g of bulbs used in the extraction 100 mL of mobile phases are used, respectively. Methanol is then evaporated and the dry residue is frozen to be freeze-dried. After freeze-drying the yield of alkaloid fraction is of approximately 4% relative to the dry bulbs.

[0069] To examine the identity of the isolated fraction High-Performance Liquid Chromatography with ultraviolet (UV) and/or mass spectrometry (MS) detection is used. In FIGS. 2 and 3 we disclose the MS and UV chromatograms of the isolated fraction, respectively. As can be noted in FIG. 3, the alkaloid fraction comprises a single substance, which is shown in the peak at 1.13 minutes. In FIG. 4 we depict the mass spectrum of the isolated substance where the values of calculated mass=301,1314 Da; calculated mass (+1)=302.1392 Da; confirm the experimental mass=302.1403 Da of the alkaloid montanine, confirming the product's identity. Difference=3.64 ppm, below 5 ppm of exact mass demonstrates the identity of montanine

[0070] To solubilize montanine in 0.9% physiological saline, the solution was placed in an ultra-sound bath for 2 hours for it to be used as a treatment.

[0071] Description of the Experiments

[0072] The present invention further proposes the montanine activity, as described in FIG. 1, to be an anti-inflammatory, such as an inhibitor of lymphocyte migration, such as an inhibitor of fibroblast migration/invasion and inhibitor of T-lymphocyte proliferation; as a modifier of inflammatory disease, especially that related to the osteoarticular system, with emphasis on rheumatoid arthritis without causing changes or depression of the patient's immune system when administered systemically.

[0073] Below we describe experiments that led to the conclusion that montanine is effective as an anti-inflammatory agent.

[0074] In Vivo Experiments

[0075] Antigen-Induced Arthritis (AIA) Model

[0076] Antigen-induced arthritis (AIA) is an animal model of arthritis that is broadly described and used in worldwide literature. One of the inducing antigens in this model in Balb-C mice is methylated bovine serum albumin (mBSA), which, after systemic immunization (subcutaneous injection) is injected into the joint. AIA is an immune-mediated (T cell-dependent) joint inflammation, whose histopathology shows many similarities with rheumatoid arthritis in humans (Grespan et al. 2008).

[0077] Briefly, mice were immunized in three steps with mBSA (day-0, day-7 and day-14), the first step comprising 500 mg of mBSA protein in an emulsion of 0.1 mL of complete Freund's adjuvant's (CFA) and 0.1 mL of sterile saline solution (0.9% sodium chloride), the second and third steps were performed with the same protein concentration, the same sterile saline solution concentration and the same adjuvant concentration, but in these steps incomplete Freund's adjuvant (IFA) was used; 3 weeks after the first immunization (day 21) mBSA at a concentration of 30 ug/mL were injected into the femorotibial joint (knee) and the contralateral knee was administered with sterile saline only and serves as the control of the experiment (Grespan et al. 2008). The injected joint developed an acute inflammation within a few hours (characterized by massive granulocyte infiltration and fibrin exudate) and from the behavioral point of view, mice showed pronounced mechanical hyperalgesia (pain) in the inflamed knee. Treatment with montanine was given twice daily, one day before intra-articular challenge, at the day of the challenge and at day of death, by intraperitoneal route and diluted in saline. Doses tested were 0.3; 1; 3 mg/kg. One group of animals received the treatment vehicle (saline) for control purposes, said group being designated positive control. The experiment is terminated at day 22 (24 hours after challenge and treatment) when animals were euthanized to collect the joint lavage fluid. Lavage was performed using a PBS-EDTA solution and then the number of leukocytes existing in the joint was counted in a Neubauer chamber at a 1:2 dilution with Turk's solution. Furthermore, pain of the animals was assessed in a digital analgesia-meter (von Frey) prior to intra-articular injection at time 0 (zero), 3, 5 and 24 hours after intra-articular challenge.

[0078] Montanine exhibited reduction in pain at all tested doses (FIG. 5), but the dose of 3 mg/kg showed a decrease as of 3 hours. In addition, the total leukocyte migration was significantly reduced at all three doses (FIG. 5) compared with the group receiving saline solution only.

[0079] Collagen-Induced Arthritis (CIA) Model with Prophylactic Treatment

[0080] The chronic arthritis model most widely used in the world literature is the model of collagen type II-induced arthritis (CIA). Said model shares many pathological features with the disease, such as type II collagen (CM), the major cartilage protein and one of the potential RA self-antigens.

[0081] CIA has been widely used to identify the potential pathogenic mechanisms of autoimmunity, including the role of different cell types, individually, at the beginning and during the progression of the disease, as well as to test and develop new therapies.

[0082] Briefly, CIA was induced in DBA/1J mice (8-12 weeks, average weight of 20 g) which were immunized by 50 L of an emulsion containing equal volumes of bovine type II collagen (2 mg/ml) and complete Freund's adjuvant (CFA) by intradermal injection (i.d.) at the base of the tail on day-0 and on day-18 after this first immunization a boost was given with an emulsion of IFA and CU, injected below the first site of injection.

[0083] After the boost injection, treatment at dosages of 0.05; 0,25 and 0.5 mg/kg was started and continued for 15 days (twice daily, intraperitoneally) and animals were monitored daily for clinical signs of the disease.

[0084] The following techniques for assessment of chronic arthritis and the effect of montanine on this model were analyzed:

[0085] Arthritis Clinical Score

[0086] Animals were monitored daily for analyzing the clinical signs of arthritis by means of the severity score as follows: 0no signs of disease; 1mild erythema and edema; 2moderate erythema and edema; 3severe erythema and edema extending from the knee to the metatarsus; 4severe erythema and edema with loss of function. The total score is the average of the scores on the legs from the onset of the disease.

[0087] Histological Analysis

[0088] The tibio-tarsal joints of DBA/1 J animals were isolated and immersed in 10% buffered formalin for fixation for 24 hours. Then, tissues were decalcified in 10% trichloroacetic acid (TCA) for approximately 18 hours. These tissues were dehydrated and embedded in paraffin blocks. Six pm-thick cuts were arranged on a microscope slide. Slides were stained with hematoxylin and eosin staining technique for assessment of the following parameters: synovial inflammation: five high-power magnification fieldsHMF was analyzed for the percentage of infiltrating mononuclear cells: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); synovial hyperplasia: 0-absent, 1-mild (5-10 cell layers), 2-moderate (11-20 layers), 3-severe (>20 layers);extent of pannus formation: 0-absent, 1-mild, 2-moderate, 3-severe; synovial fibrosis: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); synovial vascularization (angiogenesis): sum of the number of vessels in five HMF of synovial tissue; cartilage erosion: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); bone erosion: 0-absent, 1-minor erosion(s) observed only in HMF, 2-moderate erosion(s) observed at low magnification, 3-severe transcortical erosion(s) as previously described by Oliveira et al. 2011 and for assessment of cartilage degradation analysis was performed using the safranin-O staining technique. All the cuts were assessed under a microscope by two blinded observers, and the images captured by digital camera.

[0089] Prophylactic treatment with montanine exhibited reduced clinical score of the disease at doses of 0.25 and 0.5 mg/kg from day 8 of treatment (FIG. 6). Furthermore, treatment at a dose of 0.5 mg/kg improved all the histological parameters of the joint, except for synovial hyperplasia (FIG. 8).

[0090] Collagen-Induced Arthritis (CIA) Model with Therapeutic Treatment

[0091] CIA induction is performed by following the aforementioned protocol.

[0092] After the boost injection, animals were monitored daily for clinical signs of the disease and treatment with montanine at 0.5 and 1.5 mg/kg is started on the day of clinical detection of CIA. Animals given sterile saline, the treatment vehicle, were deemed as the positive control group, i.e. untreated. The drug was administered twice daily for 10 days by the intraperitoneal route.

[0093] The following techniques for assessment of chronic arthritis and the effect of montanine on this model were analyzed:

[0094] Arthritis Clinical Score

[0095] Animals were monitored daily for analyzing the clinical signs of arthritis by means of the severity score as follows: 0-no signs of disease; 1-mild erythema and edema; 2-moderate erythema and edema; 3-severe erythema and edema extending from the knee to the metatarsus; 4-severe erythema and edema with loss of function. The total score is the average of the scores on the legs from the onset of the disease.

[0096] Articular Nociception

[0097] Articular hypernociception was assessed as previously described by Pinto LG et al. 2010. For this model, a polypropylene tip was adapted to the manual force transducer, a force was applied on the subplantar surface of the paw producing tibiotarsal bending motion. The automatic pressure meter recorded the strength of the applied force when the paw is removed. The assay is repeated 3 times sequentially to provide a consistent measurement, the limiar being expressed in grams (g).

[0098] Histological Analysis

[0099] The tibio-tarsal joints of DBA/1 J animals were isolated and immersed in 10% buffered formalin for fixation for 24 hours. Then, tissues were decalcified in 10% trichloroacetic acid (TCA) for approximately 18 hours. These tissues were dehydrated and embedded in paraffin blocks. Six pm-thick cuts were arranged on a microscope slide. Slides were stained with hematoxylin and eosin staining technique for assessment of the following parameters: synovial inflammation: five high-power magnification fieldsHMF will be analyzed for the percentage of infiltrating mononuclear cells: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); synovial hyperplasia: 0-absent, 1-mild (5-10 cell layers), 2-moderate (11-20 layers), 3-severe (>20 layers); extent of pannus formation: 0-absent, 1-mild, 2-moderate, 3-severe; synovial fibrosis: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); synovial vascularization (angiogenesis): sum of the number of vessels in five HMF of synovial tissue; cartilage erosion: 0-absent, 1-mild (1-10%), 2-moderate (11-50%), 3-severe (51-100%); bone erosion: 0-absent, 1-minor erosion(s) observed only in HMF, 2-moderate erosion(s) observed at low magnification, 3-severe transcortical erosion(s) as previously described by Oliveira et al. 2011 and for assessment of cartilage degradation analysis the safranin-O staining technique was performed. All the cuts were assessed under a microscope by two blinded observers, and the images captured by digital camera.

[0100] Therapeutic treatment with montanine at the dosage of 0.5 mg/kg exhibited reduced pain (FIG. 7A), as well as a reduction of the clinical score from the third day of treatment (FIG. 7B). Furthermore, treatment at a dose of 0.5 mg/kg improved all the histological parameters, except for synovial hyperplasia (FIG. 8).

[0101] Immunosuppression Assay

[0102] An immunosuppression assay in healthy animals was performed to assess the role of montanine as an immunosuppressive agent. 20 male, healthy mice of 8-12 weeks of age were used for examining the whole blood (complete blood countparameters of: total of white blood cells (WBC); % lymphocytes (Lin %), lymphocytes/mm3 (Lin mm30; % monocytes (Mon %); monocytes/mm3 (Mon mm3); % granulocytes (Gran %); granulocytes/mm3 (Gran/mm3); total of red blood cells (RBC); hemoglobin (HGB) and total of platelets (PLQ)) of animals, which were divided as follows: healthy animals (with no treatment given) (n=4), animals treated with 0.5 or 1.5 mg/kg de montanine/12-12 hours (n=8/group). Whole blood was collected at day 0 for basal analysis of the blood parameters of the complete blood count in all animals, they were then treated for 3 days with the two doses and on the third day blood was collected again for complete blood count analysis; also, animals were still alive for three more days without any treatment for a new blood collection (FIG. 9).

[0103] This result has demonstrated that montanine does not lead to immunosuppression in any of the tested dosages as compared with the control e with each other.

[0104] In Vitro Experiments

[0105] Lymphocyte Viability Test

[0106] A lymphocyte viability test was performed to evaluate the cytotoxicity of montanine using a colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, as described by Tomita T. et al. 2006. Animals were euthanized and their draining lymph nodes (popliteal and inguinal) were removed aseptically. A single cell suspension was prepared, grown in triplicate (5105 cells/well in a 96 well plate) and treated with montanine at the dosages (0.01 M, 0.1 M, 1 M, 10 M and 100 M) for 48 hours at 37 C. at 5% CO.sub.2 in RPMI medium. After incubation, MTT (0.5 mg/ml) was added to each well and the plate was placed in the stove again and after 4 hours the supernatant was removed and 50 I of dimethyl sulphoxide (DMSO, Sigma) were added. After the plate was shaken to dissolve the MTT formazan crystals, optical density (OD) of each well was measured at 570 nm using a microplate ELISA reader. Montanine at doses of 0.01 M, 0.1 M and 1 M did not alter cell viability (FIG. 10). There is no significant difference between the cell group and the group treated with the montanine at 10 M, however, decreased cell viability can be seen, which was higher in the 100 M dose. From the results obtained in this assay, the dosage of 1 M was chosen as the test dosage for subsequent in vitro assays, as this was the highest dose that did not exhibit any cytotoxic effects on the cells.

[0107] Lymphocyte Proliferation Assay

[0108] The in vitro lymphocyte proliferation was performed using the MTT colorimetric assay described by Tomita T et al. 2006. BALB/c mice were euthanized and their draining lymph nodes were removed aseptically. A single cell suspension was prepared, grown in triplicate (5105 cells/well in a 96 well plate) and treated with montanine at the dosages of 1 M for 48 h at 37 C. at 5% CO.sub.2 in RPMI medium containing 10 mg/mL lipopolysaccharide (LPS) or 5 mg/mL of conconavalin A (ConA) or RPMI medium alone as a culture control. After incubation, MTT (0.5 mg/ml) was added to each well and the plate was placed in the stove again and after 4 hours the supernatant was removed and 50 I of dimethyl sulphoxide (DMSO, Sigma) were added. The plate was placed in the stove again and the supernatant was then removed and 50 I of DMSO (Sigma) were added. After the plate was shaken to dissolve the formazan crystals, optical density (OD) of each well was measured at 570 nm.

[0109] Both ConA and LPS are molecules that stimulate the lymphocyte proliferation, but they exhibit differences in terms of specificity. LPS acts primarily on the B cell receptor and the Toll-like receptor 4 (TLR4), molecules that are present on the surface of B lymphocytes, thus acting on these cells. ConA acts on several receptors containing glycoproteins or lipoproteins, stimulating both lymphocytes both but acting preferably on T lymphocytes.

[0110] In the performed experiments (FIG. 11), montanine had no effect on lymphocyte proliferation stimulated by LPS, but significantly decreased lymphocyte proliferation stimulated by ConA. From these data it can be concluded that montanine has preferential activity on T lymphocytes.

[0111] Synovial Fibroblast Invasion Test

[0112] To evaluate the invasion of synovial fibroblasts into matrigel inserts (collagen matrix) a BD kit (Franklin Lakes, N.J., USA) was used, and the test was performed in accordance with the manufacturer specifications.

[0113] When cells reached 70-80% confluence, they were trypsinized with trypsin-EDTA for digestion. Then, 210.sup.4 cells were resuspended in 500 I of free culture medium of fetal calf serum and placed on top of the insert. Montanine at a dose of 1 M or the same concentration of vehicle (DMSO) was added on top of the insert with the cells. At the lower compartment 750 I of culture medium with 10% fetal bovine serum was added. The plate was incubated at 37 C. for 24 h in a stove with 5% CO.sub.2. After the incubation period, the top of the chamber was cleaned with a swab, stained with crystal violet dye, and the total number of cells that invaded the Matrigel membrane was counted in an optical microscope at 100 magnification. Experiments were performed in duplicate.

[0114] This procedure allows one to compare the normal condition of cell migration and the effect of drugs on this ability. The obtained results demonstrated that montanine reduced the invasion of synovial fibroblasts of the five lines tested (FIGS. 12A and 12B).

[0115] These in vivo and in vitro results demonstrated the potential of montanine as a potential anti-inflammatory drug. The property of montanine of being able to reduce fibroblast migration and T-lymphocyte proliferation can be understood as proof of concept that montanine is able to modify the disease. Specifically when treating rheumatoid arthritis, a class of drugs can be mentioned, which is known as disease-modifying antirheumatic drug (DMARDs), which has the ability to prevent disease progression (from inflammatory to deformant) and not only to treat their symptoms (the particular case of montanine). Montanine, however, can be used as an active capable of modifying the disease when applied to the osteoarticular system diseases. Probably the aforementioned mechanisms of action also act on the treatment and prevention of other diseases having inflammation and/or increased number of fibroblasts in a localized manner as etiopathogenesis, in particular inflammatory and fibrotic lung and kidney diseases, Castleman's disease, psoriatic arthritis and juvenile rheumatoid arthritis. The use of montanine to inhibit fibroblast migration can be associated with intra-articular diseases as well as diseases involving fibroblast dysfunction as a cause or effect, through migration thereof and/or exacerbated or ectopic production of matrix, including fibrosis of organs such as the lungs and kidney.

REFERENCES

[0116] 1. Grespan R, Fukada S Y, Lemos H P, Vieira S M, Napimoga M H, Teixeira M M, Fraser A R, Liew F Y, McInnes I B, Cunha F Q. CXCR2-specific chemokines mediate leukotriene B4-dependent recruitment of neutrophils to inflamed joints in mice with antigen-induced arthritis. Arthritis Rheum. 200; 58(7):2030-40. 5 2. Tomita T, Kakiuchi Y, Tsao P S. THR0921, a novel peroxisome proliferator-activated receptor gamma agonist, reduces the severity of collagen-induced arthritis. Arthritis Res Ther. 2006;8(1):R7.

[0117] 3. Pinto L G, Cunha T M, Vieira S M, Lemos H P, Verri W A Jr, Cunha F Q, Ferreira S H. IL-17 mediates articular hypernociception in antigen-induced arthritis in mice. Pain. 2010;148(2)-.247-56.

[0118] 4. Oliveira P G, Grespan R, Pinto L G, Meurer L, Brenol J C, Roesler R, Schwartsmann G, Cunha F Q, Xavier R M. Protective effect of RC-3095, an antagonist of the gastrin-releasing peptide receptor, in experimental arthritis. Arthritis Rheum. 2011; 63(10):2956-65.