Bioorganic & Medicinal Chemistry Letters 25 (2015) 2686–2689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Therapeutic effects of hybrid liposomes with downregulation of inflammatory cytokine for model mice of rheumatoid arthritis in vivo Hideaki Ichihara, Shuichi Yamasaki, Motoki Hino, Ryuichi Ueoka, Yoko Matsumoto ⇑ Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1, Ikeda, Nishi-ku, Kumamoto 860-0082, Japan
a r t i c l e
i n f o
Article history: Received 19 February 2015 Revised 14 April 2015 Accepted 22 April 2015 Available online 1 May 2015 Keywords: Hybrid liposomes Rheumatoid arthritis Collagen-induced arthritis mouse models Chemotherapy In vivo
a b s t r a c t Therapeutic effects of HL for a collagen-induced arthritis (CIA) mouse models of HL-23 composed of 95 mol % L-a-dimyristoylphosphatidylcholine (DMPC) and 5 mol % polyoxyethylenedodecylether (C12(EO)23) in vivo were examined. Remarkably high therapeutic effects of HL-23 for CIA mouse models were obtained on the basis of clinical assessment of arthritis. The reduction of hyperplastic synovial membrane (pannus tissue) and destruction of the cartilage and bone by HL-23 was revealed on the basis of hematoxylin and eosin (HE) and safranin O staining. Furthermore, the downregulation of inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models treated with HL-23 were investigated. Remarkably high therapeutic effects without joint swelling were obtained in CIA mouse models treated with HL-23. Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints, affecting up to 1% of the population worldwide. It is characterized by an inflammation of joints, hyperplastic synovium with pannus formation, leading to bone and cartilage destruction in the joints. The affected synovial tissues contain activated macrophages, fibroblasts, T lymphocytes, and B lymphocytes, which release proinflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6.1,2 Current clinical chemotherapy of RA using disease-modifying antirheumatic drugs (DMARDs) and anti-TNF-a antibody reduce inflammation by inhibiting or halting the immune process.1–5 However, potentially negative side effects of anti-RA drug such as digestive organs dysfunction, liver dysfunction, kidney dysfunction, stomatitis, depilation, respiratory related symptoms including pneumonitis, and myelosuppression have been reported.1,2,5–7 Furthermore, tolerance to anti-RA drug in patients have been observed.1,2,8,9 Therefore, anti-RA drug that would be effective for suppressing the inflammation of joints in RA without side effects is highly desirable to improve the quality of life for RA patients. We have produced hybrid liposomes (HL) which can be prepared by just the sonication of vesicular and micellar molecules in a buffer solution.10,11 HL contain no organic solvent unlike
⇑ Corresponding author. Tel.: +81 96 326 3965; fax: +81 96 323 1331. E-mail address: [email protected] (Y. Matsumoto).
conventional liposomes and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature, and hydrophobicity can be controlled by changing the constituents and compositional ratios of the HL. The therapeutic effects of HL composed of L-a-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(20) sorbitan monolaurate (Tween 20) including antitumor drugs such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been observed on the growth of glioma cells in vivo.12 On the other hand, high inhibitory effects of HL composed of DMPC and polyoxyethylene(n) dodecyl ethers (C12(EO)n) without any drugs on the growth of tumor cells in vitro13,14 and in vivo15,16 along with the induction of apoptosis have been obtained. After receiving the approval of the Bioethics Committee, successful clinical chemotherapy with drug-free HL to patients with lymphoma has been reported.17 More recently, we have reported that HL inhibits the growth of human RA fibroblast-like synoviocytes (HFLS-RA) cells along with apoptosis in vitro.18 However, the research for the effects of HL using SKG mice as models of RA and regulation of inflammatory cytokines is very limited, because it takes more than three months until symptoms are observed. To overcome this disadvantage, in this study, we examined the therapeutic effects of HL-23 composed of 95 mol % L-a-dimyristoylphosphatidylcholin (DMPC) and 5 mol % polyoxyethylenedodecylether (C12(EO)23) for a collageninduced arthritis (CIA) mouse models19 of RA in vivo. HL-23 were prepared by using sonication (VELVO VS-N300, 300W, Velvo-clear, Tokyo, Japan) of a mixture containing
http://dx.doi.org/10.1016/j.bmcl.2015.04.083 0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689
Figure 1. Therapeutic effects of HL-23 for CIA mouse models with rheumatoid arthritis using clinical score method. (A) Rheumatic clinical score changes for 7 weeks after the booster injection. Data presented are mean ± S.D. Dose for DMPC; 136 mg/kg. (B) Photographs of finger at 7 weeks after booster injection.
90 mol % L-a-dimyristoylphosphatidylcholine (DMPC, NOF Co., Tokyo, Japan, purity>99%) and 10 mol % polyoxyethylenedodecyl ether (C12(EO)23, Sigma–Aldrich Japan, Tokyo, Japan) in 5% glucose solution at 45 °C with 300 W, followed by filtration with a 0.20 lm filter. Physical properties of HL-23 composed of 95 mol % DMPC, 5 mol % C12(EO)23 were examined. Apparent mean hydrodynamic diameters (dhy) of HL were measured using light scattering spectrometer (ELS-8000, Otsuka Electronics Co. Ltd, Japan) with He-Ne laser light source (633 nm). The diameter was calculated by Stokes–Einstein equation (dhy = (jT)/(3pgD)), where j is
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Boltzmann constant, T is absolute temperature, g is viscosity and D is diffusion coefficient. Hydrodynamic diameter (dhy) of HL-23 was under 100 nm, which were preserved for a period remaining stable for more than one month. On the other hand, DMPC liposomes were unstable and precipitated after 14 days. We examined the therapeutic effects of HL-23 using CIA (collagen-induced arthritis) mouse models of RA, which are the most commonly studied autoimmune models of rheumatoid arthritis.19 The mice were handled in accordance with the guidelines for animal experimentation set out in Japanese law. The animal studies were approved by the Committee on Animal Research of Sojo University. Joint swelling was monitored by inspection and scored.20 The results are shown in Figure 1. No detection of rheumatic clinical score indicating joint swelling and deformity of fingers was obtained in the CIA mouse models treated with HL-23, although increased rheumatic score were observed in the control group from 4 weeks after booster injection (Fig. 1A). No joint swelling was obtained in the mice treated with HL-23 on the basis of macroscopic inspection, although joint swelling and deformity of fingers in all feet were observed in the control group (Fig. 1B). It is noteworthy that remarkable therapeutic effects were obtained in the CIA mouse models of RA intraperitoneally treated with HL-23 without drugs. Then, we histologically evaluated the therapeutic effects of HL23 using the finger tissues for the CIA mouse models in vivo. The finger sections were stained with hematoxylin-eosin (HE) or safranin O protocol, and observed by optical microscope (Nikon TS-100, Tokyo, Japan).21 The results are shown in Figure 2. Pannus as thickening synovial tissue that covers articular cartilage were observed in the finger tissues of the untreated control group using HE staining (Fig. 2A). In contrast, no abnormal findings were observed in the finger tissues of the group treated with HL-23 as the same as the normal group (Fig. 2A). Cartilage proteoglycan positive cells (red color) of the fingers tissue in the group treated with HL-23 (Fig. 2B) increased compared with that of the control group (Fig. 2B), although severe cartilage loss characterized by cartilage degradation were observed in the finger tissues in the untreated control group (Fig. 2B). These results indicate that HL-23 are remarkably effective for inhibiting the pannus formation and destruction of cartilage and bone in CIA mouse models of RA. Furthermore, we carried out immunostaining using inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor
Figure 2. Therapeutic effects of HL-23 for CIA mouse models with rheumatoid arthritis on the basis of HE and safranin O staining of tissue sections in finger. (A) Histochemical stain using the HE protocol. (B) Histochemical stain using the safranin O protocol. Dose for DMPC; 136 mg/kg. Scale bar: 50 lm.
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H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689
Figure 3. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of IL-1b. Dose for DMPC; 136 mg/kg. Arrows indicate IL-1b positive cells. Scale bar: 50 lm.
Figure 4. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of TNF-a. Dose for DMPC; 136 mg/kg. Arrows indicate TNF-a positive cells. Scale bar: 50 lm.
Figure 5. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of IL-6. Dose for DMPC; 136 mg/kg. Arrows indicate IL-6 positive cells. Scale bar: 50 lm.
(TNF)-a, and IL-6 to establish the therapeutic effects of HL-23.22 The immunostained sections were counterstained with hematoxylin, rinsed in distilled water and mounted. The stained finger sections were observed by optical microscope. As shown in Figure 3, many IL-1b positive cells (brown color) were observed in the finger tissues near the cartilage and bone of the nontreated control group. On the other hand, few IL-1b positive cells were observed in the normal and the group treated with HL23. Numerous TNF-a positive cells (brown color) were observed in the finger tissues near the cartilage and bone in the control group as shown in Figure 4, although TNF-a negative cells of the finger tissue in the group treated with HL-23 were almost the same as that of the normal group. In addition, many IL-6 positive cells were observed in the inflammation subcutaneous tissue of fingers of the control group. On the other hand, no IL-6 positive cells were observed in the normal and the group treated with HL-23 as shown in Figure 5. These results indicate that HL have remarkable downregulation effects for inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models. In conclusion, we clearly demonstrated that HL-23 showed therapeutic effects for the CIA mouse models of RA in vivo along with downregulation of inflammatory cytokines. The noteworthy aspects are as follows. (a) Remarkably high therapeutic effects of HL-23 for CIA mouse models were obtained on the basis of clinical
assessment of arthritis. (b) The reduction of hyperplastic synovial membrane (pannus tissue) and destruction of the cartilage and bone by HL-23 was revealed on the basis of hematoxylin and eosin (HE) and safranin O staining. (c) The downregulation of inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models treated with HL-23 were obtained. It is noteworthy that remarkable high inhibitory effects of HL-23 without drug along with downregulation of inflammatory cytokines for the CIA mouse models of RA were obtained for the first time. Acknowledgments We thank Ryo Sakamoto and Arisa Kikumoto for technical assistance. This work was supported in part by a Grant-in-Aid for Science Research from the Ministry of Education, Science, and Culture of Japan (Nos. 25289299, 25420843 and 15k12527).
References and notes 1. Feldmann, M.; Brennan, F. M.; Maini, R. N. Cell 1996, 85, 307. 2. Scott, D. L.; Wolfe, F.; Huizinga, T. W. Lancet 2010, 376, 1094. 3. Caporali, R.; Caprioli, M.; Bobbio-Pallavicini, F.; Montecucco, C. Autoimmun. Rev. 2008, 8, 139. 4. Katchamart, W.; Trudeau, J.; Phumethum, V.; Bombardier, C. Ann. Rheum. Dis. 2009, 68, 1105.
H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689 5. Hopkins, A. M.; O’Doherty, C. E.; Foster, D. J.; Suppiah, V.; Upton, R. N.; Spargo, L. D.; Cleland, L. G.; Proudman, S. M.; Wiese, M. D. J. Clin. Pharm. Ther. 2014, 39, 555. 6. Janssen, N. M.; Genta, M. S. Arch. Intern. Med. 2000, 160, 610. 7. van Roon, E. N.; Jansen, T. L.; Houtman, N. M.; Spoelstra, P.; Brouwers, J. R. Drug Safety 2004, 27, 345. 8. Tebib, J.; Mariette, X.; Bourgeois, P.; Flipo, R. M.; Gaudin, P.; Le Loët, X.; Gineste, P.; Guy, L.; Mansfield, C. D.; Moussy, A.; Dubreuil, P.; Hermine, O.; Sibilia, J. Arthritis Res. Ther. 2009, 11, R95. 9. Yokota, K.; Akiyama, Y.; Asanuma, Y.; Miyoshi, F.; Sato, K.; Mimura, T. Rheumatol. Int. 2009, 29, 459. 10. Ueoka, R.; Matsumoto, Y.; Moss, R. A.; Swarup, S.; Straus, G.; Murakami, Y. J. Am. Chem. Soc. 1985, 107, 2185. 11. Ueoka, R.; Matsumoto, Y.; Moss, R. A.; Swarup, S.; Sugii, A.; Harada, K.; Kikuchi, J.; Murakami, Y. J. Am. Chem. Soc. 1988, 110, 1588. 12. Kitamura, I.; Kochi, M.; Matsumoto, Y.; Ueoka, R.; Kuratsu, J.; Ushio, Y. Cancer Res. 1996, 56, 3986. 13. Matsumoto, Y.; Iwamoto, Y.; Matsushita, T.; Ueoka, R. Int. J. Cancer 2005, 115, 377. 14. Iwamoto, Y.; Matsumoto, Y.; Ueoka, R. Int. J. Pharm. 2005, 292, 231. 15. Shimoda, S.; Ichihara, H.; Matsumoto, Y.; Ueoka, R. Int. J. Pharm. 2009, 372, 162. 16. Ichihara, H.; Hino, M.; Umebayashi, M.; Matsumoto, Y.; Ueoka, R. Eur. J. Med. Chem. 2012, 57, 143. 17. Ichihara, H.; Nagami, H.; Kiyokawa, T.; Matsumoto, Y.; Ueoka, R. Anticancer Res. 2008, 28, 1187. 18. Ichihara, H.; Hino, M.; Makizono, T.; Umebayashi, M.; Matsumoto, Y.; Ueoka, R. Bioorg. Med. Chem. Lett. 2011, 21, 207. 19. David, D. B.; Kary, A. L.; Edward, F. R. Nature Protocols 2007, 2, 1269. 20. Mice were reared under room temperature 25 ± 1 °C, the condition of 50 ± 10% of humidity. Female DBA/1J mice (DBA/1J JmsSLc; 5 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). After a quarantine and acclimatization period, the mice were randomly grouped on the basis of body weight using the stratified randomization method. The number of mice was five in each group. DBA/1 mice were injected subcutaneously at the base of the tail with 100 lg of bovine type II collagen (CII; Chondrex, Inc., WA, USA) emulsified in Freund’s complete adjuvant (Santa Cruz Biotechnology, Inc. CA, USA) as previously described.23,24 On day 21 post-injection, a booster injection of 100 lg CII in
21.
22.
23.
24. 25.
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phosphate buffered saline (PBS) was subcutaneously injected. HL-23 (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) were intraperitoneally administered in mice once each day for 14 days, and then HL-23 in 5% glucose solution or 5% glucose solution alone (control) was administered once every two days for 5 weeks. Joint swelling was monitored by inspection and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of wrist or ankle; and 1.0, severe swelling of wrist or ankle. Scores for all fingers, toes, wrists, and ankles were totaled for each mouse.24,25 HL-23 (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) were intraperitoneally administered in mice once each day for 14 days after booster injection, and then HL-23 in 5% glucose solution or 5% glucose solution alone (control) was administered once every two days for 5 weeks. The fingers were removed from anaesthetized mice immediately after the treatment with HL-23, and fixed in 10% formalin solution. The fingers were embedded in paraffin and sectioned at 5 lm of thickness. The finger sections were stained with hematoxylin-eosin (HE) or safranin O protocol, and observed by optical microscope (Nikon TS-100, Tokyo, Japan). Paraffin-embedded sections were cut, dewaxed in xylene and rehydrated through a series of ethanol to water. Finger sections were heated at 120 °C for 10 min for antigen activation and were blocked with a solution PBS and 1% H2O2 for 5 min. The sections were washed with PBS() and incubated with anti human/rat/mouse IL-1b antibody (NOVUS, CO, USA), TNF-a antibody (R&D Systems, MN, USA) or IL-6 antibody (R&D Systems, MN, USA) in a humidified box at 4 °C for over night. The sections were washed twice with PBS, immunostained with rabbit anti-goat immunoglobulins polyclonal antibody (HRP and Biotinylated, DAKO, Corp., Glostrup, Denmark) for over night at 4 °C. Finally, the detection of the antigen-antibody link was made through immunoperoxydase followed by 3,30 -DAB chromogen. The sections were counterstained with hematoxylin, rinsed in distilled water and mounted. Yoshitomi, H.; Sakaguchi, N.; Kobayashi, K.; Brown, G. D.; Tagami, T.; Sakihama, T.; Hirota, K.; Tanaka, S.; Nomura, T.; Miki, I.; Gordon, S.; Akira, S.; Nakamura, T.; Sakaguchi, S. J. Exp. Med. 2005, 201, 949. Juarranz, Y.; Abad, C.; Martinez, C.; Arranz, A.; Gutierrez-Canas, I.; Rosignoli, F.; Gomariz, R. P.; Leceta, J. Arthritis Res. Ther. 2005, 7, 1034. Zhenlin, H.; Qing, J.; Jieping, D.; Fang, L.; Runhui, L.; Lei, S.; Huawu, Z.; Junping, Z.; Weidong, Z. Arthritis Rheum. 2011, 63, 949.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Therapeutic effects of hybrid liposomes with downregulation of inflammatory cytokine for model mice of rheumatoid arthritis in vivo Hideaki Ichihara, Shuichi Yamasaki, Motoki Hino, Ryuichi Ueoka, Yoko Matsumoto ⇑ Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1, Ikeda, Nishi-ku, Kumamoto 860-0082, Japan
a r t i c l e
i n f o
Article history: Received 19 February 2015 Revised 14 April 2015 Accepted 22 April 2015 Available online 1 May 2015 Keywords: Hybrid liposomes Rheumatoid arthritis Collagen-induced arthritis mouse models Chemotherapy In vivo
a b s t r a c t Therapeutic effects of HL for a collagen-induced arthritis (CIA) mouse models of HL-23 composed of 95 mol % L-a-dimyristoylphosphatidylcholine (DMPC) and 5 mol % polyoxyethylenedodecylether (C12(EO)23) in vivo were examined. Remarkably high therapeutic effects of HL-23 for CIA mouse models were obtained on the basis of clinical assessment of arthritis. The reduction of hyperplastic synovial membrane (pannus tissue) and destruction of the cartilage and bone by HL-23 was revealed on the basis of hematoxylin and eosin (HE) and safranin O staining. Furthermore, the downregulation of inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models treated with HL-23 were investigated. Remarkably high therapeutic effects without joint swelling were obtained in CIA mouse models treated with HL-23. Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints, affecting up to 1% of the population worldwide. It is characterized by an inflammation of joints, hyperplastic synovium with pannus formation, leading to bone and cartilage destruction in the joints. The affected synovial tissues contain activated macrophages, fibroblasts, T lymphocytes, and B lymphocytes, which release proinflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6.1,2 Current clinical chemotherapy of RA using disease-modifying antirheumatic drugs (DMARDs) and anti-TNF-a antibody reduce inflammation by inhibiting or halting the immune process.1–5 However, potentially negative side effects of anti-RA drug such as digestive organs dysfunction, liver dysfunction, kidney dysfunction, stomatitis, depilation, respiratory related symptoms including pneumonitis, and myelosuppression have been reported.1,2,5–7 Furthermore, tolerance to anti-RA drug in patients have been observed.1,2,8,9 Therefore, anti-RA drug that would be effective for suppressing the inflammation of joints in RA without side effects is highly desirable to improve the quality of life for RA patients. We have produced hybrid liposomes (HL) which can be prepared by just the sonication of vesicular and micellar molecules in a buffer solution.10,11 HL contain no organic solvent unlike
⇑ Corresponding author. Tel.: +81 96 326 3965; fax: +81 96 323 1331. E-mail address: [email protected] (Y. Matsumoto).
conventional liposomes and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature, and hydrophobicity can be controlled by changing the constituents and compositional ratios of the HL. The therapeutic effects of HL composed of L-a-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(20) sorbitan monolaurate (Tween 20) including antitumor drugs such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been observed on the growth of glioma cells in vivo.12 On the other hand, high inhibitory effects of HL composed of DMPC and polyoxyethylene(n) dodecyl ethers (C12(EO)n) without any drugs on the growth of tumor cells in vitro13,14 and in vivo15,16 along with the induction of apoptosis have been obtained. After receiving the approval of the Bioethics Committee, successful clinical chemotherapy with drug-free HL to patients with lymphoma has been reported.17 More recently, we have reported that HL inhibits the growth of human RA fibroblast-like synoviocytes (HFLS-RA) cells along with apoptosis in vitro.18 However, the research for the effects of HL using SKG mice as models of RA and regulation of inflammatory cytokines is very limited, because it takes more than three months until symptoms are observed. To overcome this disadvantage, in this study, we examined the therapeutic effects of HL-23 composed of 95 mol % L-a-dimyristoylphosphatidylcholin (DMPC) and 5 mol % polyoxyethylenedodecylether (C12(EO)23) for a collageninduced arthritis (CIA) mouse models19 of RA in vivo. HL-23 were prepared by using sonication (VELVO VS-N300, 300W, Velvo-clear, Tokyo, Japan) of a mixture containing
http://dx.doi.org/10.1016/j.bmcl.2015.04.083 0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689
Figure 1. Therapeutic effects of HL-23 for CIA mouse models with rheumatoid arthritis using clinical score method. (A) Rheumatic clinical score changes for 7 weeks after the booster injection. Data presented are mean ± S.D. Dose for DMPC; 136 mg/kg. (B) Photographs of finger at 7 weeks after booster injection.
90 mol % L-a-dimyristoylphosphatidylcholine (DMPC, NOF Co., Tokyo, Japan, purity>99%) and 10 mol % polyoxyethylenedodecyl ether (C12(EO)23, Sigma–Aldrich Japan, Tokyo, Japan) in 5% glucose solution at 45 °C with 300 W, followed by filtration with a 0.20 lm filter. Physical properties of HL-23 composed of 95 mol % DMPC, 5 mol % C12(EO)23 were examined. Apparent mean hydrodynamic diameters (dhy) of HL were measured using light scattering spectrometer (ELS-8000, Otsuka Electronics Co. Ltd, Japan) with He-Ne laser light source (633 nm). The diameter was calculated by Stokes–Einstein equation (dhy = (jT)/(3pgD)), where j is
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Boltzmann constant, T is absolute temperature, g is viscosity and D is diffusion coefficient. Hydrodynamic diameter (dhy) of HL-23 was under 100 nm, which were preserved for a period remaining stable for more than one month. On the other hand, DMPC liposomes were unstable and precipitated after 14 days. We examined the therapeutic effects of HL-23 using CIA (collagen-induced arthritis) mouse models of RA, which are the most commonly studied autoimmune models of rheumatoid arthritis.19 The mice were handled in accordance with the guidelines for animal experimentation set out in Japanese law. The animal studies were approved by the Committee on Animal Research of Sojo University. Joint swelling was monitored by inspection and scored.20 The results are shown in Figure 1. No detection of rheumatic clinical score indicating joint swelling and deformity of fingers was obtained in the CIA mouse models treated with HL-23, although increased rheumatic score were observed in the control group from 4 weeks after booster injection (Fig. 1A). No joint swelling was obtained in the mice treated with HL-23 on the basis of macroscopic inspection, although joint swelling and deformity of fingers in all feet were observed in the control group (Fig. 1B). It is noteworthy that remarkable therapeutic effects were obtained in the CIA mouse models of RA intraperitoneally treated with HL-23 without drugs. Then, we histologically evaluated the therapeutic effects of HL23 using the finger tissues for the CIA mouse models in vivo. The finger sections were stained with hematoxylin-eosin (HE) or safranin O protocol, and observed by optical microscope (Nikon TS-100, Tokyo, Japan).21 The results are shown in Figure 2. Pannus as thickening synovial tissue that covers articular cartilage were observed in the finger tissues of the untreated control group using HE staining (Fig. 2A). In contrast, no abnormal findings were observed in the finger tissues of the group treated with HL-23 as the same as the normal group (Fig. 2A). Cartilage proteoglycan positive cells (red color) of the fingers tissue in the group treated with HL-23 (Fig. 2B) increased compared with that of the control group (Fig. 2B), although severe cartilage loss characterized by cartilage degradation were observed in the finger tissues in the untreated control group (Fig. 2B). These results indicate that HL-23 are remarkably effective for inhibiting the pannus formation and destruction of cartilage and bone in CIA mouse models of RA. Furthermore, we carried out immunostaining using inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor
Figure 2. Therapeutic effects of HL-23 for CIA mouse models with rheumatoid arthritis on the basis of HE and safranin O staining of tissue sections in finger. (A) Histochemical stain using the HE protocol. (B) Histochemical stain using the safranin O protocol. Dose for DMPC; 136 mg/kg. Scale bar: 50 lm.
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H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689
Figure 3. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of IL-1b. Dose for DMPC; 136 mg/kg. Arrows indicate IL-1b positive cells. Scale bar: 50 lm.
Figure 4. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of TNF-a. Dose for DMPC; 136 mg/kg. Arrows indicate TNF-a positive cells. Scale bar: 50 lm.
Figure 5. Therapeutic effects of HL for CIA mouse models with rheumatoid arthritis in tissue sections in finger using immunostaining of IL-6. Dose for DMPC; 136 mg/kg. Arrows indicate IL-6 positive cells. Scale bar: 50 lm.
(TNF)-a, and IL-6 to establish the therapeutic effects of HL-23.22 The immunostained sections were counterstained with hematoxylin, rinsed in distilled water and mounted. The stained finger sections were observed by optical microscope. As shown in Figure 3, many IL-1b positive cells (brown color) were observed in the finger tissues near the cartilage and bone of the nontreated control group. On the other hand, few IL-1b positive cells were observed in the normal and the group treated with HL23. Numerous TNF-a positive cells (brown color) were observed in the finger tissues near the cartilage and bone in the control group as shown in Figure 4, although TNF-a negative cells of the finger tissue in the group treated with HL-23 were almost the same as that of the normal group. In addition, many IL-6 positive cells were observed in the inflammation subcutaneous tissue of fingers of the control group. On the other hand, no IL-6 positive cells were observed in the normal and the group treated with HL-23 as shown in Figure 5. These results indicate that HL have remarkable downregulation effects for inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models. In conclusion, we clearly demonstrated that HL-23 showed therapeutic effects for the CIA mouse models of RA in vivo along with downregulation of inflammatory cytokines. The noteworthy aspects are as follows. (a) Remarkably high therapeutic effects of HL-23 for CIA mouse models were obtained on the basis of clinical
assessment of arthritis. (b) The reduction of hyperplastic synovial membrane (pannus tissue) and destruction of the cartilage and bone by HL-23 was revealed on the basis of hematoxylin and eosin (HE) and safranin O staining. (c) The downregulation of inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 for CIA mouse models treated with HL-23 were obtained. It is noteworthy that remarkable high inhibitory effects of HL-23 without drug along with downregulation of inflammatory cytokines for the CIA mouse models of RA were obtained for the first time. Acknowledgments We thank Ryo Sakamoto and Arisa Kikumoto for technical assistance. This work was supported in part by a Grant-in-Aid for Science Research from the Ministry of Education, Science, and Culture of Japan (Nos. 25289299, 25420843 and 15k12527).
References and notes 1. Feldmann, M.; Brennan, F. M.; Maini, R. N. Cell 1996, 85, 307. 2. Scott, D. L.; Wolfe, F.; Huizinga, T. W. Lancet 2010, 376, 1094. 3. Caporali, R.; Caprioli, M.; Bobbio-Pallavicini, F.; Montecucco, C. Autoimmun. Rev. 2008, 8, 139. 4. Katchamart, W.; Trudeau, J.; Phumethum, V.; Bombardier, C. Ann. Rheum. Dis. 2009, 68, 1105.
H. Ichihara et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2686–2689 5. Hopkins, A. M.; O’Doherty, C. E.; Foster, D. J.; Suppiah, V.; Upton, R. N.; Spargo, L. D.; Cleland, L. G.; Proudman, S. M.; Wiese, M. D. J. Clin. Pharm. Ther. 2014, 39, 555. 6. Janssen, N. M.; Genta, M. S. Arch. Intern. Med. 2000, 160, 610. 7. van Roon, E. N.; Jansen, T. L.; Houtman, N. M.; Spoelstra, P.; Brouwers, J. R. Drug Safety 2004, 27, 345. 8. Tebib, J.; Mariette, X.; Bourgeois, P.; Flipo, R. M.; Gaudin, P.; Le Loët, X.; Gineste, P.; Guy, L.; Mansfield, C. D.; Moussy, A.; Dubreuil, P.; Hermine, O.; Sibilia, J. Arthritis Res. Ther. 2009, 11, R95. 9. Yokota, K.; Akiyama, Y.; Asanuma, Y.; Miyoshi, F.; Sato, K.; Mimura, T. Rheumatol. Int. 2009, 29, 459. 10. Ueoka, R.; Matsumoto, Y.; Moss, R. A.; Swarup, S.; Straus, G.; Murakami, Y. J. Am. Chem. Soc. 1985, 107, 2185. 11. Ueoka, R.; Matsumoto, Y.; Moss, R. A.; Swarup, S.; Sugii, A.; Harada, K.; Kikuchi, J.; Murakami, Y. J. Am. Chem. Soc. 1988, 110, 1588. 12. Kitamura, I.; Kochi, M.; Matsumoto, Y.; Ueoka, R.; Kuratsu, J.; Ushio, Y. Cancer Res. 1996, 56, 3986. 13. Matsumoto, Y.; Iwamoto, Y.; Matsushita, T.; Ueoka, R. Int. J. Cancer 2005, 115, 377. 14. Iwamoto, Y.; Matsumoto, Y.; Ueoka, R. Int. J. Pharm. 2005, 292, 231. 15. Shimoda, S.; Ichihara, H.; Matsumoto, Y.; Ueoka, R. Int. J. Pharm. 2009, 372, 162. 16. Ichihara, H.; Hino, M.; Umebayashi, M.; Matsumoto, Y.; Ueoka, R. Eur. J. Med. Chem. 2012, 57, 143. 17. Ichihara, H.; Nagami, H.; Kiyokawa, T.; Matsumoto, Y.; Ueoka, R. Anticancer Res. 2008, 28, 1187. 18. Ichihara, H.; Hino, M.; Makizono, T.; Umebayashi, M.; Matsumoto, Y.; Ueoka, R. Bioorg. Med. Chem. Lett. 2011, 21, 207. 19. David, D. B.; Kary, A. L.; Edward, F. R. Nature Protocols 2007, 2, 1269. 20. Mice were reared under room temperature 25 ± 1 °C, the condition of 50 ± 10% of humidity. Female DBA/1J mice (DBA/1J JmsSLc; 5 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). After a quarantine and acclimatization period, the mice were randomly grouped on the basis of body weight using the stratified randomization method. The number of mice was five in each group. DBA/1 mice were injected subcutaneously at the base of the tail with 100 lg of bovine type II collagen (CII; Chondrex, Inc., WA, USA) emulsified in Freund’s complete adjuvant (Santa Cruz Biotechnology, Inc. CA, USA) as previously described.23,24 On day 21 post-injection, a booster injection of 100 lg CII in
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phosphate buffered saline (PBS) was subcutaneously injected. HL-23 (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) were intraperitoneally administered in mice once each day for 14 days, and then HL-23 in 5% glucose solution or 5% glucose solution alone (control) was administered once every two days for 5 weeks. Joint swelling was monitored by inspection and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of wrist or ankle; and 1.0, severe swelling of wrist or ankle. Scores for all fingers, toes, wrists, and ankles were totaled for each mouse.24,25 HL-23 (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) were intraperitoneally administered in mice once each day for 14 days after booster injection, and then HL-23 in 5% glucose solution or 5% glucose solution alone (control) was administered once every two days for 5 weeks. The fingers were removed from anaesthetized mice immediately after the treatment with HL-23, and fixed in 10% formalin solution. The fingers were embedded in paraffin and sectioned at 5 lm of thickness. The finger sections were stained with hematoxylin-eosin (HE) or safranin O protocol, and observed by optical microscope (Nikon TS-100, Tokyo, Japan). Paraffin-embedded sections were cut, dewaxed in xylene and rehydrated through a series of ethanol to water. Finger sections were heated at 120 °C for 10 min for antigen activation and were blocked with a solution PBS and 1% H2O2 for 5 min. The sections were washed with PBS() and incubated with anti human/rat/mouse IL-1b antibody (NOVUS, CO, USA), TNF-a antibody (R&D Systems, MN, USA) or IL-6 antibody (R&D Systems, MN, USA) in a humidified box at 4 °C for over night. The sections were washed twice with PBS, immunostained with rabbit anti-goat immunoglobulins polyclonal antibody (HRP and Biotinylated, DAKO, Corp., Glostrup, Denmark) for over night at 4 °C. Finally, the detection of the antigen-antibody link was made through immunoperoxydase followed by 3,30 -DAB chromogen. The sections were counterstained with hematoxylin, rinsed in distilled water and mounted. Yoshitomi, H.; Sakaguchi, N.; Kobayashi, K.; Brown, G. D.; Tagami, T.; Sakihama, T.; Hirota, K.; Tanaka, S.; Nomura, T.; Miki, I.; Gordon, S.; Akira, S.; Nakamura, T.; Sakaguchi, S. J. Exp. Med. 2005, 201, 949. Juarranz, Y.; Abad, C.; Martinez, C.; Arranz, A.; Gutierrez-Canas, I.; Rosignoli, F.; Gomariz, R. P.; Leceta, J. Arthritis Res. Ther. 2005, 7, 1034. Zhenlin, H.; Qing, J.; Jieping, D.; Fang, L.; Runhui, L.; Lei, S.; Huawu, Z.; Junping, Z.; Weidong, Z. Arthritis Rheum. 2011, 63, 949.
