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Objective:To determine the effects of pentoxifylline-tocopherol-clodronate combination (PENTOCLO) protocol in the treatment of localized temporal bone osteoradionecrosis (TBORN).Methods:A retrospective analysis was conducted on the clinical data of 21 patients, who suffered localized TBORN (23 ears) and were treated with the PENTOCLO protocol in Sun Yat-sen Memorial Hospital, Sun Yat-sen University from November 2020 to April 2021. The curative effects of the PENTOCLO protocol were evaluated based on the changes in ear symptoms and the extent of exposed bone before and after treatment.Results:The PENTOCLO protocol was applied for (506 ± 48) d on average. As a result, 19 ears (82.6%) became free of earache and purulent ear discharge, and two ears (8.7%) showed alleviation of symptoms. Moreover, nine ears (39.1%) exhibited re-epithelialization in the ear canal, 11 ears (47.6%) showed a decrease in exposed ear canal bone, and the osteonecrosis of three ears (13.0%) was stable.Conclusions:PENTOCLO has encouraging treatment effects on TBORN, and thus can be used as an effective nonsurgical option for localized TBORN.
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Objective To investigate the protective effect of clodronate SPIO liposomes on liver injury of rats with severe acute pancreatitis(SAP)and the role of MRI in evaluating the extent of liver injury.Methods Superparamagnetic Fe3O4 nanoparticles were prepared by chemical coprecipitation.Clodronate-SPIO-containing liposomes was prepared by the thin-film method.SAP models were prepared by a uniform injection of sodium taurocholate(2 ml/kg body weight)into the subcapsular space of the pancreas.SD rats were randomly divided into control group,SAP plus SPIO group, and clodronate-SPIO-containing liposome group.Six hours after SAP models were available,T2-weighted MRI scanning(in the same plane)of the liver of rats in each group were performed.At the end of the scanning,blood samples were taken from the supcrior mesenteric vein to measure the contents of serum ALT and AST.Meanwhile, The pathological changes in the liver and pancreas were observed.Results Transmission electron microscopic examination showed that liposomes had a uniform size.No changes in the pancreas of rats in control group were noted.The pathological changes in the pancreas and liver of rats in SAP plus clodronate-SPIO-containing liposome group were significantly milder than those in SAP plus SPIO liposome group.The contents of serum ALT and AST in rats in SAP plus SPIO liposome group were significantly higher than those in control group(P＜0.01), while the contents of serum ALT and AST in rats in SAP plus clodronate-SPIO-containing group were significantly lower than those in SAP plus SPIO liposome group(P＜0.01).The MRI signal intensity of the liver in SAP plus SPIO liposome group and SAP plus clodronate-SPIO-containing liposome group was significantly lower than that in control group.The significant changes in the MRI signal intensity of the liver in SAP plus SPIO liposome group and SAP plus Clodronate-SPIO liposome group were noted(P＜0.01).Conclusion Clodronate-containing liposomes have protective effects against liver injury in SAP rats and SPIO can be used as a tracer for MRI examination.
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Objective To explore the preparation of liposomal clodronate and investigate its inducing effects on the apoptosis of peritoneal macrophages in rats after severe acute pancreatitis (SAP).Methods Liposomal clodronate was prepared by means of thin film. SAP rat model was established by retrograde injection of 5% sodium taurocholate into the pancreatic duct. The peritoneal macrophages were obtained from SAP rats. After exposure to different doses of liposomal clodronate (50, 100,150 μl), the PM proliferation was determined by MTT colourimetry. The apoptosis of PM was measured by flow cytometry and agarose gel electrophoresis, respectively. Results The prepared liposomal clodronate had a suitable encapsulation efficiency of clodronate (5.8%) with an average size of 200 nm. The spherical shape of liposome was confirmed by transmission electron microscope. Exposed to liposomal clodronate of different doses resulted in a obvious growth depression (P＜0.01). The apoptotic rate of the PM was (10.32±0.34) %, (18.16±0.49)% and (29.87±0.35)% in three different dose groups and the difference was marked (P＜0.01). 1.2% of agarose gel electrophoresis of DNA extracted from apoptotic macrophages induced by liposomal clodronate showed clearer and characteristic ladder following the liposomal clodronate concentration. Conclusion Liposomal clodronate has a definite effect on peritoneal macrophages in SAP rats.
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Objective: To observe the therapeutic effect of 153Sm-clodronate on nude mice orthotopically transplanted with human osteosarcoma. Methods: Severe combined immune deficiency (SCID) mice were subcutaneously inoculated with human osteosarcoma cell line HOS-8603. The formed tumors were then transplanted into BALB/c nude mice at the tibia for passage; the tumors were harvested for establishing orthotopical mice model of human osteosarcoma. Mice treated with 153Sm-EDTMP and clodronate group served as controls. The distribution of 153Sm-clodronate was determined 24 h later. The appearance, tumor weights, X-ray examination, histological observation, and survival of mice were compared between the 3 groups. Results: It showed that the distribution of 153Sm-clodronate in the lesion bone was much higher than that in other organs, with the radioactive ratio of lesion bone to blood being 4 627.1 : 1, lesion bone to normal bone being 10.8 : 1, and normal bone to liver being 8. 4 : 1. 153Sm clodronate treated mice had a slighter osteolytic reaction and a longer survival period, but the tumor mass was not significantly different compared with those of the control group. Conclusion: 153Sm-clodronate gathers at the lesion bone and it has good therapeutic effect on the mice model of human oscosarcoma, making it a promising agent for treatment of bone tumor.
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Objective To study the effects of elodronate-liposome on inducing apoptosis of alve-olar macrophages from rats with acute neetotizing pancreatitis (ANP).Methods The AMs of eight rats with ANP were isolated, purified then incubated from broehoalveolar lavage by the differing rates of attachment of the various cell types in a forty-well cell culture plate.Then they were randomized in-to five groups including control group,blank liposome group( 50 μ1, 100 μ1),clodronate-liposome group (50μ1,100μ1).Values of OD were determined by MTT.AO fluorescence and haematoxylin dye were employed to determine the apoptosis of the AMs.Results There were no significant differences be-tween control group and blank liposome group(50 μ1, 100 μ1).Significant differences were found be-tween control group and clodronate-liposome group(50 μ1, 100 μ1).There were no marked differences between blank liposome group(50μ1, 100 μ1)and clodronate-liposome group(50 μ1,100μ1).AO fluo-rescence and haematoxylin dye were available to define the apoptosis of the AMs.Conclusion Clodr-onate-liposome can effectively induce the apoptosis of the AMs.
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Objective To investigate the protective effect of lipsomal clodronate against hepatic injury in rats with acute necrotizing pancreatitis (ANP). Methods 48 SD rats were randomly divided into control group, ANP group and lipsomal clodronate group, respectively. The models of ANP were established by injection of sodium taurocholate into the pancreatic capsule. Lipsomal clodronate was prepared by means of thin film. Blank liposomes and clodronate-containing liposomes was injected via caudal vein in ANP group and lipsomal clodronate group, respectively. The rats were sacrificed at 2, 6 h after ANP induction, the serum levels of ALT, AST and AMS, IL-6,IL-12 were measured, and pathologic changes of liver and pancreas were observed. Results At 6 h, serum level of ALT was (73 ± 11) U/L, (257 ± 33) U/L and (184 ± 29) U/L in control group, ANP group and lipsomal clodronate group, respectively;serum levels of AST were (190 ± 32)U/L, (590 ± 70)U/L and (430±52)U/L, respectively;serum levels of AMS were (814±80)U/L, (5031 ± 471) U/L and (2843 ± 236) U/L, respectively, serum levels of IL-6 were (26.7 ± 5.7) pmol/L, (218.0 ±4.7)pmol/L and (112.3 ± 8. O) pmol/L, respectively;serum levels of IL-12 were (4. 2 ± 1.0) pmol/L,(309.5 ± 8.5) pmol/L and (153.7 ± 6.3) pmol/L. The values in ANP group and lipsomal clodronate group were significantly higher than those in control group, while the values in lipsomal clodronate group were significantly lower than those in ANP group (P < 0. 01). Pathologic changes of liver and pancreas were significantly attenuated in lipsomal clodronate group. Conclusions Intravenous liposomal clodronate could exert protective effects on the hepatic injury in rats with ANP.
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A new ion chromatography was developed for the determination of clodronate disodium and its related substances. A suppressed conductivity detection was used. The analytical column was an IonPac AS11-HC anion-exchange column. The column temperature was maintained at 30 ℃. The potassium hydroxide solution was used as the eluent at a flow rate of 1.2 mL/min. For the determination of content and related substances,an isocratic elution program and a gradient elution program were performed, respectively.A good linear relationship was observed in the concentration range of clodronate disodium of 0.0568-0.1895 g/L(r=0.9999). The average recoveries of the injection and the capsules were 100.1%(RSD=0.7%) and 98.9%(RSD=0.6%),respectively. The limit of detection was 0.3 ng. The related substances gained a completely chromatography separation with the principal peak. The present method is accurate, sensitive and simple, which provides a new reliable analytical method for the quality control of clodronate disodium and its preparations.
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Objective To investigate the protective effects of lipsomal clodronate on renal injury in rats with severe acute pancreatifis and the assessment of renal injury. Method Totally 48 rats were randomly divided into three group:normal control group (C);SAP group, in which rats were treated with pure liposomal (P);treatment group, in which SAP rats were treated with liposomal clodronate disodium(T). The SAP model of rat was induced by injection of 5 % sodium taurochohte beneath the pancreatic membrane. Rats of normal control group received isovolumetric injections of 0.9% physiological saline solution instead of sodium taurocholate. Blood samples were collected to measure AMS,BUN,Cr,IL-6 and IL-12 at 2 hors, 6 hours after SAP. At the same time, the samples of pancreatic and renal tissues were taken for observing the pathological changes. Results Compared with controlgroup, serious renal and pancreatic damages were found in group P, and the AMS, BUN, Cr levels elevated signifi-candy (P < 0.01). Compared with group P,the renal and pancreatic damages were attenuated in group T, and the levels of Cr and AMS decreased significantly (P < 0.01), and the IL-6, IL-12 were decreased at 2 hours and 6 hours (P < 0.01). The BUN decreased significantly at6 hours (P < 0.05). Conciusions Excessive release of inflammatory mediator play an important role in renal injury in SAP. Lipsomal clodronate disodium can alleviate the damage of pancreas and kidney.
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Objective To investigate the apoptosis of Kupffer cell (KC) induced by lipsomal clodronate in rat with acute necrotizing pancreatitis (ANP). Methods Lipsomal clodronate was prepared by means of thin film, the model of ANP was established by injection of 5% sodium taurocholate of 4 ml/kg into the pancreatic capsule. The Kupffer cells were obtained from ANP rat. After exposure to different doses of lipsomal clodronate (0, 50, 100, 150 μl) , then the proliferation and apoptosis of KC was measured by MTT, flow cytometry and agarose gel electrophoresis of DNA. Results The prepared lipsomal clodronate had an average size of 100～200 nm, the spherical shape of liposome was uniform and confirmed by transmission electron microscope. When exposed to different concentration of lipsomal clodronate for 24 h, the growth suppression rate was 17. 4% , 24. 2% and 31. 1% , respectively, while the apoptosis rate of the KC was (14. 12 ±0.37)% , (18.74±0.43)% and (27.51 ±0.39)%, respectively; the difference was statistically significantly (P<0. 01) , the DNA of KC began degradation and gradually showed clear and characteristic ladder. Conclusions Lipsomal clodronate could induce apoptosis and suppress the growth of Kupffer cells in ANP rats.
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BACKGROUND AND OBJECTIVES: Clodronate liposomes deplete phagocytic cells, thereby suppressing inflammation after vascular injury. We compared the effect of clodronate liposomes on macrophage depletion and neointimal formation in apolipoprotein E-deficient mice [ApoE (-) mice]. MATERIALS AND METHODS: ApoE (-) mice were randomly assigned to the clodronate liposomes group (Clodronate Group, n=7) and the vehicle liposomes group (Control Group, n=7). Clodronate (0.1 mL/10 g) was injected via the tail vein starting 2 days (d-2) before left common carotid artery injury. RESULTS: The percentage of blood monocytes was subsequently decreased after clodronate injection (14.0+/-7.4% at baseline, 6.8+/-4.9% at 24 hours and 0.7+/-0.3% at 1 week after the clodronate liposome injection). The percentage of macrophages in the plaque area was significantly lower in the clodronate group at week 2 (32.0+/-6.5 vs. 68.7+/-7.6%, respectively, p<0.05) and at week 4 (37.3+/-8.5 vs. 62.6+/-9.4%, respectively, p<0.05). The interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha concentrations were significantly decreased in the clodronate group at week 4 (12.3+/-2.5 vs. 22.9+/-3.5 pg/mL, respectively, p<0.05 for IL-6 and 16.6+/-2.2 vs. 43.6+/-6.1 pg/mL, respectively, p<0.05 for TNF-alpha). The plaque volume was significantly greater in the control group at week 2 (0.345+/-0.063 vs. 0.153+/-0.053 mm2, respectively, p<0.05) and at week 4 (0.320+/-0.027 vs. 0.167+/-0.070 mm2, respectively, p<0.05). CONCLUSION: Intravenous administration of clodronate liposomes depleted monocytes and macrophages, and so this reduced the inflammatory markers and neointimal formation in ApoE (-) mice.
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Animals , Mice , Administration, Intravenous , Apolipoproteins , Apolipoproteins E , Carotid Arteries , Carotid Artery Injuries , Carotid Artery, Common , Clodronic Acid , Inflammation , Interleukin-6 , Interleukins , Liposomes , Macrophages , Monocytes , Phagocytes , Tumor Necrosis Factor-alpha , Vascular System Injuries , VeinsABSTRACT
Free clodronate has a very poor ability to permeate cell membrane and an extremely short half-life in circulation. However, it can be encapsulated with liposomes, and then can be phagocytosed by monocytes/macrophages. Clodronate is released in the cells and be metabolized to a toxic ATP analog. By this way, monocytes/macrophages can be effectively depleted. The study showed that the prepared liposomes had a negative charge (-40mV) on the surface and a high encapsulation efficiency of clodronate (17.6%～19.0%) with an average size of 200nm. The spherical shape of liposome was confirmed both by transmission electron microscope and laser scanning confocal microscope. Neither free clodronate nor liposome clodronate inhibited vascular endothelial cell and smooth muscle cell proliferation. Clodronate, once encapsulated in liposomes, significantly reduced macrophage proliferation in a dose dependent manner, while free clodronate or empty liposomes had no effect on macrophages. With laser scanning confocal microscope observation, rhodamine labeled liposomes were found to penetrate and accumulate inside monocytes and macrophages, but not into the smooth muscle cells. Furthermore, rhodamine labeled liposomes without encapsulating clodronate was found to accumulate inside macrophages, but causing no damage to cells. The macrophages which engulfed rhodamine labeled clodronate liposomes would manifest a morphological structure resembling apoptotic state. The results suggest that monocytes/macrophages can be depleted via phagocytosis of liposome encapsulated clodronate without affecting non-phagocytotic cells.