Ito M, Chokki M, Ogino Y, Satomi Y, Azuma Y, Ohta T, Kiyoki M
Pharmacological Research Department, TEIJIN Institute for Bio-Medical Research, Tokyo, Japan.
Calcif Tissue Int. 1998 Aug;63(2):143-7. doi: 10.1007/s002239900505.
We compared the cytotoxic effects of alendronate (ALN) and incadronate (YM175) on isolated rabbit osteoclasts in vitro and on rats in vivo. In the in vitro experiment, each bisphosphonate was added to the culture of isolated osteoclasts at the final concentration of 3 x 10(-5), 3 x 10(-4), or 3 x 10(-3) M, and the amount of creatine phosphokinase (CPK) released into the medium was taken as an index of cytotoxicity at 5, 10, and 24 hours after the treatment. Also viability of osteoclasts, measured in terms of trypan blue exclusion, was assessed at 24 hours after the treatment. In YM175-treated groups, CPK activity in the medium increased in a concentration-dependent manner with time, and phase-contrast microscopic observation revealed damaged cell membranes and nuclear deterioration in YM175-treated osteoclasts. As a result, the viability of the osteoclasts was decreased at the concentrations of 3 x 10(-4) and 3 x 10(-3) M. However, in the ALN-treated groups, neither CPK activity nor viability of isolated osteoclasts changed significantly compared with control levels even at 3 x 10(-3) M for up to 24 hours. In the in vivo experiment, each bisphosphonate was administered separately to normal rats (7 weeks old, Sprague-Dawley) by intravenous injection at 1, 5, or 25 nmol/kg. Two days after the injection, the animals were euthanized, and the plasma Ca concentration and total CPK activity were measured. In YM175-injected rats, the CPK activity increased at 25 mmol/kg, and a slight decrease in the plasma Ca level was seen at this dose. In contrast, in ALN-injected rats, CPK activity did not increase even at 5 or 25 mmol/kg, and the plasma Ca level did decrease significantly compared with controls. Isozyme analysis revealed that, not only was CPK activity increased in the BB type in YM175-injected rats, it was also increased in the MB and MM types. In conclusion, alendronate, unlike YM175, does not have any cytotoxic effects on osteoclasts either in vitro or in vivo.
我们比较了阿仑膦酸盐（ALN）和因卡膦酸盐（YM175）在体外对分离的兔破骨细胞以及在体内对大鼠的细胞毒性作用。在体外实验中，将每种双膦酸盐以3×10⁻⁵、3×10⁻⁴或3×10⁻³ M的终浓度添加到分离的破骨细胞培养物中，并将释放到培养基中的肌酸磷酸激酶（CPK）量作为处理后5、10和24小时细胞毒性的指标。此外，在处理后24小时，通过台盼蓝排斥法评估破骨细胞的活力。在YM175处理组中，培养基中的CPK活性随时间呈浓度依赖性增加，相差显微镜观察显示YM175处理的破骨细胞膜受损且细胞核退化。结果，在3×10⁻⁴和3×10⁻³ M浓度下破骨细胞的活力降低。然而，在ALN处理组中，即使在3×10⁻³ M浓度下处理长达24小时，分离的破骨细胞的CPK活性和活力与对照水平相比均无显著变化。在体内实验中，将每种双膦酸盐分别以1、5或25 nmol/kg的剂量通过静脉注射给予正常大鼠（7周龄，Sprague-Dawley）。注射两天后，对动物实施安乐死，并测量血浆钙浓度和总CPK活性。在注射YM175的大鼠中，CPK活性在25 mmol/kg时增加，且在此剂量下血浆钙水平略有下降。相比之下，在注射ALN的大鼠中，即使在5或25 mmol/kg时CPK活性也未增加，且血浆钙水平与对照组相比确实显著下降。同工酶分析显示，不仅注射YM175的大鼠中BB型CPK活性增加，MB型和MM型CPK活性也增加。总之，与YM175不同，阿仑膦酸盐在体外和体内对破骨细胞均无任何细胞毒性作用。
