EFFECT OF LOW-DOSE TACROLIMUS COADMINISTERED WITH RALOXIFENE ON THE SKELETAL SYSTEM IN MALE RATS

Abstract

Polish Pharmaceutical Society Tacrolimus, a calcineurin inhibitor, is used as an immunosuppressive drug to prevent organ transplant rejection. Numerous clinical and experimental reports indicate that administration of immunosuppressants leads to development of osteopenia (1-3). Experimental osteopenia induced by calcineurin inhibitors (cyclosporin and tacrolimus) has been reported to be associated with high bone turnover (3). They accelerate bone remodeling, and net bone loss is caused by bone resorption prevailing over bone formation. However, there are also reports demonstrating bone formation inducing effect of tacrolimus in experimental models. Tacrolimus induced bone formation in demineralized bone matrix implants (isogeneic and xenogeneic) and in implants containing bone marrow cells (allografts and isografts) The aim of the present study was to investigate whether low-dose tacrolimus (0.3 mg/kg po daily for 4 weeks) affects bone mineralization and bone Immunosuppresants are known to unfavorably affect the osseous system. However, in our previous study on bone histomorphometric parameters we observed that low-dose tacrolimus intensified bone formation. The aim of the present study was to investigate the effects of low-dose tacrolimus on bone mechanical properties and mineralization in male rats. The effects of concurrent administration of tacrolimus and raloxifene were also studied. Raloxifene is a selective estrogen receptor modulator, used in the prevention and treatment of postmenopausal osteoporosis. In male rats raloxifene induces moderate intensification of bone mineralization. The experiments were carried out on mature male Wistar rats. The animals were divided into four groups (n = 7): control rats, rats treated with tacrolimus (0.3 mg/kg po), rats treated with raloxifene hydrochloride (5 mg/kg po) and rats treated with tacrolimus and raloxifene hydrochloride concurrently at abovementioned doses. The drugs were administered daily for 4 weeks. Body mass, bone mass and bone mineral content in the tibia, femur and L-4 vertebra, as well as mechanical properties of the whole femur (extrinsic stiffness, ultimate and breaking load, deformation caused by the applied load) and the femoral neck (load at fracture) were examined. Administration of tacrolimus at a dose of 0.3 mg/kg po for 4 weeks did not affect bone mechanical properties and mineralization. Concurrent administration of tacrolimus and raloxifene resulted in changes similar to those caused by raloxifene alone (statistically significant increases in the bone mass/body mass ratio, bone mineral content/body mass ratio and bone mineral content/bone mass ratio in comparison with the control rats, and no effect on bone mechanical properties). Results of the present study do not support the hypothesis that tacrolimus may be useful as a drug stimulating bone formation in skeletal diseases. mechanical properties in male rats. The effects of concurrent administration of tacrolimus and raloxifene on the sketetal system of male rats were also studied. Raloxifene is a selective estrogen receptor modulator, used in the prevention and treatment of postmenopausal osteoporosis. Raloxifene has been also considered as a potential drug used in osteoporosis in men. Here we used raloxifene as a reference drug inducing moderate intensification of bone mineralization in male rats (8). PHARMACOLOGY EFFECT OF LOW-DOSE TACROLIMUS Keywords EXPERIMENTAL The experiments were carried out on mature male Wistar rats (body mass at the beginning of the experiment: 250ñ300 g), fed a standard diet ad libitum. The rats were obtained from the Center of Experimental Medicine, Medical University of Silesia. The procedure of the experiments on animals was approved by the Ethical Commission, Katowice. The animals were divided into four groups (n = 7): I ñ control rats; II ñ rats treated with tacrolimus (Prograf, Fujisawa, 0.3 mg/kg po); III ñ rats treated with raloxifene hydrochloride (Evista, Eli Lilly, 5 mg/kg po); IV ñ rats treated with tacrolimus (0.3 mg/kg po) and raloxifene hydrochloride (5 mg/kg po). Raloxifene was suspended in 0.25% water solution of carboxymethylcellulose, tacrolimus was suspended in distilled water. The drugs were administered by a stomach gavage (po) once daily for 4 weeks. The control rats received the vehicle in the same volume of 2 mL/kg po daily. The animals were weighed every second day. After 4 weeks of drug administration, the animals were sacrificed and the right and left tibial and femoral bones and L-4 vertebra were excised. In the isolated left bones, mass and macrometric parameters were determined (length, diameter of the diaphysis in the mid-length). Mass of the liver, testicles, kidneys and spleen was also determined. Bone mechanical properties were assessed using the set constructed at the Department of Pharmacology, Medical University of Silesia, in cooperation with Hottinger Baldwin Messtechnik GmbH. Mechanical properties of the whole femur and the femoral neck were examined, as previously described (9, 10). Mechanical properties of whole left femurs were studied using a bending test with three-point loading. The load was applied perpendicularly to the long axis of the femur in the mid-length of the bone supported on its epiphyses. The load increased at a rate of 100 N/min. The load was measured by the sensor with a strain gauge, the deformation was measured by an inductive sensor. The signals sent by the sensors were amplified and registered using the XY recorder. The load-deformation curves, obtained for each bone, representing the relationships between load applied to the bone and deformation in response to the load, were analyzed. The load-deformation curve can be divided into the elastic deformation region and the plastic deformation region. Within the elastic deformation region, the slope of load-deformation curve, representing the extrinsic stiffness of bone, was tested. Within the plastic deformation region, the ultimate load and the breaking load were determined. The ultimate load is the maximum load sustained by the bone. The breaking load is the load at which the bone actually breaks. Deformation caused by the applied loads was also measured. Mechanical properties of the femoral neck were studied using a compression test. The load was applied to the head of the femur along the long axis of the femur. The bone was prepared to the measurement by fixing the diaphysis, which was cut in 1.7 cm from the proximal end of the right femur, in a metacrylate plate. The load causing the fracture of the femoral neck was determined. In order to determine the content of mineral substances in bones, the L-4 vertebra, left tibia and femur were mineralized at the temperature of 640 O C for 48 h and weighed. Results are presented as the mean ± SEM. Statistical estimation was performed using ANOVA followed by post-hoc Duncanís test. When appriopriate (lack of homogeneity of variance), KruskalWallis ANOVA, followed by Mann-Whitney U test, was used to determine specific differences. RESULTS Administration of tacrolimus at a dose of 0.3 mg/kg po daily for 4 weeks did not affect body mass gain, the mass of liver and testicles Tacrolimus did not statistically significantly affect the length and diameter of the investigated long bones The results concerning the effects of administration of raloxifene at a dose of 5 mg/kg po on the Effect of low-dose tacrolimus coadministered with raloxifene... 20