Experiment Study Of Target Antitumor Conjugate Methotrexate-bisphosphonates

Objective This is a study of : 1. the method to synthetize Methotrexate-bisphosphonates conjugate which has characteristics of bone target tropism and cytotoxicity of chemotherapeutics. Nuclear magnetic resonance,etc. will be applied to ensure molecular structure alike active compound. 2. the conjugate's effect on osteoblastic vigor, flate plate clone and MTT reduction assay with different concentration.3. the conjugate's effect on osteogenic sarcoma to inhibit proliferation or induce apoptosis. 4. the conjugate's toxic or side effect on important organs. Method 1. The methotrexate- bisphosphonates conjugate was made from hydrogenization of methotrexate- bisphosphonates benzyl ester which had been made from coupled reaction of bisphosphonates benzyl ester and methotrexate. 2.A. A adult flank bone was cut to cultivate osteoblast in vitro. The 5× 104/ml cultivated osteoblast was seeded in culture flask. 24,48 and 96 hours after seeding, different densities of cultivated osteoblast were dyed by 0.4% trypanblau and calculated in terms of living and dead cells. 1000 living osteoblast were seeded in 6-orifice with the effect of different concentration of methotrexate- bisphosphonates conjugate. 24 hours later, the cell adherence rate were observed, and 10 days later, clone formation wererecorded. 2*104 osteoblast were seeded per bore in 96-orifice.Different concentration of methotrexate- bisphosphonates conjugate were added every 4 bores. Everyday the optical density(OD) values of osteoblast were recorded over 24-96 hours to assess cell multiplication.2.B. MG-63 cell strain preserved in liquid nitrogen(LIN) were made into 2><105 cell suspension which were seeded in 96-orifice with the effect of different concentration of methotrexate-diphosphate conjugate. The optical density(OD) values of osteogenic sarcoma cell were recorded everyday over 24~96 hours to assess cell multiplication.48 hours later, the nutrient solution was removed, and the osteogenic sarcoma cell was rediluted and dyed by acridine orange to assess the cell's apoptosis. After the effect of 1500ug/ml emthexate or 2000ug/ml methotrexate-diphosphate conjugate, MG-63 osteogenic sarcoma cell were cultured for 24-96 hours and detected by flow cytometer to calculate the cell's apoptosis rate. 2><105/ml MG-63 cell suspension were added in 6—orifice which was provided with cover glass. The different concentrations of methotrexate- bisphosphonates conjugate(20,200 and 2000 ug/ml) were added in 6—orifice when the MG-63 cells was properly cultured.24~96 hours later, the cover glass was removed, and the MG-63 cells were dyed by TUNEL. The induction time of apoptosis and dosage effect of methotrexate- bisphosphonates conjugate were detected. Eventually the apopotosis cell was observed with electron microscope.3. 10 days after osteogenic sarcoma's animal model was made from BALB/C-nu/nu athymic mouse, the mouse was randomly assigned into one of three groups: Experiment group was received 2000ug/ml methotrexate- bisphosphonates conjugate; Control group was received 1500jig/ml methotrexate; andNegative control group was received saline.4 weeks later, the mouse was executed, and the neoplasma was weighed. The restraining-neoplasma rate was calculated and the mouse'histomorphology of heart,lung,liver,spleen and kidney was observed to assess the conjugate's toxic or side effect. Result l.The optical spectrum data of methotrexate- bisphosphonates conjugate was consistent with the standard by analyzing its structure with nuclear magnetic resonance.2. A. The AKP in the first, second and third generation was 81.2%, 84.3% and 87.2%, respectively. The different concentration of methotrexate- bisphosphonates conjugate had no significant effect on osteoblast cell's adherence rate, vigor and clone formation. The OD values of all osteoblast cell groups were increased de dieindiem, and there was no significant difference among them. 2.B. After the effect of 1500ug/ml emthexate or 2000ug/ml or 3000ug/ml methotrexate-bisphosphonates conjugate and being cultured for 24-96 hours, the OD values of MG-63 osteogenic sarcoma cell were decresed. There was no significant difference among them (P>0.05) .The acridine orange dying tests implied that: with the increasing of drug concentration and the prolongation of time, the osteogenic sarcoma cell was decreased in volume. Meanwhile, hyperchromatic and flavovirens drum dyeing was seen in cell nucleus. Moreover, bubble-form ecptoma in cell membrane and apopototic body emerged. The detection of flow cytometry implied that: after the effect of 2000ug/ml methotrexate- bisphosphonates conjugate and 1500ug/ml methotrexate, the apopotosis rate increased gradually over 24~96 hours. The TUNEL dyeing implied that: with the increasing of drug concentration and the prolongation of time, the positive brown cells increased. However, nobrown granula in cellular nucleus was seen in control group. The electron microscope implied that: Integrity and regularity of osteogenic sarcoma cellular nuleus was seen in control group, with the increasing of drug concentration and the prolongation of time, the typical apoptosis cells emerged in experiment groups.3. After the effect of emthexate or methotrexate- bisphosphonates conjugate, the new growth of athymic mouse was significantly depressed. The restraining-neoplasm rate was significant lower than that in negative control group. The food ,drink and pneuma, ect. were significantly better with naked eyes than those in control group. The tumorous cellular density in experiment and control groups was significantly decreased than that in negative control group. Meanwhile, apomorphosis and necrosis emerged in treated groups. However, the tumor cell growth in non-treated group was active and rapid.Conclusion 1. The new conjugate's structure is consistent with the active compound. 2. The toxic effect of new conjugate on human osteoblast is light. Moreover, the new conjugate has strong induction of MG-63 osteogenic sarcoma cell apoptosis. 3. The new conjugate and emthexate can both significantly inhibit athymic mouse'osteosarcoma growth. However, the pathobiologic examination implys that the damage of the new conjugate to liver and kidney was slighter compared with that of emthexate. The new conjugate can be regarded as one of new anti-bone-tumor drugs and has potential value in clinical application.