Targeting Antitumor Conjugate Phenamine Acid Caryolysine-hexanedioic Acid Bridge Grafting-bisphosphonates: Targeting And Therapeutic Effect

Object: In this research we will connect the phenamine acid caryolysine which is a chemotherapeutic drug to bisphosphonates which is used as bone-seeking vehicle, and then combine this compounds to radioactive nuclide ¹⁵³Sm to get a new bone targeting chemotherapeutic and radioactive drug for bone cancer specific treatment. Then we will validate the effect of this new drug by in vitro and in vivo methods to provide some theoretical evidence for clinical application in the future.Method: 1. Preparation and identification of the coupled diphosphonate compounds.Firstly, combined the phenamine acid caryolysine with the diphosphonate to get a new kind of conjugate. And then ¹⁵³Sm was connected to the conjugate.The composition was purified and analyzed by using high performance liquid chromatograph. The elemental analysis, infrared spectra analysis and ¹H-nuclear magnetic resonance (¹H-NMR) analysis was got by using nuclear magnetic resonance analyzer, infrared spectrometer and mass chromatographic analysis respectively. Hydroxyapatite crystal adsorbing experiment. To test the bone-seeking of the conjugate initially. The conjugate was marked with nuclide ¹⁵³Sm and was injected into nude mice bearing bone cancer. Then the nuclide concentration of region of interest was tested by using radioisotope scanning. 2. Cytotoxicity test. osteoplast were observed with the effect of different concentration of phenamine acid caryolysine- bisphosphonates conjugate in vitro.Using MTT, Cell viability analysis,Acridine orange stain, Flow cytometry and Scanning electron microscope to observe MG-63 cell multiplication and apoptosis with the effect of different concentration of phenamine acid caryolysine- bisphosphonates conjugate in vitro. 3. Untitumor test in vivo.First, to establish BALB/C-nu/nu nude mice osteosarcoma orthotopic model. Second targeting test. different concentration of phenamine acid caryolysine- bisphosphonates conjugate were injected from tail vein.some time later.usingγ-immune events-per-unit-time meter and high performance liquid chromatogram to analyze concentration in each tissue, ten day later, the mice were executed, and the neoplasma was weighed. The restraining-neoplasma rate was calculated and the histomorphology of heart, lung, liver, and spleen of mice was observed to assess the conjugate's toxic or side effect. Design primer and Detected RT-PCR and western blot to analyze caspase-3 gene, C-myc gene expression different between two group.Result: 1. The optical spectrum data of phenamine acid caryolysine - bisphosphonates conjugate was consistent with the standard by analyzing its structure with nuclear magnetic resonance. 2. The ALP and type 1 collagen was stained positively. The different concentration of phenamine acid caryolysine - 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. After the effect of different concentration of phenamine acid caryolysine - 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 cell viability analysis show all vital cell were decreased de dieindiem. There was significant difference among them (P＜0.05)on twenty-two and forty-eight hour.All this imply the phenamine acid caryolysine- bisphosphonates conjugate can induce apoptosis with with the increasing of drug concentration and the prolongation of time. The acridine orange dye 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 40μg/ml phenamine acid caryolysine - bisphosphonates conjugate and 20μg/ml phenamine acid caryolysine, the apopotosis rate increased gradually over 24～96 hours. 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 phenamine acid caryolysine or phenarnine acid caryolysine - 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 significant 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 phenamine acid caryolysine 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 phenamine acid caryolysine. The new conjugate can be regarded as one of new anti-bone-tumor drugs and has potential value in clinical application.