Acute Toxicity And Drug Releas Study In Vivo Of Methotrexate Modified By The Peptide

To identify the change of acute toxicity, and initially evaluate the release of methotrexate from the conjugate in vivo in model animals based on the comparison between methotrexate modified by the receptor-binding peptide (LHRH-MTX) and the parent drug.The acute toxicity of LHRH-MTX in healthy Kunming mice was observed by intravenous injections. The human breast cancer MCF-7 cells(1×107/ml,0.2ml) were inoculated hypodermically in BALB/cA nude mice. The pathology and immunology methods were applied to confirm whether LHRH receptors were highly expressed in tumor sites in the tumor-bearing nude mice. HPLC method was used to analyse the release of MTX from LHRH-MTX in vivo in the tumor-bearing nude mice compared with MTX injection, which was used to evaluate the capacity of this conjugate to delivery the drug.The median lethal dose of LHRH-MTX in kunming mice was 278.4mg/kg by intravenous injections,and its 95% confidence limit was 232.2―333.8mg/kg.The success rate of hypodermic tumor inoculation was 97.5%,and the tumors measured about 1.5cm in diameter 30 days after inoculation. LHRH receptors were highly expressed in tumor sites in the tumor-bearing nude mice. A HPLC analysis method was developed to measure concentrations of MTX in plasma and other tissues of the tumor-bearing nude mice . The recoveries of this method were more than 85%,and the relative standard deviations of the intra-day and inter-day were less than 10%.The concentrations of MTX in plasma,heart,liver,spleen, kidney and tumor tissues were detected after administration at 1h,2h,4h,12h,24h,respectively. Compared with MTX group, the concentrations of MTX of LHRH-MTX group were higher in tumor tissues, but lower in plasma, liver and kidney(p<0.05).The acute toxicity of LHRH-MTX decreased evidently compared with MTX. LHRH-MTX significantly changed the distribution of MTX in the tumor-bearing nude mice. The concentrations of MTX evidently increased in the tissues where LHRH receptors were highly expressed, but decreased in plasma, liver and kidney.