Toxicological And Stability Assessment Of A Novel Small Peptide RWR

Objectives:To assess the toxicology and stability of a novel small peptide RWR according to the requirements of preclinical research for innovative drugs. This will provide theoretical and experimental basis for further pharmacokinetic study and structure optimization of RWR.Methods:1. Preparation and identification of RWR RWR was synthesized by Fmoc solid phase method, purified by reverse phase HPLC and dried with vacuum freezing. The purity was identified by Electrospray ionization mass(ESI- MS).2. Toxicology analysis of RWR2.1 Acute toxicity test in mice The administration dose was designed based on preliminary pharmacokinetics data(see the thesis of Zhao Haixia). The maximum tolerated dose(MTD) was determined according to the death amount of mice.2.2 Mice bone marrow micronucleus assay RWR was injected intraperitoneally at dose of 24, 96 and 120 mg/kg. The bone marrow micronucleus was counted to determine the mutagenicity of RWR.2.3 Mice sperm abnormality test RWR was injected intraperitoneally at dose of 24, 96 and 120 mg/kg. The aberrated sperm was counted to determine the genetic toxic effect of RWR.2.4 Chronic toxicity test RWR was injected intraperitoneally to rat at dose of 4.5, 18.8 and 75 mg/kg. The toxicity reaction of RWR was assessed by recording body mass, food utilization, main organ coefficient and blood routine indexes, as well as the alteration of lung, liver, spleen,kidney and heart. The nontoxic reaction dose was also confirmed.3. RWR stability evaluation In order to obtain the chemical environment which RWR can stably exist in plasma,LC-MS/MS method was used to detect RWR in the fresh and 4-hour solution which was prepared in 50% methanol, SD rat plasma anticoagulated by EDTA, as well as in EDTA plasma with various stabilizer.4. Design and stability research of RWR derivative In order to obtain more stable RWR derivative, omega-amino capylic acid was added on the N-terminal of RWR. The yielded product showed same activity compared with RWR. The stability of RWR derivative in methanol and plasma was detected by the methods mentioned above. This may provide an idea for design and development of antithrombotic peptide drugs with more stability.Results:1. The amount of synthesized RWR is one gram which can meet the need of subsequent experiments. The purity could reach 99.006%.2. The MTD of acute toxicity test for rats was larger than 120 mg/kg. The results of bone marrow micronucleus assay, mice sperm abnormality test showed no significance between all doses. Thirty-day feeding test had no adverse effects on body weight, food utility rate, blood routine indexes and organ coefficient of rats. There were also no obvious histological alterations related to RWR in rat organs.3. The data showed that the stability of RWR was between 40~70% in different conditions. RWR exhibited poor stability in 50% methanol solution and plasma which was judged by the standard of 85~115%.4. The stability of RWR derivative was between 86~90%, which demonstrates that it can exist steadily in methanol solution and plasma.Conclusion:Our data showed that RWR meet the drug safety toxicology evaluation standard,demonstrating that RWR holds the promise for further drug development. RWR holds poor stability in methanol solution and plasma. Stability can be improved by adding an ω-amino capylic acid onto RWR. This research lays the theoretical and technical foundation for developing more stable antithrombotic small peptide drugs.