The Comparisons Of The Effects Between Enzyme Staining And Biochemical Methods In Drug Safety Evaluation And Ranolazine Genotoxicity Study

Drug toxicity is one of the main causes leading to failure of drug development. In order to decrease high cost from the drug researches and developments, it is very important to make certain the potential toxicity of drugs in early state of drug developments. The toxicity of livers and kidneys is the most important in the drug toxicities. The liver is not only the main site of biotransformations and detoxification but also the sensitive target organ of the drug toxicity. It is reported that drug-induced liver disease or liver damage in adverse drug reactions accounted for 10%-15% [1].Therefore, the early detection of drug toxicities is crucial for drug development. In the present study, Beagle dogs were used as a model, and Low Molecular Weight Fucoidan(LMWF) was conducted as the research object. According to the change of the sensitivity index and the toxicity reaction of livers, by different test methods,we performed the early analysis of drug toxicities so as to predict the possible toxicity and improve the accuracy predicting sensitive indicators of the vital organs toxicity induced by drug.1. The comparisons of the effects between enzyme staining and biochemical methods in drug safety evaluationObjective:to elucidate the effects of LMWF regarding serum biochemistry and enzymatic histochemistry in Beagle dogs.Methods:1) experimental animal grouping and medication:16 Beagle dogs including 8 male and 8 female were stochastically divided into 4 groups:control group (Veh), low-dose group (L,200 mg/kg/d), medium-dose group (M,800 mg/kg/d) and high-dose group (H,3000 mg/kg/d). Each group contained 4 Beagle dogs with 2 male and 2 female. They were fed using stomach lavaging continuously for 6 days followed by 1-day suspension until the thirteenth week.2) index determination:Syndrome observation was conducted every day. The weight of each Beagle dog was measured every week. Serum biochemical index were tested prior to the first time LMWF treatment and after the last treatment. The Beagle livers were finally taken and weighted to calculate organ coefficient. liver was taken and frozen in liquid nitrogen, sectioned and stained, the enzyme histochemical detection. The remaining 4% neutral formaldehyde into the liver were fixed, paraffin-embedded sections after, HE staining, optical microscopy.Results:1) syndrome observation:Before and during the experimental period, no abnormal syndromes were observed in either LMWF treated groups or Veh group (i.e. live in a good state with normal diet).2) body weight:The results shown that, compared to Veh group, there was no significant weight change for LMWF treated groups (P>0.05).3) serum biochemistry test:compare with Veh group, serum biochemistry index of LMWF treated groups didn't show obvious deviation before, during and after treatment (P>0.05).4) Pathological assessment:liver fresh weights and organ coefficients didn't show outstanding difference compared with Veh group (P>0.05); no evident pathological changes was found in LMWF treated liver histomorphology by microscopy observation.5) Both ATPase(adenosine triphosphatase, ATPase) and SDH(Succinate dehydrogenase,SDH) liver qualitative observation and quantitative analysis,3000 mg/kg/d dose activity decreased significantly, compared with the Veh group, was statistically significant (P<0.05).Conclusions:Beagle dogs were treated for 13 weeks with LMWF. 1)There is no significant changes in terms of serum biochemical and pathology found during LMWF treatment.2) Histochemical detection, LMWF 3000 mg/kg/d dose is significantly reduced in ATPase and SDH activities, indicating that the liver mitochondrial function has been impacted, also show that enzyme staining method sensitive than biochemical methods.2. Experimental study on Ranolazine genotoxicityObjective:Toassess the drug safety of Ranolazine by investigating its genotoxicity.Methods:The genotoxicity of Ranolazine was evaluated by using the methods of Ames test, Micronucleus test of mice bone marrow polychromatic erythrocytes and sperm shape abnormality test respectively.There were 3 dosing groups for Ranolazine in the Micronucleus test of mice bone marrow polychromatic erythrocytes and sperm shape abnormality test assay in mice,222,111 and 55 mg/kg, with a negative control with saline and a positive control with 40 mg/kgCyclophosphamide. Salmonella reverse mutation assay adopted two methods including with and without S9 mixture. Test strains TA97, TA98, TA 100 and TA102 were used in this study. There were six dose groups for extraction as follows:5000,2500,1000,500,250 and 125 mg/plate, with a negative control and a posilave control.Results:Compared with the control group, there were no statistical difference (P>0.05) in number of colony with back mutation per vessel in TA 97, TA 98, TA 100 and TA 102 strains treated with Ranolazine at<5000μg per plate with or without S9 activation., and no dosage-dependent relation was shown. Mmoreover, within the dosage of 55～222 mg/kg, both the induced micronuclei of mice bone marrow polychromatic erythrocytes and the sperm shape abnormality rate showed no obvious differences(P>0.05) between Ranolazine treated and untreated groups in mice.Conclusion:Ranolazine in the dose range measured, not shown genetic toxicity.