| HYY-014, a new disaccharide anthracycline with the carbohydrate moiety, is synthesized based essentially on the hypothesis that the elongation of the carbohydrate moiety, and its modification, might result in improved compounds. In particular, it was supposed that an increased number of atomic contacts of the sugar moieties within the minor groove of the DNA binding site would increase the stability and the DNA sequence selectivity in the formation of the ternary molecular complex with DNA and topoisomerase II.Moreover, the absence of the amino group in the moiety directly Linked to the planar chromophoric portion of the molecule would abolish the refusal of guanine residue in the first base pair adjacent to the intercalation site. The potential of HYY-014 to ameliorate the results of other anthracyclines was confirmed by in vitro testing in tumour cell culture systems and in vivo experiments on a wide spectrum of human tumour xenografts in nude immunodepressed mice. The major metabolite for HYY-014 appears to be unmodified compound, and the half-life is shorter than Doxorubicin and Epirubicin. C13-ol-metabolite, a toxic metabolite of HYY-014, is lower than Doxorubicin, which predict a high probability for safety. There is a significant synergistic effect was observed when HYY-014 was administered before cisplatin treatment.Objective:NTo develop a LC-MS/MS method for the determination of HYY-014 and HYY-M3 in biological samples, study pharmacokinetic and tissue distribution in rats, which can provide valuable evidence and more information for preclinical studies.Methods:NN1.NValidationNofNAnNanalyticalNHPLC-MS/MS-methodNorNheNdeterminationNofN HYY-014NandNHYY-M3NnNatNlasmaNChromatographic conditions are:Chromatographic column:Phenomenex Luna 5μ Ci8(2) 150mm×2.0mm; Mobile phase:Acetonitrile:water(containing 0.1% Formic acid)=28:72, Flow rate:0.2 ml/min; Column temperature:40℃; injection volume:10 μl. MS conditions are:LC-ESI-MS/MS was performed in the selected ion monitoring (SRM) mode using target ions at m/z 644.0'331.0(33eV) for HYY-014, at m/z 646.0' 332.7(27eV) for HYY-M3 and at m/z 544.0'396.8(15eV) for HYY-014 Internal standard Doxorubicin. Spray voltage:4.7kV; Capillary temperature:320℃;Source CID:-8V; Sheath gas pressure:30Arb; Aux gas pressure:3Arb; Collision pressure:1.0; scaning time is 0.5s.2.NPharmacokineticNofTHYY-0141NnNatsNSingleNadministration:24 rats were equally divided randomly into 3 groups(with half males and half females). The plasma concentrations of HYY-014 and HYY-M3 were detected by LC-MS/MS method after intravenous administration of HYY-014(0.75mg/kg,1.5 mg/kg,3.0mg/kg). Blood samples(approximately 0.2ml) were collected from post-orbital venous plexus at the following times:pre-dose, post-dose 5,15,30 min,1,2,4,8,16,24,36,48,72,96 h. Blood was collected into heparinized tubes and centrifuged at 3000 r/min for 15 min, the plasma was separated immediately and stored at-70℃ until analysis.MultipleNadministration:N30 rats were equally divided randomly into 3 groups(with half males and half females). The plasma concentrations of HYY-014 and HYY-M3 were detected by LC-MS/MS method after multiple dosing intravenous administration of HYY-014(0.75mg/kg,1.5 mg/kg,3.0mg/kg) by once every two weeks. Blood samples(approximately 0.2ml) were collected from post-orbital venous plexus at the following times:pre-dose of the fourth、fifth、sixth and post-dose 5,15,30 min,1,2,4,8,16,24,36,48,72,96h. Blood was collected into heparinized tubes and centrifuged at 3000 r/min for 15 min, the plasma was separated immediately and stored at-70 ℃ until analysis.3.NTissue Noistribution NofiHYY-014NnNatsN64 rats were equally divided randomly into 8 groups(with half males and half females). The tissues concentrations of HYY-014 and HYY-M3 were detected by LC-MS/MS method after intravenous administration of 1.5 mg/kg HYY-014. The animals were executed at the following times:pre-dose and post-dose 5min,1,4,10,24, 72,120 h. Tissues,such as, heart、liver、spleen、lung、kidney、stomach、brain、intestine、 muscle、fat、skin、bone、testicle (ovary),etc, were collected after executed, dried by filter paper, sheared about 0.5g cut into pieces, and homogenized with physiological saline(w:v=1:4).Results:N1. The method was validated successfully to study HYY-014 in rat plasma.2. Single administration:The pharmacokinetic parameters were calculated by the procedure of DAS 3.0. HYY-014:Cmax(ng/ml):816.1±333.9,1628.6±618.6, 3495.5±1138.2; AUCo-t(ng/ml*h):1259.4±221.7,2219.5±276.9,4287.1±529.2; T1/2(h): 33.3±8.9,24.4±3.6,24.5±2.8; MRTo-t(h):18.2±1.3,15.1±2.4,15.6±2.1; Vd(L/kg): 27.1±8.6,23.4±5.2,24.2±4.4; CL(L/h/kg):0.57±0.11,0.66±0.08,0.68±0.09. In low dose group most plasma concentration of HYY-M3 were lower than LLOQ, so the group wasn’t analyzed. The pharmacokinetic parameters of HYY-M3 of other groups are:Cmax(ng/ml):2.2±0.6,3.1±0.6; AUCo-t(ng/ml*h):103.1±13.7,174.4±19.5; the ratio with the same dose HYY-014 was between 4% and 5%. Between 0.75mg/kg and 3.0 mg/kg, AUCo-t and Cmax of HYY-014 has good dose-effect relationship. The pharmacokinetic parameters were not statistically significant in gender in different dose groups using SPSS 17.0 statistical software.Multiple administration:The pharmacokinetic parameters were calculated by the procedure of DAS 3.0. In high dose group, the most animals were dead, so the data from the group was excluded from the analysis. The pharmacokinetic parameters of HYY-014 of other groups are:Cmax(ng/ml):1514.8±181.0,2569.2±473.5; AUCo-t(ng/ml*h):1463.5±246.7,2923.6±617.1; T1/2(h):16.8±3.5、25.3±4.3; MRTo-t(h): 10.5±1.8,16.3±1.6; Vd(L/kg):12.2±3.7,18.3±3.1; CL(L/h/kg):0.50±0.08,0.51±0.12; HYY-M3:Cmax(ng/ml):1.6±0.3,3.5±1.1; AUCo-t(ng/ml*h):88.1±21.7,195.4±64.7; the ratio with the same dose HYY-014 is between 6% and 7%. The pharmacokinetic parameters were not statistically significant in gender in different dose groups using SPSS 17.0 statistical software(except AUCo-t and CL).3. The resultN of tissue distribution shows:(1) HYY-014 distributes to tissues rapidly and widely following intravenous administration.. At 5min, the concentration of HYY-014 were:spleen> kidney>lung>intestine>heart>ovary>liver>stomach>bone>muscle>fat> skin>testicle>brain, the tissues with the highest drug concentrations were in lungs, kidneys, spleen; From 1 to 24 hours, the concentration gradually declined in all tissues(except intestine, muscle and fat. From 72-120 h, the concentration in all tissues were steady but still detectable. After 120h, the concentration in spleen and lung were still high. The concentration in skin, testicle and brain were all very low. (2)From 5 min-10h, the concentration of HYY-M3 gradually increased in all organs, and, at 5min HYY-M3 cannot detected in spleen, bone, muscle and brain. From 10-24 h, the highest concentration was detected in most tissues and then gradually declined, and after 120h still detectable. (3) The tissue distribution of HYY-014 and HYY-M3 had obvious targets, mainly distributed in spleen, kidney, lung, heart, liver and slowing elimination in spleen and lung. (4) HYY-014 cannot cross the blood-brain barrier.Conclusion:N1. The method was validated successfully to study HYY-014 in rat plasma2. Between 0.75mg/kg and 3.0 mg/kg, AUC and Cmax of HYY-014 has good dose-effect relationship.3. The tissue distribution of HYY-014 had obvious targets, mainly distributed in spleen, kidney, lung, heart, liver. HYY-014 cannot cross the blood-brain barrier. |
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