| Cancer and obesity are two major diseases that threaten human health in twenty-first Century, so the development of anti-tumor drugs and lipid-lowering drugs has broad prospects. HL-13 and C-11 are small molecular compounds synthesized by our group, which have respectively shown good anti-tumor and lipid-lowering activity in the preliminary pharmacological studies. On the basis of previous pharmacological study, the study on their pharmacokinetic characteristics could provide early information about the absorption, distribution and metabolic conversion and lay basis on the potential safety and efficacy.Pharmacokinetics is an essentiall part of drug discovery and development which include absorption, distribution, metabolism and excretion. In the early stages of drug development, the early information on metabolic conversion and drug-drug interaction of compounds in vivo could be obtained by exploring the metabolic properties mediated by CYP 450 in vitro. The research on the absorption, distribution and metabolism profile of drug candidate in vivo could reveal its dynamic changes and excretion pathway.This study was consisted of two parts; one was investigation of the metabolism in vitro, bioavailability and tissue distribution in vivo of HL-13, an anti-tumor compound, the other was metabolites identification in rats of C-11, an anti-obesity compound.1. The study on the in vitro and in vivo metabolism of HL-13HL-13 was a small molecule compound synthesized by our laboratory, and it has been proved that HL-13 had a good anti-tumor activity in various cell types. Because of its significant bioactivities, HL-13 was investigated as a potential anticancer drug candidate. The first part of this paper researched the metabolic characteristics in vitro, oral bioavailability in rats and tissue distribution in tumor bearing mice of HL-13. The main contents of our study including:(1) The development and validation of a method for quantitative analysis of HL-13 in liver microsomal incubation systemA rapid, sensitive ultra-performance liquid chromatography-tandem triple quatrupole mass spectromrtry method was developed and verified for quantitation of HL-13 in liver microsomal incubation system. The results of method validation suggested that the specific, sensitivity, accuracy and repeatability all met the general criteria for biological sample analysis methods. So this method was employed to quantitative analysis of HL-13 in liver microsomal incubation system.(2) The metabolic stability of HL-13 in liver microsomes of different speciesThe metabolic stability of HL-13 in rat, human, Beagle dog and monkey liver microsomes in vitro was investigated.The half-time in vitro of HL-13 in rat, human, Beagle dog and monkey liver microsomes was 176.79,75.33,40.13 and 30.77 min, respectively. The intrinsic clearance in vitro was 0.005,0.012,0.023 and 0.030 mL/min/mg protein, respectively. The results indicated that HL-13 was a slow metabolizer and stable in liver microsome. Therefore, it is assumed that the intrinsic clearance in vivo was 11.88?9.00?34.16 and 44.55 mL/min/kg, the hepatic clearance 7.45? 7.92?17.99 and 22.14 mL/min/kg. In addition, the clearance rate of HL-13 in rat was similar to that in human.(3) Identification of CYP450 enzyme isoforms involving mtabolizing HL-13 in human liver microsomeThe CYP450 enzyme iso forms involving mtabolizing HL-13 was researched by using the specific chemical inhibitors. The results showed CYP1A2 contributed most to the metabolism of HL-13, which indicated CYP1A2 may be the main metabolic enzyme involved in HL-13, while the contributiom of polymorphisms CYP2C19, CYP2D6 was small.(4) Inhibitory effect of HL-13 on the main CYP450 enzymesThe inhibitory effects of HL-13 on main CYP450 enzymes in vitro were examined using specific probe substrates. Using phenacetin, omeprazole, tolbutamide, chiorzoxazone and dextromethorphan as a substrate and comparing the concentration of metabolites with different concentrations of HL-13, the effects of HL-13 on CYP1A2, CYP2C19, CYP2C9, CYP2E1, CYP2D6 and CYP3A4 activities were obtained. The results showed that HL-13 exhibited middle inhibitory effects on CYP2C9 and CYP2C19 activity with IC50 value of 1.077 ?M and 8.153 ?M CYP2E1, while it exhibited strong inhibitory effect on CYP2E1 with IC5o value of 0.299?M.(5) The development and validation of a method for quantitative analysis of HL-13 in plasma sampleWe established a method for quantitative analysis of HL-13 in plasma sample on the basis of method developed in vitro above. We validated specificity, sensitivity, linearity, accuracy, precision, recovery, stability, and matrix effect of the method according to the guidelines of FDA and SFDA on bio-analytical method validation. The results showed that it was satisfactory in terms of specificity, sensitivity, linearity, accuracy and precision in the detection of HL-13 in rat plasma. Furthermore, we verified the linearity of processing methods of tissue distribution samples, and it was exciting that all the results met the requirements.(6) The bioavailability of HL-13 in ratsIn this part, SD rats were treated once with 5 mg/kg i.v. and 10 mg/kg p.o. HL-13 respectively for the drug-time curve study. Blood samples were collected at different time points in which the concentration of HL-13 was determined by using established UPLC-MS/MS method. The pharmacokinetic software DAS2.1 was employed to calculate pharmacokinetic parameters and oral bioavailability of HL-13. The results indicated that AUC(0-t) of intravenous and oral administration were 1904.25±459.94 and 578.34±32.80 respectively, resulting in the oral bioavailability of 15.19% of HL-13 in rats.(7) Tissue distribution of HL-13 in Balb/c miceA total of twenty Balb/c tumor bearing mice were randomly divided into four groups. Mice were sacrificed at different times after treated with 10 mg/kg HL-13 by intravenous injection (i.v.). Heart, liver, spleen, lung, kidney, brain, stomach, small intestine, mulsle and tumor were collected and homogenized in 30 min,4 h,8 h, and 10 h after taking blood sample, then the concentration of HL-13 were detected and calculated by tissue calibration curve. The results showed that HL-13 was mainly distributed in small intestine, liver, kidney and tumor, secondly in heart, spleen, stomach and lung, almost none in brain and mulsle. Specially, the concentration of HL-13 in tissue was much higher than that in plasma. More importantly, the content of HL-13 in tumor tissue was not significantly decreased in 4-10 hour demonstrating HL-13 had affinity to tumor issues, which played an important role in the treatment of tumor.2. Metabolites identification of C-11 in ratsThe second part of this paper was identification of C-11 in rats in vivo. C-11 is a small molecule compound that is sythesized on the basis of the structure of anti-diabetic drugs rosiglitazone. The pharmacological experiment has proved C-11 have optimal anti-obesity activity. In this study, a sensitive and reliable approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) was applied to characterize the metabolic profile of HL-13 in rat plasma, urine and feces after intravenous administration of HL-13 (30 mg/kg). Totally twenty-five metabolites were discovered and tentatively identified in rats containing phase ? and phase ? metabolites. All the phase ? and phase ? metabolites were found in feces. The phase ? biotransformation pathways of C-11 included N-depyridinylation, N-demethylation, mono-hydroxylation and di-hydroxylation. N-depyridinylation and N-demethylation metabolites could be detected in all biological samples (plasma, urine, feces) in rats. Meanwhile, metabolic pathways involved phase ? metabolism contained glucuronidation and sulfation. Two glucuronidated conjugates were found in plasma and none of phase ? metabolites was detected in urine. These results indicated that the fecal clearance was the major elimination route of C-11 as well as its metabolites in rat. |
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