ObjectiveTo develop a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain(FLT3-TKD)of acute myeloid leukemia(AML)and apply to the monitor of minimal residual disease(MRD).MethodsRecombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed respectively and mixed at certain ratios.The obtained standard plasmids with mutation rates of 50%,1%,0.1% and 0% respectively were determined by restriction fragment length polymorphism(RFLP)in combination with Sanger method.The plasmid DNA standards and blood DNA standards,at various FLT3-D835Y mutation rates,were determined by the developed method to verify the sensitivity.The genomic DNA samples of patients with AML before and after treatment were determined by the developed method to monitor the MRD.ResultsSequencing proved that both the recombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed correctly.The sensitivity of developed method increased to 0.1% through Sanger method combined with digestion with EcoR Ⅴ/Xho Ⅰ and recovery of mutant fragments in determination of purified plasmid DNA and collected blood DNA samples.MRD was detected in the peripheral blood sample of a patients with AML in complete remission period by the developed method but not by Sanger method.ConclusionA highly sensitive method for detection of FLT3-TKD mutation was developed,which was of an important clinical significance in guiding the treatment of AML and monitoring the MRD in complete remission period.

The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The Km, Vmax, CLint and T1/2 of thienorphine obtained from human liver microsomes were (4.00 ? 0.59) ?mol?L-1, (0.21 ? 0.06) ?mol?L-1?min-1, (117 ? 3.19) mL?min-1?kg-1 and (223 ? 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 ? 0.69) and (3.28 ? 0.50) ?mol?L-1, (0.18 ? 0.04) and (0.14 ? 0.04) ?mol?L-1?min-1, (213 ? 1.06) and (527 ? 7.79) mL?min-1?kg-1, (244 ? 1.21) and (70.7 ? 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differencesobserved were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.

The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The K(m), V(max), CL(int) and T1/2 of thienorphine obtained from human liver microsomes were (4.00 +/- 0.59) micromol x L(-1), (0.21 +/- 0.06) micromol x L(-1) x min(-1), (117 +/- 3.19) mL x min(-1) x kg(-1) and (223 +/- 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 +/- 0.69) and (3.28 +/- 0.50) micromol x L(-1), (0.18 +/- 0.04) and (0.14 +/- 0.04) micromol x L(-1) x min(-1), (213 +/- 1.06) and (527 +/- 7.79) mL x min(-1) x kg(-1), (244 +/- 1.21) and (70.7 +/- 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differences observed were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.

Objective To study the safety and efficacy of selective head cooling (SHC) with mild systemic hypotherrnia in neonates with HIE. Methods Fifty-four term infants with severe neonatal HIE were randomly assigned to the head cooling group (n=27) and control group (n=27). Forty-one infants in 96 h after admission were eligible for the study(SHC group n=21, control group n=20). In SHC group, the naso-pharyngeal temperature was maintained at (34.0±0.2) ℃ and rectal temperature maintained at 34～35 ℃ for 72 h, then rewarmed spontaneously. In control group, normal rectal temperature was maintained. During the period of the study, the infants of two groups were monitored on nasopharyngeal temperature, heart rate, respiratory rate,transcutaneous arterial oxygen saturation and blood pressure. Primary adverse effects inclu-ding severe arrhythmia, venous thrombosis or hemorrhage and severe hypotension were observed. The efficacy indicators including rate of death and severe disability, exercise and cognition development index were as-sessed. Results Severe arrhythmia, hypotension and renal failure were not found in both groups. Follow-up was conducted until postnatal 18 months and was not available in 6 babies (3 in SHC group and 3 in control group respectively). Death and severe disability occurred in 4 of 18 infants (22.2%)in SHC group and in 9 of 17 infants(52.9% ) in the control group respectively (P<0.05). Conclusion SHC for 72 h with mild systemic hypothermia in neonates with HIE is safe and effective. The therapy could reduce the risk of disabili-ty and handicap significantly.

OBJECTIVE To investigate and compare the enzyme kinetic characters of psoralen (PRN)and isopsoralen(IPRN)in rat and human liver microsomes. METHODS PRN and IPRN in liver microsomes incubates were determined using LC-MS/MS. The enzyme kinetic and metabolic stability of PRN and IPRN were investigated by employing the optimized rat and human liver microsomes incubations. The Vmax and Km values were calculated using the nonlinear regression method. RESULTS The quanti?tative method showed good linearity within the range of 0.1-50.0 μmol · L-1 and was suitable for the assay in biological samples. The in vitro elimination was linear with the substrate concentrations lower than 1 μmol,the protein concentration within 0.5 g · L-1,and the incubation time within 40 min. The t1/2 values of PRN and IPRN in rat and human liver microsomes were 74.5,95.0,74.5 and 173.3 min, respectively. The Vmax values of PRN in rat and human liver microsomes were(1.140±0.080)μmol·min-1·g-1 protein,(0.620±0.060)μmol·min-1·g-1 protein,while Km values of PRN in rat and human liver microsomes were (12.9 ± 0.3)μmol · L- 1,(7.4 ± 1.3)μmol · L- 1,respectively. The Vmax values of IPRN in rat and human liver microsomes were(0.251±0.012)and(0.103±0.014)μmol·min-1·g-1 protein,while Km values of IPRN in rat and human liver microsomes were (3.0 ± 0.4)μmol · L-1,(3.4 ± 0.7)μmol · L-1,respectively. CONCLUSION The enzyme kinetic characters and metabolic stability of PRN and IPRN show species and chemical structures related differences. Interestingly,the metabolic eliminations of PRN and IPRN are similar in rats. However,the metabolic elimination of IPRN in humans involved in CYP enzymes may be much slower than that of PRN.

In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.

Rotundine (1 micromol L(-1)) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS. CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine. The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively. The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS2 spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MS(n) spectra of LC-iTrap-MS(n). The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation. A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite. The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine. Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were explored by using LC/Q-TOF-MSMS and LC/iTrap-MS(n) techniques.

OBJECTIVE To explore the absorption mechanism of thiophenorphine, and its effect on P-glycoprotein (P-gp) expression by using Caco-2 cell monolayer model. METHODSThe LC-MS-MS method was applied to determine thiophenorphine concentration in millicell system. The bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption. P-gp inhibition was studied by flow cytometry using calcein-AM as P-gp substrate.The expression of P-gp was evaluated using Western blotting. RESULTSThiophenorphine transport in Caco-2 cells was in time-dependent manner. Its average apparent permeability coefficient (P_(app)) was 2.338×10~(-6) cm·s~(-1). P_(app) was increased 2.8 folds by P-gp inhibitor ciclosporin A, and 2.3 folds by mulitdrug resistance-associated protein2 (MRP2) inhibitor MK571. The accumulation of calcein-AM and the expression of P-gp in Caco-2 cell line wasn't changed noticeably by thiophenorphine. CONCLUSION Thiophenorphine is a common substrate of P-gp and MRP2 and it shows normal transport in millicell system. The expression of P-gp doesn't induce by thiophenorphine.

Aim To investigate the inhibitory and in-duction effects of the active components of SIWU de-coction on cytochrome P450 enzymes ( CYP ) and as-sess the CYP based drug interaction. Methods Ac-tive components fructose, ferulic acid, paeoniflorin, li-gustrazine and their combinations were incubated sepa-rately with human liver microsomes ( HLM) and probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS to assess the inhibitory activities on human CYP1 A2 , 2 B6 , 2 C9 , 2 C19 , 2 D6 and 3 A4 . Sandwich-cultured rat hepatocytes model was used to evaluate the CYP1 A2 and CYP3 A1/2 induc-tion. Results The inhibitory rates on CYP1A2, 2B6, 2 C9 and 2 C9 by the test groups at 100 μmol · L-1 were all < 62%, while the activities of CYP3 A4 and 2D6 were not affected. The CYP1A2 activities in the test groups of peoniflorin and its combinations ( 50μmol·L-1 ) were significantly enhanced, with the in-creasing fold more than 40% of positive control group. No significant induction on rat CYP3 A1/2 was ob-served for four principles and their combinations. Con-clusions The active components of SIWU decoction do not show significant inhibitory effects on six CYP isoforms. Peoniflorin could induce the CYP1A2 activity in rat hepatocytes. The induction activity is enhanced by the concomitant use of peoniflorin with ferulic acid and/or ligustrazine.

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.