| In recent years,the overall incidence of malignant tumors is increasing all over the world.Leukemia is a common hematological disease and belongs to malignant tumor.It is mainly manifested in the unrestricted proliferation of a large number of leukemic cells in bone marrow and other hematopoietic tissues and released to the peripheral blood which induce the production of normal blood cells is significantly inhibited.Acute myeloid leukemia?AML?is the most common adult acute leukemia.Now,the research on the pathogenesis and prognostic factors of AML has been increasing.But until now,nearly one third of adult AML can not be cured.Recurrent refractory leukemia and resistance to drug are the difficulties in the treatment of leukemia.By methods of cell biology and molecular biology,it is a trend to analyze the pathogenesis of different types of leukemia and find the corresponding key targets for precise targeted therapy.Histone acetylation and deacetylation play an important role in maintaining the dynamic balance between tumor genes and antitumor genes.Histone deacetyIases?HDACs?play an important role in tumorigenesis and development by depolarizing the acetylation of lysine-disabled acetyl groups at the histone end,resulting in"relative densification"of chromatin conformation,inhibiting the transcription of related genes.It is considered to be one of the most potential drug targetsof anti-tumor in recent years.Histone deacetylation inhibitors?HDACIs?can affect the biological behavior of tumors in many ways,mainly in promoting cell differentiation,blocking cell cycle,inducing apoptosis and regulating the transduction of related anti-tumor genes.Its therapeutic mechanisms for leukemia include inducing cell differentiation,especially for ATRA-resistant patients,restoring the sensitivity of leukemic cells to ATRA,inducing apoptosis and inhibiting proliferation of leukemic cells,and blocking cell cycle.Retinoids,a group of compounds similar to vitamin A in structure,are the first-line drugs for the treatment of APL.All-trans-retinoic acid?ATRA?can form a dimer with RAR or RXR and increase the expression of target genes by acting on retinoic acid responsive element?RARE?in promoter,thereby inducing cell differentiation,inhibiting proliferation and inducing apoptosis.Tamibarotene?AM80?is a new selective RAR?agonist,which was launched in Japan in 2005.It can be used in the treatment of ATRA-resistant and relapsed refractory APL.Malignant tumors,including leukemia,are multi-factor-induced and multi-pathogenic diseases.Traditional medicine has many disadvantages,such as low efficacy,multi-drug resistance and toxic side effects.Combination therapy is usually more effective and has fewer side effects than a single drug.HDACIs,for example,can be used in combination with RAR agonists to enhance efficacy and reduce toxic and side effects in leukemia.At the same time,it has become an important strategy in the field of drug design to design multi-target drugs or synergistic prodrugs for multiple pathogenesis of malignant tumors and multiple pathological links.WJD-A-1,N1-?6-?hydroxyamino?-6-oxobutyl?-N4-?5,5,8,8-tetramethyl-5,6,7,8-tetrah ydronaphthalen-2-yl?terephthalamide,is a novel HDAC inhibitor designed and synthesized by ester bonding or amide bonding,in which the RAR?selective agonist AM80 is used as hydrophobic group and the hydroxamic acid and carboxyl group are used as zinc ion chelating groups.When the target compound enters the body,it can inhibit the proliferation,promote the differentiation and induce apoptosis of tumor cells by inhibiting the activity of HDAC.At the same time,it can release AM80 under the catalysis of amidase,and AM80 acts on RARa receptor to induce the differentiation and inhibit the proliferation of leukemia cells,thus realizing the relationship with precursor.The synergistic effect of drugs can play the role of multitarget drug therapy.Previous cell test in vitro showed that WJD-A-1 had better proliferation inhibition activity on three kinds of leukemia cells,and the anti-proliferation activity on NB4 cells was significantly higher than that on AM80,but the anti-proliferation activity on HL-60 and K562 cells was similar to that of AM80.The purposes of this study were to establish an analysis method in vivo and investigate the pharmacokinetics,tissue distribution,plasma protein binding,metabolism and excretion of WJD-A-1 in rats.The possible inhibition to human liver cytochrome CYP450 enzyme were also revealed.The study gives the useful information for a comprehensive evaluation of the efficacy and safety of WJD-A-1.The results are as follows:1 The pharmacokinetic parameters of intravenous administration of WJD-A-1 in rats were determined1.1 Plasma concentrations of WJD-A-1 and AM80 in rats were measured by PLC-MS/MS.After a simple sample preparation procedure by protein precipitation with methanol and acetonitrile,WJD-A-1,AM80 and the internal standard were chromatographed on an ACQUITY UPLCTMM BEH C188 column.The mobile phase consisted of methanol-0.1%formic acid?80:20,v/v?and the flow rate was 0.20 mL/min.The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring?MRM?mode via electrospray ionization?ESI?source.Each plasma sample was chromatographed within 2.6 min.The linear calibration curves for WJD-A-1 and the AM80 were obtained in the concentration range of 5.405.40×103and 5.085.08×103 ng/mL,respectively?r?0.99?.The intra-and inter-day precision?relative standard deviation,RSD?values were from 1.55%4.02%and 0.60%5.09%,and the accuracy?relative error,RE?was from-2.55%6.80%,determined from quality control?QC?samples for the analytes.This UHPLC-MS/MS method is simple,accurate and rapid for the quantification of WJD-A-1 and AM80 in rat plasma.These results showed that this method was easy to be operated,with highly sensitivity,specific and with good accuracy.1.2 WJD-A-1 and AM80 were not detected in all the plasma samples of rats after intragastric administration of 8.0,16.0,32.0 mg/kg,which indicated that WJD-A-1 and AM80 were poorly absorbed in vivo and had very low bioavailability and were not suitable for oral use.1.3 To evaluate the linear pharmacokinetics of WJD-A-1 in rats,the established UHPLC-MS/MS method was used.Blood samples were collected at specific time points after a single intravenation administration of WJD-A-1 75,150,300?g/kg to rats.The main pharmacokinetic of WJD-A-1were as follows:the value of t1/2/2 was?0.10±0.05?h??0.24±0.02?h??0.51±0.06?h,AUC?0-t?were?150.60±21.69??g·h/L??883.07±235.68??g·h/L??3311.59±418.15??g·h/L,respectively.AUC?0-t?was increased proportionally as dose escalation,and the correlation coefficient>0.9 indicating a good linear relationship between AUC?0-t?and dose.The main pharmacokinetic of AM80 after intravenation administration were as follows:the value of t1/2/2 was?0.08±0.01?h??0.13±0.01?h??0.30±0.07?h,AUC?0-t?were?14.36±1.97??g·h/L??65.71±3.27??g·h/L??315.11±27.89??g·h/L,respectively.AUC?0-t?was increased proportionally as dose escalation,the correlation coefficient>0.9 indicating a good linear relationship between AUC?0-t?and dose.2 The tissue distribution of intravenous administration of WJD-A-1 and AM80 in rats was studiedThe tissue distribution of WJD-A-1 after intravenous administration in rats was investigated by LC-MS/MS,which was established to detect the concentration of WJD-A-1 in plasma samples.The aim of this study was to evaluate the targeting ability of compound WJD-A-1 in rats and the accumulation in various tissues.After intravenation administration of WJD-A-1 150?g/kgto rats,the distribution of WJD-A-1and AM80 in tissues were extensive and the elimination rates in all tissues were relatively fast,indicating unlikely drug accumulation in rats.Concentrations of WJD-A-1and AM80 in plasma,stomach wall and liver were higher than in others organs.Concentrations of WJD-A-1 and AM80 in brain were all below the LLOQ.The protein binding interaction of WJD-A-1 with rat and human plasma was investigated using equilibrium dialysis method.After being equilibrated for 48 h,the average plasma protein binding ratio in the concentration of 50200 ng/mL were?84.16±1.84?%??84.35±2.63?%??82.39±2.11?%in rat plasma and?86.12±2.58?%??83.10±2.01?%??80.86±1.88?%in human plasma.The plasma protein binding ratio of trantinterol was at moderate level and showed concentration-independent.3 The metabolism and excretion of WJD-A-1 in rats were determinedThe cumulative bile and urine samples of rats were collected before and 012 h after intravenous administration of WJD-A-1.The metabolites were studied by mass spectrometry after treatment.The main metabolite was identified as AM80 by combination the study of metabolites with the structural characteristics of WJD-A-1.The excretion of WJD-A-1 and AM80 in rats was studied by LC-MS/MS.The results showed that the combined determination method of WJD-A-1 and AM80 in plasma was also suitable for the determination of two compounds in excreta?bile,feces,urine?with satisfactory linearity,accuracy and precision.It was found that after intravenous administration of WJD-A-1,the excretion of prototype and AM80 was rapid and the cumulative excretion was over 80%within 12 hours.Bile is the most important excretion pathway,and the cumulative excretion volume is about 97%of the dosage.Urine accounts for about 1.35%of the total excretion volume.Prototype drug is the most important excretion form,followed by metabolite AM80,which accounts for 63.3%and22.5%of the dosage respectively.4 The effect of WJD-A-1 on human liver cytochrome CYP450 enzyme was confirmedCYP1A2,CYP3A4,CYP2C19,CYP2C9 and CYP2D6 are closely related to liver drug metabolism.The possibility of WJD-A-1 to inhibit human liver CYP450 activities was evaluated in vitro in HLM.The IC500 values of every CYP450 enzymes were determined to evaluate the inhibition capability.WJD-A-1 did not inhibit CYP2D6?CYP1A2?CYP2C19 and CYP2C9 activities(IC50>10?mol/L)and inhibit CYP3A4slightly(IC50=8.56?mol/L).The inhibition of WJD-A-1 on five main CYP enzymes showed that WJD-A-1 had weak inhibitory effects on CYP2D6,CYP1A2,CYP2C19 and CYP2C9 indicating there was no clinical significance,may inhibit CYP3A4 activities.The significance of this study is to determine the concentration of WJD-A-1 and its metabolite AM80 in biological samples by LC-MS/MS.The pharmacokinetic experiments of WJD-A-1 in rats were carried out systematically.The dynamic changes and characteristics of WJD-A-1 in rats were discussed.The role of CYP450 in the metabolism of WJD-A-1 was determined.These results will provide important theoretical basis and application value for the research and development of WJD-A-1drugs,the prediction of drug interactions and the guidance of clinical safety and rational drug use. |
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