Study On The Metabolites Of Dihydroartemisinin And Their Distribution In Mice

Artemisinin(ART),a sesquiterpene lactone type antimalarial drug,was isolated from Artemisia annua L.by Youyou Tu's group,ART is highly effective,quick-acting,low-toxic and safe,and overcomes the problem of resistant malaria.Dihydroartemisinin(DHA)was developed by introducing a hydroxyl group into the structure of ART.Subsequently,a series of potent antimalarial derivatives were developed,such as artemether(AM),arteether(AE),artesunate(AS)and etc.Currently,artemisinin-based combination therapy(ACT)is recommended by WHO as the fist-line treatment for malaria.The clinical application of ART derivatives has saved millions of lives in the past decades.As new antimalarial drugs,the metabolism of ART derivatives in vivo have attracted extensive attention.As a main active metabolite in the metabolism of AM,AE and AS in vivo,DHA plays a vital role in revealing the metabolism mechanism of ART drugs.Thus,DHA is selected to reveal the metabolism mechanism in vivo.Currently,a large number of studies focused on the metabolic characteristics of DHA in vivo,ignoring the metabolic characteristics of DHA inside RBC.Considering that the plasmodium is a protozoan that develops inside RBC,and DHA could kill the blood stage parasites.Therefore,it is very important to elucidate the metabolic characteristics of DHA inside RBC for further revealing its mechanism.Early studies have shown that 3H-DHA is enriched inside RBC,and the enrichment effect inside plasmodium infected RBC was stronger than healthy RBC.To our knowledge,no study has been reported so far on the metabolites of DHA inside RBC.In summary,this study aimed at the metabolites of DHA inside RBC,and conducted the study on the metabolic process and metabolic mechanism of DHA in mice,which made up for the deficiencies of previous studies.1 Study on the metabolites of DHA in mice by intragastric administrationIn this study,the fragmentation patterns of DHA and some metabolites in mass spectrometry were summarized.On this basis,a UPLC-QTOF/MS method was established for the identification of DHA metabolites in mouse blood samples.Combined with UNIFI platform,a total of 34 metabolites were identified,including 28 phase ? metabolites and 6 phase ? metabolites.Phase ? metabolites mainly include ART,1-deoxy ART,1-deoxy DHA,7 hydroxylation metabolites,2 deoxygenation hydroxylation metabolites,7 dehydrogenation hydroxylation metabolites and 9 dehydration hydroxylation metabolites;phase ? metabolites mainly include ?-DHA?-glucuronide,2 hydroxylation glucuronidation metabolites and 3 dehydrogenation glucuronidation metabolites.On this basis,the metabolic pathways of DHA in mice via intragastric administration was established.By adding protective agents,a UPLC-QTOF/MS method was established for the detection of DHA and metabolites inside RBC for the first time.A total of 15(22)metabolites were identified from infected(healthy)RBC.Combined with reference substances,the prototype drug and 4 metabolites were structurely identified,such as 1-deoxy DHA,3?-hydroxy deoxy DHA and 1-deoxy ART and ?-DHA ?-glucuronide.The metabolites of DHA inside RBC were consistent with that of blood and plasma samples,indicating that DHA and metabolites could enter into RBC,which laid the foundation for further revealing the mechanism of action.A total of 31 DHA metabolites were identified in plasmodium infected mice blood samples,including 26 phase ? metabolites and 5 phase ? metabolites.In comparison,a total of 34 metabolites of DHA were identified in healthy mice blood samples,including 28 phase ? metabolites and 6 phase ? metabolites.By comparing the differences of DHA metabolites between infected and healthy mice,it was found that the types of DHA metabolite in infected blood samples were consistent with that of healthy blood samples,but the number and peak area of metabolites in infected blood samples were less than that of helthy blood.It was speculated that plasmodium affects the activity of metabolic enzymes in the liver.2 Study on the metabolites of DHA in mice by tail intravenous injectionIn order to further reveal the metabolic mechanism of DHA in vivo,the tail intravenous injection group was conducted in this study.The UPLC-QTOF/MS method was used for the detection of DHA metabolites in mice blood samples.Combined with UNIFI platform,a total of 14 metabolites were identified,including 13 phase ? metabolites and 1 phase ? metabolites.Phase ? metabolites mainly include 1-deoxy ART,1-deoxy DHA,2 hydroxylation metabolites,1 deoxygenation hydroxylation metabolites and 2 dehydrogenation hydroxylation metabolites;phase ?metabolite is ?-DHA ?-glucuronide.Further semi-quantitative analysis showed that?-DHA ?-glucuronide was the main metabolites of DHA in mice blood and plasma.DHA and phase I metabolites could enter into RBC,which is helpful to further reveal the mechanism of action.In this study,the difference of DHA metabolites in mice blood samples were compared between intragastric administration and tail intravenous injection.Results showed that DHA was metabolized by gastrointestinal tract and more metabolites can be produced in vivo,such as(hydroxylation of DHA)DHA+O,(dehydrogenation hydroxylation of DHA)DHA-H2+O,(dehydrogenation of DHA)DHA-H2,(dehydration hydroxylation)DHA-H2O+O and etc.In this study,a total of 7 metabolites of DHA were identified in infected mice blood samples,including 6 phase ? metabolites and 1 phase ? metabolite.In comparason,a total of 14 metabolites were identified in healthy mice blood samples,including 13 phase ? metabolites and 1 phase ? metabolite.By comparing the differences of DHA metabolites in blood samples between infected and healthy mice,it was found that the types of DHA metabolite in infected blood samples were consistent with that of healthy blood samples,but the number and content of metabolites in infected blood samples were less than that of helthy blood.It was speculated that plasmodium affects the activity of metabolic enzymes in the liver.However,in the intragastric administration group,as DHA was metabolized in gastrointestinal tract,the number of metabolites in infected mice blood samples is not much different with that in healthy mice blood samples.3 Study on DHA metabolites in mice blood samples based on DBS DESI-MSI technologyBased on dry blood spot(DBS)and DESI-MSI technology,a DBS DESI-MSI method was established for the detection of DHA and metabolites in mice blood samples.With this rapid,intuitively and high throughput method,3 new metabolites of DHA were discovered,which made up for the missing metabolites information during the sample pretreatment process of LC-MS analysis.Combined with UPLC-QTOF/MS method,the DBS samples were extracted and detected.Comparing the results of two groups,it was speculated that some metabolites were degraded during the extraction process of the blood samples with organic solvent.4 Study on the distribution of DHA and metabolites in miceIn this study,frozen section technology and DESI-MSI technology were combined to establish the whole-body mass spectrometry imaging(MSI)method.On this basis,the in situ distribution of DHA and metabolites were characterized in vivo for the first time.As time went by,the metabolism process of various metabolites were different:T1,T6,T7 and T9 were mainly distributed in gastrointestinal tract and gradually disappeared;T2,T4 and T8 were mainly distributed in gallbladder and colon,with the characteristics of gradually increasing and widly distribution;T3,T5 and T10 were mainly distributed in small intestine,with the characteristics of increases first and then descrases.In this study,the UPLC-QTOF/MS method was employed to identify the metabolites of DHA in the main organs and tissues of mice.Results showed that a total of 30 metabolites were identified.There were 17,22,14,18,14,10,22,18,20 and 8 metabolites were identified from heart,liver,spleen,lung,kidney,brain,stomach,large intestine,small intestine and gallbladder,respectively.Results showed that DHA and metabolites were widely distributed in mice.Further semi-quantitative analysis showed that DHA prototype was mainly distributed in the gastrointestinal tract,phase I metabolites were mainly distributed in the liver,such as 1-deoxy DHA,1-deoxy ART,DHA-H2O+O,etc.And phase ? metabolite,?-DHA ?-glucuronide,was mainly distributed in the gallbladder.With the combination of UPLC-QTOF/MS and DESI-MSI methods,the metabolic process of DHA in mice was described for the first time.(?)DHA could be transformed into metabolites in the gastrointestinal tract via intragastric administration,such as DHA+O,DHA-O+O,DHA-H2O+O and etc.(?)DHA and some metabolites in gastrointestinal tract were rapidly absorbed and metabolized into various types of metabolites in the liver.Phase I metabolites were mainly distributed in liver and lung,such as 1-deoxy DHA,1-deoxy ART,DHA+O,DHA-H2O+O and etc.Phase ? metabolite,?-DHA ?-glucuronide,was mainly excreted into the intestinal system through the gallbladder.(?)The residual DHA and metabolites enters into the intestinal tract along with the stomach contents and continues to be absorbed or degraded or transformed by the intestinal flora.5 Study on the microbial transformation of DHAIn order to further clarify the structure of DHA metabolites inside RBC,a total of 9 microbial strains were selected to simulate the mammalian metabolites of DHA.The UPLC-QTOF/MS method was employed to identify the biotransformation products.By comparing the conversion rate of strains to DHA and the number of mammalian metabolites,MT9(Cunninghamella elegans ATCC 9245)was selected as the best transformation model to prepare mammalian metabolites of DHA in further research.In summary,from the perspective of DHA metabolites,the UPLC-QTOF/MS and DESI-MSI techniques were employed to identify the metabolites of DHA inside RBC and their in situ distribution in mice for the first time.A comparative analysis of the DHA metabolites between infected and healthy mice blood samples by different route of administration was carried out,and the metabolites of DHA in mice were partially clarified.In order to further structurely identify the DHA metabolites inside RBC,a microbial transformation model was established,which has certain reference significance for subsequent research.