| ObjectivesFuzi, which is the treated lateral roots of Aconitum carmichaeli, has been used as a famous Chinese medicinal herb for the treatment of colds, polyarthralgia, diarrhea, heart failure, beriberi, and edema for thousands of years. Raw aconite roots are used only after processing due to their toxicity, which results in side effects and adverse clinical reactions such as severe arrhythmia. However, poisoning may still cause adverse reaction and limit its clinical application to a large extent. Thus, the determination of the safe application of Fuzi is urgent and essential. In order to develop and enlarge the usage of Fuzi, it is necessary to do the research on the absorption, distribution, metabolism and excretion (ADME) in vivo and in vitro. The aim of this thesis is to elucidate the toxicokinetics, absorption and metabolic mechanism of aconitine, which is helpful to evaluation of drug therapy, drug toxicity and clinical drug safety.Methods1. The Toxicokinetics of Aconitine as Targeted Marker of Fuzi Following Single and Multiple Doses 1.1 Determination of safety window of aconitine in miceThe death rate and general states of mice was used to evaluate the acute toxic reaction and the median lethal dose (LD50) of the main diterpenoid alkaloids. The writhing response induced by acetic acid (0.7%) in mice was used to evaluate the analgesia actions.1.2 The toxicokinetics of pure aconitine and aconitine in Fuzi in ratsPrevious studies have shown that Fuzi has a narrow therapeutic index because of the aconitine alkaloids, which are known for their high toxicity and toxicokinetics activity as well as being the target markers of the crude malcohol extracts of Fuzi. Therefore, the main purpose of the present study is to characterise the toxicokinetics behaviours of aconitine as the targeted marker of Fuzi. The prolonged administration of Fuzi for the treatment of chronic diseases, such as bradycardia sexarrhythmia, increases the danger of toxic reaction over time. Thus, elucidating the toxicokinetics behaviour of Fuzi following multiple administrations is very essential. No literatures published the toxicokinetics after administiation of multiple doses of pure aconitine or Fuzi extracts, let alone the comparison of toxicokinetics characteristics of pure aconitine and Fuzi extracts or the comparison between single dose and multiple doses. In this thesis, we investigated the toxicokinetics behaviours of aconitine following single and multiple administrations of pure aconitine or raw Fuzi extracts in order to compare the toxicokinetics characteristics of aconitine between pure aconitine and Fuzi extracts as well as compare the difference at single dose and multiple doses. Gaining information on the toxic and pharmacological effects of Fuzi is of utmost importance.A sensitive and fast UPLC-MS/MS method was successfully developed, which could analyze AC and its six metabolites in CYP enzymes and in rat plasma. After the last administration of aconitine or alcohol isolated aconite extract, blood samples were taken from eyes. The plasma concentration was detected by UPLC/MS/MS and analysed by 3P97.1.3 Plasma protein bindingAlthough some basic information about the bioavailability of AC is readily accessible, no data on its plasma protein binding are presently available. The pharmacological activity of a compound depends on its pharmacokinetic as well as its pharmacodynamic properties, which may be affected by plasma protein binding. Hence, the AC protein binding mechanism should be revealed to gain further insight into the bioactivity of Fuzi extract.2.The Absorption of Aconitine in RatsRat intestinal perfusion model, an in situ model with intact circulation, was used to study regional intestinal absorption. One-way ANOVA with post hoc was used to determine the differences of the amounts of aconitine absorptive from different regions of the intestine when 10M aconitine was perfused.3.The Metabolism of Aconitine by Human CYP IsoformsIt was reported that aconitine could be mainly metabolized by cytochrome P450 in rat liver microsomes. However, the role of CYP in aconitine metabolism is unknown in humans, also is unknown in the aspect of in-depth understanding of which CYP isoform(s) responsible for producing each metabolites, in other words, the CYP-mediated metabolism pathway is unknown. It has been estimated that 90% of human drug metabolism can be attributed to seven main enzymes (CYP 1A2,2C9, 2C8,2C19,2D6,2E1 and 3A4/5). Therefore, an in vitro study was conducted to identify the metabolites of aconitine in recombinant human cytochrome P450 enzymes by ultra performance liquid chromatograph-tandem mass spectrometry (UPLC/MS/MS). These results would provide useful and urgent information of aconitine that is useful in toxicology, pharmacology, and clinical use. Results1. The LD50 of aconitine, mesaconitine and hypaconitine was 3.17 mg/kg,2.11 mg/kg and 3.11 mg/kg; aconitine, mesaconitine and hypaconitine can obviously decrease the writhing reaction of mice induced by acetic acid compared to the control group (p<0.05).2. Aconitine was absorbed after i.v administration at 40μg/kg. The half life of elimination (T1/2) was 80.98±6.40 min and the AUC was 3201.89±338.91 (ng/ml)*min. The absolute bioavailability of aconitine in rat was 8.31±0.89%.3. Pure AC exhibited similar pharmacokinetic behaviors after single and multiple administrations. No significant variations in the pharmacokinetics parameters of single and multiple doses were observed (ANOVA, P>0.05).4. Multiple administrations of Fuzi extract resulted in variations in its pharmacokinetic behavior.A statistically significant difference in Tmax between single and multiple oral admininistrations of Fuzi extracts was observed probably because of the other complex ingredients in Fuzi that could affect the pharmacokinetics behavior of this compound after prolonged exposure. In addition, the AUC, MRT, Tmax, and T1/2 were insignificant compared with those of a multiple dose of pure AC (ANOVA, P>0.05). In addition, the absolute bioavailability of aconitine in rats after single administration of Fuzi extract and the total alkaloids were 4.72±2.66% and 5.79±0.82%.5. The plasma protein binding rates of aconitine at low, middle and high concentrations were 31.9%±1.69%,27.4%±1.71% and 23.9%±2.51%, respectively.6. There were no significant differences (one-way ANOVA with post hoc test, P> 0.05) for the aconitine absorption in different regions of the intestine when 10M aconitine was perfused. The absorbed amount of aconitine in duodenum, jejunum, ileum and colon in rats were 29.18±8.64%,22.60±3.00%,25.03±6.97% and 15.02±2.75%, respectively.7. Recombinant CYP3A4 produced M1, M2, M3 and M5; CYP3A5 metabolized aconitine to all of the six metabolites. Recombinant CYP2D6 produced all the six metabolites except M5. Recombinant CYP2C8 could only produce M1 and CYP2D9 produce M2 and M3. Additionally, single CYP enzyme experiments showed that CYP1A2 and 2E1 cannot metabolism aconitine at all.Conclusions1. Aconitine, mesaconitine and hypaconitine have excellent effect on airritation with high toxicity, which is used to cause adverse effects and limit clinical application of fuzi.2. The absorption of AC was very fast both for pure AC and Fuzi extract. Moreover, AC was rapidly eliminated with a short T1/2 (i.v.,80.98±6.40 min). Pure AC exhibited similar pharmacokinetic behaviours after single and multiple administrations. In contrast, Fuzi exhibited variations in its pharmacokinetic behaviour after multiple administrations of extracts probably due to its other complex ingredients.3. The main metabolic pathway (O-de-methylation,N-de-ethylation, de-hydrogenation and hydroxylation) of aconitine was conducted by CYP 3A4/5 and CYP 2D6. |
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