Synthesis, Pharmacokinetics And Tissue Distributions Of MPEG-AZT Conjugate

Objectives: Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). The reverse transcriptase (RT) plays a crucial role in the replication of HIV life cycle, and is responsible for the conversion of the single-stranded RNA viral genome into double-stranded DNA that subsequently integrates into the host DNA. Up to now, 22 drugs have been approved by FDA and applied in clinical therapy, including 7 kinds of nucleoside reverse transcriptase inhibitors (NRTIs), 3 kinds of nucleotide reverse transcriptase inhibitors (NtRTIs), 3 kinds of non-nucleoside reverse transcriptase inhibitors (NNRTIs), 8 kinds of protease inhibitors (PIs) and 1 kind of fusion inhibitor. As the first drug of anti-HIV therapy, NRTIs have become irreplaceable, and are the major choice in clinic. However, the serious side effects, emergent resistance to the virus strains and bad pharmacokinetics properties of this kind of drugs limited their application in clinic. For example, pharmacokinetics parameters of t_1/2 are only 1.2h for both AZT and ddI. So, to improve the feature of drug's pharmacokinetics is a major concern in HIV NRTIs research.In this thesis, according to the principle of prodrug in drug design, pegylation technique was used to modify the AZT molecule to form the mPEG-AZT conjugate. The synthetic routes were optimized to fit for the production of industrial scales. Also, the release experiment in vitro, the pharmacokinetic natures and tissue distribution of mPEG-AZT conjugate in two types of administration routes, were systemically investigated for the evaluation of its improved pharmacokinetics feature, which lie solid foundation for further drug development.Methods: The synthesis of mPEG-AZT conjugate was carried out starting from the acylating reaction of mPEG2000 with succinic anhydride to produce mPEG-SA by introduced succinic acid (SA) at the terminal hydroxyl group of mPEG The important intermediate mPEG-SA was used to react with AZT in following two ways. (1) mPEG-AZT was obtained by the esterification of mPEG-SA with 5'-hydroxy of AZT catalyzed by DCC and DMAP. (2) mPEG-AZT was prepared by ester exchange reaction of AZT with one active ester, which was synthesized by acylating reaction of mPEG-SA with N-hydroxylsuccinimide (NHS). Finally, the best synthesis route of mPEG-AZT for industrial production was screened by comparative studies of the procedures, yields, characters and purities of mPEG-AZT product.mPEG-AZT release character in different kinds of release media was evaluated by dialysis bag diffusion technique. Some mathematical models, including zero level kinetic equation, one level kinetic equation, Higuchi equation and Weibull equation were adopted to simulate the release behavior in vitro. The pharmacokinetic properties in vivo were investigated after oral and intravenous (i.v.) administration of mPEG-AZT, and AZT was used as controlling drug for comparative purpose. At the same time, the tissue distribution in heart, liver, spleen, lung, kidney, brain and blood was detected after i.v.. In addition, the targeting effect of mPEG-AZT was evaluated by parameters of tissue distribution.Results: The structure of mPEG-AZT conjugate was confirmed by FT-IR,¹H-NMR and ¹³C-NMR spectrum, and its molecular weight distribution was inferred by EI-MS spectrum; The reaction manipulation was simplified, and the reaction condition was optimized with an excellent yield of 95%.Results of release experiments in different kinds release media were in accord with mathematical models, which is expressed by the Higuchi equation: Q=13.1580t^1/2-5.5911 (r=0.9977) while in PBS (pH6.8) media, followed by expression of Weibull equation LnLn (1-Q)^-1=0.7513Lnt-1.6040(r=0.9918) while in dilute hydrochloric acid solution (pH1.2). The release quantities of AZT can be described by zero level kinetic equation in both release media.The parameters of pharmacokinetics of mPEG-AZT in mice administrated by oral and i.v. could be described by double phase kinetics model. By oral administration, t_1/2β of mPEG-AZT is prolonged to 2.083h and AUC (0-12h) is increased to 96.20 mg/L·h in comparison to AZT( t_1/2β =1.161h and AUC = 89.72mg/L·h). By i.v administration, the half life of AZT is 1.091h, and mPEG-AZT becomes 2.032h in the same case, the half life is nearly doubled increase(P<0.05), AUC(0-12h)is improved from to 74.613 in contrary to AZT (63.578). The data showed that mPEG-AZT has longer MRT and better absorption than AZT, and the distribution feature of mPEG-AZT has obviously altered by the changed tissue distribution. The relative ingest rate of both liver and spleen exceeds 1, to be 2.076 and 1.829, which means that mPEG-AZT has the targeting feature to liver and spleen. At the same time the relative ingest rate of heart, kidney and brain is 0.751, 0.486 and 0.626 respectively, all less than 1.These parameters indicate that the prodrug could decrease the toxicity to heart, kidney and nerve to some extend.Conclusion: The stability of the drug has been improved by modifying AZT to be mPEG-AZT. The results of mPEG-AZT release in vitro revealed notable slow-release, and the pharmacokinetics parameters in vivo showed that mPEG-AZT has a longer half life time and better bioavailability than AZT. mPEG-AZT is one promising prodrug of HIV-1 NRTIs and worth to be further developed as an anti-AIDS drug. This study has not been reported in the literature at domestic and aboard.