| Cardiotonic steroids(CTS) are a kind of steroids that possesses strong cardiotonic effect. Chemically, cardiac steroids are divided into two types namely cardenolides and bufadienolides. Recently, cardiotonic steroids have been shown to exhibit great potential in inhibiting the growth of malignant tumor cells and inducing cancer cells apoptosis. Therefore researchers have given much attention to their prospects in the prevention and treatment of cancer. Digoxinum, Strophanthin G and UNBS1450(semi-synthetic drug) have been studied, however they were found to be pharmacologically in effective.The clinical use of cardiotonic steroids in the treatment of cancer is limited due to setbacks such as cardiac toxicity, narrow therapeutic window, low drug delivery rate and poor tumor targeting. Hence, there is the need to develop lead compounds without cardiac toxicity and increase antitumor activity. In this study, six kinds of cardiotonic steroids were screened and separated from cortex periplocae and Semen Lepidii. In vitro anti-tumor experimental results proved that all the cardiotonic steroids demonstrated beneficial antitumor effect.The extraction rate,difficulty of synthesis and purification of cardiotonic steroids, influenced the selection of periplogenin(glycoligands of cardiotonic steroids) and periplocymarin(cardiacglycoside) isolated from cortex periplocae for the experiments. Targeting conjugate prodrug and targeting nano-prodrug were formulated and their antitumor activities were evaluated in vitro and in vivo. They might be useful as targeting agents and carriers in improving the safety profile of cardiac glycosides for cancer therapy.Chapter 1: Research advances in anti-tumor activity of cardiotonic steroids and prodrug strategies to improve tumor-targetablity and efficiencyThis review focused on the anti-cancer mechanism of cardiotonic steroids and advances of prodrug design in increasing the tumor-targetablity and efficiency.Meanwhile, the research progress of periplogenin and periplocymarin were also expounded. With respect to the problem in the application of periplogenin and periplocymarin, this study has offered the solutions and established legislation basis,the design idea and conception. This part laid a good theoretical foundation for the development of the experimental work.Chapter 2: Screening and structural analysis of antitumor active ingredients isolated from natural productsIn this chapter, cortex periplocae and Semen Lepidii was chosen as research objective. The six cytotoxic compounds were isolated from cortex periplocae and Semen Lepidii by bioactive tracking method. Structures of the bioactive compounds were elucidated using spectroscopic analysis(1H,13 C nuclear magnetic resonance data;infra red data; electron ionization mass spectrometry).The growth inhibitory activity was measured using MTT bioassay.1. The screening of anti-tumor active components from natural products: The powder Semen Lepidii was extracted by water and was then concentrated to give a dark brown residue. After adding ethanol to filter proteins, the filtered liquor was concentrated through extraction using ethyl ether, dichloromethane and n-butanol accordingly. In vitro antitumor activities of different fractions were evaluated. The results showed that dichloromethane and n-butanol fraction possessed relatively high anti-tumor activity. Through positive and negative silica gel column chromatography,compound 1 was purified from dichloromethane fraction with yield of 0.0013%,compound 2 and 3 were purified from n-butanol fraction with yields of 0.00024% and0.000043%, respectively. Using the same screening method as in Semen Lepidii, Compoud 4 was isolated and purified from n-butanol extraction of cortex periplocaewith yields of 0.00024%. Compound 5 and 6 were obtianed from compound 4 by acid and enzyme hydrolyses, respectively. The yields of compounds isolated from cortex periplocae were signficantly higher than compounds isolated from Semen Lepidii.2. Structural identification and activity comparison of active compounds:Compounds 1-3 were identified to be helveticoside, evonoside and evobioside respectively using spectrum, spectroscopy and mass spectra after their isolation fromSemen Lepidii for first time. Compounds 4-6 from cortex periplocae were identified to be periplocin, periplogenin, and periplocymarin with all them being recognized to be cardiotonic steroids. Then In vitro antitumor activities of different compounds were evaluated. The results showed that all the compounds exhibited strong cytotoxic activities against the human cancer cell lines with periplocymarin emerging as the most effective compound(IC50=0.019±0.012 ?g/m L, in MCF-7). Structure-activity relationship indicated that type and amount of glycans have some influences on the antitumor activity of cardiotonic steroids. Cardiotonic steroids had stronger toxicity for tumor cells than 5-Fu, while it showed lower toxicity towards normol cells than its5-Fu counterparts.Chapter 3:Studies on octreotide-periplogenin conjugates for hepatoma targeting and treatmentOctreotide(OCT) has been applied in the treatment and diagnosis of malignant tumor with high SSTRs expression. Following its successful application on radio-oncology, OCT has aslo been applied in the anti-tumor drug delivery system. In this part,the yield of periplogenin(PPG, yield: 0.048%) was enhanced by isolating and purifing it from n-butanol extraction of cortex periplocae via acid hydrolysis method. Periplogenin was then chosen as research objective, and various octreotide-periplogenin conjugates were synthesized and their cytotoxicity determined.HPLC method for the determination of periplogenin concentration in the mice biological sample was established and biodistribution of octreotide-periplogenin conjugates was investigated. Again, transplanted hepatoma(H22) solid tumor model was established while in vivo anti-tumor effects, cardiotoxicity and hepatotoxicity of octreotide-periplogenin conjugates were evaluated.1. Synthesis of octreotide-periplogenin conjugates and in vitro cytotoxicity evaluation: periplogenin was prepared by acid hydrolysis method, and verified by IR,MS and NMR data. Succinic anhydride was selected as linker while various octreotide-periplogenin conjugates were synthesized and these include OCT(Phe)-S-PPG, OCT(Lys)-S-PPG and OCT-2S-2PPG, which were characterizedby LC-MS, IR, MS and NMR. Cytotoxicity of OCT(Phe)-S-PPG, OCT(Lys)-S-PPG and OCT-2S-2PPG were measured using MTT bioassay and the results showed that IC50 of OCT(Phe)-S-PPG in Hep G2 cell was significantly lower than PPG, however,IC50 of OCT(Phe)-S-PPG in L-02 cell was significantly higher than PPG. It posits that the cytotoxicity of PPG on Hep G2 was enhanced and the toxicity in L-02 cell was reduced after conjugation with OCT. Additionally, the influence of OCT on the cytotoxicity of OCT(Phe)-S-PPG was investigated and the results revealed that OCT significantly reduced cytotoxicity of OCT(Phe)-S-PPG on Hep G2. This means that the enhancement of cytotoxicity is related to endocytosis mediated by SSTRs receptor.Furthermore, cellular uptake experiment proved that OCT(Phe)-S-PPG could enhance cellular uptake of PPG in Hep G2.2. The biodistribution and pharmacodynamics studies of OCT(Phe)-S-PPG:HPLC method for the determination of periplogenin concentrationin the mice plasma,while tissue homogenate and transplanted hepatoma(H22) solid tumor model was established. The biodistribution and pharmacodynamics of OCT(Phe)-S-PPG was also investigated. After single intravenous injection, OCT(Phe)-S-PPG enhanced the distribution of PPG in tumor tissue, while reducing its distribution into heart and liver.Pharmacodynamics results showed that inhibition rate of OCT(Phe)-S-PPG was significantly higher than PPG(69.4% vs 52.18%, P < 0.01). However, results of pathological section revealed an irregularlyoriented cardiocyte and inflammatory cell infiltration in the heart. Moreso, liver cell disorders, vacuolar degeneration, necrosis,wider hepaticsinusoidal and increased hepatic sinusoidal cells were observed. It shows that OCT(Phe)-S-PPG can reduce the cardiotoxicity and hepatotoxicity of PPG.The above results have shown that the cancer cell and umor targeting of periplogenin would be improved by conjugating with octreotide, while their in vivo toxicity was reduced.Chapter 4: Studies on octreotide-periplocymarin conjugates for hepatoma targeting and treatmentIn this section, periplocymarin was isolated from cortex periplocae by enzymehydrolysis method with higher cytotoxicity than periplogenin. Optimization of the periplocymarin preparation was carried. Octreotide-periplocymarin conjugates were synthesized while cytotoxicity of various conjugates was determined by MTT assay.HPLC method for the determination of periplocymarin in the mice plasma as well as tissue homogenate was established. The biodistribution of octreotide-periplocymarin conjugates was investigated. Transplanted hepatoma(H22) solid tumor model was established, and in vivo anti-tumor effects, cardiotoxicity and hepatotoxicity of octreotide-periplocymarin conjugates were evaluated.1. Optimization of the periplocymarin was preparated by enzymatic hydrolysis:Conversion efficiency was selected as indicator. The influence of temperature, p H,substrate concentration, enzyme amount, and reaction time were investigated. The optimum condition was obtained as followed, temperature: 50 °C, reaction medium:citrate-citric acid trisodium buffer solution(p H=5.0), substrate concentration: 50 g/L,enzyme / substrate =0.6: 1, reaction time: 24 h. Molecular weight of the product was534.63, and it is verified by NMR data to be periplocymarin(yield: 0.075%).2. Synthesis of octreotide-periplocymarin conjugates and in vitro cytotoxicity evaluation: Succinic anhydride was selected as linker and various octreotide-periplocymarin conjugates were synthesized, which included OCT(Phe)-S-PPM, OCT(Lys)-S-PPM and OCT-2S-2PPM. HPLC-UV, IR and NMR were used to characterized them and they demonstrated purity of 95%. Cytotoxicity of various octreotide-periplocymarin conjugates was determined by MTT method.Results demonstrated that the cytotoxicity of OCT(Phe)-S-PPM was highest in MCF-7 and Hep G2, and significantly higher than PPM. On the contrary, toxicity of OCT(Phe)-S-PPM in L-02 was significantly lower than PPM. All results indicated that OCT(Phe)-S-PPM could improve the antitumor activity with lower hepatotoxicity and selectivity.3. The biodistribution and pharmacodynamics studies of OCT(Phe)-S-PPM:After single intravenous injection, PPM mainly distributed in liver, kidney and heart,but few distributed in tumor. On the contrary, OCT(Phe)-S-PPM enhanced thedistribution of PPM in tumor tissue, while educing its distribution in the heart and liver. It indicates that OCT(Phe)-S-PPM could increase the drug delivery and its targeting ability into tumor. The results of pharmacodynamics showed that inhibition rate of OCT(Phe)-S-PPM was significantly higher than PPM(69.4% vs 52.18%, P <0.01). The results of pathological section showed irregularlyoriented cardiocyte and inflammatory cell infiltration in the heart. Moreso, liver cell disorders, vacuolar degeneration, necrosis, wider hepaticsinusoidal and increase in hepatic sinusoidal cells were also observed. However, no significant change was observed in the OCT(Phe)-S-PPM group. Liver function test also proved that hepatotoxicity of OCT(Phe)-S-PPM was obviously lower than PPM. It indicates that OCT(Phe)-S-PPM could minimize the cardiotoxicity and hepatotoxicity of PPM. Dense tumor cell and hyperchromia were observed in blank control group, yet significant fewer tumor cell and necrocytosiswere were observed in PPM group and OCT(Phe)-S-PPM group. It was obviously found in OCT(Phe)-S-PPM group, indicating that OCT(Phe)-S-PPM could improve in vivo antitumor activity of PPM. The above results showed that after conjugating with OCT, PPM exhibited better cancer cell and tumor targeting coupled with markedly lowering toxcity as well as improving its therapeutic effects compared with free periplocymarin. This experiment laid the theoretical foundation and innovative ideas for the key technology of targeting cardiac steroid prodrug.Chapter 5: The study of oxidative-reductive sensitive PEGylated self-assembly prodrug nanoparticlesIn this section, periplocymarin and vitamin E were conjugated with dithiodiglycolic acid to form prodrug. Then the prodrug was dissolved in ethanol with methylether polyethylene glycol 2000–linoleate(m PEG2000-LD), to prepare oxidative-reductive sensitive PEGylated self-assembly Vitamin E-periplocymarin nano-prodrug nanomedicine by precipitation method(MPSSV-NPs). Characterization,stability and in vitro release studies of MPSSV-NPs were investigated.Pharmacokinetics parameters, biodistribution in tumor(H22) and in vivo antitumor activity were evaluated.1. Synthesis and characterization of PEGylated self-assembly Vitamin E-periplocymarin nano-prodrugnanomedicine by precipitation method(MPSSV-NPs):dithiodiglycolic acid was selected as linker to synthesize Vitamin E-periplocymarin conjugate(PPM-S-S-VE). Esterification was used to synthesizemethyl ether polyethylene glycol 2000-linoleate conjugate(m PEG2000-LD). Target products PPM-S-S-VE and m PEG2000-LD were confirmed by ESI-MS and NMR data.Subsequently, PPM-S-S-VE nanoparticles(PSSV-NPS) was prepared by nanoprecipitation in water of PPM-S-S-VE. PEGylated PPM-S-S-VE nanoparticles(MPSSV-NPs) were obtained by co-nanoprecipitation in water of PPM-S-S-VE with various amount of m PEG2000-LD. The results of mean particle size and Zeta potential showed that the smaller size could be obtained with the increase in m PEG2000-LD amount, and polydispersity index of nanoparticles were smaller than 0.2.Transmission electron microscopy showed that PSSV-NPs and MPSSV-NPs were spherical and homogeneous particles. Stability study demonstrated that PSSV-NPs was not stable in PBS(p H=7.4), but MPSSV-NPs showed no significant changes in mean particle size during the storage period for 1 month. In vitro release study results showed that the release of PPM from MPSSV-NPs and the degradation of conjugates were oxidative-reductive sensitive.2. Pharmacokinetics, biodistribution and pharmacodynamics of MPSSV-NPs:After single intravenous injection of MPSSV-NPs in rat, Cmax was reduced significantly, and Tmax, t1/2 and AUC0-24 h were increased obviously to 8, 91.86 and21.68 times respectively as comparated with PPM. Compared to PSSV-NPs, Cmax,Tmax, t1/2 and AUC0-24 h were enhanced obviously to 4.43, 2, 8.99 and 9.25 times respectively. The distribution results in H22 tumor mice showed that PSSV-NPs and MPSSV-NPs were mainly distributed in liver, spleen and lung, and significantly higher than PPM. The distribution of MPSSV-NPs in tumor was obviously higher than PSSV-NPs and PPM, but there was no significant difference between PSSV-NPs and MPSS-NPs in heart distribution. Pharmacodynamics demonstrated that the solid tumor volume of PPM, PSSV-NPs and MPSSV-NPs were significantly smaller thanblank control group. The inhibition rate of MPSSV-NPs was 66.41%, significantly higher than PPM and PSSV-NPs. Pathological section showed that irregularlyoriented cardiocyte, myocardial fibrosis and lymphocytes infiltration in heart were observed in PPM group. However, no obvious histopathological effects on hearts were observed on PSSV-NPs, MPSSV-NPs and PBS groups. For the PPM group, the liver sections exhibited thinner hepatic cell, dilation of hepaticsinusoids, increased atrophyin liver,but there were only slight damage in PSSV-NPs and MPSSV-NPs groups. Tumor cell mitosis index, cell density and round or oval vesicular nuclei were observed in blank control group. But for PPM and PSSV-NPs groups, there were fewer tumor cell and larger cell necrosis area than in the control group. It was found more obviously in MPSSV-NPs group. This indicates that MPSSV-NPs could improve in vivo antitumor activity of PPM. The above results proved that redox-responsive PEGylated self-assembled prodrug-nanoparticles could exhibit improved blood circulation, tumor distribution and tumor suppression effects. Therefore, this study provides new method and thinking for the risk-benefit ratio and nano targeted drug delivery of PPM. |
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