In the present study, we describe the synthesis and physicochemical properties of a novel pH- and thermoresponsive micellar drug delivery system for an anticancer ellipticinium derivative based on the triblock copolymer poly(ethylene oxide)-block-[tert-butylacrylamide-co-6-(N-methacryloylamino)hexanoic acid hydrazide]-block-poly(ethylene oxide). The system was designed to meet the basic criteria required for drug carrier systems, namely, solubility in water (overcoming the insolubility of ellipticine), satisfactory drug loading, particle size suitable for an efficient enhanced permeability and retention effect and adequate stability in blood plasma (pH 7.4) followed by rapid drug release in tumors or tumor cell endosomes (pH<6.5). The copolymer in the form of a unimer can be eliminated by kidneys because the weight-average molecular weight of 21 kDa is sufficiently below the renal threshold. The half-life of drug release in a pH 5.0 buffer solution (pH of a late endosome) was ~45 h, but a negligible amount of the free ellipticine derivative was detected at pH 7.4 (pH of blood). Consequently, this supramolecular polymer conjugate is a good candidate for the delivery of ellipticine-based drugs and will therefore be subjected to more detailed studies.
- MeSH
- Ellipticines administration & dosage blood therapeutic use MeSH
- Hydrogen-Ion Concentration MeSH
- Micelles * MeSH
- Neoplasms drug therapy MeSH
- Half-Life MeSH
- Polymers chemistry MeSH
- Antineoplastic Agents administration & dosage blood therapeutic use MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The (32)P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system.
- MeSH
- DNA Adducts chemistry metabolism MeSH
- Ellipticines administration & dosage chemistry pharmacokinetics MeSH
- Rats MeSH
- Metabolic Clearance Rate MeSH
- Micelles MeSH
- Organ Specificity MeSH
- Rats, Wistar MeSH
- Drug Compounding methods MeSH
- Antineoplastic Agents administration & dosage chemistry pharmacokinetics MeSH
- Tissue Distribution MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds. METHODS: HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot. RESULTS: The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line. CONCLUSION: The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.
- MeSH
- Models, Biological MeSH
- Ellipticines administration & dosage pharmacology MeSH
- Histone Deacetylase Inhibitors administration & dosage pharmacology MeSH
- Microsomes, Liver drug effects metabolism MeSH
- Rats MeSH
- Valproic Acid administration & dosage pharmacology MeSH
- Hydroxamic Acids administration & dosage pharmacology MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Brain Neoplasms drug therapy genetics pathology MeSH
- Neuroblastoma drug therapy genetics pathology MeSH
- DNA Damage MeSH
- Drug Evaluation, Preclinical MeSH
- Antineoplastic Combined Chemotherapy Protocols pharmacology therapeutic use MeSH
- Treatment Outcome MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action with promising brain tumor specificity. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP) - and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. The toxicity of ellipticine to U87MG glioblastoma cells and mechanisms of its action to these cells are aims of this study. METHODS: Ellipticine metabolites formed in U87MG cells were analyzed using HPLC. Covalent DNA modifications by ellipticine were detected by 32P-postlabeling. CYP enzyme expression was examined by QPCR and Western blot. RESULTS: U87MG glioblastoma cell proliferation was efficiently inhibited by ellipticine. This effect might be associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP1A1, 1B1 and 3A4, lactoperoxidase and cyclooxygenase 1, the enzymes expressed in U87MG cells. Moreover, by inducing CYP1B1, 3A4 and 1A1 enzymes in U87MG cells, ellipticine increases its own enzymatic activation, thereby enhancing its own genotoxic and pharmacological potential in these cells. Ellipticine concentration used for U87MG cell treatment is extremely important for its pharmacological effects, as its metabolite profiles differed substantially predicting ellipticine to be either detoxified or activated. CONCLUSION: The results found in this study are the first report showing cytotoxicity and DNA adduct formation by ellipticine in glioblastomas.
- MeSH
- DNA Adducts metabolism MeSH
- Autoradiography MeSH
- Ellipticines administration & dosage metabolism pharmacology MeSH
- Glioblastoma drug therapy metabolism pathology MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Cell Line, Tumor MeSH
- Polymerase Chain Reaction MeSH
- Cell Proliferation drug effects MeSH
- Antineoplastic Agents administration & dosage metabolism pharmacology MeSH
- Phosphorus Radioisotopes MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- Cell Survival drug effects MeSH
- Chromatography, High Pressure Liquid MeSH
- Dose-Response Relationship, Drug MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- DNA Adducts analysis chemistry MeSH
- Ellipticines administration & dosage pharmacokinetics therapeutic use MeSH
- Research Support as Topic MeSH
- Micelles MeSH
- Models, Animal MeSH
- Rats, Wistar MeSH
- Antineoplastic Agents administration & dosage pharmacokinetics therapeutic use MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- MeSH
- Ellipticines administration & dosage pharmacology MeSH
- Research Support as Topic MeSH
- Histone Deacetylases administration & dosage pharmacology MeSH
- Valproic Acid administration & dosage pharmacology MeSH
- Cell Line, Tumor MeSH
- Neuroblastoma therapy MeSH
- In Vitro Techniques MeSH
- Publication type
- Congress MeSH
- MeSH
- DNA Adducts MeSH
- Ellipticines administration & dosage chemistry MeSH
- Research Support as Topic MeSH
- Rats MeSH
- Antineoplastic Agents administration & dosage chemistry MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Congress MeSH
