Potassium Chloride → Topotecan

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Camptothecin drugs reversibly complexed to synthetic oligonucleotides in their lactones forms were produced in phosphate-buffered-saline using the following procedure. The Phosphate-buffered saline (PBS) buffer contained 8 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 3 mM KCl (pH 7.4). The oligonucleotides d(CGTACG) were synthesized on an automated DNA synthesizer at the Genetic Facility of University of Illinois at Urbana Champaign. In addition, (dG-dC)15 and (dA-dT)15 were purchased from IDT (Coralville, Iowa). The CPT-11 used in the study was generously provided by Yakult (Tokyo) and topotecan (TPT) was obtained from the National Cancer Institute. The drug lactone stock solutions were prepared in aqueous solution at 2 mM, pH 5. Solutions of drug-DNA complexes were prepared by mixing the appropriate amounts of drug stock solution and DNA stock solution in PBS, followed by pH adjustment to the desired value. Drug-DNA complexes solutions were vacuum-dried in a SpeedVac at room temperature, and then dissolved in 0.5 ml 99.8% D2O for 1D 1H NMR spectra; or in 0.5 ml 90% H2O/10%/D2O for 1D H2O spectra. The final DNA duplex concentrations ranged between 0.7 to 1 mM for all 1D NMR spectra. 1D H2O NMR spectra were collected using the 1-1 pulse sequence (Sklenar, V.; Brooks, B. R.; Zon, G.; Bax, A. FEBS Lett 1996, 216, 249-252). The NMR spectra were recorded on a Varian VXR500 (University of Illinois, Urbana, Ill.) and Varian Inova 500 (University of Kentucky, Lexington, Ky.) 500 MHz spectrometers. The chemical shifts (in ppm) were referenced to the HDO peak which was calibrated to a 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) external standard at different temperatures. The NMR data were processed with the program FELIX v. 1.1 (Hare Research, Woodinville, Wash.) or FELIX 95.0 (MSI) on Silicon Graphics workstations. The UV spectrum of a drug-DNA complex was obtained by subtracting the spectrum of free DNA from the spectrum of the complex. The hydrolysis kinetics of both drugs were determined by the quantitative reversed-phase high-performance liquid chromatographic (HPLC) methods as decribed previously [Mi, Z.; Malak, H.; Burke, T. G. Biochemistry 1995, 34, 13722-13728; Warner, D. L.; Burke, T. G. J. Chromatogr. B 1997, 691, 161-171]. DNA stock solutions were prepared by dissolving the oligos in PBS at a concentration of ˜30 mM base concentration, with adjustment of pH to 7.4. 5 μg1 of 1 mM drug stock solutions (either lactone form or carboxylate form) were then added to 0.5 ml pH 7.4 PBS or DNA stock solutions and assayed by HPLC to determine lactone stability or relactonization parameters, respectively. We observed a stronger hypochromic effect shift in the UV spectra of TPT in the presence of (dG-dC)15 vs. (dA-dT)15 is consistent with a stronger interaction with the former, albeit differences in transition state dipole interactions could also account for the observed effect. 2D-NOESY experiments were also completed for both CPT-11 and TPT complexed to d(CGTACG) at different drug:DNA ratios of 1:1, 2:1, and 3:1. The drug-DNA complexes formed in this manner could be delivered in a number of different manners.

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