Physico-chemical characterization of 10-hydroxycamptothecin and formulation approach towards improving its solution stability and solubility.

摘要

Naturally occurring alkaloid, 10-hydroxy-camptothecin (10HC) is a promising structural derivative of camptothecin which possesses the ability to inhibit a wide range of human tumors. However, its anti-tumor potential has not been fully realized owing to the pH dependent hydrolytic instability and poor aqueous solubility.To obtain the kinetic and thermodynamic parameters of hydrolysis, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of 10HC. Validation of the analytical method was done with respect to reproducibility, % recovery, and level of detection. Hydrolysis of the lactone ring of 10HC followed a 1st order decay with a rate constant equal to (0.0281 +/- 0.001) min-1 in PBS at pH 7.4 and at a temperature of 37 °C. The activation energy for the hydrolysis reaction as calculated from the Arrhenius equation was (79.41 +/- 0.92) kJ mol -1, whereas the enthalpy and entropy of hydrolysis of 10-hydroxy-camptothecin were on average 12.45 kJ mol-1 and 52.37 J K-1 mol-1, respectively. The positive enthalpy and entropy values of the 10HC-lactone hydrolysis indicate that the reaction is endothermic and entropically driven.Physicochemical characterization was carried out to fully characterize the poor aqueous solubility and solid-state properties of 10-hydroxy-camptothecin (10HC). Molecular and system properties were determined from titration, partition and solubility studies using UV and fluorescence spectroscopy, while solid state characterization of the 10HC was carried out with x-ray, DSC and TGA. The enthalpies of solution of the unionized and ionized forms of 10HC, as deduced from isothermal and iso-pH equilibrium solubility measurements, were 45.4 kJ·mol-1 and 22.7 kJ·mol-1, respectively. The pKa of 10HC was determined to be 1.42 at 25 °C, while the basicity of the quinoline group of 10HC was shown to decrease with increasing temperature due to a positive enthalpy of deprotonation of 23.6 kJ.mol-1. The intrinsic partition coefficient of 10HC was determined to be 6.49, which is significantly smaller than that of the parent camptothecin. Evidently, the hydroxyl substitution on the A ring of camptothecin renders the molecule considerably more polar, though still hydrophobic and sparingly soluble in aqueous media. Dissolution studies supported by x-ray and thermal analysis revealed polymorphism and serious metastability of the 10HC anhydrous form in aqueous solutions. The aqueous solubility of 10HC-lactone monohydrate was found to be pH and temperature dependent with an estimated intrinsic solubility of 1.81 +/- 0.215 muM. Contrary to the low intrinsic solubility, the solubility of 10HC in extremely acidic media increased by more than 3 orders of magnitude.Furthermore, the purpose of the present work was to study the pH-solubilization behavior of 10HC, and to systematically discern the improved solubility and stability of 10-hydroxy-camptothecin in presence of pharmaceutically acceptable co-solvents and surfactants. Drug concentrations following equilibration of 10HC in various pH solutions and at different weight fractions of the cosolvents PEG400, PEG1450, PEG6000, PEG8000, glycerol and propylene glycol and, surfactants F-68, F127, Gelucire and Sodium Dodecyl Sulfate (SDS) were determined using HPLC. The kinetic and thermodynamic parameters of hydrolysis were determined by monitoring the drug hydrolysis using a derivative spectroscopic method. Association of drug to micelles was studied using fluorescence anisotropic technique. Based on the pH-solubility profile, the pKa of the phenol group of 10HC was determined to be 7.6 +/- 0.023. The presence of 10HC-lactone at highly basic pH was attributed to the pseudo- equilibrium between the anionic 10HC-lactone and 10HC-carboxylate. An exponential and a linear increase in the solubility of 10HC was observed with increasing concentrations of the cosolvents and the surfactants, respectively, with PEG400 and SDS showing the maximum solubilization efficiency. (Abstract shortened by UMI.)