Physicochemical characterization of 10-hydroxy-camptothecin (10-HC) was carried out to fully characterize the poor aqueous solubility and hydrolytic instability that severely limit its efficient delivery and pharmacological activity. Molecular and system properties were determined from titration, partition, and solubility studies using UV and fluorescence spectroscopy, while solid state characterization of the 10-HC was carried out with X-ray, DSC, and TGA. The enthalpies of solution of the unionized and ionized forms of 10-HC, as deduced from isothermal and iso-pH equilibrium solubility measurements, were 45.4 kJ·mol−1 and 22.7 kJ·mol−1, respectively. The pK a of 10-HC was determined to be 1.42 at 25 °C, while the basicity of the quinoline group of 10-HC 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 10-HC 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 10-HC anhydrous form in aqueous solutions. The aqueous solubility of 10-HC-lactone monohydrate was found to be pH and temperature dependent with an estimated intrinsic solubility of (1.81 ± 0.2) μM. Contrary to the low intrinsic solubility, the solubility of 10-HC in extremely acidic media increased by more than 3 orders of magnitude. This feature can be used to facilitate fabrication of highly efficient drug delivery systems for 10-HC systemic administration.

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To accurately derive the kinetic and thermodynamic parameters governing the hydrolysis of the lactone ring at physiological pH, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of camptothecin (CPT). The hydrolysis of the CPT-lactone and the lactonization of CPT-carboxylate at 310.15 K followed a first-order decay with apparent rate constants equal to 0.0279 ± 0.0016 min −1 and 0.0282 ± 0.0024 min −1 , respectively. The activation energy associated with the hydrolysis of the CPT-lactone and the lactonization of the CPT-carboxylate as calculated from the Arrhenius equation was 89.18 ± 0.84 and 86.49 ± 2.7 kJ mol −1 , respectively. The enthalpy and entropy of the thermodynamically favored hydrolysis reaction were on average 10.49 kJ mol −1 and 48.00 J K −1 mol −1 , respectively. The positive enthalpy and entropy values of the CPT-lactone hydrolysis indicate that the reaction is endothermic and entropically driven. The stability of CPTlactone in the presence of human serum albumin (HSA) was also analyzed. Notwithstanding the much faster hydrolysis of the CPT-lactone in the presence of HSA at various temperatures, the energy of activation was determined to be similar to the one estimated in the absence of HSA, suggesting that HSA does not catalyze the hydrolysis reaction, but it merely binds, sequesters, and stabilizes the CPT-carboxylate species. C 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 704-

Kinetic and thermodynamic analysis of the 9-nitrocamptothecin (9NC) hydrolysis reaction in the presence and absence of human serum albumin (HSA) in phosphate-buffered saline (PBS) of pH 7.4 was carried out by first derivative absorption spectroscopy. The thermodynamic parameters determined in these studies provided a mechanistic explanation toward the endothermic but yet thermodynamically favorable hydrolysis of 9NC at physiological temperature and pH. In the presence of HSA, the apparent rate constant of 9NC hydrolysis was 3-3.5 times higher than in 9NC solutions alone, whereas the apparent equilibrium constant of 9NC hydrolysis was found to increase at a higher extent in the presence of HSA than in PBS with increasing temperature, reaching almost complete hydrolysis of the 9NC to the 9NC-carboxylate at 315.15 K. Importantly, the E a of the 9NC hydrolysis reaction in the presence of HSA was determined to be on average 17 kJ mol −1 lower than the E a determined in plain PBS. Moreover, analysis of binding isotherms constructed for the HSA interaction with 9NC, using infinitely cooperative and independent binding models, demonstrated an incredibly higher binding constant for the 9NC-carboxylate form as compared to the very weak and concentration-dependent binding for the 9NC-lactone species at 310.15 K. Taken together, the preferential association of the carboxylate form with HSA and the lower E a of 9NC hydrolysis in the presence of HSA provide a mechanistic explanation for the shift of lactone-carboxylate equilibria toward the carboxylate product under physiological conditions of pH and ionic strength. C

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