1. Introduction
Doxycycline (DOX) is a drug effective against a large variety of bacteria. In addition, it has antiprotozoal actions and may be administered in conjunction with quinine in the management of falciparum malaria. Unfortunately DOX is quite susceptible to light, thus diverse strategies towards increasing its photostability are envisioned. A commonly applied approach to increase the stability of drugs is the formation of complexes with macromolecules, of which molecular encapsulation with β-cyclodextrin (βCD) constitutes an alternative for the development of new pharmaceutical dosage forms. The objective of this work was to investigate the possibility of obtaining an inclusion complex between DOX and βCD in order to enhance this drug stability, by combining both experimental and theoretical approaches.
2. Methods
For experimental studies, nuclear magnetic resonance techniques (1H NMR and 2D ROESY) were determined in solution (D2O, pD=2.3). All experiments were performed on a Bruker® Avance II High Resolution Spectrometer. Theoretical studies were performed applying molecular modeling and docking techniques using Gaussian03 and Autodock3 packages. Molecular dynamics simulations and energetic analyses were applied using the Amber9 software suite.
3. Results
The minimum energy conformation of DOX was obtained by simmulated annealing analyses, which was afterwards employed for the molecular docking calculations. Docking results suggested the formation of an inclusion complex between DOX and βCD, with a unique cluster being predicted. In this model, the aromatic ring of DOX (ring D, Fig. 1) was inserted into βCD hydrophobic cavity, with a mean docked energy of -11.03 Kcal/mol. Molecular dynamics simulations in explicit solvent conditions showed that the inclusion complexed is maintained throughout the simulation (5 ns), with ring D deeply buried into the βCD cavity and ring A oriented toward the wide ring of βCD. Energetic component analyses demonstrated that the complex is mainly stabilized by hydrophobic contacts, with a minor contribution of electrostatic interactions.
The 1H NMR studies evidenced a marked shielding effect on internal βCD protons, which confirms the inclusion complex formation. 2D ROESY assays showed correlation between the internal protons of βCD and aromatic protons of DOX, suggesting that ring D is inserted into βCD cavity.
4. Conclusion
We were able to successfully combine molecular modeling studies with experimental spectroscopic assays, in order to elucidate the molecular basis of DOX-βCD interaction. The work performed allow us to study the complexation between DOX and βCD at a molecular level.
Acknowledgments
The authors thanks Consejo Nacional de Investigaciones Científicas y Tecnológicas de la Nación (CONICET), Argentina for financial support.
