Enhanced Stability and Bioactivity of Natural Anticancer Topoisomerase I Inhibitors through Cyclodextrin Complexation

Abstract

The use of cyclodextrins as drug nano-carrier systems for drug delivery is gaining importance in the pharmaceutical industry due to the interesting pharmacokinetic properties of the resulting inclusion complexes. In the present work, complexes of the anti-cancer alkaloids camptothecin and luotonin A have been prepared with β-cyclodextrin and hydroxypropyl-β-cyclodextrin. These cyclodextrin complexes were characterized by nuclear magnetic resonance spectroscopy (NMR). The variations in the ¹H-NMR and ¹³C-NMR chemical shifts allowed to establish the inclusion modes of the compounds into the cyclodextrin cavities, which were supported by docking and molecular dynamics studies. The efficiency of the complexation was quantified by UV-Vis spectrophotometry and spectrofluorimetry, which showed that the protonation equilibria of camptothecin and luotonin A were drastically hampered upon formation of the inclusion complexes. The stabilization of camptothecin towards hydrolysis inside the cyclodextrin cavity was verified by the quantitation of the active lactone form by reverse phase liquid chromatography fluorimetric detection, both in basic conditions and in the presence of serum albumin. The antitumor activity of luotonin A and camptothecin complexes were studied in several cancer cell lines (breast, lung, hepatic carcinoma, ovarian carcinoma and human neuroblastoma) and an enhanced activity was found compared to the free alkaloids, particularly in the case of hydroxypropyl-β-cyclodextrin derivatives. This result shows that the cyclodextrin inclusion strategy has much potential towards reaching the goal of employing luotonin A or its analogues as stable analogues of camptothecin.

Keywords: anticancer agents; cavitands; drug stabilization; molecular recognition; supramolecular complexes.

Figure 1

Structures and numbering of camptothecin and luotonin A.

Figure 2

Initial studies that prove the formation in solution of inclusion complexes of camptothecin and luotonin A with β-CD and HP-β-CD. Fluorescence emission spectra of camptothecin in the presence of: (A) increasing β-CD concentrations; (B) increasing HP-β-CD concentrations. Fluorescence emission spectra of luotonin A in the presence of: (C) increasing β-CD concentrations; (D) increasing HP-β-CD concentrations. The complexes were prepared in acidic media.

Figure 3

(A) Changes observed in selected signals of the camptothecin ¹H-NMR spectrum upon complexation with β-cyclodextrin, showing significant changes in the position of the signals in (a) and (c) and only slight changes in (b) and (d). (B) Normalized (0–1) chemical shift changes in the ¹H-NMR and ¹³C-NMR spectra of camptothecin following complexation with β-cyclodextrin and hydroxypropyl-β-cyclodextrin. (C) Model of inclusion of camptothecin inside the β-CD cavity according to NMR data. (D) Two alternative models of inclusion of luotonin A inside the β-CD cavity according to NMR data.

Figure 4

Docking of camptothecin and luotonin A inside the cyclodextrin cavities: (A) camptothecin–β-CD complex; (B) camptothecin-HP-β-CD complex; (C) luotonin A–β-CD complex; (D) luotonin A-HP-β-CD complex.

Figure 5

Molecular dynamics studies: (A) RMSD for the camptothecin-β-CD and luotonin A-β-CD complexes; (B) evolution of the distance between the center of mass (COM) of β-CD and the center of mass of camptothecin, and similar study of the center of ring A; (C) evolution of the distance between the center of mass of β-CD and the center of mass of luotonin A, and similar study of the center of ring E.

Figure 6

(A) Effect of the addition of increasing volumes (10 μL per addition) of 10 M HCl on the UV-Vis absorption spectra of camptothecin in aqueous solution. (B) Ratiometric titration of camptothecin. λ = 366 nm corresponds to the neutral form and λ = 401 nm corresponds to the protonated form. (C) Effect of the addition of increasing volumes (10 μL per addition) of 10M HCl on the UV-Vis absorption spectra of the camptothecin-β-CD complex in aqueous solution. Inset: Ratiometric titration of the camptothecin-β-CD complex. (D) Effect of the addition of increasing volumes (10 μL per addition) of 10M HCl on the UV-Vis absorption spectra of the camptothecin-HP-β-CD complex in aqueous solution. Inset: Ratiometric titration of the camptothecin-HP-β-CD complex.

Figure 7

Ratiometric titration of luotonin A and its complexes with β-CD and HPβ-CD upon addition of increasing volumes (20 μL per addition) of 1.0 M HClO₄.

Figure 8

Chromatograms showing that CD complexation protects camptothecin from hydrolysis. (A) Hydrolysis of the camptothecin lactone E ring in the presence of aqueous ammonia. (B) The same reaction followed by separation in the presence of luotonin A as an internal standard. (C) Campothecin hydrolysis in the presence of β-cyclodextrin. (D) Campothecin hydrolysis in the presence of HP-β-CD.