Hydroxy-Propil-β-Cyclodextrin Inclusion Complexes of two Biphenylnicotinamide Derivatives: Formulation and Anti-Proliferative Activity Evaluation in Pancreatic Cancer Cell Models

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with poor outcomes largely due to its unique microenvironment, which is responsible for the low response to drugs and drug-resistance phenomena. This clinical need led us to explore new therapeutic approaches for systemic PDAC treatment by the utilization of two newly synthesized biphenylnicotinamide derivatives, PTA73 and PTA34, with remarkable antitumor activity in an in vitro PDAC model. Given their poor water solubility, inclusion complexes of PTA34 and PTA73 in Hydroxy-Propil-β-Cyclodextrin (HP-β-CD) were prepared in solution and at the solid state. Complexation studies demonstrated that HP-β-CD is able to form stable host-guest inclusion complexes with PTA34 and PTA73, characterized by a 1:1 apparent formation constant of 503.9 M^-1 and 369.2 M^-1, respectively (also demonstrated by the Job plot), and by an increase in aqueous solubility of about 150 times (from 1.95 µg/mL to 292.5 µg/mL) and 106 times (from 7.16 µg/mL to 762.5 µg/mL), in the presence of 45% w/v of HP-β-CD, respectively. In vitro studies confirmed the high antitumor activity of the complexed PTA34 and PTA73 towards PDAC cells, the strong G2/M phase arrest followed by induction of apoptosis, and thus their eligibility for PDAC therapy.

Keywords: biphenylnicotinamide derivatives; cyclodextrin inclusion complex; pancreatic ductal adenocarcinoma; phase solubility studies; preformulation studies.

Figure 1

Chemical structures of PTA34, PTA73, hydroxy-propil-β-cyclodextrin (HP-β-CD) and graphical representation of the inclusion complex.

Figure 2

Phase solubility diagrams. (a) PTA34/HP-β-CD; (b) PTA73/HP-β-CD.

Figure 3

Job plots. (a) PTA 34/HP-β-CD; (b) PTA 73/HP-β-CD.

Figure 4

Dissolution profiles of PTA/HP-β-CD solid complexes.

Figure 5

Dose/effect plots of the mean of three different cell proliferation experiments, conducted in PDAC cell lines incubated for 72 h with HP-β-CD (Ctrl HP-β-CD), PTA34, and PTA34/HP-β-CD (panel (a)) or with HP-β-CD (Ctrl HP-β-CD, PTA73, and PTA73/HP-β-CD (Panel (b)). Dose was expressed in each graph as 0.01–10 µM concentration range, in terms of PTA34 or PTA73, corresponding to 2.9–2900 µg/mL concentration range for HP-β-CD alone.

Figure 5

Dose/effect plots of the mean of three different cell proliferation experiments, conducted in PDAC cell lines incubated for 72 h with HP-β-CD (Ctrl HP-β-CD), PTA34, and PTA34/HP-β-CD (panel (a)) or with HP-β-CD (Ctrl HP-β-CD, PTA73, and PTA73/HP-β-CD (Panel (b)). Dose was expressed in each graph as 0.01–10 µM concentration range, in terms of PTA34 or PTA73, corresponding to 2.9–2900 µg/mL concentration range for HP-β-CD alone.

Figure 6

Effects on cell cycle of PDAC cells by CD (Ctrl-CD), complexed PTA34 and PTA73 (PTA34/HP-β-CD and PTA73/HP-β-CD, respectively) and PTA34 and PTA73, alone. The modulation of cell cycle phases was evaluated by flow cytometry (FCM) analysis after staining cells with propidium iodide. In panel (a), a representative analysis of three independent experiments is reported for all cell lines investigated. In panel (b) the bar graph shows cell population percentage in each phase of the cell cycle.

Figure 7

(a) Representative apoptosis analysis of PDAC cells after 24 h treatment with HP-β-CD alone (Ctrl HP-β-CD), PTA34, PTA34/HP-β-CD, PTA73, and PTA73/HP-β-CD. Apoptosis was evaluated by using Annexin V/PI staining followed by FCM analysis. (b) Fold change of Annexin V positive cells (early plus late apoptosis) in treated samples versus their corresponding reference compounds (Ctrl cells and Ctrl HP-β-CD for PTAs and complexed-PTAs treated cells, respectively). The results are the mean ± SD of three independent experiments (* p < 0.05; *** p < 0.001).