Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

Abstract

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA). These bile acid-tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The in vitro anticancer activities of these bile acid-tamoxifen conjugates show that the free amine headgroup based cholic acid-tamoxifen conjugate (CA-Tam3-Am) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid-tamoxifen conjugates. The cholic acid-tamoxifen conjugate (CA-Tam3-Am) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor -ve breast cancer cell lines. The enhanced anticancer activity of CA-Tam3-Am is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G0 phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome c, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER -ve cells. In vivo studies in the 4T1 tumor model showed that CA-Tam3-Am is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid-drug conjugates.

Dihydroartemisinin–Bile Acid Hybridization as an Effective Approach to Enhance Dihydroartemisinin Anticancer Activity

“…The lipophilicity of BAs has also been exploited to improve the bioavailability of chemotherapeutics by conjugating drugs to endogenousB As through ac ovalentl inkage. [19] The discovery of growth inhibitory effects of endogenous BAs on several cancer cell lines [17] through, among others, apoptosis, membrane alterations, modulation of nuclear receptors and oxidative stress, points to their potentiala nticancer activity.U DCA is knownt oe xert cytoprotectivee ffects against the toxicity of other BAs [20] anda nticarcinogenic effects against hepatocellular carcinomaand colon cancers. [21] However,endogenousBAs display ar elativelyl ow cytotoxicity (IC 50 > 100 mm), therefore the search for new derivatives of BAs with higherc ytotoxic activity has received much attention.…”

Nature of the charged head group dictates the anticancer potential of lithocholic acid-tamoxifen conjugates for breast cancer therapy

“…We discovered that ineffective anticancer activity of lithocholic acidtamoxifen amphiphile is due to weak electrostatic interactions with cell membranes, causing minimum membrane perturbations. 18 In this manuscript, we hypothesize that therapeutic efficacy of lipid-drug conjugates can be modulated by introduction of variable charged head groups, 19 where hydrophobicity of lipid molecule will help in translocation of lipid-drug conjugate across the membrane, and variable charged head group might work as another pharmacophore by enhancing the activity of anticancer drug (Fig. 1a).…”

“…As observed previously, conjugation of a primary amine (soft-charge) to the amphiphile makes tamoxifen ineffective against breast cancer cell lines. 18 LCA-Tam-DMAP was most potent, having IC 50 values of 6.5, 12.72, 3.13 and 7.0 μM in MDA-MB-231, MCF-7, MDA-MB-468 and T47D cell lines, respectively (Table 1). Variations in toxicity of LCA-Tam-DMAP among the ER +ve or ER −ve cell lines might be due to different genomic and proteomic natures of cell lines.…”

Spectroscopic methods and theoretical studies of bromoacetic substituted derivatives of bile acids

“…35 In the case of sodium azide, the resulting products are used as substrates for click chemistry reactions. Singh et al employed a series of chloro substituted derivatives of bile acids that were used in the synthesis of cationic bile acid-based facial amphiphiles featuring trimethyl ammonium head groups.…”

“…Bile acids can react with halogenoacetic acid halides and potassium carbonate in chloroform or dichloromethane, with calcium or sodium hydride and tetrabutylammonium bromide (TEBA) in toluene, as well as pyridine or 4-(dimethylamino)pyridine (DMAP) in toluene. [27][28][29][30][31][32][33][34][35] These compounds were used in nucleophilic reactions with N-, S-or O-nucleophiles. In many cases bromo-or chloroacetyl substituted derivatives of bile acids react with pyrimidines (e.g.…”