New tariquidar-like ABCB1 modulators in cancer chemotherapy:Preclinical pharmacokinetic, pharmacodynamic investigations and computational studies
Year: 2010
Type: dissertation
Abstract: The ABCB1 transporter (P-glycoprotein), an ATP-driven efflux protein, strongly influences the absorption, distribution and excretion of various drugs. As an essential component of the blood-brain barrier, P-glycoprotein limits the penetration of potent cytostatic agents (e.g. paclitaxel) into the central nervous system (CNS). Furthermore, over-expression of ABCB1 transporters in tumor cells is one of the major mechanisms of classical multidrug resistance (MDR) in cancer. Hence, inhibition of P glycoprotein is an attractive strategy to improve the therapy of both, tumors in the CNS and MDR malignancies in the periphery.\nAccordingly, a series of potent inhibitors derived from the 3rd generation P-glycoprotein modulator tariquidar was investigated in vitro. A fluorescence-based efflux-assay using human Kb-V1 cells was established at the microtiter plate format. The obtained data were in good agreement with the validated flow cytometric method while throughput was substantially increased. Additionally, cells expressing the murine transporter were generated to assure activity at rodent P-glycoprotein. Further biopharmaceutical investigations (concerning toxicity, stability, plasma albumin binding and inhibition of cytochrome P450 isoenzymes) confirmed the suitability of tariquidar and the three most promising derivatives (bearing polar residues such as a methoxyethoxyethylamino (ME27-4), a morpholino (ME30-1) or an ethoxyethyloxy (ME33-1) moiety) for further in vivo studies.\nIn order to determine the concentration of the selected modulators in brain and plasma samples, a solid-phase extraction procedure followed by RP-HPLC analysis was established and validated. The method yielded reproducible high recoveries, a low limit of quantification as well as high accuracy and precision. The concomitant administration of paclitaxel in successive bio-distribution studies in NMRI mice revealed favorable bioavailability and high metabolic stability of the tariquidar-like compounds. As desired, the modulators significantly increased the brain AUC of paclitaxel. The observed increase by a factor of two to four correlated with the previously determined in vitro potency of the various inhibitors. In contrast to the co-administration of the 2nd generation modulator valspodar, the plasma AUC of paclitaxel was unaffected, indicating lower systemic toxicity.\nTo investigate if the increased paclitaxel brain concentration is of relevance for the therapy of malignant brain tumors, an intracerebral xenograft model in nude mice was refined. Human U 118 MG glioblastoma cells were transfected with the genes encoding for the far-red fluorescent protein or the luciferase2 (Luc2) enzyme to facilitate non-invasive determination of tumor burden by means of fluorescence or bioluminescence imaging (BLI). Tumors originating from intracerebral injection of luciferase2 expressing cells were detectable in the living animal. Unfortunately, the growth behavior was unsuitable for the intended in vivo investigations. Alternatively, highly tumorigenic U 87-Luc2 xenografts were utilized for subsequent treatment experiments evaluating the combination of paclitaxel with the most potent modulators ME33-1 or tariquidar. Since the obtained light signals largely correlated with the histological determined tumor burden, BLI revealed a moderate retardation of tumor progression when paclitaxel was combined with an ABCB1 modulator.\nFurthermore, the therapeutic value of the co-administration of ABCB1 modulators with paclitaxel in the treatment of MDR tumors was investigated. For this purpose, ABCB1 transporter over-expressing Kb-V1 cells were extensively characterized in vitro followed by subcutaneous implantation into nude mice. The resulting malignancies showed high tumorigenicity, reproducible growth kinetics and maintained transporter expression in vivo. In subsequent treatment experiments ME30-1, ME33-1 and tariquidar were investigated. In contrast to the other modulators, only the combination of ME30-1 with paclitaxel significantly slowed down the growth of Kb-V1 tumors compared to paclitaxel mono-therapy.\nAdditionally, molecular modeling investigations were performed based on the very recently published x-ray structure of the murine Abcb1a transporter. Conformational analysis of the tariquidar-like modulators provided the starting conformation for successive docking experiments. The location of the subsequently identified binding region is in accordance with the suggested transport mechanism and experimental data from the literature. The proposed binding modes largely agree with the inhibitory potencies of the investigated ABCB1 modulators and explain the negligible ABCB1 activity of tariquidar-derived, selective ABCG2 modulators. The findings facilitate the structure based design of optimized ABCB1 modulators and may contribute to improve the adjuvant cancer chemotherapy of glioblastomas and MDR tumors.
Author: Peter Höcherl
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W189363407