Development of Combination Therapies of Pazopanib Through a PK/PD Approach

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2018-05

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Abstract

Objective: The increased efflux of tyrosine kinase inhibitors (TKIs) by multidrug resistance-related ATP-binding cassette (ABC) transporters represents an important mechanism of TKIs resistance. Interestingly, many TKIs have also been found to be able to serve as inhibitors of ABC transporters. Therefore, the overall goal is to develop a combination therapy of two different TKIs to reverse efflux transporter-mediated resistance against the coadministered TKI, thus improving the overall treatment outcomes synergistically. To approach this goal, the objectives of this project were 1) to use a systematic in vitro approach to rapidly identify a TKI that can modulate the transport of TKI pazopanib, the chosen model substrate; 2) to investigate the mechanism of various modulators on the exposure and cytotoxicity of pazopanib in vitro using wild-type and prolonged pazopanib-exposed 786-O cells; and 3) to investigate the impact of the novel combination on the pharmacokinetic of pazopanib after oral administration in mice. Methods: For objective 1), in vitro transcellular transport experiments in Caco-2 cell monolayers were used as a screening tool for identifying a TKI that can reverse the transporter-mediated efflux of pazopanib. For objective 2), MTT proliferation assays and uptake studies were performed in both wild-type and prolonged pazopanib-exposed 786-O cells. For objective 3), pharmacokinetic studies of pazopanib in the presence or absence of the most effective inhibitor (TKI) identified in objective 1) were performed in FVB wild-type mice and FVB P-glycoprotein (P-gp) knockout mice. Results: 1) In the presence of zosuquidar (LY335979, 1.2 µM), a known inhibitor of P-gp, the efflux ratio of pazopanib decreased from 12.9 to 1.2, accompanied with a 6-fold increase of the intracellular accumulation of pazopanib in Caco-2 cells. Nilotinib can function as an effective efflux transporter inhibitor to reverse the efflux and increase the intracellular accumulation of pazopanib in Caco-2 cells. Concentration-dependent studies showed that nilotinib inhibited the efflux of pazopanib with an IC50 value of 0.4 μM. 2) Pazopanib-resistant cell line was not developed in the current studies. The prolonged exposure of 786-O cells to pazopanib was accompanied by an increased intracellular accumulation (~100-fold) of pazopanib and formation of autolysosomes compared to the wild-type 786-O cells. The co-incubation with nilotinib can increase the sensitivity of the cells to pazopanib, especially the co-incubation with 50 μM nilotinib caused at least 70% death of both cell lines in the tested pazopanib concentration range of 3-500 μM. 3) Compared to wild-type mice, the AUC0-∞ of pazopanib was significantly increased in MDR1a/b-/- mice (~1.5 folds, p < 0.01) at a dose of 1 mg/kg, but not at a higher dose of 10 mg/kg. The pharmacokinetic studies in the wild-type mice showed that the Cmax and AUC0–∞ of pazopanib (2 mg/kg) were slightly increased in the presence of nilotinib (10 mg/kg). However, the plasma Cmax and AUC0–∞ of pazopanib (1 mg/kg) was substantially increased by 1.9 and 3.1 folds after coadministration of nilotinib at 20 mg/kg, respectively. Conclusions: The results showed that the in vitro sensitivity and systemic exposure of pazopanib can be increased by nilotinib, suggesting that the combined PK/PD approach developed here can be used to identify clinically useful novel TKI combinations to enhance intracellular concentrations of a targeted TKI and improve its therapeutic efficacy. The role of P-gp in the resistance development and distribution of pazopanib needs to be further validated. In addition to the increased efflux by ABC transporters, the cellular sequestration also appears to be a resistance mechanism for pazopanib which deserves further validation. Further in vivo efficacy studies are warranted to evaluate the anticancer effects of this new combination.

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Tyrosine kinase inhibitors, Multidrug resistance, P-gp, Combination therapy, PK/PD

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