Date of Award

12-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Clinton F. Stewart, Pharm.D.

Committee

Michael A. Dyer, Ph.D. R. Kiplin Guy, Ph.D. Bernd Meibohm, Ph.D. Victor M. Santana, M.D.

Keywords

ABC transporter, BCRP, Nutlin-3a, Pharmacokinetics, PBPK

Abstract

Nutlin-3a is an MDM2-p53 interaction antagonist that is under investigation in preclinical models for a variety of pediatric malignancies, including neuroblastoma, retinoblastoma, leukemia, and rhabdomyosarcoma. In the current research, we conducted preclinical pharmacology studies of nutlin-3a to evaluate the synergistic effect of the nutlin-3a and topotecan combination on neuroblastoma cell growth, to assess the effect of nutlin-3a on breast cancer resistance protein (BCRP), and to characterize the disposition of nutlin-3a in the mouse plasma and multiple tissues.

Activating the p53 pathway might offer a new therapy for neuroblastoma. In the first part of the study, we assessed the effect of nutlin-3a on the cell viability of neuroblastoma both as a single agent and in combination with topotecan. We showed that targeting MDM2-p53 interaction using nutlin-3a reduced cell growth in neuroblastoma cells. p53 wild-type cells were much more sensitive to nutlin-3a treatment compared to p53 mutant cells. When nutlin-3a was combined with topotecan, a synergistic effect on neuroblastoma cell growth was observed. To explore the mechanism of synergy, we performed quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis and found reduction of P-gp expression at both the message level and protein level in p53 wild-type neuroblastoma cells. This is the first study showing the synergistic effect of nutlin-3a in combination with topotecan in neuroblastoma cells and the reduction of P-gp expression by nutlin-3a in p53 wild-type cells.

Although nutlin-3a is currently under pre-clinical investigation as a p53 reactivation agent, it has been recently demonstrated also to have p53 independent actions in cancer cells. In the second part of the study, we first reported that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually "inactive" in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.

Understanding drug disposition is critical in preclinical drug development. In the third part of the study, we used physiologically-based pharmacokinetic (PBPK) modeling to characterize the disposition of nutlin-3a in mice. Plasma protein binding and blood partitioning were assessed by in vitro studies. After intravenous (10 and 20 mg/kg) and oral (50, 100, and 200 mg/kg) dosing, tissue concentrations of nutlin-3a were determined in plasma, liver, spleen, intestine, muscle, lung, adipose, bone marrow, adrenal gland, brain, retina, and vitreous fluid. The PBPK model was simultaneously fit to all pharmacokinetic data using NONMEM. Nutlin-3a exhibited nonlinear binding to murine plasma proteins, with the unbound fraction ranging from 0.7 to 11.8%. Nutlin-3a disposition was characterized by rapid absorption with peak plasma concentrations at approximately 2 h and biphasic elimination consistent with a saturable clearance process. The final PBPK model successfully described the plasma and tissue disposition of nutlin-3a. Simulations suggested high bioavailability, rapid attainment of steady state, and little accumulation when administered once or twice daily at dosages up to 400 mg/kg. The final model was used to perform simulations of unbound tissue concentrations to determine which dosing regimens are appropriate for preclinical models of several pediatric malignancies.

In conclusion, our results showed that nutlin-3a synergistically inhibited the growth of neuroblastoma cells when combined with topotecan. Nutlin-3a reversed BCRP-mediated drug resistance by inhibiting the function of BCRP. A PBPK model was successfully established to describe the disposition of nutlin-3a in plasma and tissues of interest for pediatric malignancies.

DOI

10.21007/etd.cghs.2011.0372

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