Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53- deficient breast cancer treatment

Seung Woo Chung, Gui Chul Kim, Seho Kweon, Hanul Lee, Jeong Uk Choi,
Foyez Mahmud, Hyo Won Chang, Ji Won Kim, Woo-Chan Son, Sang Yoon Kim, Youngro Byun

PII: S0142-9612(18)30561-1

DOI: 10.1016/j.biomaterials.2018.08.007

Reference: JBMT 18813

To appear in: Biomaterials

Received Date: 26 January 2018

Accepted Date: 03 August 2018

Please cite this article as: Seung Woo Chung, Gui Chul Kim, Seho Kweon, Hanul Lee, Jeong Uk Choi, Foyez Mahmud, Hyo Won Chang, Ji Won Kim, Woo-Chan Son, Sang Yoon Kim, Youngro Byun, Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53-deficient breast cancer treatment, Biomaterials (2018), doi: 10.1016/j.biomaterials.2018.08.007

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Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53-deficient breast cancer treatment

Seung Woo Chung1,2,3, Gui Chul Kim4, Seho Kweon5, Hanul Lee1, Jeong Uk Choi1, Foyez Mahmud5, Hyo Won Chang4, Ji Won Kim4, Woo-Chan Son6,7, Sang Yoon Kim4*, and Youngro Byun1,5*

1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, South Korea, 2Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21231, United States, 3Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, United States, 4Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea, 5Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergent Science and Technology, Seoul National University, Seoul 08826, South Korea, 6Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea, 7Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea

Short title: Metronomic oral doxorubicin and MK-8776 combination therapy

*Correspondence to: Youngro Byun, Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergent Science and Technology, Seoul National University, Seoul 08826, South Korea, Tel.: +82-2-880-7866, E-mail: [email protected], and Sang Yoon Kim, Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea, Tel.: +82-2-3010-3715, E-mail: [email protected]

First Authors: Seung Woo Chung and Gui Chul Kim have equally contributed to the study as the first authors.

Keywords: Oral chemotherapy; Metronomic chemotherapy; Chk1 inhibitor; p53-deficient breast cancer; Doxorubicin; Synthetic lethality; MK-8776; Breast cancer

Abstract

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long- term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53- deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

1.Introduction

Despite significant advances in cancer therapeutics, cytotoxic chemotherapy still remains as the frontline treatment option for many types of cancer. Cytotoxic drugs are typically administered at maximum tolerated dose (MTD) with extended drug-free periods between treatment cycles to allow patients to recover from chemotherapy-induced toxicities [1]. However, the patients suffer from severe adverse effects and more importantly, the discontinuation of therapy enables the regrowth of tumor cells, leading to the treatment failure [2]. Recently, metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has increasingly gained interests as an alternative of MTD [3]. Such dosing regimen can reduce acute toxicities and prevent tumor regrowth that could occur during therapy breaks by continuously providing therapeutic pressure to the tumor.
Metronomic chemotherapy has been extensively investigated in clinical studies on breast cancer patients and proved their clinical benefits over conventional MTD therapy [4-6]. Its mechanism of action has been considered multi-faceted, including antiangiogenic effects, immunomodulation, and direct cytotoxic effects [3]. However, the direct cytotoxic effect plays only minor role attributed to the lowered dose that inevitably attenuates its potency. Nevertheless, the cytotoxic effect is the principle working mechanism of cytotoxic drugs and therefore, augmenting the direct cytotoxic effect while maintaining the low systemic toxicities of metronomic chemotherapy would be undoubtedly beneficial in improving the clinical outcome of the patients.
Current efforts to improve the therapeutic effect of cytotoxic drugs include the combination of cell cycle checkpoint inhibitors. Cell cycle checkpoints maintain the genomic integrity upon genotoxic stress by arresting the cell cycle progression to allow the cells to repair their DNA damage prior to entry into mitosis [7]. Their disruption, therefore, induces premature entry into mitosis, promoting the mitotic catastrophe by the accumulation of DNA damages in the presence of genotoxic stress. Checkpoint kinase 1 (Chk1) is the particularly important kinase that initiates G2/M checkpoint and has been recognized as a promising molecular target for sensitizing cancer cells to cytotoxic drugs [8-10]. In normal cells, tumor suppressor p53 plays a crucial role as a checkpoint regulator and arrests cell cycle in both G1 and G2/M upon DNA damage [7, 11]. However, p53 is

frequently mutated in human cancer cells and their cell cycle arrest mostly depends on Chk1 [7, 12]. Hence, Chk1 inhibition could selectively induce synthetic lethality with cytotoxic drugs to the p53-deficient cancer cells, while normal cells being protected by the p53-mediated G1 checkpoint. Such selectivity might be particularly important for metronomic chemotherapy regarding its multiple mechanisms in exerting an anticancer effect, since p53-deficient cancer cells but not the p53- proficient endothelial cells or immune cells would be affected by the Chk1 inhibition, thus enhancing only the direct cytotoxic effect without modulating any other possible mechanisms.
Currently, breast cancer is the most common cancer and second leading cause of cancer death among women in the US [13]. It has been known that about 20-30% of breast cancers harbor mutated p53, which is associated with poor prognosis [14, 15]. In this study, we sought to enhance the therapeutic index of metronomic doxorubicin on p53-deficient breast cancer by combining it with MK-8776, which is a selective Chk1 inhibitor currently being investigated in clinical trials [16- 18]. Doxorubicin, a commonly used cytotoxic drug for breast cancer, was formulated as an electrostatic complex with deoxycholic acid (DOCA) to allow its oral administration since oral drugs are highly favored in metronomic chemotherapy due to the frequent and potential long-term administration. The preparation of the oral doxorubicin formulation was based on the earlier reports that demonstrated the complex formation with bile acid could facilitate the intestinal absorption of the counter drug [19-21]. The oral absorption of the oral doxorubicin was confirmed using in vitro assays as well as in vivo using pharmacokinetic and pharmacodynamic studies. The transition of cell cycle checkpoint pathway and cell cycle arrest, as well as apoptosis after treatment of low- concentration doxorubicin with or without MK-8776, were investigated in vitro using several breast carcinomas with different p53 status. Finally, the anticancer effects of metronomic low-dose oral doxorubicin combined with MK-8776 were evaluated in vivo.

2.Materials and Methods

2.1.Cell lines

MDA-MB-231, MCF-7, Caco-2, and SCC7 were purchased from ATCC. HCC1937 and HCC1954 were purchased from Korea Cell Line Bank. MDA-MB-231, MCF-7, Caco-2, and SCC7 were grown in DMEM (Gibco). HCC1937 and HCC1954 were grown in RPMI 1640 (Gibco). Both cell culture media were supplemented with 10% FBS (Gibco) and 1% Pen-Strep (Sigma-Aldrich). Non-essential amino acid (Sigma-Aldrich) was additionally added (1%) to the medium for Caco-2. The cells were maintained in a humidified 5% CO2 incubator at 37°C. The cells were authenticated using STR analysis and were tested mycoplasma negative.

2.2.Preparation of oral doxorubicin

Doxorubicin HCl (100 mg; Sangon Biotech) and sodium deoxycholate (75 mg, 1.05 eq; Sigma-Aldrich) were dissolved in DW (10 ml) and the solution was adjusted to pH 7.0 using 0.1 N NaOH solution. The formed precipitate was collected by centrifugation and dried under reduced pressure to obtain oral doxorubicin (DOX/DOCA complex) as a red solid.

2.3.Determination of octanol-water partition coefficient

The shake-flask method was used to determine the partition coefficient of doxorubicin and DOX/DOCA complex. The substances (100 μg) were dissolved (or suspended) in n-octanol (200 μl) followed by the addition of DW (200 μl). The mixture was vigorously vortexed for 3 h and then kept steady to allow the phase separation. The fluorescent intensity of doxorubicin was measured from each phase using a microplate reader (Synergy HT, BioTek Instruments) at 470/580 nm to determine the concentration of the substances. The partition coefficient was calculated according to the following equation:
[Compound]
organic

log