Targeting and Treatment of Tumor Hypoxia by Newly Designed

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Targeting and treatment of tumor hypoxia by newly designed prodrug possessing high permeability in solid tumors Yutaka Ikeda, Hikaru Hisano, Yuji Nishikawa, and Yukio Nagasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00011 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 22, 2016

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Molecular Pharmaceutics

Targeting and treatment of tumor hypoxia by newly designed prodrug possessing high permeability in solid tumors Yutaka Ikeda1, Hikaru Hisano1, Yuji Nishikawa2, Yukio Nagasaki1, 3, 4*

1

Department of Materials Science, Master’s School of Medical Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305-8573, Japan. 2

Division of Tumor Pathology, Department of Pathology, Asahikawa Medical University, Midorigaoka Higashi-2-jyo 1-1-1, Asahikawa, Hokkaido 078-8510, Japan 3

Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan. 4

Satellite Laboratory, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305-8573, Japan. KEYWORDS . Tumor hypoxia, prodrug, solid tumor, drug penetration

ABSTRACT

Tumor hypoxia, which is associated with poor prognosis in cancer, is known to lead to resistance to radiotherapy and anticancer chemotherapy. Impaired drug penetration in hypoxic regions has been recognized as an essential barrier to drug development in solid tumors. Here, we propose

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novel hypoxia-activated prodrugs, which drastically improved the penetration property of commonly used anticancer drugs in the hypoxic region. In this design, conventional anticancer drugs were modified with 2-nitroimidazole derivatives. The most important point of this study was that the prodrug designed formed a 6-membered cyclic structure to allow liberation of the active drug in the hypoxic region. This design markedly increased the selectivity of the hypoxiatargeted prodrug, resulting in significant reduction of adverse effects in the normoxic region. In vitro studies confirmed the selective activation under hypoxic conditions. In vivo studies showed drastic reduction of adverse effects associated with conventional anticancer drugs and improvement of the survival rate of mice. Immunofluorescence analyses confirmed that the designed prodrug had a tendency to localize at the hypoxic region, in contrast to conventional anticancer drugs, which localize only at the normoxic region.

Introduction In spite of the development of various kinds of anticancer drugs, there are still unmet medical needs in cancer treatment. Clinical application of anticancer drugs is limited by problems such as lack of tissue specificity; therefore, enhanced targeted delivery of anticancer drug to tumors represents an important approach to these issues. Especially, limited penetration and selectivity of anticancer drugs for hypoxic tumor cells located distant from tumor blood vessels is a serious problem in cancer chemotherapy (1). Hypoxia is a feature of solid tumors and is well known to promote mutagenesis and invasiveness; it is associated with a poor prognosis in cancer (2). A strategy for targeting the hypoxic region of tumors is highly required; however, the resistance of hypoxic tumors to chemo- and radiation therapy has hampered drug development (3). To develop hypoxia-targeting anticancer drugs, issues associated with drug resistance need to be overcome. Several studies that investigated the drug resistance of hypoxic tumors focused on the molecular


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mechanism of resistance; however, recent studies proposed that poor penetration of anticancer drugs in the hypoxic region is another serious issue to be considered in the treatment of solid tumors (4). The hypoxic region is located distant from tumor blood vessels and is considered as an important characteristic of the tumor microenvironment, since it increases extensive extracellular matrix (ECM) components and promotes a disordered vascular architecture, limiting the accessibility of anticancer drugs to hypoxic areas. Therefore, during the development of hypoxia-targeting anticancer drugs, poor drug penetration in the hypoxic region should be considered (5). Hypoxia-activated prodrugs (HAPs) have been considered as a promising strategy for the treatment of tumor hypoxia (6). The HAP is inactive under normoxic conditions but is activated in the hypoxic region; hence, HAPs are less toxic in normoxic conditions, resulting in the reduction of adverse effects. While designing the HAP, the physiological differences between normoxic and hypoxic conditions are taken into account because these differences can trigger structural changes and activation of the prodrug. Since hypoxic tumor tissues are known to overexpress several endogenous reductive enzymes, several prodrugs that can be activated by reduction have been reported (6). Several functional groups such as nitroaromatic, quinone and N-oxide groups that undergo reduction in the hypoxic environment can be employed in the development of HAPs. Specific reducing reactions in the tumor hypoxic region activate these prodrugs and afford toxic compounds. 2-Nitroimidazole (NIM) analogues are a family of wellknown compounds, which respond to tumor hypoxic environments. They effectively undergo an enzymatic reducing reaction of the nitro group, accompanied by the formation of a covalent bond with thiol-containing proteins in hypoxic cells (7). One of the applications of this unique property is in the development of imaging probes. For example, pimonidazole, which is a


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derivative of NIM, has been used as a tumor hypoxia marker in animal and human studies (8). Further, the property of NIM in hypoxic environment has been evaluated for the design of prodrugs. For example, Hay et al. designed NIM-conjugated doxorubicin and para-aminophenyl mustard (9, 10). Although this is a novel idea, the clinical applications of this drug are controversial since drug release caused by reduction was not observed in hypoxic cell cultures and the activities did not differ significantly between the hypoxic and normoxic culture conditions. In order to improve the efficiency of HAP, it is important to enhance the selectivity of drug release and drug penetration in the hypoxic area. However, to our knowledge, no study has clearly established the impact of increased penetration of HAP on therapeutic effect. To improve the specificity of drug release and penetration efficiency of drugs in the hypoxic area, we designed a new 2-nitroimidazole-based prodrug, and observed that during hypoxic reducing reactions, a 6-membered ring was formed, accompanied by liberation of active drugs in the hypoxic region. The newly designed prodrug could increase the efficiency of drug release and be applied to the amide linkage between the 2-nitroimidazole group and conventional antitumor drugs. Since the amide bond is stable in normoxic regions compared with that of other linkages, including ester and carbamate, our prodrug can suppress unwanted drug release in the normoxic region, resulting in selective targeting of hypoxic tumor cells.

Experimental section Synthesis Synthesis of all compounds used in this study is described in supporting information.

Analysis


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ESI- MS analysis and LC/MS/MS analysis were carried out by API 2000 (AB SCIEX). Ultra high-speed liquid chromatograph system was carried out by LaChrom ULTRA (HITACHI) (column: lachrom ultra C18, eluent: acetonitrile/ 0.1 % trifluoroacetic acid = 0/100 (0 min) ~ (5 min) then, 0/100 (5 min) ~ 5/95 (20 min)).

Cell culture Cells were cultured in DMEM medium (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen) at 37°C under 5% CO2. Oxygen concentration was controlled by hypoxia workstation InvivO2 400 system (Baker Ruskinn).

Drug release analysis in vitro Human pancreatic cancer cells (MIA PaCa-2) were seeded (1 × 104 per well) on a 96-well flatbottom plate and 100 µM gemcitabine prodrug, 5, was added and cultured under normoxic or hypoxic conditions. After incubation, cells were collected by trypsin treatment and freeze-dried. Acetonitrile (100 mL) was added to the residues, followed by sonication to extract the released drug. Products were analyzed by LC/MS/MS (column: LaChrom Ultra C18, eluent: acetonitrile/0.1% trifluoroacetic acid = 0/100 (0 min) ~ (5 min) and then 0/100 (5 min) ~ 5/95 (20 min)). Quantification was performed in multiple reactions monitoring (MRM) mode using transitions of m/z 264.1/112.1. Release amount of gemcitabine per well was calculated by the calibration curve for gemcitabine. The accuracy was assessed by the replicate analysis (n = 5,separate cultures in the same experiment). Calibration curve to determine the concentration of gemcitabine is shown in supporting information (Figure S1).


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Cytotoxicity assay in vitro Cytotoxicity assay in vitro Human pancreatic cancer cells (MIA PaCa-2) were seeded (5 x 103 per well) on a 96-well flatbottom plate and prodrug 4 or 5 was added at the concentration indicated in Figure 6 and incubated for 6 h or 1 h, respectively, under hypoxic (oxygen concentration: 0.1%) or normoxic conditions. After drug exposure, drug was removed by washing cells twice with PBS. Then, cells were cultured in the medium without the prodrug for 48 h. Cell viability was determined using a WST-1 assay (Dojindo, Kumamoto, Japan). The accuracy was assessed by the replicate analysis (n = 5,separate cultures in the same experiment).

Animals The histological study and anticancer activity were conducted with BALB/c mice (4 weeks old, male, approximately 20 g) and acute toxicity studies were carried out using ICR mice (5 weeks old, male, approximately 25 g). All mice were purchased from Charles River, Japan, Inc. The mice were housed in the experimental animal facilities at the University of Tsukuba in a temperature and humidity controlled environment with a 12-h light/12-h dark cycle. All mice were fed commercial chow and water ad libitum. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the University of Tsukuba.

Histological and immunofluorescence studies BALB/c mice were subcutaneously injected with Colon 26 cells, and when the size of the tumors reached 1 cm in diameter, doxorubicin (60 mg/kg) or doxorubicin prodrug (60 mg/kg) was


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administered intraperitoneally. Tumor samples were collected at 0.5, 1, and 2 h following administration and subjected to histological and immunofluorescence analyses. Pimonidazole (60 mg/kg) was intravenously injected 1 h before sacrifice. For the detection of hypoxic areas, 4% paraformaldehyde-fixed frozen sections were incubated with anti-pimonidazole rabbit antisera (1:200; Hypoxiprobe, Inc., Burlington, MA) and then with anti-rabbit antibody conjugated with Alexa Fluor 488 (1:200).

Anticancer activity in vivo Colon 26 tumor-bearing mice were randomly divided into three groups (6 mice/group). Solution containing 50% poly(ethylene glycol) (400 Da) was used as the vehicle. Doxorubicin or doxorubicin prodrug was injected intraperitoneally when the tumor grew to 1 cm in diameter (day 0). According to the MSDS, LD50 of doxorubicin are 1.245 mg/kg (I.V.) and 11.16 mg/kg (I.P.). Because our prodrug, 4, is less toxic in addition to the poor water solubility, we employed I.P route. Doxorubicin (4 mg/kg) was injected on day 0, and doxorubicin prodrug (16 mg/kg) was administered on days 0, 2, 4, 6, and 8. Antitumor activity was evaluated in terms of the volume of tumor, according to the method described in previous studies (11).

Statistical analysis In all experiments, statistical analyses were performed using the Student’s t-test. A P-value of less than 0.05 was considered statistically significant for all analyses.

Results Design and synthesis of novel hypoxia-activated prodrugs


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The nitro group of NIM has been reported to undergo enzymatic reducing reactions in hypoxic conditions to produce hydroxylamine. Due to the strong reactivity of the resulting hydroxylamine intermediate, it can react with a thiol-containing protein in hypoxic cells to afford stable adduct (8). As stated above, our design involved the use of this reaction for the development of a prodrug. We designed intramolecular nucleophilic cleavage of an acyl linkage by the amino groups. Hydroxylamine intermediate could also promote cyclization and drug release under hypoxic reducing conditions. In order to increase the efficiency and specificity of the reaction in hypoxic tumor environment, we employed a 6-membered cyclization reaction, as shown in Figure 1. According to this reaction, conventional antitumor drugs can be released under hypoxic conditions.

Figure 1. Design of hypoxia-activated prodrug

To evaluate the effect of the linker structure between 2-nitroimidazole and the drug on the efficiency of the intramolecular cyclization reaction described in Figure 1, two model compounds, methyl 2-nitroimidazole-1-propioate, 1, and methyl 2-nitroimidazole-aceate, 2, which differ in linker length (Fig. 2), were synthesized according to a previously reported method (9). The nitro group was chemically reduced over palladium on carbon (Pd/C) in water at


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37 °C, and crude products were subjected to 1H NMR and mass spectrometry analyses. The reaction conditions are described in the supporting information. It is interesting to note that only cyclic compound 3 was observed when 1 was reduced (Fig. 3), whereas methyl 2aminoimidazole-1-propionate was not detected as an intermediate, indicating that the spontaneous cyclization reaction occurred just after the chemical reducing reaction. On the contrary, methyl 2-aminoimidazole derivative was detected during the reaction of 2 (See Fig. S2 in supporting information) and cyclization products were not observed. In addition, mass spectrometric analysis indicated a cyclic product in the case of 1 but not in 2, as shown in Figure 3 and Figure S2 in the supporting information. These results clearly suggest that the intramolecular cyclization reaction accelerated the deacylation reaction of 1 during the reducing reaction of the nitro group in 1 via formation of a six-membered ring, as described in Figure 1. Further, the length of the linker is important for efficient cyclization reaction. Surprisingly, this intramolecular cyclization reaction also occurred in the 2-nitroimidazole derivative in which the compound was stably linked to 2-imidazole via amide bond. For example, the naphthylmethylamide moiety (S2 in supporting information) instead of methyl ester in compound 1 also led to the formation of the same cyclic compound as compound 3 after the chemical reducing reaction shown in Figure S3. On the basis of the results obtained, we concluded that this strategy is widely applicable for efficient release of drug containing amine and/or hydroxyl group, attributed to the stable amide bond in response to reducing conditions. This is very important because several anti-cancer drugs possess amino group(s) and hydroxyl group(s) in the active site.


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Figure 2. Structure of the 2-nitroimidazole derivatives used in this study.

Figure 3. Evaluation of spontaneous cyclization reaction. Hydrogenation of 1 afforded product 3 via intramolecular cyclization reaction. 1H-NMR of 3 is also shown.

In order to confirm our novel design of potential anticancer prodrugs, we synthesized two compounds, 4 and 5, using doxorubicin and gemcitabine, which are conventional anticancer drugs, in which their amino groups accounts for their anticancer activities (10,12). The structures of these prodrugs are shown in Figure 4 (See supporting information for detailed synthetic


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methods and analytic data). Since the active amino groups were covered by a 2-nitroimidazolyl moiety via an amide linkage, it was anticipated that their activities would reduce under normoxic conditions.. N

N

O2N

O HO NH O O

OH

O

OH

O

OMe

HO O HO

4 Figure 4. Structure of hypoxia-activated doxorubicin prodrug (4) and gemcitabine prodrug (5).

Drug release and anti-cancer activity in vitro To demonstrate selective drug release under hypoxic conditions, human pancreatic cancer cells (MIA PaCa-2) were incubated with gemcitabine prodrug, 5, under normoxic or hypoxic (0.1% O2) conditions, and the released gemcitabine was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). As shown in Figure 5, liberated gemcitabine was detected at the retention time of free gemcitabine (14 min), and the amount was much higher under the hypoxic conditions than that observed under normoxic conditions, indicating effective drug release with our prodrug under hypoxic conditions. Although it was difficult to confirm drug release after the chemical reductions due to the low stability of drug under strong reducing condition, in vitro cell experiments clearly observed the liberated drugs, indicating a robust property of our designed prodrug.


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Figure 5. Enhanced release of gemcitabine under hypoxic cellular condition. Typical LC/MS charts (left). Dotted line: hypoxic condition, solid line: normoxic condition. And time course analysis of released gemcitabine per well (right). Diamond: normoxic condition, square: hypoxic condition (0.1% O2). *p

Targeting and Treatment of Tumor Hypoxia by Newly Designed

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