- Email: [email protected]
Preclinical Evaluation of Promitil, a Radiation-Responsive Liposomal Formulation of Mitomycin C Prodrug, in Chemoradiotherapy
Accepted Manuscript Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy Xi Tian, Ph.D., Samuel B. Warner, Kyle T. Wagner, B.S., Joseph M. Caster, M.D., Ph.D., Tian Zhang, M.D., Patricia Ohana, Ph.D., Alberto A. Gabizon, M.D., Ph.D., Andrew Z. Wang, M.D. PII:
S0360-3016(16)32808-5
DOI:
10.1016/j.ijrobp.2016.06.2457
Reference:
ROB 23705
To appear in:
International Journal of Radiation Oncology • Biology • Physics
Received Date: 18 March 2016 Revised Date:
18 June 2016
Accepted Date: 23 June 2016
Please cite this article as: Tian X, Warner SB, Wagner KT, Caster JM, Zhang T, Ohana P, Gabizon AA, Wang AZ, Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy, International Journal of Radiation Oncology • Biology • Physics (2016), doi: 10.1016/j.ijrobp.2016.06.2457. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy.
Xi Tian, Ph.D.1, Samuel B. Warner1, Kyle T. Wagner, B.S.1, Joseph M. Caster, M.D., Ph.D.1, Tian Zhang, M.D.2, Patricia Ohana, Ph.D.3, Alberto A. Gabizon, M.D., Ph.D.3,4,
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Andrew Z. Wang, M.D.1*
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1. Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center,
Carolina Center for Cancer Nanotechnology Excellence
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Carolina Institute of Nanomedicine
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 2. Division of Medical Oncology, Department of Medicine
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Duke Cancer Institute
Duke University Medical Center
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Durham, NC 27705
3. Lipomedix Pharmaceuticals Ltd. POB 39262
Jerusalem 9139102, Israel
4. Shaare Zedek Medical Ctr., Jerusalem 91031, Israel
Short title: Promitil as a radiosensitizer
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*Corresponding Author:
Department of Radiation Oncology
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Lineberger Comprehensive Cancer Center
(P) 919-966-7700, (F) 919-966-7681
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Email: [email protected]
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CB 7512, UNC Chapel Hill Chapel Hill, North Carolina, 27514
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Andrew Zhuang Wang
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Summary Promitil was evaluated preclinically for chemoradiotherapy in colorectal cancer models. We demonstrated that Promitil can be safely administered with concurrent radiation at doses not
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achievable with free MMC. More importantly, Promitil enhanced the antitumor efficacy of 5Fluorouracil-based chemoradiotherapy in human colorectal tumor xenografts, whereas equitoxic doses of MMC did not. Our data supports clinical investigations utilizing Promitil in
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chemoradiotherapy of solid tumors.
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Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy.
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Abstract Purpose: Mitomycin C (MMC) is a potent chemotherapeutic and radiosensitizer, but its use has been limited by toxicity. Promitil is a pegylated liposomal formulation of a MMC lipid-based
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prodrug, which has shown a significantly lower toxicity profile in preclinical and phase I clinical investigations. In this study, we examined the effect of radiation on in vitro drug release and
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examined the efficacy and toxicity of Promitil with concurrent radiation in colorectal cancer models.
Methods and Materials: Promitil was obtained from LipoMedix under a research agreement. We tested the effects of radiation on release of active MMC from Promitil in vitro. We next examined the radiosensitization effect of Promitil in vitro. We further evaluated the toxicity of a
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single injection of free MMC or Promitil when combined with radiation by assessing the effects on blood counts and in-field skin and hair toxicity. Finally, we compared the efficacy of MMC
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and Promitil in chemoradiotherapy using mouse xenograft models. Results: MMC was released from Promitil in a controlled release profile, and the rate of release
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was significantly increased in medium from previously irradiated cells. Both Promitil and MMC potently radiosensitized HT-29 cells in vitro. Toxicity of MMC (8.4 mg/kg) was substantially greater than equivalent doses of Promitil (30 mg/kg). Mice treated with human equivalent doses of MMC (3.3 mg/kg) experienced comparable levels of toxicity as Promitil-treated mice. Promitil improved the antitumor efficacy of 5-Fluorouracil-based chemoradiotherapy in mouse xenograft models of colorectal cancer; equitoxic doses of MMC did not.
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Conclusions: We demonstrated that Promitil is an attractive agent for chemoradiotherapy because it demonstrates a radiation-triggered release of active drug. We further demonstrated that Promitil is a well-tolerated and potent radiosensitizer at doses not achievable with free
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Keywords: Mitomycin C, Radiation, Nanoparticle, Toxicity
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MMC. These results support clinical investigations utilizing Promitil in chemoradiotherapy.
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Introduction Chemoradiotherapy (CRT) is a key treatment paradigm in the curative management of many cancers, including rectal cancer[1]. Among the chemotherapeutic agents that are given as
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part of a CRT regimen, mitomycin C (MMC) is one of the most potent and effective[2-4]. Unfortunately, MMC has significant toxicity; its use is associated with several significant side effects including hemolytic uremic syndrome (HUS), pulmonary fibrosis, and delayed
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myelosuppression, which can be both severe and prolonged. At present, MMC is not widely utilized in many clinical applications, outside of CRT for anal cancer and intravesicular
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instillation for superficial bladder cancer, due to its potential toxicity.
Recently, Gabizon et al. developed a liposomal formulation of a MMC prodrug, currently known as Promitil, to minimize MMC toxicity and retain activity against multidrug-resistant tumors[5]. Promitil is a pegylated liposomal formulation of a MMC lipid-based prodrug (MLP)
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that has shown a significantly lower toxicity profile in preclinical and phase I clinical investigations[6]. Promitil has a maximum tolerated dose (MTD) of 3 mg/kg every 4 weeks, which is 2.7-fold greater than the MTD of free MMC in humans[6]. Promitil is designed to
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release active MMC mainly in tumors. MMC is linked to glycerol lipids through a cleavable dithiobenzyl bridge and conversion of the prodrug to active MMC requires cleaving this
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dithiobenzyl bridge by reducing agents, which are found in particularly high concentrations within tumors[7].
Given the favorable toxicity profile of Promitil, we hypothesized that it is more effective
and less toxic than MMC in a CRT regimen. Nanoparticle (NP) formulations of chemotherapeutic drugs impart a number of physical advantages, which can improve their therapeutic index in CRT. NPs preferentially accumulate in tumors as a result of the enhanced
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permeability and retention (EPR) effect[8]. In addition, Promitil’s slow MMC release potentiates a synergistic effect when combined with radiotherapy. Several preclinical studies have demonstrated the potential for nanoformulation to improve the therapeutic index of CRT[9-12].
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Lastly, we theorized that radiotherapy-induced cancer cell death can also trigger MMC release, further providing synergy between MMC and radiation. Therefore, we examined Promitil-based
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CRT using mouse xenograft models of colorectal cancer.
Materials and Methods
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Materials
Promitil was provided by Lipomedix Pharmaceuticals Ltd. (Jerusalem, Israel). 5 mg/ml Promitil is equivalent to 1.4 mg/ml MMC. MMC was purchased from Fisher Scientific (Fair Lawn, NJ),
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and 5-Fluorouracil (5-FU) was purchased from Sigma-Aldrich.
Mice
Nu/Nu mice (female, 8-10 weeks old) were obtained from the animal colony at XXXX. 57BL/6J
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mice (male, 8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed in accordance with guidelines provided by XXX
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Institutional Animal Care and Use Committee under an approved protocol.
Cell Culture
Human colorectal cancer cell lines HT-29 and SW480 were obtained from the XXX. HT-29 and SW480 cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (Life Technologies), supplemented with 10% (vol/vol) fetal bovine serum (Life
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Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37oC in 5% CO2 humidified atmosphere.
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X-Ray Irradiation
Cells and mice were irradiated using a Precision X-RAD 320 (Precision X-Ray, Inc) machine operating at 320 kVp and 12.5 mA with a source surface distance of 47 cm at a dose rate of 110
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cGy/min. During irradiation, mice were shielded with 4 mm of lead while the tumor on the left
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flank remained exposed.
Ellman’s Test
Free thiol concentrations in culture medium were measured by Ellman’s test. One mL of culture medium from 3 x 105 HT-29 cells was collected 48 h after cells were irradiated with 0 (control)
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or 20 Gy ionizing radiation. Medium was centrifuged at 1,500 rpm for 2 min, and supernatant was collected for analysis. Culture medium without cells was used as blank control. Ten µL of 100 mM DNTB, 100 µL of 10x Tris, 390 µL ddH2O, and 500 µL of medium was mixed and
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poured into a disposable cuvette. Absorbance at 412 nm was measured after 5 min. The molarity
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of thiol groups was calculated using the extinction coefficient ɛ = 13,600 M−1cm−1.
Stability of Promitil in Medium or PBS Twenty µL of 5 mg/mL Promitil was mixed with 980 µL of either complete cell culture medium (10% FBS and 1% Penicillin/Streptomycin) or 1X PBS at 37oC. Samples were collected at 0, 24, 48, or 72 h of incubation for HPLC analysis.
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Drug Release Profile A microdialysis method was utilized to determine the effects of radiation on MMC release from Promitil in vitro. Schematic representation of the experimental design is shown in Figure 1A.
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Culture medium from HT-29 cells was collected 48 h after cells were irradiated with 0 (control) or 20 Gy ionizing radiation. Promitil (5 mg/ml) was mixed with medium at 1:1 ratio. The sample solution (100 µL) was then loaded into a Slide-A-Lyzer mini dialysis unit (2,000 MWCO,
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Thermo Scientific) against 1 x PBS buffer (1 mL) in a vial with gentle stirring at 37oC. Cleavage of the prodrug and release of MMC from the liposomal composition was determined as a
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function of time quantifying MMC levels in PBS surrounding the dialysis unit utilizing a Shimadzu HPLC unit (C18, 150 x 4.6 mm column with a 85/15 water/acetonitrile mobile phase at 1 mL/min flow rate) with a UV detector at 360 nm wavelength.
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Cell Viability Assay
Cells were seeded at 2,000 cells/well in 96-well plates overnight and treated with Promitil, MMC, or PBS (control) for 48 hours, washed with PBS after incubation and incubated in fresh complete
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medium for 48 hours. Following incubation, MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]
cell
proliferation
assays
were
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performed according to manufacturer protocols using the CellTiter 96® Aqueous One Solution Cell Proliferation assay kit (Promega).
Clonogenic Assay
Plating efficiency (PE) of each cell line was determined. Monolayer HT-29 or SW480 cells were treated with 10nM Promitil (MMC-equivalents), 10nM MMC, or PBS (control) for 48 hours,
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washed 3 times with fresh medium and then irradiated with 0, 1, 2, 4, 6, and 8 Gy. Following irradiation, cells were trypsinized and plated into 25mL flasks at densities ranging from 100 to 250,000 cells per flask. Cells were incubated for 13 days and then fixed and stained with 4%
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formaldehyde /80% methanol /0.25% crystal violet (Fisher Scientific). All colonies containing 30 or more cells were counted. The surviving fraction (SF) was calculated using the formula /(# )() . SF was plotted against the radiation dose on a log scale.
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Linear-quadratic formula SF= was used to generate survival curves. The sensitization enhancement ratio (SER) was calculated by dividing the dose of radiation required to kill 90% of
In vivo Toxicity Assessments
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untreated cells by the dose of radiation required to kill 90% of drug-treated cells.
C57BL/6J mice were intravenously injected with 30 mg/kg of Promitil (8.8 mg/kg MMC-
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equivalents), a nearly equivalent dose of MMC (8.4 mg/kg), a lower dose of MMC (3.3 mg/kg), or PBS (control) followed by 3 daily doses of radiation to the left flank (5 Gy x 3). Overall survival was summarized in a table. For acute hematologic toxicity, blood samples were
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collected via cardiac puncture after 1 or 7 days of single dose injection. Animals were anesthetized with a ketamine/xylazine solution prior to the cardiac puncture procedure. A 100 µL
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sample of whole blood was stored in EDTA coated tubes at 4°C prior to analysis at the XXX. Weight loss was assessed after a single dose of 15 Gy in all groups except for high dose MMC (8.4 mg/kg) in which no animals survived long enough for long-term analysis. Mice were weighed every 2-3 days to assess for weight loss.
In Vivo Drug Efficacy
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HT-29 and SW480 cells (1 × 106 cells in 200 uL 1:1 DMEM:F12 and Matrigel) were subcutaneously injected into the left dorsal flank of 8 week old male Nu/Nu mice. Treatment began approximately ten days after tumor inoculation when tumors reached volumes of 100 to
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150 mm3. Mice were weighed and randomly sorted into treatment groups (8-10 mice per group). A single dose of Promitil (30 mg/kg) or MMC (3.3 mg/kg) with or without 5-FU (20 mg/kg) were administered by tail-vein I.V. injection. This dose of Promitil (30 mg/kg) was selected as it
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is a safe (sub-MTD) dose of Promitil in mice., It delivers an equivalent dose of 8.8 mg/kg MMC. The dose of 3.3 mg/kg MMC was selected as it is equivalent to clinical dosing in humans of 10
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mg/m2 upon conversion to body surface-based dose. Starting one hour after IV injection, the xenografts were treated with 3 fractions of radiation (500 cGy each) spaced 24 hours apart. Tumor volumes were determined by measuring two perpendicular diameters, a and b, using the formula V = 0.5 x a x b2, with a and b being the longer and shorter diameters respectively.
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Measurements were recorded every 3 days using a digital caliper. The relative tumor size was defined by V/V0, with V as the volume calculated and V0 being the tumor volume measured on the initial day of treatment. Mice were euthanized when tumors achieved a maximum dimension
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Statistical Analysis
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of 2 cm or 10 times the initial volume.
Statistical tests were performed using GraphPad Prism or R software. Results from drug release, MTS, body weight, and blood toxicity were compared using one-way ANOVA followed by posthoc analysis when significant main effects or interactions were observed. Results from tumor growth data were analyzed using area under the curve analysis as previously described[9]. For
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clonogenic assay, the linear-quadratic cell survival curves were compared with the CFAssay
Results Radiation enhances MMC release from Promitil
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function cellsurvLQdiff (R software). P-values less than 0.05 were considered significant.
We examined the stability of Promitil in culture medium (without cells) or PBS. As
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shown in Figure 1B, there was no degradation of Promitil in either solution, even after 72 hours of incubation. The MMC prodrug in Promitil was designed to be cleaved by increased levels of
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reducing agents. We hypothesized that radiation-induced cell damage or cell death releases many cellular components; many of which are reducing agents that can in turn increase MMC release from Promitil. We tested this hypothesis by measuring the concentration of free thiol groups in cell medium containing irradiated or non-irradiated cells and found that radiation significantly
1C).
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increased (~2.6 fold) the concentration of free thiol groups in culture medium (p<0.001) (Figure
We next quantified the drug release profile of Promitil in culture medium from irradiated
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or non-irradiated (control) cells. Experimental set up is shown in Figure 1A. As seen in Fig 1D, MMC release is significantly faster when Promitil is exposed to medium from irradiated cells
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than that of non-irradiated cells. By 72 hours, Promitil in non-irradiated medium had released approximately 50% of its payload compared to almost 80% in irradiated medium.
In vitro cytotoxicity of MMC and Promitil To determine Promitil’s potential in CRT, we first compared the in vitro cytotoxic effects of Promitil and MMC using colorectal cancer cell lines HT-29 and SW480. As shown in Figure
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2A, both cell lines demonstrated a dose-dependent response to Promitil and MMC. Using these dose-response curves, we selected the IC90 of free MMC (10 nM) for radiosensitization studies.
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HT-29 and SW480 cells were exposed to varying doses of radiation after drug treatment (10 nM MMC-equivalents). Radiation survival curves demonstrated that both Promitil and MMC produced significant radiosensitization in HT-29 cells (p<0.001); however, there was no
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significant sensitization in SW480 cells (Figure 2B). Sensitization enhancement ratios (10% cell survival) were approximately 1.3 for MMC and 1.4 for Promitil in HT-29 cells. For SW480 cells,
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the SER was 1.13 and 1.16 for MMC and Promitil respectively.
Promitil-based chemoradiotherapy is well tolerated in mice
We then compared the toxicity of CRT with Promitil (30 mg/kg), an equivalent dose of
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MMC (8.8 mg/kg), and a clinically based dose of MMC (3.3 mg/kg). The combination of Promitil and flank radiation was well tolerated. Only one animal died 14 days following treatment, and it did not show any obvious signs of treatment toxicity prior to death. In contrast,
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animals treated with near-equivalent doses of MMC (8.4 mg/kg) and flank radiation (developed oral mucositis, rapid weight loss, and 100% died within 20 days of administration (Figure 3A).
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The clinically based dose of MMC was well tolerated with 100% survival and transient weight loss, which was similar to Promitil-treated mice (Figure 3B). Hematologic toxicity is a significant effect of MMC. To assess hematologic toxicity of
Promitil-based chemoradiotherapy, we assessed peripheral blood counts on days 1 and 7 after treatment with a single injection of a drug and irradiation in non-tumor bearing mice. As seen in Figure 3C, white blood cell (WBC) counts decreased in all treatment groups. This effect was
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most pronounced in the high-dose MMC group. While WBC counts decreased in Promitiltreated animals, they were still inside the normal range for mice and returned to normal within 7 days. In contrast, WBCs remained low after 7 days following high dose (8.4 mg/kg) MMC.
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MMC also affects platelet counts. After seven days, animals in all treatment groups had platelet
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Promitil is more effective than MMC in chemoradiotherapy
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To investigate the efficacy of Promitil-based CRT regimens, we utilized two murine flank xenograft models of colorectal cancer. Mice received a single injection of Promitil (30 mg/kg) or MMC (3.3 mg/kg) with fractionated radiotherapy (5 Gy x 3) +/- 5FU. High-dose MMC was not utilized because animals would not have survived long enough to sufficiently
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evaluate tumor growth. Promitil-based CRT significantly prolonged tumor growth delay in both HT-29 (Promitil+XRT vs MMC+XRT: p-value = 0.02786) and SW480 (5-FU+CRLX101+XRT vs CRLX101+XRT: p-value = 0.02331) tumor xenografts (Figure 4A and 4B). The combination
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of 5-FU + Promitil further enhanced therapeutic efficacy in HT-29 (5FU+Promitil+XRT vs 5FU+MMC+XRT: p-value=0.001234), but not SW480 xenografts (5FU+Promitil+XRT vs
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5FU+MMC+XRT: p-value=0.3176) (Figure 4A and 4B). MMC did not significantly prolong tumor growth delay in either tumor model and there was no benefit to the addition of MMC to 5FU. Our results demonstrate that Promitil is more effective than tolerable doses of MMC in CRT. Discussion There is strong interest in broadening the clinical utility of MMC in CRT because it is a potent radiosensitizer that can potentially improve clinical outcomes for patients with solid tumor
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malignancies. Novel approaches, which decrease the toxicity of MMC, are needed to accomplish this goal. Promitil is a well-tolerated liposomal formulation of a MMC prodrug in early phase clinical trials. Once Promitil is stable in circulation within the tissues, exposure to reducing
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agents cleaves and activates the prodrug, which releases MMC from Promitil. We theorized that radiotherapy-induced cell death will transiently enrich the tumor microenvironment with reducing agents and trigger the release of MMC from Promitil; this will allow tumor-specific
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prodrug activation. Finally, we hypothesized that Promitil would be more effective and less toxic
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than MMC for use in CRT.
The development of radiation-triggered drug release vectors holds great potential for improving the therapeutic index of CRT. Our in vitro studies support our hypothesis that radiation enhances MMC release from Promitil by increasing levels of thiol reducing agents within the tumor microenvironment (Figure 5). By stimulating drug release within tumors,
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radiation can directly enhance the therapeutic efficacy of Promitil. The slow release from MMC in non-irradiated tissues helps to further mitigate the off-target effects of particles which do interact with normal tissues. Thus, the concurrent use of radiation helps to maximize the
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therapeutic advantages of Promitil.
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As a chemotherapeutic, Promitil was less toxic to both HT-29 and SW480 tumor cells than free MMC in vitro because pegylated liposomes are very stable and uptake by tumor cells is minimal[13]. This is consistent with the delayed release of active MMC in the absence of radiation (Figure 1C). However, Promitil was an equally potent radiosensitizer to free MMC in HT29 cells in vitro. When combined with radiation, controlled release from nanoparticles can prolong the time for synergistic interaction between radiation and drug, which enhances
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cytotoxicity. The triggered release of Promitil by radiation combines the pharmacologic benefits of rapid drug onset and prolonged drug release, making it a potent radiosensitizer. We demonstrated that Promitil is a potent radiosensitizer in vivo. A single injection of
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Promitil potently sensitized colorectal tumor xenografts to fractionated radiotherapy; however, a single injection of equitoxic free MMC +/- 5-FU did not. There are several likely explanations for improved efficacy with Promitil. Animals treated with Promitil received over twice the
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equivalent dose of MMC than animals in the free MMC groups (8.4 mg/kg vs 3.3 mg/kg). A therapeutic comparison between Promitil and MMC at this high dose was not possible because
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all of the animals in the MMC arm died of treatment-related toxicity. The well-established pharmacokinetic advantages of nanoformulation likely also contribute to the improved efficacy of Promitil. These include increased circulation time (t1/2 0.5 hours for free MMC vs 8-9 hours for Promitil), controlled sustained release, and preferential accumulation within tumors.
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Extended intratumoral release from particles may be particularly important because we utilized a fractionated radiation schedule delivered over 3 days. In the present study, human equivalent doses of MMC (3.3 mg/kg) did not radiosensitize
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tumor xenografts in vivo. Several studies have previously reported radiosensitizing effects of MMC in established tumors, but there are significant differences between those studies and the
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present one. Several studies did not assess in vivo tumor growth delay but rather ex vivo measures of clonogenic potential or cell survival[14,15]. Those that did assess tumor growth utilized higher doses of MMC (4-6 mg/kg) or high dose single fraction radiation (15-48 Gy)[1619]. High dose regimens such as these are rarely utilized in CRT, and our current dose/fractionation schedule more closely mimics common clinical practice. Our results suggest
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that in a clinically relevant treatment schema, Promitil provides enhanced efficacy not attainable with free MMC. In conclusion, we demonstrated that Promitil is more efficacious and less toxic than
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MMC in CRT. Importantly, radiation can trigger the release of MMC from Promitil, resulting in tumor-specific drug delivery. Our data support the development of early phase clinical trials
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evaluating Promitil-based CRT.
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1. Seiwert TY, Salama JK, Vokes EE. The concurrent chemoradiation paradigm—general principles. Nature Clinical Practice Oncology 2007;4:86-100. 2. Budach W, Paulsen F, Welz S, Classen J, Scheithauer H, Marini P, Belka C, Bamberg M. Mitomycin c in combination with radiotherapy as a potent inhibitor of tumour cell repopulation in a human squamous cell carcinoma. Br J Cancer 2002;86:470-476. 3. Volpe A, Racioppi M, D'Agostino D, Cappa E, Filianoti A, Bassi PF. Mitomycin c for the treatment of bladder cancer. Minerva urologica e nefrologica = The Italian journal of urology and nephrology 2010;62:133-144. 4. Ajani JA, Winter KA, Gunderson LL, Pedersen J, Benson AB, 3rd, Thomas CR, Jr., Mayer RJ, Haddock MG, Rich TA, Willett C. Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: A randomized controlled trial. Jama 2008;299:1914-1921. 5. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 6. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 7. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 8. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the epr effect in macromolecular therapeutics: A review. Journal of controlled release : official journal of the Controlled Release Society 2000;65:271-284. 9. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 10. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 11. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 12. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 13. Amitay Y, Shmeeda H, Patil Y, Gorin J, Tzemach D, Mak L, Ohana P, Gabizon A. Pharmacologic studies of a prodrug of mitomycin c in pegylated liposomes (promitil((r))): High stability in plasma and rapid thiolytic prodrug activation in tissues. Pharmaceutical research 2016;33:686-700. 14. Rockwell S. Cytotoxicities of mitomycin c and x rays to aerobic and hypoxic cells in vitro. International journal of radiation oncology, biology, physics 1982;8:1035-1039. 15. Rockwell S, Kennedy KA. Combination therapy with radiation and mitomycin c: Preliminary results with emt6 tumor cells in vitro and in vivo. International journal of radiation oncology, biology, physics 1979;5:1673-1676. 16. Rauth AM, Mohindra JK, Tannock IF. Activity of mitomycin c for aerobic and hypoxic cells in vitro and in vivo. Cancer research 1983;43:4154-4158. 17. Rockwell S. Effects of mitomycin c alone and in combination with x-rays on emt6 mouse mammary tumors in vivo. Journal of the National Cancer Institute 1983;71:765-771. 18. von der Maase H, Overgaard J, Vaeth M. Effect of cancer chemotherapeutic drugs on radiation-induced lung damage in mice. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 1986;5:245-257. 19. Spremulli EN, Leith JT, Bliven SF, Campbell DE, Dexter DL, Glicksman AS, Calabresi P. Response of a human colon adenocarcinoma (dld-1) to x irradiation and mitomycin c in vivo. International journal of radiation oncology, biology, physics 1983;9:1209-1212.
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Figures and Legends Figure 1: In vitro drug release profile. (A) Schematic diagram of the experimental design for in vitro drug release. (B) Percent of intact MLP in culture medium and PBS at 37oC over 72 h incubation. (C) Relative levels of free thiol group in medium of irradiated (Radiation) or nonirradiated (Control) HT-29 cells 48 h after radiation. (D)Release profile of Promitil when mixed with culture medium of irradiated (Radiation) or non-irradiated (Control) HT-29 cells in PBS at 37 °C.
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Figure 2: In vitro cytotoxicity of Promitil and MMC in colorectal cancer cell lines. (A) MTS assays in HT-29 and SW480 cells 48 hours following treatment with Promitil or MMC. (B) Clonogenic assay of HT-29 and SW480 cells after treatment with radiation and Promitil or MMC.
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Figure 3: Toxicity profile of Promitil as a radiosensitizer. (A) Table summarized the survival rate of C57BL/6J mice after treatment by MMC (8.4 mg/kg), MMC (3.3 mg/kg) or Promitil (30 mg/kg) with the combination of irradiation (3 x 5 Gy) within 20 days of administration. (B) After treatment, mice body weight was measured every 2-3 days over one month. (C) Peripheral WBC, lymphocyte, platelet, and RBC count 1 or 7 days after treatment with saline, 8.4 mg/kg MMC, 3.3 mg/kg MMC, or Promitil and fractionated radiotherapy. Dashed lines represent upper and lower limits of normal range for healthy mice.
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Figure 4: In vivo antitumor activity of Promitil as a radiosensitizer for colorectal cancer cell xenograft models. Mice bearing flank tumor xenografts (A) HT-29 and (B) SW480 were intravenous injected with a single dose of Promitil (30 mg/kg), or MMC (3.3 mg/kg), and/or with 5-FU (20 mg/kg) followed by radiation therapy (XRT). Tumors were irradiated in 3 daily fractions of 5 Gy followed by chemotherapy injection.
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Figure 5: Schematic presentation demonstrating effect of radiation on MMC release from Promitil. (A) Promitil can passively target tumors through EPR effect. (B) Radiation induces cell death which releases reducing thiol groups. (C) The prodrug MLP consists of MMC and glycerol lipid, which are linked through a cleavable dithiobenzyl bridge, which is cleaved by the released reducing agents following radiation.
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S0360-3016(16)32808-5
DOI:
10.1016/j.ijrobp.2016.06.2457
Reference:
ROB 23705
To appear in:
International Journal of Radiation Oncology • Biology • Physics
Received Date: 18 March 2016 Revised Date:
18 June 2016
Accepted Date: 23 June 2016
Please cite this article as: Tian X, Warner SB, Wagner KT, Caster JM, Zhang T, Ohana P, Gabizon AA, Wang AZ, Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy, International Journal of Radiation Oncology • Biology • Physics (2016), doi: 10.1016/j.ijrobp.2016.06.2457. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy.
Xi Tian, Ph.D.1, Samuel B. Warner1, Kyle T. Wagner, B.S.1, Joseph M. Caster, M.D., Ph.D.1, Tian Zhang, M.D.2, Patricia Ohana, Ph.D.3, Alberto A. Gabizon, M.D., Ph.D.3,4,
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Andrew Z. Wang, M.D.1*
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1. Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center,
Carolina Center for Cancer Nanotechnology Excellence
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Carolina Institute of Nanomedicine
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 2. Division of Medical Oncology, Department of Medicine
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Duke Cancer Institute
Duke University Medical Center
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Durham, NC 27705
3. Lipomedix Pharmaceuticals Ltd. POB 39262
Jerusalem 9139102, Israel
4. Shaare Zedek Medical Ctr., Jerusalem 91031, Israel
Short title: Promitil as a radiosensitizer
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*Corresponding Author:
Department of Radiation Oncology
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Lineberger Comprehensive Cancer Center
(P) 919-966-7700, (F) 919-966-7681
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Email: [email protected]
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CB 7512, UNC Chapel Hill Chapel Hill, North Carolina, 27514
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Andrew Zhuang Wang
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Summary Promitil was evaluated preclinically for chemoradiotherapy in colorectal cancer models. We demonstrated that Promitil can be safely administered with concurrent radiation at doses not
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achievable with free MMC. More importantly, Promitil enhanced the antitumor efficacy of 5Fluorouracil-based chemoradiotherapy in human colorectal tumor xenografts, whereas equitoxic doses of MMC did not. Our data supports clinical investigations utilizing Promitil in
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chemoradiotherapy of solid tumors.
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Preclinical evaluation of Promitil®, a radiation-responsive liposomal formulation of mitomycin C prodrug, in chemoradiotherapy.
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Abstract Purpose: Mitomycin C (MMC) is a potent chemotherapeutic and radiosensitizer, but its use has been limited by toxicity. Promitil is a pegylated liposomal formulation of a MMC lipid-based
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prodrug, which has shown a significantly lower toxicity profile in preclinical and phase I clinical investigations. In this study, we examined the effect of radiation on in vitro drug release and
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examined the efficacy and toxicity of Promitil with concurrent radiation in colorectal cancer models.
Methods and Materials: Promitil was obtained from LipoMedix under a research agreement. We tested the effects of radiation on release of active MMC from Promitil in vitro. We next examined the radiosensitization effect of Promitil in vitro. We further evaluated the toxicity of a
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single injection of free MMC or Promitil when combined with radiation by assessing the effects on blood counts and in-field skin and hair toxicity. Finally, we compared the efficacy of MMC
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and Promitil in chemoradiotherapy using mouse xenograft models. Results: MMC was released from Promitil in a controlled release profile, and the rate of release
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was significantly increased in medium from previously irradiated cells. Both Promitil and MMC potently radiosensitized HT-29 cells in vitro. Toxicity of MMC (8.4 mg/kg) was substantially greater than equivalent doses of Promitil (30 mg/kg). Mice treated with human equivalent doses of MMC (3.3 mg/kg) experienced comparable levels of toxicity as Promitil-treated mice. Promitil improved the antitumor efficacy of 5-Fluorouracil-based chemoradiotherapy in mouse xenograft models of colorectal cancer; equitoxic doses of MMC did not.
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Conclusions: We demonstrated that Promitil is an attractive agent for chemoradiotherapy because it demonstrates a radiation-triggered release of active drug. We further demonstrated that Promitil is a well-tolerated and potent radiosensitizer at doses not achievable with free
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Keywords: Mitomycin C, Radiation, Nanoparticle, Toxicity
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MMC. These results support clinical investigations utilizing Promitil in chemoradiotherapy.
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Introduction Chemoradiotherapy (CRT) is a key treatment paradigm in the curative management of many cancers, including rectal cancer[1]. Among the chemotherapeutic agents that are given as
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part of a CRT regimen, mitomycin C (MMC) is one of the most potent and effective[2-4]. Unfortunately, MMC has significant toxicity; its use is associated with several significant side effects including hemolytic uremic syndrome (HUS), pulmonary fibrosis, and delayed
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myelosuppression, which can be both severe and prolonged. At present, MMC is not widely utilized in many clinical applications, outside of CRT for anal cancer and intravesicular
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instillation for superficial bladder cancer, due to its potential toxicity.
Recently, Gabizon et al. developed a liposomal formulation of a MMC prodrug, currently known as Promitil, to minimize MMC toxicity and retain activity against multidrug-resistant tumors[5]. Promitil is a pegylated liposomal formulation of a MMC lipid-based prodrug (MLP)
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that has shown a significantly lower toxicity profile in preclinical and phase I clinical investigations[6]. Promitil has a maximum tolerated dose (MTD) of 3 mg/kg every 4 weeks, which is 2.7-fold greater than the MTD of free MMC in humans[6]. Promitil is designed to
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release active MMC mainly in tumors. MMC is linked to glycerol lipids through a cleavable dithiobenzyl bridge and conversion of the prodrug to active MMC requires cleaving this
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dithiobenzyl bridge by reducing agents, which are found in particularly high concentrations within tumors[7].
Given the favorable toxicity profile of Promitil, we hypothesized that it is more effective
and less toxic than MMC in a CRT regimen. Nanoparticle (NP) formulations of chemotherapeutic drugs impart a number of physical advantages, which can improve their therapeutic index in CRT. NPs preferentially accumulate in tumors as a result of the enhanced
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permeability and retention (EPR) effect[8]. In addition, Promitil’s slow MMC release potentiates a synergistic effect when combined with radiotherapy. Several preclinical studies have demonstrated the potential for nanoformulation to improve the therapeutic index of CRT[9-12].
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Lastly, we theorized that radiotherapy-induced cancer cell death can also trigger MMC release, further providing synergy between MMC and radiation. Therefore, we examined Promitil-based
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CRT using mouse xenograft models of colorectal cancer.
Materials and Methods
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Materials
Promitil was provided by Lipomedix Pharmaceuticals Ltd. (Jerusalem, Israel). 5 mg/ml Promitil is equivalent to 1.4 mg/ml MMC. MMC was purchased from Fisher Scientific (Fair Lawn, NJ),
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and 5-Fluorouracil (5-FU) was purchased from Sigma-Aldrich.
Mice
Nu/Nu mice (female, 8-10 weeks old) were obtained from the animal colony at XXXX. 57BL/6J
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mice (male, 8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed in accordance with guidelines provided by XXX
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Institutional Animal Care and Use Committee under an approved protocol.
Cell Culture
Human colorectal cancer cell lines HT-29 and SW480 were obtained from the XXX. HT-29 and SW480 cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (Life Technologies), supplemented with 10% (vol/vol) fetal bovine serum (Life
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Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37oC in 5% CO2 humidified atmosphere.
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X-Ray Irradiation
Cells and mice were irradiated using a Precision X-RAD 320 (Precision X-Ray, Inc) machine operating at 320 kVp and 12.5 mA with a source surface distance of 47 cm at a dose rate of 110
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cGy/min. During irradiation, mice were shielded with 4 mm of lead while the tumor on the left
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flank remained exposed.
Ellman’s Test
Free thiol concentrations in culture medium were measured by Ellman’s test. One mL of culture medium from 3 x 105 HT-29 cells was collected 48 h after cells were irradiated with 0 (control)
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or 20 Gy ionizing radiation. Medium was centrifuged at 1,500 rpm for 2 min, and supernatant was collected for analysis. Culture medium without cells was used as blank control. Ten µL of 100 mM DNTB, 100 µL of 10x Tris, 390 µL ddH2O, and 500 µL of medium was mixed and
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poured into a disposable cuvette. Absorbance at 412 nm was measured after 5 min. The molarity
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of thiol groups was calculated using the extinction coefficient ɛ = 13,600 M−1cm−1.
Stability of Promitil in Medium or PBS Twenty µL of 5 mg/mL Promitil was mixed with 980 µL of either complete cell culture medium (10% FBS and 1% Penicillin/Streptomycin) or 1X PBS at 37oC. Samples were collected at 0, 24, 48, or 72 h of incubation for HPLC analysis.
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Drug Release Profile A microdialysis method was utilized to determine the effects of radiation on MMC release from Promitil in vitro. Schematic representation of the experimental design is shown in Figure 1A.
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Culture medium from HT-29 cells was collected 48 h after cells were irradiated with 0 (control) or 20 Gy ionizing radiation. Promitil (5 mg/ml) was mixed with medium at 1:1 ratio. The sample solution (100 µL) was then loaded into a Slide-A-Lyzer mini dialysis unit (2,000 MWCO,
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Thermo Scientific) against 1 x PBS buffer (1 mL) in a vial with gentle stirring at 37oC. Cleavage of the prodrug and release of MMC from the liposomal composition was determined as a
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function of time quantifying MMC levels in PBS surrounding the dialysis unit utilizing a Shimadzu HPLC unit (C18, 150 x 4.6 mm column with a 85/15 water/acetonitrile mobile phase at 1 mL/min flow rate) with a UV detector at 360 nm wavelength.
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Cell Viability Assay
Cells were seeded at 2,000 cells/well in 96-well plates overnight and treated with Promitil, MMC, or PBS (control) for 48 hours, washed with PBS after incubation and incubated in fresh complete
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medium for 48 hours. Following incubation, MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]
cell
proliferation
assays
were
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performed according to manufacturer protocols using the CellTiter 96® Aqueous One Solution Cell Proliferation assay kit (Promega).
Clonogenic Assay
Plating efficiency (PE) of each cell line was determined. Monolayer HT-29 or SW480 cells were treated with 10nM Promitil (MMC-equivalents), 10nM MMC, or PBS (control) for 48 hours,
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washed 3 times with fresh medium and then irradiated with 0, 1, 2, 4, 6, and 8 Gy. Following irradiation, cells were trypsinized and plated into 25mL flasks at densities ranging from 100 to 250,000 cells per flask. Cells were incubated for 13 days and then fixed and stained with 4%
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formaldehyde /80% methanol /0.25% crystal violet (Fisher Scientific). All colonies containing 30 or more cells were counted. The surviving fraction (SF) was calculated using the formula /(# )() . SF was plotted against the radiation dose on a log scale.
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Linear-quadratic formula SF= was used to generate survival curves. The sensitization enhancement ratio (SER) was calculated by dividing the dose of radiation required to kill 90% of
In vivo Toxicity Assessments
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untreated cells by the dose of radiation required to kill 90% of drug-treated cells.
C57BL/6J mice were intravenously injected with 30 mg/kg of Promitil (8.8 mg/kg MMC-
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equivalents), a nearly equivalent dose of MMC (8.4 mg/kg), a lower dose of MMC (3.3 mg/kg), or PBS (control) followed by 3 daily doses of radiation to the left flank (5 Gy x 3). Overall survival was summarized in a table. For acute hematologic toxicity, blood samples were
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collected via cardiac puncture after 1 or 7 days of single dose injection. Animals were anesthetized with a ketamine/xylazine solution prior to the cardiac puncture procedure. A 100 µL
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sample of whole blood was stored in EDTA coated tubes at 4°C prior to analysis at the XXX. Weight loss was assessed after a single dose of 15 Gy in all groups except for high dose MMC (8.4 mg/kg) in which no animals survived long enough for long-term analysis. Mice were weighed every 2-3 days to assess for weight loss.
In Vivo Drug Efficacy
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HT-29 and SW480 cells (1 × 106 cells in 200 uL 1:1 DMEM:F12 and Matrigel) were subcutaneously injected into the left dorsal flank of 8 week old male Nu/Nu mice. Treatment began approximately ten days after tumor inoculation when tumors reached volumes of 100 to
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150 mm3. Mice were weighed and randomly sorted into treatment groups (8-10 mice per group). A single dose of Promitil (30 mg/kg) or MMC (3.3 mg/kg) with or without 5-FU (20 mg/kg) were administered by tail-vein I.V. injection. This dose of Promitil (30 mg/kg) was selected as it
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is a safe (sub-MTD) dose of Promitil in mice., It delivers an equivalent dose of 8.8 mg/kg MMC. The dose of 3.3 mg/kg MMC was selected as it is equivalent to clinical dosing in humans of 10
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mg/m2 upon conversion to body surface-based dose. Starting one hour after IV injection, the xenografts were treated with 3 fractions of radiation (500 cGy each) spaced 24 hours apart. Tumor volumes were determined by measuring two perpendicular diameters, a and b, using the formula V = 0.5 x a x b2, with a and b being the longer and shorter diameters respectively.
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Measurements were recorded every 3 days using a digital caliper. The relative tumor size was defined by V/V0, with V as the volume calculated and V0 being the tumor volume measured on the initial day of treatment. Mice were euthanized when tumors achieved a maximum dimension
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Statistical Analysis
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of 2 cm or 10 times the initial volume.
Statistical tests were performed using GraphPad Prism or R software. Results from drug release, MTS, body weight, and blood toxicity were compared using one-way ANOVA followed by posthoc analysis when significant main effects or interactions were observed. Results from tumor growth data were analyzed using area under the curve analysis as previously described[9]. For
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clonogenic assay, the linear-quadratic cell survival curves were compared with the CFAssay
Results Radiation enhances MMC release from Promitil
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function cellsurvLQdiff (R software). P-values less than 0.05 were considered significant.
We examined the stability of Promitil in culture medium (without cells) or PBS. As
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shown in Figure 1B, there was no degradation of Promitil in either solution, even after 72 hours of incubation. The MMC prodrug in Promitil was designed to be cleaved by increased levels of
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reducing agents. We hypothesized that radiation-induced cell damage or cell death releases many cellular components; many of which are reducing agents that can in turn increase MMC release from Promitil. We tested this hypothesis by measuring the concentration of free thiol groups in cell medium containing irradiated or non-irradiated cells and found that radiation significantly
1C).
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increased (~2.6 fold) the concentration of free thiol groups in culture medium (p<0.001) (Figure
We next quantified the drug release profile of Promitil in culture medium from irradiated
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or non-irradiated (control) cells. Experimental set up is shown in Figure 1A. As seen in Fig 1D, MMC release is significantly faster when Promitil is exposed to medium from irradiated cells
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than that of non-irradiated cells. By 72 hours, Promitil in non-irradiated medium had released approximately 50% of its payload compared to almost 80% in irradiated medium.
In vitro cytotoxicity of MMC and Promitil To determine Promitil’s potential in CRT, we first compared the in vitro cytotoxic effects of Promitil and MMC using colorectal cancer cell lines HT-29 and SW480. As shown in Figure
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2A, both cell lines demonstrated a dose-dependent response to Promitil and MMC. Using these dose-response curves, we selected the IC90 of free MMC (10 nM) for radiosensitization studies.
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HT-29 and SW480 cells were exposed to varying doses of radiation after drug treatment (10 nM MMC-equivalents). Radiation survival curves demonstrated that both Promitil and MMC produced significant radiosensitization in HT-29 cells (p<0.001); however, there was no
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significant sensitization in SW480 cells (Figure 2B). Sensitization enhancement ratios (10% cell survival) were approximately 1.3 for MMC and 1.4 for Promitil in HT-29 cells. For SW480 cells,
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the SER was 1.13 and 1.16 for MMC and Promitil respectively.
Promitil-based chemoradiotherapy is well tolerated in mice
We then compared the toxicity of CRT with Promitil (30 mg/kg), an equivalent dose of
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MMC (8.8 mg/kg), and a clinically based dose of MMC (3.3 mg/kg). The combination of Promitil and flank radiation was well tolerated. Only one animal died 14 days following treatment, and it did not show any obvious signs of treatment toxicity prior to death. In contrast,
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animals treated with near-equivalent doses of MMC (8.4 mg/kg) and flank radiation (developed oral mucositis, rapid weight loss, and 100% died within 20 days of administration (Figure 3A).
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The clinically based dose of MMC was well tolerated with 100% survival and transient weight loss, which was similar to Promitil-treated mice (Figure 3B). Hematologic toxicity is a significant effect of MMC. To assess hematologic toxicity of
Promitil-based chemoradiotherapy, we assessed peripheral blood counts on days 1 and 7 after treatment with a single injection of a drug and irradiation in non-tumor bearing mice. As seen in Figure 3C, white blood cell (WBC) counts decreased in all treatment groups. This effect was
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most pronounced in the high-dose MMC group. While WBC counts decreased in Promitiltreated animals, they were still inside the normal range for mice and returned to normal within 7 days. In contrast, WBCs remained low after 7 days following high dose (8.4 mg/kg) MMC.
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MMC also affects platelet counts. After seven days, animals in all treatment groups had platelet
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Promitil is more effective than MMC in chemoradiotherapy
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To investigate the efficacy of Promitil-based CRT regimens, we utilized two murine flank xenograft models of colorectal cancer. Mice received a single injection of Promitil (30 mg/kg) or MMC (3.3 mg/kg) with fractionated radiotherapy (5 Gy x 3) +/- 5FU. High-dose MMC was not utilized because animals would not have survived long enough to sufficiently
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evaluate tumor growth. Promitil-based CRT significantly prolonged tumor growth delay in both HT-29 (Promitil+XRT vs MMC+XRT: p-value = 0.02786) and SW480 (5-FU+CRLX101+XRT vs CRLX101+XRT: p-value = 0.02331) tumor xenografts (Figure 4A and 4B). The combination
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of 5-FU + Promitil further enhanced therapeutic efficacy in HT-29 (5FU+Promitil+XRT vs 5FU+MMC+XRT: p-value=0.001234), but not SW480 xenografts (5FU+Promitil+XRT vs
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5FU+MMC+XRT: p-value=0.3176) (Figure 4A and 4B). MMC did not significantly prolong tumor growth delay in either tumor model and there was no benefit to the addition of MMC to 5FU. Our results demonstrate that Promitil is more effective than tolerable doses of MMC in CRT. Discussion There is strong interest in broadening the clinical utility of MMC in CRT because it is a potent radiosensitizer that can potentially improve clinical outcomes for patients with solid tumor
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malignancies. Novel approaches, which decrease the toxicity of MMC, are needed to accomplish this goal. Promitil is a well-tolerated liposomal formulation of a MMC prodrug in early phase clinical trials. Once Promitil is stable in circulation within the tissues, exposure to reducing
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agents cleaves and activates the prodrug, which releases MMC from Promitil. We theorized that radiotherapy-induced cell death will transiently enrich the tumor microenvironment with reducing agents and trigger the release of MMC from Promitil; this will allow tumor-specific
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prodrug activation. Finally, we hypothesized that Promitil would be more effective and less toxic
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than MMC for use in CRT.
The development of radiation-triggered drug release vectors holds great potential for improving the therapeutic index of CRT. Our in vitro studies support our hypothesis that radiation enhances MMC release from Promitil by increasing levels of thiol reducing agents within the tumor microenvironment (Figure 5). By stimulating drug release within tumors,
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radiation can directly enhance the therapeutic efficacy of Promitil. The slow release from MMC in non-irradiated tissues helps to further mitigate the off-target effects of particles which do interact with normal tissues. Thus, the concurrent use of radiation helps to maximize the
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therapeutic advantages of Promitil.
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As a chemotherapeutic, Promitil was less toxic to both HT-29 and SW480 tumor cells than free MMC in vitro because pegylated liposomes are very stable and uptake by tumor cells is minimal[13]. This is consistent with the delayed release of active MMC in the absence of radiation (Figure 1C). However, Promitil was an equally potent radiosensitizer to free MMC in HT29 cells in vitro. When combined with radiation, controlled release from nanoparticles can prolong the time for synergistic interaction between radiation and drug, which enhances
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cytotoxicity. The triggered release of Promitil by radiation combines the pharmacologic benefits of rapid drug onset and prolonged drug release, making it a potent radiosensitizer. We demonstrated that Promitil is a potent radiosensitizer in vivo. A single injection of
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Promitil potently sensitized colorectal tumor xenografts to fractionated radiotherapy; however, a single injection of equitoxic free MMC +/- 5-FU did not. There are several likely explanations for improved efficacy with Promitil. Animals treated with Promitil received over twice the
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equivalent dose of MMC than animals in the free MMC groups (8.4 mg/kg vs 3.3 mg/kg). A therapeutic comparison between Promitil and MMC at this high dose was not possible because
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all of the animals in the MMC arm died of treatment-related toxicity. The well-established pharmacokinetic advantages of nanoformulation likely also contribute to the improved efficacy of Promitil. These include increased circulation time (t1/2 0.5 hours for free MMC vs 8-9 hours for Promitil), controlled sustained release, and preferential accumulation within tumors.
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Extended intratumoral release from particles may be particularly important because we utilized a fractionated radiation schedule delivered over 3 days. In the present study, human equivalent doses of MMC (3.3 mg/kg) did not radiosensitize
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tumor xenografts in vivo. Several studies have previously reported radiosensitizing effects of MMC in established tumors, but there are significant differences between those studies and the
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present one. Several studies did not assess in vivo tumor growth delay but rather ex vivo measures of clonogenic potential or cell survival[14,15]. Those that did assess tumor growth utilized higher doses of MMC (4-6 mg/kg) or high dose single fraction radiation (15-48 Gy)[1619]. High dose regimens such as these are rarely utilized in CRT, and our current dose/fractionation schedule more closely mimics common clinical practice. Our results suggest
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that in a clinically relevant treatment schema, Promitil provides enhanced efficacy not attainable with free MMC. In conclusion, we demonstrated that Promitil is more efficacious and less toxic than
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MMC in CRT. Importantly, radiation can trigger the release of MMC from Promitil, resulting in tumor-specific drug delivery. Our data support the development of early phase clinical trials
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evaluating Promitil-based CRT.
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1. Seiwert TY, Salama JK, Vokes EE. The concurrent chemoradiation paradigm—general principles. Nature Clinical Practice Oncology 2007;4:86-100. 2. Budach W, Paulsen F, Welz S, Classen J, Scheithauer H, Marini P, Belka C, Bamberg M. Mitomycin c in combination with radiotherapy as a potent inhibitor of tumour cell repopulation in a human squamous cell carcinoma. Br J Cancer 2002;86:470-476. 3. Volpe A, Racioppi M, D'Agostino D, Cappa E, Filianoti A, Bassi PF. Mitomycin c for the treatment of bladder cancer. Minerva urologica e nefrologica = The Italian journal of urology and nephrology 2010;62:133-144. 4. Ajani JA, Winter KA, Gunderson LL, Pedersen J, Benson AB, 3rd, Thomas CR, Jr., Mayer RJ, Haddock MG, Rich TA, Willett C. Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: A randomized controlled trial. Jama 2008;299:1914-1921. 5. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 6. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 7. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 8. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the epr effect in macromolecular therapeutics: A review. Journal of controlled release : official journal of the Controlled Release Society 2000;65:271-284. 9. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 10. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 11. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 12. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. 13. Amitay Y, Shmeeda H, Patil Y, Gorin J, Tzemach D, Mak L, Ohana P, Gabizon A. Pharmacologic studies of a prodrug of mitomycin c in pegylated liposomes (promitil((r))): High stability in plasma and rapid thiolytic prodrug activation in tissues. Pharmaceutical research 2016;33:686-700. 14. Rockwell S. Cytotoxicities of mitomycin c and x rays to aerobic and hypoxic cells in vitro. International journal of radiation oncology, biology, physics 1982;8:1035-1039. 15. Rockwell S, Kennedy KA. Combination therapy with radiation and mitomycin c: Preliminary results with emt6 tumor cells in vitro and in vivo. International journal of radiation oncology, biology, physics 1979;5:1673-1676. 16. Rauth AM, Mohindra JK, Tannock IF. Activity of mitomycin c for aerobic and hypoxic cells in vitro and in vivo. Cancer research 1983;43:4154-4158. 17. Rockwell S. Effects of mitomycin c alone and in combination with x-rays on emt6 mouse mammary tumors in vivo. Journal of the National Cancer Institute 1983;71:765-771. 18. von der Maase H, Overgaard J, Vaeth M. Effect of cancer chemotherapeutic drugs on radiation-induced lung damage in mice. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 1986;5:245-257. 19. Spremulli EN, Leith JT, Bliven SF, Campbell DE, Dexter DL, Glicksman AS, Calabresi P. Response of a human colon adenocarcinoma (dld-1) to x irradiation and mitomycin c in vivo. International journal of radiation oncology, biology, physics 1983;9:1209-1212.
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Figures and Legends Figure 1: In vitro drug release profile. (A) Schematic diagram of the experimental design for in vitro drug release. (B) Percent of intact MLP in culture medium and PBS at 37oC over 72 h incubation. (C) Relative levels of free thiol group in medium of irradiated (Radiation) or nonirradiated (Control) HT-29 cells 48 h after radiation. (D)Release profile of Promitil when mixed with culture medium of irradiated (Radiation) or non-irradiated (Control) HT-29 cells in PBS at 37 °C.
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Figure 2: In vitro cytotoxicity of Promitil and MMC in colorectal cancer cell lines. (A) MTS assays in HT-29 and SW480 cells 48 hours following treatment with Promitil or MMC. (B) Clonogenic assay of HT-29 and SW480 cells after treatment with radiation and Promitil or MMC.
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Figure 3: Toxicity profile of Promitil as a radiosensitizer. (A) Table summarized the survival rate of C57BL/6J mice after treatment by MMC (8.4 mg/kg), MMC (3.3 mg/kg) or Promitil (30 mg/kg) with the combination of irradiation (3 x 5 Gy) within 20 days of administration. (B) After treatment, mice body weight was measured every 2-3 days over one month. (C) Peripheral WBC, lymphocyte, platelet, and RBC count 1 or 7 days after treatment with saline, 8.4 mg/kg MMC, 3.3 mg/kg MMC, or Promitil and fractionated radiotherapy. Dashed lines represent upper and lower limits of normal range for healthy mice.
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Figure 4: In vivo antitumor activity of Promitil as a radiosensitizer for colorectal cancer cell xenograft models. Mice bearing flank tumor xenografts (A) HT-29 and (B) SW480 were intravenous injected with a single dose of Promitil (30 mg/kg), or MMC (3.3 mg/kg), and/or with 5-FU (20 mg/kg) followed by radiation therapy (XRT). Tumors were irradiated in 3 daily fractions of 5 Gy followed by chemotherapy injection.
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Figure 5: Schematic presentation demonstrating effect of radiation on MMC release from Promitil. (A) Promitil can passively target tumors through EPR effect. (B) Radiation induces cell death which releases reducing thiol groups. (C) The prodrug MLP consists of MMC and glycerol lipid, which are linked through a cleavable dithiobenzyl bridge, which is cleaved by the released reducing agents following radiation.
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