Original Contribution
Sonodynamically Induced Antitumor Effects of 5-Aminolevulinic Acid and Fractionated Ultrasound Irradiation in an Orthotopic Rat Glioma Model

https://doi.org/10.1016/j.ultrasmedbio.2012.07.026Get rights and content

Abstract

The sonodynamically induced selective antitumor effects of 5-aminolevulinic acid (5-ALA) on a C6 glioma that was implanted in a rat brain were evaluated. One week after the inoculation of the brains with the C6 rat glioma cells, glioma development was monitored using a 1.5 T MRI. Brains both with and without intravenous administration of 5-ALA (60 mg/kg body weight) or Radachlorin (40 mg/kg body weight) were insonated by a 1 MHz ultrasound at a dose of 2.65 W/cm2. Irradiation was performed in a fractionated manner to avoid any thermal effects in the tissue due to the focused ultrasound; 16 min of irradiation were followed by a 3 min recess, then 4 min of resumed irradiation. Mean tumor sizes, measured after the rats were sacrificed 2 weeks post treatment, were 122.48 ± 39.64 mm3 in sham-operated rats, 87.42 ± 21.40 mm3 in rats receiving ultrasound without 5-ALA, 10.50 ± 8.20 mm3 in rats receiving ultrasound with 5-ALA, and 56.42 ± 12.48 mm3 in rats receiving ultrasound with Radachlorin. The tumor size was significantly smaller in the therapy group receiving sonodynamic 5-ALA than in any of the other groups (p < 0.05). This experimental rat model showed that sonodynamic therapy can be useful in the treatment of deep-seated malignant gliomas.

Introduction

Human malignant glial tumors, particularly glioblastomas, strongly invade neighboring tissue and cannot be completely resected surgically, even with up-to-date technologies such as neuronavigation and photodynamic diagnosis. Fewer than half of the patients with glial tumors survive more than 1 year, and the 5-year survival rate is only 5% to 8%, even when all existing treatment options are used (Lacroix et al. 2001). To eradicate the unresectable nests of tumor cells invading the adjacent normal brain tissue, it is necessary to activate any tumor-selective sensitizing agent used through external-body irradiation without damaging the surrounding normal tissue.

Sonodynamic therapy (SDT) has the potential to be a useful and noninvasive treatment, destroying deep-seated brain tumors by sonication through the human skull while avoiding damage to the surrounding normal brain tissue. By adjusting the acoustic intensity of the ultrasound, hazardous effects to the surrounding tissues can be minimized (Sasaki et al. 1998). In addition, a photosensitizing hematoporphyrin derivative or antitumor drugs (Saad and Hahn 1992; Suzuki et al. 2007), which have been found to localize selectively in some tumor cells, could be activated by ultrasound to generate reactive oxygen species from the cavitation effect, thereby yielding antitumor effects. Because ultrasound can penetrate both bone and soft tissues, it may have a great advantage over visible light, which has limited penetration and is employed only in an intraoperative manner in tumor photodynamic therapy (PDT). In contrast, the energy of ultrasound can be delivered into deep-seated lesions and can be focused into a small volume of tissue (ter Haar et al. 1991; ter Haar and Robertson 1993; Fry 1993).

A natural porhyrin precursor, 5-aminolevulinic acid (5-ALA), has already been used in fluorescent-assisted resection and PDT for human gliomas (Olzowy et al. 2002). Therefore, it is easy to apply to the treatment of human brain tumors. If we find a sonodynamically induced therapeutic effect of the administration of 5-ALA, SDT can be a treatment option that is an alternative to the current method of fluorescent-guided resection. Our treatment could potentially target a tumor infiltrating normal tissue because 5-ALA can accumulate in such tissues, which is a situation that cannot be remedied effectively by any existing method. Although a variety of photosensitizers have been tested on experimental tumors to determine whether any sonodynamically induced antitumor effects occurred, a comparison of the sonodynamic effects of various photosensitizer compounds on the same tumor model has not been well documented. It is important to note that SDT such as photodynamic therapy requires frequent optimization of a variety of experimental parameters to increase antitumor efficacy, including the drug-to-treatment interval and the dosages of both the ultrasound irradiation rate and the drug administration. In this study, we investigated the antitumor effects of SDT in experimental rat gliomas by using focused ultrasound in combination with the systemic administration of 5-ALA under optimized treatment parameters, and we compared those results with the antitumor effects of SDT from the systemic administration of Radachlorine.

Section snippets

Preparation of the sonodynamically active agent

The 5-ALA hydrochloride (Sigma Chemical Company, St. Louis, MO, USA) was dissolved in 0.01 M PBS (pH 7.4), sterilized, aliquoted, and stored in the dark at 4°C. All other reagents were commercial products of analytic grade. Radachlorine was purchased from the RADA-PHARMA group (RADA-PHARMA Co. Ltd., Moscow, Russia), which was stable in solution and in the dark.

Animal model preparation

Intracranial gliomas were prepared by inoculating 5 × 106 C6 glioma cells stereotactically 5 mm deep into the frontal lobe of the left

Tumor imaging by magnetic resonance imaging

MR images were made at a 1.5 T MRI unit (GE 1.5 T, Milwaukee, WI, USA) for selected rats to confirm the formation of gliomas at 7 or 10 days after the implantation of C6 cells into the brain tissue. Brain axial T1-weighted images were acquired using a wrist coil to monitor the formation of a tumor mass after injecting a contrast agent at a dose of 1 mg/kg. Tumor growth was typically 5 to 7 mm in diameter at either day 7 or day 10. Observations were made at the right frontal lobe in front of the

Discussion

Focused ultrasound with an intensity of less than 10 W/cm2 can penetrate deeply into tissue and affect a deep-seated lesion without damaging the surrounding tissue (Nonaka et al. 2009). However, the apparent advantage of the deep penetration in focused ultrasound over the use of laser light in PDT may be compromised by potent thermal damage to the tissue, as shown in the significant temperature elevation from single-dose irradiation in vivo (Fig. 3), unless the sonication is regulated. A

Conclusion

SDT with optimized 5-ALA pharmacokinetics and focused ultrasound irradiation was effective for the treatment of intracranial gliomas in rats. Selective tumor destruction and tumor growth inhibition were obtained by the nonthermal effects of weak ultrasound irradiation in a fractionated manner. The ultrasound was enhanced by 5-ALA and did not cause damage to the surrounding normal brain tissue. Great tumor regression through a single noninvasive treatment of brain gliomas, as shown in this

Acknowledgments

This work was supported by a grant in aid from the Catholic University of Daegu, Korea.

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