Metronomic photodynamic therapy using an implantable LED device and orally administered 5-aminolevulinic acid

概要

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Metronomic photodynamic
therapy using an implantable LED
device and orally administered
5‑aminolevulinic acid
Izumi Kirino1,2, Katsuhiko Fujita3, Kei Sakanoue4, Rin Sugita5, Kento Yamagishi6,
Shinji Takeoka7, Toshinori Fujie6,8, Shinji Uemoto2 & Yuji Morimoto1*
Metronomic photodynamic therapy (mPDT) is a form of PDT that induces cancer cell death by
intermittent continuous irradiation with a relatively weak power of light for a long duration (several
days). We previously developed a wirelessly powered, fully implantable LED device and reported a
significant anti-tumor effect of mPDT. Considering application in clinical practice, the method used
for repeated administrations of photosensitizers required for mPDT should not have a high patient
burden such as the burden of transvenous administration. Therefore, in this study, we selected
5-aminolevulinic acid (ALA), which can be administered orally, as a photosensitizer, and we studied
the antitumor effects of mPDT. In mice with intradermal tumors that were orally administered ALA
(200 mg/kg daily for 5 days), the tumor in each mouse was simultaneously irradiated (8 h/day for
5 days) using a wirelessly powered implantable green LED device (532 nm, 0.05 mW). Tumor growth in
the mPDT-treated mice was suppressed by about half compared to that in untreated mice. The results
showed that mPDT using the wirelessly powered implantable LED device exerted an antitumor effect
even with the use of orally administered ALA, and this treatment scheme can reduce the burden of
photosensitizer administration for a patient.
One of the effective cancer treatments is photodynamic therapy (PDT). PDT uses a photosensitizer and light in
well-oxygenated cancer tissue to generate reactive oxygen species (ROS), resulting in cancer cell d
­ eath1. Tumorselective treatment is possible when light can be directed only to the tumor, and PDT therefore spares patients
from many of the adverse effects associated with chemotherapy, radiation, and ­surgery2. Since optical devices
such as fiber optics to directly irradiate the cancer tissues are used in PDT, the clinical applications of PDT are
limited to specified cancers, including skin cancers (which can be directly viewed), cancers in the upper aerodigestive tract (which can be endoscopically accessible), and brain tumors (which can be directly accessed via
an operation microscope). Although PDT for tumors in deeply located organs such as the liver, pancreas, and
ovaries is possible in principle, it has not been clinically applicable. This may be because the procedure involves
surgical invasion, such as laparotomy, and because of concerns about complications such as organ damage and
postoperative adhesion due to the high irradiation intensity (> 100 mW/cm2) used in conventional PDT.
A new modality of PDT for treating tumors in deeply located organs is therefore needed. One new modality
that has been focused on is metronomic PDT(mPDT), in which the photosensitizer is administered repeatedly
and light is irradiated during an extended period at low fluence r­ ates3, resulting in apoptosis-dominant cell
­death4. The key to the therapeutic efficacy of mPDT is that the number of tumor cells dying over time must
exceed the tumor’s regrowth r­ ate5. This implies that mPDT with a single administration of photosensitizer and
low-intensity light irradiation for a long time will not induce adequate tumor cell death to achieve tumor control.
Hence, repeated photosensitizer administration will be r­ equired5.

1

Department of Physiology, National Defense Medical College, Namiki 3‑2, Tokorozawa, Saitama  359‑8513,
Japan. 2Division of Hepatobiliary‑Pancreatic Surgery and Transplantation, Department of Surgery, Graduate
School of Medicine, Kyoto University, Kyoto, Japan. 3Institute for Materials Chemistry and Engineering, Kyushu
University, Fukuoka, Japan. 4Pleiades Technologies LLC, Fukuoka, Japan. 5Graduate School of Advanced Science
and Engineering, Waseda University, Tokyo, Japan. 6Research Organization for Nano & Life Innovation, Waseda
University, Tokyo, Japan. 7Faculty of Science and Engineering, Waseda University, Tokyo, Japan. 8School of Life
Science and Technology, Tokyo Institute of Technology, Tokyo, Japan. *email: gazy246@gmail.com
Scientific Reports |

(2020) 10:22017

| https://doi.org/10.1038/s41598-020-79067-7

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Figure 1.  Temporal changes in PPIX fluorescence after ALA administration. A mouse with intradermal
tumors (both sides of the back, indicated by dashed-line circles in the black-and-white (BW) photo) was
transesophageally administered 300 mg/kg of ALA, and PPIX-derived fluorescence was measured every hour up
to 10 h after administration.
Conventional PDT requires a light source that is capable of delivering about 100 mW/cm2 of irradiation
intensity, whereas mPDT only requires a low intensity of about 0.1 mW/cm2, thus making it possible to reduce
the size of the light source. This may allow implantation of the light source inside the body and treatment of
tumors in deeply located organs in body cavities (cranial, thoracic, and abdominal cavities), which has not been
possible with conventional PDT. We have recently developed a micro LED light source (11 × 7 × 0.8 mm) for
­mPDT6. It is wirelessly powered with a near-field communication (NFC) system and can be completely implanted
inside the body.
Since the first report on anti-tumor effects of mPDT by Wilson et al. in 2­ 0043, more than 10 studies have
been ­published3,7–9. A clinical benefit of mPDT has also been s­ hown10. However, the previously reported antitumor effects in most of the animal studies are qualitative without statistical evidence. On the other hand, using
the above-mentioned wirelessly powered LED device, we achieved complete eradication of more than 50% of
the tumors in model mice in our previous ­study6. In that study, photofrin was used as a photosensitizer and
was administered intravenously every three days. However, considering application in clinical practice, such a
repeated transvenous medication protocol (regimen) would be a burden for patients.
Almost all photosensitizers, including clinically used porphyrin derivatives and phthalocyanine derivatives,
are currently used by transvenous administration. The only exception is 5-aminolevulinic acid (ALA), which
is a photosensitizer (or more precisely a precursor to a photosensitizer) that can be administered orally. Hence,
considering the need for repeated administrations, ALA is the most desirable photosensitizer among the currently available photosensitizers.
Therefore, in this study, we verified the anti-tumor effect of mPDT using our established light system when
ALA was orally administered to mice.
In the present study, a green (532 nm) LED was used since our previous study showed that the anti-tumor
effect of mPDT using a green (532 nm) LED was greater than that of mPDT using a red (630 nm) ­LED6.

Results

Temporal changes in protoporphyrin IX (PPIX) after ALA administration.  After administration

to the body, ALA is transferred into cells and is metabolized into protoporphyrin IX (PPIX), which functions as
a photosensitizer. Administered ALA is known to accumulate more selectively in tumor tissues than in normal
­tissues11.
To determine the amount of ALA required to exert an antitumor effect in mPDT, we investigated the relationship between ALA dose and the amount of PPIX produced. In addition, to determine the optimal irradiation
period in mPDT, we investigated temporal changes in the amount of PPIX in tumors after ALA administration.
Since PPIX is also a fluorescent substance, accumulation of PPIX in the tumor can be determined indirectly by
fluorescence measurement, in which the relative intensity of fluorescence of PPIX from tumors can be evaluated.
An intradermal tumor model was made by implanting cancer cells at two locations in the backs of mice.
ALA solution was administered transesophageally using a sonde. Figure 1 shows the temporal distribution of
PPIX-derived fluorescence in the body skin of mice after administration of ALA (300 mg/kg), and Fig. 2 shows
the temporal change in relative intensity of PPIX-derived fluorescence at the tumor site when the amount of
ALA administered was varied.
The fluorescence intensity in systemic skin increased from 1 h after administration, and the fluorescence
intensity at the tumor site was stronger and larger amounts of PPIX were produced in tumors. The fluorescence
intensity at the tumor site reached the highest value at 2–3 h after ALA administration and then decreased
gradually. This fluorescence trend was observed when ALA doses of 133 mg/kg or more were used, but at doses
of 88.9 mg/kg, there was no significant increase in PPIX fluorescence in tumors. Peak PPIX fluorescence values
were almost the same at ALA doses of 133 to 300 mg/kg, but the decline of PPIX fluorescence after the peak was
more gradual with larger ALA doses.
The failure to detect PPIX fluorescence at an ALA dose of 88.9 mg/kg may be due to the experimental method
in which tumor-derived fluorescence is captured through the skin. Fluorescence of PPIX produced in skin tissues
became background noise, probably having masked the weak fluorescence from PPIX produced inside tumors.
Based on the above-described results, the ALA dose was set to 200 mg/kg in subsequent mPDT experiments. ...

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