Abstract
The effect of different static magnetic field strengths, 1 T or 7 T, on the quality of nitroxyl radical-based magnetic resonance redox imaging (MRRI) was examined. A stable nitroxyl radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MC-PROXYL), was used as a T1 contrast agent. Phantoms and animals were scanned at 1 T and 7 T using a similar gradient echo sequence. The quality of T1-weighted images and susceptibility of T1-weighted signals were compared. The nitroxyl radical-based T1-weighted signal enhancement ratio was higher at 1 T compared with at 7 T when the identical phantom was scanned using a similar gradient echo sequence. The gradient echo scanning at 7 T was sensitive to movement and/or flux of the sample solution, which could result in the distortion of baseline T1-weighted signals. No such wobbling of the signal was observed when the experiment was done at 1 T. The detection at the lower field is less affected by voltex flow in the sample, much stable T1-weighted signal detection is available at the lower field. The visual characteristics of in vivo nitroxyl decay profiles were similar between the 1 T and 7 T experiments, except noises were large at 1 T. The correlation trends of in vivo decay constants among brain regions also similar between 1 T and 7 T experiments. Nitroxyl radical-based MRRI could be an adequate theranostic tool when performed on clinically popular low magnetic field MRI instruments.
Keywords: Redox-sensitive contrast agent, magnetic resonance imaging, nitroxyl radical, T1-weighted contrast, gradient echo, signal decay rate
Introduction
To provide safe and efficient radiation therapy, the observation of patho-physiological information from diseased tissue is very important. Oxygen concentration and/or redox status in target tissues, which can affect prognosis, is beneficial information for planning radiation therapy. Noninvasive imaging techniques to provide such bio-functional information have been developed on magnetic resonance based imaging modalities, such as electron paramagnetic resonance imaging (EPRI), magnetic resonance imaging (MRI), and Overhauser MRI (OMRI; also known as proton-electron double resonance imaging [PEDRI]).
The redox imaging technique, which can estimate tissue redox status non-invasively, utilizes nitroxyl radicals as a redox-sensitive contrast agent. Nitroxyl radicals are stable free radical species; however, nitroxyl radicals can be enzymatically reduced to their corresponding hydroxyl amines in living cells. The in vivo reduction rate of the nitroxyl radical was changed in pathological redox conditions. Numerous pathophysiological cases changing in vivo redox states were reported by electron paramagnetic resonance (EPR; also known as electron spin resonance [ESR]) spectroscopic experiments [1-12]. For example, inflammation models in rat stomach mucosa [1,2], which providehydroxyl radicals and/or superoxides, can accelerate the nitroxyl reduction rate, or the Se-deficient rat model [3], which generates hydrogen peroxide-rich conditions, decreased the nitroxyl reduction rate. It was reported using 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MC-PROXYL), a blood-brain-barrier permeable amphiphilic nitroxyl radical, as a redox probe that X-irradiation to mouse whole body modified tissue redox status and make the reduction rate slower [4].
The redox imaging technique was initially developed in the field of EPR [13,14]. EPR can directly detect paramagnetic species through the absorption of microwave and/or radio frequencies by the electron spins existing in a magnetic field. EPR can be combined with magnetic resonance based imaging to map stable paramagnetic compounds, such as nitroxyl radicals [15-17]. Several studies of in vivo EPRI, including pioneering work on redox imaging, were reported in the late 1990s [18-24]. EPRI, however, has some problems that need to be overcome, including low spatial resolution, low temporal resolution, no anatomical information, and volume limitation of the subject.
From the middle 2000s, MRI-based high-resolution redox imaging techniques have been reported [25]. MRI can also detect nitroxyl radicals thorough proton-T1 shortening effects of the paramagnetism of nitroxyl radicals. In other words, nitroxyl radicals can give contrast enhancement on T1-weighted MR images. By utilizing MRI to detect nitroxyl radicals, most of difficulties associated with EPRI were resolved. In addition, MRI has become a widely used diagnostic tool in clinical use, although the most-popular clinical MRI instruments work at relatively low field strengths of <3 T. However, most animal experiments were done at higher field strengths >4.7 T to obtain higher signal-to-noise ratios (SNR) and resulting higher spatial resolution [25-27]. In addition to the clinical systems, several low field preclinical MRI systems using 1 T permanent magnet have been developed and improved for in vivo measurements. In general, T1-contrast agents show higher T1-relaxivity at lower magnetic field [28].
High field instruments can give increasing SNR, which can be a significant advantage on the image based diagnosis. In homogeneity of RF field associated with high field operation requires proper shimming, which lead a technical complexity and difficulty of manipulation. In addition, high field magnetic resonance requires corresponding higher frequency and RF power, which makes specific absorption ratio (SAR) larger. Low field instruments face the issue of lower SNR; however, can provide much easy manipulating, human-body-friendly and wallet-friendly diagnostics. Especially for redox imaging, which is a kind of dynamic imaging and requires sequence of scans, low field instruments, which can keep SAR lower, may be suitable, when the redox information can be obtained adequately. Therefore, in this paper, the basic features of 1 T and 7 T MRI on nitroxyl-induced contrast were compared to consider the feasibility of redox imaging at low magnetic field strengths.
Materials and methods
Chemicals
3-Methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MC-PROXYL) was synthesized in accordance with the method reported previously [22,29]. The physical, chemical, and biological properties of MC-PROXYL are arranged in Table 1. Deionized water (Milli-Q system, Merck Millipore, Billerica, MA, USA) was used for preparing MC-PROXYL water solutions.
Table 1.
Physical, chemical, and biological properties of MC-PROXYL
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Animals
Seven-week-old female C3H mice were supplied by Japan SLC, Inc. (Shizuoka, Japan) Animals were housed five per cage in climatecontrolled (23 ± 1°C and 55 ± 5% humidity), circadian rhythm-adjusted (12-hr light-dark cycle) rooms and were allowed food and water ad libitum. Mice were used for experiments after a habituation period of 1 week. Experiments were carried out in compliance with and approved by the Animal Use Committee of the National Institute of Radiological Sciences.
Phantoms
Phantom-1 was composed of seven glass tubes (i.d. = 6.4 mm, o.d. = 8.0 mm) filled with 0, 0.4, 0.8, 1.4, 1.6, 2.3, 2.6 mM water solutions of MC-PROXYL, a nitroxyl contrast agent. The hexagonally arranged glass tubes were immersed in a larger outer plastic tube (Falcon, 50 mL disposal centrifuging tube) filled with pure water. The arrangement of phantom-1 is shown in Figure 1A.
Figure 1.
Schematic drawing of the phantoms. A: Phantom-1: seven hexagonally arranged glass tubes (i.d. = 6.4 mm, o.d. = 8.0 mm), containing different concentrations of MC-PROXYL-water solutions, were immersed in a larger outer plastic tube (Falcon, 50 mL disposable centrifuge tube) filled with pure water. Numbers in the figure are the concentrations of the MC-PROXYL in mM. B: Phantom-2: syringes containing the same volume of 4 mmol/L MC-PROXYL water solution or pure water were connected to a single plastic cylindrical reactor (internal diameter, 12.7 mm) through bifurcated vinyl tubing. The reactor and the connected lines were filled with pure water. The solutions in the syringes were pushed out, delivered into the reactor via the vinyl tubing, and then entered through a nozzle, which caused an unstable vortex water flow in the reactor. Surplus volume from reaction mixture was drained.
Phantom-2 (Figure 1B) was designed to observe the stability of image intensity, which can be disturbed vortexes flowing in a liquid sample. Syringes containing the same volume of 4 mmol/L MC-PROXYL water solution or pure water were connected to a single plastic cylindrical reactor (i.d. = 12.7 mm, length = 55 mm) through bifurcated vinyl tubing. The reactor and the connected lines were filled with pure water. When both syringes were pressed at the same time and at the same rate, the solutions in the syringes mixed through the vinyl tubing, and then delivered into the reactor through a nozzle, which produced an unstable vortex water flow in the reactor. Surplus volume from the reaction mixture was drained.
MRI scanner and pulse sequences
MR images were acquired using a 7 T horizontal MRI scanner (Magnet: 40 cm bore, Kobelco and Jastec, Kobe, Japan; console and electronics: Avence-I system and ParaVision® 5.1 system, BrukerBiospin, Ettlingen, Germany) and 1 T horizontal MRI scanner (permanent magnet, ParaVision® 5.1 system, BrukerBiospin). For in vitro 7 T MRI acquisition, a 35-mm inner-diameter quadrature volume coil for transmission and reception (Rapid Biomedical, Rimpar, Germany) was used. Sample temperature was maintained at room temperature (23 ± 0.5°C) using a gradient cooling system and air-conditioner. For in vitro 1 T MRI acquisition, a 35-mm inner-diameter solenoid coil (6-turned) for transmission and reception (Aspect Imaging, Shoham, Israel) was used. Sample temperature was maintained at room temperature (23 ± 0.5°C) using a fan and air-conditioner. For phantom-1, 2-dimensional, single-slice, inversion-recovery MRI was performed using a rapid acquisition with relaxation enhancement (RARE) sequence with the following parameters for T1 relaxometry: repetition time (TR) = 10,000 ms, effective echo time (TE) = 20 ms, inversion time = 52, 75, 100, 200, 400, 800, 1600, 3200, and 6400 ms, matrix size = 128 × 128, FOV = 32 × 32 mm, slice thickness (ST) = 2 mm, and number of acquisitions (NA) = 1. The slice orientation was horizontal. The total acquisition time was 5 min 20 s. For phantom-2, a set of five slices (0.75 mm thick with 0.75 mm gaps) of T1-weighted images was obtained repeatedly 70 times by multi slice FLASH sequences using the following parameters: TE = 6 ms (for 1 T) or 5.1 ms (for 7 T), TR = 75 ms, FA = 45°, matrix size = 128 × 128, FOV = 25.6 × 25.6 mm, and NA = 2. The nominal voxel resolution was 200 × 200 µm. The acquisition time for one set was 19.2 s, and the total scan time was 22 min 24 s.
For in vivo 7 T MRI acquisition, MR images were obtained using a same 7 T MRI scanner (40 cm bore, Kobelco + Jastec, and BrukerBiospin) witha 72-mm inner-diameter volume coil for transmission (BrukerBiospin) and a quadrature surface coil for reception (mouse head, Rapid Biomedical, Rimpar, Germany). Rectal temperature was maintained at 36 ± 0.5°C using a heater (Raid Biomedical). For in vivo 1 T MRI acquisition, MR images were obtained using a same 1 T MRI scanner (BrukerBiospin) with a 6-turned solenoid coil (Aspect Imaging). Rectal temperature was maintained at 36 ± 0.5°C using a warm-water circulation system. For both 7 T and 1 T in vivo MRI, same imaging parameters were used as in vitro phantom-2 study. All of the MRI data were reconstructed and analyzed using ParaVision software (Ver. 5.1, BrukerBiospin).
MRI measurement for phantoms
Phantom-1 was set on a MRI probe (volume coil for 7 T or solenoid coil for 1 T), and then scanned using multislice FLASH sequences, as described above. A single frame with five slices, which was vertical to the glass tubes, was obtained. For phantom-2, the plastic cylindrical reactor was set in the center of the MRI probe. A dynamic data set composed on 70 frames was obtained using multislice FLASH sequences. Frames were obtained every 20 s. Each frame contained five slices (1.5 mm thick with 2.5 mm center-to-center gap). After the 5th flame (i.e. from 1 min 40 s after the start of the scanning), both syringes were pushed at the same time and same rate to expel the reaction mixture. The injection took approximately 10 s. The scanning continued for further 20 min.
MRI measurement for an animal
A mouse was anesthetized using 2.0% isoflurane, and the tail vein was cannulated using a 30-gauge needle attached to a polyethylene tube (PE10, Becton Dickinson and Company, Sparks, MD, USA) for administration of MC-PROXYL. The mouse was placed on a body cradle (Rapid Biomedical) and immobilized using adhesive tape. The mouse was then placed in a proton volume radio-frequency (RF) resonator (72 mm i.d., BrukerBioSpin) with surface RF receiver (Rapid Biomedical). Rectal temperature was monitored using an optical fiber thermometer (FOT-M and FTI-10, FISO Technologies, Quebec, Canada) and electric temperature controller (E5CN, Omron, Kyoto, Japan) during MRI measurements. Breathing rate was monitored using a monitoring and gating system (Model 1030, SAII, New York, USA), and anesthesia was adjusted by watching the breathing rate on a case-by-case basis.
After 5th baseline image (1 min 40 sec), a 0.75 μmol/g b.w. (5 μL/g b.w. of 150 mM solution) of MC-PROXYL was injected intravenously. The injection, which took approximately 10 sec, was started at the beginning of the 6th scan and finished by the middle of 6th scan. The scanning continued for a further 20 min.
Image data analysis
MRI data were analyzed using ImageJ (a public domain Java image processing program that can be extended using plug-ins, http://rsb.info.nih.gov/ij/, NIH, Bethesda, MD, USA).
For dynamic imaging data, the T1-weighted signal enhancement of a given pixel at (x, y) coordination, ΔM%x,y, is as follows:
ΔM%x,y = ((Sx,y after CA injection/Sx,y before CA injection) -1) ×100 (1)
Where Sx,y is the image intensity of a pixel (x, y). CA indicate “contrast agent” used in the experiment. “Sx,y before CA injection” is an average image intensity of frame 1 to 5.
L-band EPR measurement for an animal
A mouse was anesthetized using 1.5% isoflurane, and the tail vein was cannulated for injection of MC-PROXYL. The mouse was fixed on an acrylic holder, and placed in the gap of magnet pole pieces of the L-band EPR apparatus (JEOL, Tokyo Japan). The 1.1 GHz single loop surface coil (5.5 mm i.d.) was put on the parietal region of the mouse’s head. The core body temperature in the rectum was monitored using a nonmagnetic probe (FISO Technologies Inc.), and was regulated at 37 ± 1°C using hot air and an infra-red lamp during EPR experiments. Immediately after the injection of the sterilized solution of MC-PROXYL into the tail vein (1.5 μmol/g b.w.), the lowest-field line of the triplet EPR spectra of MC-PROXYL in the head were measured repeatedly. The EPR conditions were as follows: microwave frequency = 1.04 GHz, scan rate = 10 mT/min, time constant = 0.1 sec, and field modulation frequency = 100 kHz, microwave power = 4.0 mW, and field modulation width = 1 mT. EPR data acquisition was controlled by the WIN-RAD ESR Data Analyzer System (Radical Research, Inc., Hino, Tokyo). The acquired EPR spectra were analyzed using an in-house line fitting program, and Gaussian line shape was fitted. The signal height and the line width of the fitted Gaussian line was measured and then EPR signal intensity was obtained by (signal height) × (line width)2. The EPR signal decay constants, k1 and k2, of MC-PROXYL were calculated from the slope of the signal decay curves, which were determined from semi-logarithmic plots of the EPR signal intensity.
Results and discussion
An absolutely identical phantom sample was scanned using different MR scanners, one operated at 1 T and the other at 7 T. The resolution, or perhaps it should be said SNR, of the image scanned by the 1 T scanner (Figure 2A) was discernibly worse compared with that of the 7 T scanner (Figure 2B). Both 1 T and 7 T imaging conditions, however, could clearly show the 0.8 mm wall of the glass tubes. This may be enough resolution for distinguishing the anatomical structure in a large subject, such as human, monkey, and/or relatively large rodents.
Figure 2.
Comparison of T1-weighted images of phantom-1 scanned by FLASH (TE = 5.1 ms, TR = 75 ms, FA = 45°) at 1 T and 7 T. T1-weighted MR images obtained at (A) 1 T and (B) 7 T. (C) Relations of percentage signal amplification (ΔM%) and concentrations of MC-PROXYL obtained at 1 T and 7 T. (D) Relations of T1-relaxivity (R1) and concentrations of MC-PROXYL obtained at 1 T and 7 T.
Both 1 T and 7 T images (Figure 2A and 2B, respectively) showed T1-weighted contrast differences depending on the concentration of MC-PROXYL in the glass tubes. The region of interest (ROI) was set inside each glass tube. An average intensity value in the center tube, which contained water, was obtained as the baseline value. To digitize the percentage contrast enhancement induced by MC-PROXYL in the phantom-1 experiment, ΔM%, all pixel values were divided by the baseline value, subtract 1, and then multiply 100. An average value of ΔM% and SD in each ROI was recalculated and plotted versus the concentration of MC-PROXYL (Figure 2C). The T1-weighted signal enhancement ratio, ΔM%, was larger at 1 T compared with at 7 T (Figure 2C). T1-relaxivity values of MC-PROXYL evaluated from the data observed at 1 T and 7 T were 0.270 and 0.144 mM-1s-1, respectively (Figure 2D).
Figure 3 shows the results of the signal susceptibility test using phantom-2. The time courses of ΔM% images were obtained as described in “Image Data Analysis”. A circular ROI was placed on the inside area of the cylindrical reactor (image not shown), and an average ΔM% value and SD in the ROI was calculated for every slice of each frames. Five slices were observed for each frame. The results observed for only slice-3 (the center slice of five slices) are shown in Figure 3. The upper panels in Figure 3 show the result with just flowing water, i.e. both syringes were filled with water and simply pressed simultaneously to make a vortex flow in the reactor. The lower panels show the results of mixing MC-PROXYL and water. The results obtained at 1 T and 7 T are shown in the left and right panels, respectively. The variation in values, SD error bar, appear larger at 1 T; however, the values were drifting almost horizontally (Figure 3 left panels). The result obtained at 7 T showed less variation, but the values drifted forming a slight decay (Figure 3 right panels). These results suggested that image intensity obtained at 1 T scanning was much more stable against flow patterns in the sample. These results also gave cause for concern regarding baseline fluctuation with 7 T scanning.
Figure 3.
Comparison of T1-signal stability observed using phantom-2 at 1 T and 7 T. Left and right panels are time courses of MC-PROXYL-induced T1-weighted signals observed at 1 T and 7 T, respectively. Seventy frames were obtained every 20 sec. Two syringes connected to phantom-2 were pushed simultaneously, and the reaction mixtures were injected into the cylindrical reactor during the 6th frame and taking 10 sec. The upper panels show the results with water only, i.e. both syringes were filled with water. The lower panels show the result of mixing MC-PROXYL and water. Marks and error bars indicate average ΔM% values and standard deviation (SD) in a circular region of interest (ROI) set on the 3rd (center) slice of the five slices.
Figure 4 shows T1-weighted images of a mouse head scanned before injecting MC-PROXYL. Distinguishing the anatomical structure in the mouse brain was more difficult in T1-weighted images obtained at 1 T when NA was 2, although some details were discernible (left column). Lager NA (= 8) can give better anatomical details, even though the scans were done at 1 T (center column). The T1-weighted images obtained at 7 T showed brain internal structures very clearly (right column), even when the NA was 2. For a mouse or a similar small subject, experiments using the 1 T imager may require extra processing to obtain high resolution exploratory images for estimating anatomical structures clearly.
Figure 4.
T1-weighted images of a mouse head scanned by FLASH at 1 T (left 2 columns) and 7 T (right column). Five 0.75 mm thickness slices were set on the cerebrum region with 0.75 mm gaps.
Figure 5 shows the average decay profiles of T1-weighted signals induced by MC-PROXYL in mouse brain regions. ROIs were put on the cortex, hippocampus, ventricle, and medulla area of the brain. The time course of ln (ΔM%) was plotted. The decay profiles of MC-PROXYL in each part of the brain showed basically two phases, i.e. an initial fast decay phase and a later slow decay phase. The initial fast decay reflected redox status in the tissue, and the later slow decay mostly reflected clearance [30,31]. The estimated decay constants of the initial fast decay phase, k1, showed a similar pattern in both the 1 T and 7 T experiments, where the k1 decay rates of MC-PROXYL were quite rapid in the cortex and medulla compared with lower k1 in the ventricle (Table 2). However, the estimated k1 decay rates were slight faster in the 7 T experiments compared with the 1 T experiments (Table 2). This slight faster k1 at 7 T may be due to unsteady baseline as shown in Figure 3. On the other hand, the estimated decay constants of the later slow phase, k2, were slightly faster in the 1 T experiments compared with the 7 T experiments but were within an order of magnitude.
Figure 5.
Comparison of decay profiles of MC-PROXYL-induced T1-weighted signals in several brain areas of a mouse. Left and right columns showed the decay profiles obtained at 1 T and 7 T, respectively. Four regions of interest (ROIs), i.e. cortex, hippocampus, ventricle, and medulla areas, were set on the corresponding 5 slices of scout scan. Average ln (ΔM%) values were obtained for every brain areas. Finally, average ln (ΔM%) ± standard deviation (SD) of 3 mice was plotted versus time. The time window for calculating k1 and k2 was 0.8-3.0 min and 7.5-20.0 min, respectively.
Table 2.
Comparison of in vivo decay rates of MC-PROXYL obtained by 1 T or 7 T MRI
| Brain Aria | k1 (min-1) | k2 (min-1) | ||
|---|---|---|---|---|
|
| ||||
| 1 T | 7 T | 1 T | 7 T | |
| Cortex | 0.576 ± 0.024 | 0.722 ± 0.032* | 0.061 ± 0.024 | 0.016 ± 0.006 |
| Hippocampus | 0.492 ± 0.078 | 0.641 ± 0.005 | 0.019 ± 0.017 | 0.014 ± 0.008 |
| Ventricle | 0.421 ± 0.223 | 0.447 ± 0.031 | 0.084 ± 0.018 | 0.016 ± 0.027* |
| Medulla | 0.569 ± 0.056 | 0.666 ± 0.069 | 0.073± 0.018 | 0.017 ± 0.006* |
n = 3. The time window for calculating k1 and k2 was 0.8-3.0 min and 7.5-20.0 min, respectively.
indicates significance between 1 T and 7 T by p < 0.05.
The L-band EPR experiment also showed a similar two-phase signal decay profile (data not shown), although the in vivo L-band EPR experiment was much suffered from a problem related to the broad fluctuation noises on the base line. The k1 and k2 values could be estimated as 0.504 ± 0.017 and 0.062 ± 0.045 min-1, respectively. The L-band EPR experiment using surface coil, which probably reflects comprehensive EPR signal decay in the brain cortex, skull bone, and skin regions, is only as a guide, but showed similar values as that obtained for the cortex in the 1 T MRI experiments.
From the published pharmacokinetic results, the blood clearance rate of a membrane-impermeable hydrophilic triarylmethyl-type paramagnetic probe (Oxo63) in C3H mouse can be expected as 0.03-0.04 min-1 [32]. In addition, the k2 decay rates of several nitroxyl probes have been obtained as 0.06 min-1 or faster for C3H mouse [30] and as 0.055 min-1 or faster for Wistar rat [31]. Based on those previous data, the average k2 values evaluated by the L-band EPR (0.062 min-1) and the 1 T MRI experiments except hippocampus (0.061-0.084 min-1) in this paper were, therefore, acceptable as the true physiological values. However, the k2 value obtained as 0.019 min-1 in the hippocampus by 1 T experiment was viscerally too slow to accept as a natural physiological value. Relatively large noise in 1 T experiment may sometimes make such coincidental error. On the other hand, something certain artificial error can be suspected as a cause of relatively smaller k2 values (0.014-0.017) evaluated by low noise 7 T MRI experiment in this paper.
Although higher noise levels in the 1 T instrument and lower signal stability in the 7 T instrument was obtained in in vitro experiments, both instruments could suitably estimate difference of the in vivo signal decay rate constants among different tissues/organs. Clinically popular lower field, i.e. 0.5-3.5 T, MRI instruments are inherently ready for utilization of redox imaging techniques, when a suitable clinically available redox-sensitive contrast agent becomes available. Development of a non-toxic redox-sensitive contrast agent is keenly awaited.
Conclusion
Nitroxyl radical-based redox imaging showed a satisfactory performance profile in a low magnetic field strength. Lower field, i.e. 1 T, MRI apparatus proposed higher T1-relaxivity, even though the signal to noise ratio is lower, compare to higher field, i.e. 7 T, apparatus. In addition, the detection at the lower field is less affected by voltex flow in a sample, and then much stable T1-weighted signal detection is available at the lower field. Although the shapes of in vivo nitroxyl decay curves and trends of in vivo nitroxyl decay constants among brain regions were similar between the 1 T and 7 T experiments, the decay constant values observed at 1 T experiment were rather as acceptable.
Acknowledgements
The authors are grateful to Yoshikazu Ozawa and Nobuhiro Nitta for their help with the MRI operation. This work was partly supported by JSPS KAKENHI Grant Numbers 24390011 and 26461901 (K. M.). This work was partially supported by JSPS KAKENHI Grant Number 16H01363 (K. Y.), by PRESTO from Japan Science and Technology Agency (K. Y.), and by the Platform Project for Supporting in Drug Discovery and Life Science Research from AMED, Japan, (K. Y.).
Disclosure of conflict of interest
None.
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