Feeding Time-Dependent Changes in the Preventive Effect of Empagliflozin on the Development of Peripheral Neuropathic Pain in Diabetic Model Mice

Abstract

It is known that the daily feeding cycle affects the dosing time-dependent changes in the pharmacodynamics and pharmacokinetics of many drugs. Our previous study demonstrated that administration of empagliflozin (EMPA), sodium-glucose cotransporter 2 (SGLT2) inhibitor, at the beginning of daily feeding cycle (active phase) effectively prevents the development of neuropathic pain in streptozotocin (STZ)-induced diabetic mice. Although the blood glucose levels are closely related to feeding, the relationship between the daily feeding cycle and the optimal dosing time of EMPA remains unclear. In this study, we used STZ-induced diabetic mice and implemented a daily time-restricted feeding (TRF) regimen to investigate whether the dosing time-dependent preventive effect of EMPA on the diabetic neuropathy is modulated by TRF. Animals were housed under a 12-h light/dark cycle, and were assigned to either light-phase TRF (feeding during the light phase) or dark-phase TRF (feeding during the dark phase). The hypoglycemic effect of EMPA was enhanced when the drug was administrated at the beginning of both TRF conditions. A similar influence of the daily feeding cycle on the dosing time-dependent hypoglycemic effect of EMPA was also observed in its preventive effect on the development of diabetic neuropathic pain. Further analysis revealed that dosing time-dependent variations in both the hypoglycemic effect of EMPA and its preventive effect on diabetes-induced pain hypersensitivity were attributable to corresponding changes in urinary glucose excretion. Our results support the notion that the administration of EMPA at the onset of daily feeding cycle effectively suppresses the development of diabetic peripheral neuropathy.

INTRODUCTION

Circadian rhythms in biological functions are widely observed in mammals and are regulated by a molecular clock composed of a hierarchical network of endogenous oscillators.^1,2) The master pacemaker resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, which synchronizes to the 24-h day through photic input from the visual system. The SCN then sends signals that coordinate the phase of peripheral clocks in various tissues.^3–8)

Since daily variations in biological functions influence the efficacy and/or toxicity of drugs, many medications exhibit time-of-day-dependent differences in their therapeutic effects.^9–11) These variations are primarily attributed to circadian changes in pharmacokinetic parameters such as absorption, distribution, metabolism, and elimination—or in pharmacodynamics. Although the molecular clock is a major oscillator of biological functions, the feeding schedule also plays an important role on entraining peripheral oscillators. Nocturnal mice consume approximately 80% of their total daily food during the dark phase.¹²⁾ However, subjected to feeding restricted to the light phase, the circadian rhythm of peripheral tissues, such as liver and kidney, become entrained by the feeding cycle, resulting in anti-phasic expression of clock genes relative to the normal rhythm, without altering the rhythm of SCN.¹³⁾ Therefore, the feeding schedule is thought to significantly influence the dosing time-dependent efficacy and/or toxicity of many drugs.^9–11)

Empagliflozin (EMPA) is a potent sodium-glucose cotransporter 2 (SGLT2) inhibitor used for treatment of hyperglycemia in patients with type 2 diabetes mellitus, primarily by promoting urinary glucose excretion. We previously demonstrated that the hypoglycemic effect of EMPA in streptozotocin (STZ)-induced diabetic mice varies depending on the timing of administration.¹⁴⁾ The effect was enhanced when EMPA was administered at the beginning of the dark phase, which is the active period of nocturnal animals. Furthermore, EMPA administration at the same time of day significantly suppressed the development of diabetes-induced peripheral neuropathic pain. However, it remains unclear whether feeding schedules influence these dosing time-dependent effects of EMPA. In this study, we investigated whether the preventive effects of EMPA on STZ-induced hyperglycemia and peripheral neuropathic pain are modulated by manipulation of the feeding schedule.

MATERIALS AND METHODS

Animals and Treatments

Four- to five-week-old male ICR mice were purchased from Jackson laboratory Japan (Kanagawa, Japan). They were housed in groups (5 to 8 per cage) under light and dark cycles (lights on from Zeitgeber time (ZT)0 to ZT12), with food and water available ad libitum or under time-restricted feeding (TRF) conditions (feeding between ZT0 and ZT12 or ZT12 and ZT0). The animals were adapted to the feeding conditions for 2 weeks prior to the experiments. Room temperature and humidity were controlled at 24 ± 1°C and 60 ± 10%, respectively. Diabetes-induced neuropathic pain model mice were established by intraperitoneal injection of STZ at a dose of 200 mg/kg (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Control mice were injected with an equal volume of saline. EMPA (20 mg/kg) or saline was orally administered every day after treatment with STZ. The dosage of EMPA was set based on our previous study.¹⁴⁾ All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of Kyushu University and approved by the Institutional Animal Care and Use Committee of Kyushu University (Approval Protocol ID # A23-100).

Glucose Quantification in Plasma and Urine Samples

Plasma and urine samples were prepared as previously described.¹⁴⁾ Glucose levels in samples collected from STZ-induced diabetic mice and control mice were quantified using a glucose assay kit (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer’s protocol.

Assessment of Urinary Glucose Excretion Promoting Effect and Diuretic Action

STZ-induced diabetic mice received a single oral administration of EMPA (20 mg/kg) or saline at ZT0 or ZT12 under either light phase-TRF (feeding between ZT0 and ZT12) or dark phase-TRF (feeding between ZT12 and ZT0) schedules (n = 5–6). Urinary glucose excretion, urinary glucose concentration, and urine volume were measured for up to 12 h after EMPA administration. In each STZ-induced diabetic mouse, Δ urine volume and Δ urinary glucose concentration were defined as the differences in total urine volume and urinary glucose concentration, respectively, between 12 h after EMPA administration and 12 h after saline administration. Δ urinary glucose excretion was then calculated for each mouse by multiplying the corresponding Δ urine volume by the Δ urinary glucose concentration.

Assessment of Diabetes-Induced Peripheral Neuropathic Pain

To assess diabetes-induced peripheral neuropathic pain, mice were placed individually in opaque plastic cylinders on a wire mesh and allowed to acclimate to the new environment. After 30 min, calibrated von Frey filaments (0.02–2.0 g, North Coast Medical, Morgan Hill, CA, U.S.A.) were applied five times to the plantar surfaces of the hind paws. The paw withdrawal threshold (PWT) was calculated by up and down methods as previously described.^14,15)

Quantitative RT-PCR Analysis

The kidneys were collected at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22 to extract mRNA from the renal cortex. Total RNA was extracted using RNAiso Reagent (TaKaRa Bio, Osaka, Japan). cDNA was synthesized using the ReverTra Ace qPCR Kit (Toyobo Life Science, Osaka, Japan), and amplified by PCR using the LightCycler 96 system (Roche Diagnostics, Mannheim, Germany) and THUNDERBIRD NEXT SYBR qPCR Mix (Toyobo Life Science). Gene expression was normalized using “β-actinˮ mRNAs. Primer sequences are listed in Supplementary Table 1.

Western Blotting

Western blotting was performed as previously described.¹⁴⁾ Tissue samples from the renal cortex were obtained at ZT2 and ZT14. Denatured total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. The following reagents were used for detection of β-ACTIN and SGLT2 proteins: β-ACTIN (1 : 10000; sc1616-HRP, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), SGLT2 (1 : 1000; sc393350, Santa Cruz Biotechnology), anti-mouse immunoglobulin G (IgG) (1 : 10000; ab6820, Abcam, Cambridge, U.K.), and a chemiluminescence reagent (Nacalai Tesque, Kyoto, Japan). The visualized images were scanned using an ImageQuant LAS4010 (GE Healthcare, Tokyo, Japan).

Statistical Analysis

All statistical analyses were performed using JMP pro 16 (SAS Institute Japan). Date were expressed as means with standard deviation (S.D.). Statistical significance among group was analyzed using one-way or two-way ANOVA followed by Tukey–Kramer’s post hoc tests. Student’s t-test was used for independent comparison between the two groups. A p-value of <0.05 was considered statistically significant.

RESULTS

Influence of TRF on Diurnal Variation in Drinking Water Intake and Plasma Glucose Levels in Healthy and STZ-Induced Diabetic Mice

Under ad libitum feeding conditions, we previously demonstrated that the hypoglycemic effect of EMPA and its stimulatory effect on urinary glucose excretion were enhanced when the drug was administered at the beginning of the active phase in STZ-induced diabetic mice.¹⁴⁾ Nocturnal mice typically consume approximately 80% of their daily diet during the dark phase. To investigate whether feeding time affects the preventive effect of EMPA on the development of peripheral neuropathic pain in diabetic mice, we subjected STZ-treated mice to light phase TRF or dark phase TRF for 2 weeks. During the TRF schedule, water was freely available.

We first investigated the relationship between TRF schedule and water intake, as well as between TRF patterns and plasma glucose levels in both healthy and STZ-induced diabetic mice. Under either TRF conditions, the amount of water intake showed a significant diurnal variation, increasing during feeding period (Figs. 1A, 1B). Although no obvious diurnal variation in plasma glucose levels was observed in STZ-treated diabetic mice under either TRF conditions, a transient increase in plasma glucose levels were observed at the beginning of feeding in these mice (Figs. 1C, 1D). These results suggest that plasma glucose levels in STZ-induced diabetic mice were changed in a feeding time-dependent manner.

Fig. 1. Diurnal Variations in Food Intake, Water Consumption, and Plasma Glucose Levels under TRF Conditions

Mice were housed under the light phase or dark phase TRF conditions from 2 weeks prior to intraperitoneal injection of STZ (200 mg/kg). Food intake, water consumption, and plasma glucose levels were measured 2 weeks after STZ treatment. (A, B) Temporal profiles of amount of food intake (left panel) and water consumption (right panel) in healthy and STZ-induced diabetic mice under the light phase TRF condition (A) or the dark phase TRF condition (B). (C, D) Circadian profile of plasma glucose levels in healthy and STZ-induced diabetic mice under the light phase TRF (C) or the dark phase TRF conditions (D). Values are shown as mean with S.D. (n = 6). Statistical significance was determined using one-way ANOVA.

Feeding Time Modulates the Dosing-Time Dependency of the Hypoglycemic Effect of EMPA in STZ-Induced Diabetic Mice

Next, we investigated whether TRF affects the dosing time-dependent hypoglycemic effect of EMPA in STZ-induced diabetic mice. Diabetic mice subjected to either light-phase and dark-phase TRF were orally administered EMPA (20 mg/kg) once daily at ZT0 or ZT12 for 21 d, starting the day following STZ treatment. Under the light-phase TRF condition, daily oral administration of EMPA at ZT12 (beginning of fasting period) had a negligible effect on plasma glucose levels of STZ-induced diabetic mice, whereas administration of EMPA at ZT0 (beginning of feeding period) significantly decreased their plasma glucose levels (Fig. 2A). Conversely, under dark-phase TRF condition, daily administration of EMPA at ZT0 (beginning of fasting) had a negligible effect on plasma glucose levels in diabetic mice, while administration at ZT12 (beginning of feeding) significantly reduced glucose levels (Fig. 2B). These findings suggest that the hypoglycemic effect of EMPA is significantly enhanced when the drug is administered at the onset of feeding in diabetic mice.

Fig. 2. Influence of TRF on the Dosing Time-Dependency of Hypoglycemic Effect of EMPA in STZ-Induced Diabetic Mice

Mice were housed under the light phase or dark phase TRF conditions from 2 weeks prior to STZ treatment (200 mg/kg, i.p.). After treatment with STZ, mice were orally administered with a single daily dose of EMPA at ZT0 or ZT12. (A) Dosing time-dependency of hypoglycemic effect of EMPA (20 mg/kg, p.o.) in STZ-induced diabetic mice under the light phase TRF condition. (B) Dosing time-dependency of hypoglycemic effect of EMPA (20 mg/kg, p.o.) in STZ-induced diabetic mice under the dark phase TRF condition. Values are shown as means with S.D. (n = 4–6). Statistical significance was determined using one-way ANOVA.

Effect of Feeding Time on the Preventive Effect of EMPA on Diabetic-Induced Peripheral Neuropathic Pain

Our previous study demonstrated that the hypoglycemic effect of EMPA in STZ-induced diabetic mice was enhanced when the drug was administered at the beginning of their active phase, which may explain the dosing time-dependent difference in the preventive effect of EMPA on the development of diabetic pain hypersensitivity.¹⁴⁾ Therefore, we explored whether administering EMPA at the beginning of feeding period under TRF conditions would also enhance its preventive effect on diabetes-induced peripheral neuropathic pain. Mice housed under light- or dark-phase TRF conditions were orally administered EMPA (20 mg/kg) at either ZT0 or ZT12, starting the day following STZ treatment. PWT was assessed every 3 days after the initiation of EMPA treatment. In the light-phase TRF group, administration of EMPA at ZT0 (beginning of the feeding period) significantly suppressed pain hypersensitivity in STZ-induced diabetic mice, whereas administration at ZT12 (beginning of the fasting period) had a negligible effect (Fig. 3A). Similarly, in the dark-phase TRF group, administration of EMPA at ZT12 (beginning of feeding) significantly suppressed the development of peripheral neuropathic pain (Fig. 3B). These results indicate that, consistent with its hypoglycemic effect, EMPA significantly suppresses the development of neuropathic pain hypersensitivity when the drug was administered at the beginning of the feeding period in STZ-induced diabetic mice under TRF conditions.

Fig. 3. Influence of TRF on Dosing Time-Dependency of the Preventive Effect of EMPA on the Development of Pain Hypersensitivity in STZ-Induced Diabetic Mice

Mice were housed under the light phase or dark phase TRF conditions from 2 weeks prior to STZ treatment (200 mg/kg, i.p.). After treatment with STZ, mice were orally administered with a single daily dose of EMPA at ZT0 or ZT12. (A) Dosing time-dependency of preventive effect of EMPA on the development of pain hypersensitivity in STZ-induced diabetic mice under light phase TRF condition. The PWT was assessed from ZT20 to ZT22. (B) Dosing time-dependency of preventive effect on EMPA on the development of pain hypersensitivity in STZ-induced diabetic mice under dark phase TRF condition. The PWT was assessed from ZT8 to ZT10. Values are shown as means with S.D. (n = 4–6). Statistical significance was determined using two-way ANOVA. ^**p < 0.01, ^*p < 0.05, significant difference between the two groups.

Feeding Time-Dependent Modulation of the Ability of EMPA to Promote Urinary Glucose Excretion

TRF is known to affect the expression and function of transporters, metabolic enzymes, and receptors through changes in the expression of clock genes various tissues.^9–11) To determine whether TRF alters clock gene expression in the kidney, we investigated the temporal expression profiles of clock genes in the kidneys of healthy and STZ-induced diabetic mice under ad libitum, light-phase TRF, and dark-phase TRF conditions. Both Bmal1 and Per2 mRNA levels in the kidneys of healthy and STZ-induced diabetic mice exhibited significant circadian rhythms under all feeding conditions (Fig. 4A). Under the dark-phase TRF condition, both Bmal1 and Per2 mRNA levels exhibited significant diurnal oscillations under all feeding conditions in both healthy and STZ-induced diabetic mice (Fig. 4A). Under the dark-phase TRF condition, the expression patterns were similar to those observed under ad libitum conditions. In contrast, light-phase TRF induced anti-phasic expression of Bmal1 and Per2 relative to the ad libitum condition (Fig. 4A). These results suggest that feeding schedules alter the diurnal expression of clock genes in the kidney. Although we further examined whether the protein levels of SGLT2 in the renal cortex were affected by TRF, no significant differences in SGLT2 protein levels were observed among the ad libitum, light-phase TRF, and dark-phase TRF conditions in STZ-induced diabetic mice (Fig. 4B).

Fig. 4. Influence of TRF on Dosing Time-Dependent Change in the Ability of EMPA to Promote Urinary Glucose Excretion of STZ-Induced Diabetic Mice

(A) Temporal mRNA expression profiles of circadian clock genes (Bmal1 and Per2) in the kidney of healthy or STZ-induced diabetic mice with ad libitum feeding, light phase TRF, and dark phase TRF. (B) Temporal profiles of SGLT2 protein levels in the kidney of healthy or STZ-induced diabetic mice with ad libitum feeding, light phase TRF, and dark phase TRF. (C–E) Difference in urinary glucose excretion (C), urine glucose concentration (D), and urine volume (E) in STZ-induced diabetic mice after a single oral administration of EMPA (20 mg/kg) or saline at ZT0 or ZT12 under light phase TRF or dark phase TRF condition. In each STZ-induced diabetic mouse, Δ urine volume and Δ urinary glucose concentration were defined as the differences in total urine volume and urinary glucose concentration, respectively, between 12 h after EMPA administration and 12 h after saline administration. Δ urinary glucose excretion was then calculated for each mouse by multiplying the corresponding Δ urine volume by the Δ urinary glucose concentration. Values are shown as means with S.D. (n = 5–6). For panels A and B, statistical difference was determined using one-way ANOVA with Tukey–Kramer post hoc test. For panels C, D and E, student’s t-test. ^**p < 0.01, ^*p < 0.05, significant difference between the two groups.

In our previous study, the absence of time-dependent changes in EMPA-induced urinary glucose excretion were observed in STZ-induced diabetic mice, despite the absence of time-dependent variation in SGLT2 protein levels.¹⁴⁾ Based on this, we evaluated the urinary glucose excretion-promoting and diuretic effects of EMPA under light- and dark-phase TRF conditions (methods described in Assessment of Urinary Glucose Excretion Promoting Effect and Diuretic Action.) In the dark-phase TRF group, urinary glucose excretion in STZ-induced diabetic mice was significantly increased when EMPA was administered at ZT12 (p < 0.01, Fig. 4C, left panel). In contrast, in the light-phase TRF group, EMPA administration at ZT0 significantly enhanced urinary glucose excretion (p < 0.01, Fig. 4C, right panel). Although no significant dosing time-dependent differences were observed in urinary glucose concentrations under either TRF condition (Fig. 4D), their urine volume was significantly increased when EMPA was administered at the beginning of the feeding period (p < 0.01, Fig. 4E). These results suggest that the ability of EMPA to promote urinary glucose excretion is due to its diuretic effect, and that the effect exhibits variation depending on the daily feeding cycle.

DISCUSSION

A TRF schedule is known to alter the rhythmic phase in the expression of receptors, channels, transporters, and drug-metabolizing enzymes.^9–11) The target protein of EMPA, SGLT2, is mainly expressed in the proximal renal tubular cells.¹⁶⁾ In this study, no significant time-dependent differences in SGLT2 protein levels were observed in kidneys of STZ-induced diabetic mice under either ad libitum or TRF conditions. These findings suggest that the dosing time-dependent differences in the hypoglycemic effects of EMPA and its preventive effects on peripheral neuropathic pain are unlikely to be attributable to diurnal changes in SGLT2 expression. This notion is also supported by our previous findings that when EMPA was administered to STZ-induced diabetic mice at either ZT0 or ZT12, neither EMPA concentrations in the kidney nor plasma exhibited significant dosing time-dependent difference.¹⁴⁾

Blood glucose concentration is known to fluctuate depending on feeding patterns.¹⁷⁾ In this study, STZ-induced diabetic mice under TRF conditions exhibited a transient rise in plasma glucose levels at the beginning of the feeding period. Although previous studies have shown that STZ-induced diabetic mice fed ad libitum maintain high blood glucose levels throughout the day without showing clear diurnal rhythms,¹⁸⁾ we also found that the hypoglycemic effect of EMPA is greater when the drug is administered at the beginning of the active phase.¹⁴⁾ These results collectively suggest that diurnal variation in plasma glucose levels is not the primary factor driving the dosing time-dependent changes in the hypoglycemic effect of EMPA. Although the feeding period is restricted under TRF conditions compared with that under ad libitum feeding, the total amount of daily food intake and water consumption did not differ significantly between light-phase TRF and dark-phase TRF in either normal mice or STZ-induced diabetic mice (Supplementary Fig. S1). Therefore, the enhanced hypoglycemic effect of EMPA and the increased urinary glucose excretion observed in this study are unlikely, attributable to differences in experimental conditions related to TRF, but rather reflect the pharmacological action of EMPA in combination with the feeding schedule.

We found that both urine volume and urinary glucose excretion were significantly increased when EMPA was administered at the beginning of the feeding period in mice under both TRF conditions. In both human and experimental animals, glomerular filtration rate (GFR) exhibits diurnal variation, increasing during their active period,¹⁹⁾ and is regulated by circadian clock genes in glomerular epithelial cells.²⁰⁾ In this study, light-phase TRF reversed the expression rhythms of key clock genes (Bmal1 and Per2) in the kidney compared with those under ad libitum or dark-phase TRF conditions. These results suggest that TRF alters the renal circadian clock function, which may in turn affect diurnal variation of GFR. Because EMPA acts on SGLT2 after its glomerular filtration, diurnal variations in GFR caused by feeding time may modulate its pharmacodynamic effects. Although our data are consistent with this hypothesis, further studies are needed to directly confirm the involvement of GFR in the observed dosing time-dependent efficacy of EMPA.

It has been suggested that insulin signaling contributes to the entrainment of peripheral clock gene rhythms under time-restricted feeding conditions.^21,22) Interestingly, despite the insulin-deficient state in STZ-induced diabetic mice, the expression rhythms of renal clock genes still exhibited feeding time-dependent phase shifts. Previous studies have also implicated other hormonal regulators, such as glucocorticoids and glucagon, in the modulation of peripheral clock gene rhythms.^23,24) Therefore, in STZ-induced diabetic mice, the phase and amplitude of clock gene expression rhythms may have been influenced by changes in these hormonal signals, which could partly account for the damping patterns observed in Bmal1 and Per2 rhythms under the indicated conditions.

The development of diabetic peripheral neuropathy has been linked to activation of the polyol pathway under sustained hyperglycemia, where sorbitol accumulation in neurons increases intracellular osmotic pressure and contributes to pain hypersensitivity.^25–28) We previously reported that, in STZ-induced diabetic mice, the decrease in pain threshold correlated with sorbitol accumulation in neuronal cells.¹⁸⁾ In the present study, we demonstrated that the dosing time-dependency of the urinary glucose excretion-promoting effect of EMPA varied depending on the feeding schedule, and this variation appeared to be associated with phase shifts in renal clock gene expression and circadian changes in GFR. Taken together, these results suggest that the hypoglycemic effect of EMPA suppresses activation of the polyol pathway, thereby indirectly reducing sorbitol accumulation in neurons and consequently preventing the development of pain hypersensitivity associated with diabetic peripheral neuropathy.

This study demonstrated that both the hypoglycemic effect of EMPA and its ability to prevent the development of peripheral neuropathic pain are enhanced in a feeding time-dependent manner in STZ-induced diabetic mice. Notably, EMPA administration at the onset of feeding—when glomerular glucose filtrations likely elevated—resulted in more effective inhibition of renal glucose reabsorption via SGLT2. Consequently, EMPA-induced promotion of urinary glucose excretion was also enhanced in a feeding time-dependent manner. Our findings suggest that the preventive effect of EMPA on the development of peripheral neuropathic pain is associated with feeding time and highlights the potential utility of aligning drug administration with feeding schedules to improve glycemic control and prevent diabetes complications.

Acknowledgments

We are grateful for the technical support provided by the Research Support Center, Graduate School of Medical Sciences, Kyushu University.

Funding

This study was supported in part by a Grant-in-Aid for Scientific Research B (25K02425 to S.K.), a Grant-in-Aid for Scientific Research C (25K10469 to A.T.), a Grant-in-Aid for Challenging Exploratory Research (22K18375 to S.K.) from Japan Society for the Promotion of Science and the Platform Project for Supporting Drug Discovery, Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED (Grant JP25am121031 to S.O.) and JST SPRING (Grant JPMJSP2136 to A.S.) from Japan Science and Technology Agency (JST).

Author Contributions

Ai Sato: Data curation, writing—original draft, visualization, validation, methodology, investigation, funding acquisition, formal analysis. Naho Iwanaka: Validation, visualization, methodology, investigation, formal analysis. Tomoaki Yamauchi: Writing—review & editing. Akito Tsuruta: Writing—original draft, writing—review & editing, supervision, funding acquisition. Satoru Koyanagi: Writing—review & editing, supervision, methodology, investigation, project administration, funding acquisition, conceptualization. Shigehiro Ohdo: Writing—review & editing, supervision, funding acquisition.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All the data supporting the findings of this study are contained within the paper.

Supplementary Materials

This article contains supplementary materials.

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