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The Importance of Therapeutic Time Window in the Treatment of Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability. Despite its importance in public health, there are presently no drugs to treat TBI. Many reasons underlie why drugs have failed clinical trials, one reason is that most drugs to treat TBI lose much of their efficacy before patients are first treated. This review discusses the importance of therapeutic time window; the time interval between TBI onset and the initiation of treatment. Therapeutic time window is complex, as brain injury is both acute and chronic, resulting in multiple drug targets that appear and disappear with differing kinetics. The speed and increasing complexity of TBI pathophysiology is a major reason why drugs lose efficacy as time to first dose increases. Recent Phase III clinical trials treated moderate to severe TBI patients within 4–8 h after injury, yet they turned away many potential patients who could not be treated within these time windows. Additionally, most head trauma is mild TBI. Unlike moderate to severe TBI, patients with mild TBI often delay treatment until their symptoms do not abate. Thus, drugs to treat moderate to severe TBI likely will need to retain high efficacy for up to 12 h after injury; drugs for mild TBI, however, will likely need even longer windows. Early pathological events following TBI progress with similar kinetics in humans and animal TBI models suggesting that preclinical testing of time windows assists the design of clinical trials. We reviewed preclinical studies of drugs first dosed later than 4 h after injury. This review showed that therapeutic time window can differ depending upon the animal TBI model and the outcome measure. We identify the few drugs (methamphetamine, melanocortin, minocycline plus N-acetylcysteine, and cycloserine) that demonstrated good therapeutic windows with multiple outcome measures. On the basis of their therapeutic window, these drugs appear to be excellent candidates for clinical trials. In addition to further testing of these drugs, we recommend that the assessment of therapeutic time window with multiple outcome measures becomes a standard component of preclinical drug testing.

THERAPEUTIC TIME WINDOW IS A KEY ELEMENT OF DRUGS TO TREAT TBI
Despite decades of research, there are currently no treatments for TBI other than palliative care (Diaz-Arrastia et al., 2014). The reasons for the lack of therapeutics are many; drug may have failed in clinical trials since most preclinical studies dose drugs immediately or soon after experimental TBI (Diaz-Arrastia et al., 2014). This experimental design fails to take into account the well-documented clinical phenomenon of treatment gap: the time individuals wait before seeking medical care after head trauma (Tanielian and Jaycox, 2008;Demakis and Rimland, 2010). In 1991, one quarter of an estimated 1.5 million patients in America did not seek medical care after receiving a TBI that did not result in death or long-term institutionalization (Sosin et al., 1996). The multiple reasons given to postpone or avoid treatment include perceived symptom resolution, as well as the time and cost of treatment (Demakis and Rimland, 2010). Military personnel are particularly at risk for TBI. Lack of access to safe and accessible transportation for deployed military personnel can delay treatment up to 72 h after TBI (Farmer et al., 2017). Thus, treatment gap likely contributes to negative outcomes after TBI. Despite the importance of treatment gap, we know relatively little about the time course of pathophysiological events that can be successfully targeted with drugs first dosed many hours to days following TBI.
The treatment of thromboembolic stroke using tissue plasminogen activator illustrates the importance of time window in neurodegenerative diseases with a rapid onset. Thromboembolic stroke produces a complex and rapidly evolving injury with an overlapping, yet distinct, pathophysiology to TBI. Tissue plasminogen activator (t-PA) is highly effective if dosed within 4.5 h of a stroke, yet its utility drops sharply after 4.5 h due to the increased probability of hemorrhage (Ahmed et al., 2013). Despite its established ability to prevent injury, only 2-5% of stroke patients receive t-PA (Miller et al., 2011). A major reason for the limited use of t-PA is its short time window (Miller et al., 2011). The experience of clinicians with t-PA to treat stoke suggests similar difficulties will arise if drugs to treat TBI have similarly short therapeutic time windows that fall off sharply.
Since no drug has received FDA-approval, a key unanswered question is: what is a clinically relevant therapeutic window for a TBI drug? Clinical trials at designated trauma centers have enrolled patients 4-7 h after a moderate to severe TBI. Even with the high skill of the clinical staff at these trauma centers; many patients could not be enrolled because treatment could not be initiated within 4-7 h. Less specialized hospitals are likely to have even longer treatment delays. To treat the largest number of patients, a drug or drug combination will likely need to retain high efficacy when first dosed 12 h after moderate to severe TBI. In contrast to those with severe or moderate TBI, patients with mild TBI often delay seeking medical help for days after injury until their symptoms do not abate (Sosin et al., 1996;Tanielian and Jaycox, 2008;Demakis and Rimland, 2010). Thus, drugs will need to retain efficacy when dosed days after injury to treat large numbers of patients with mild TBI. Traumatic brain injury has a complex pathophysiology whether initiated by a blunt impact, penetration through the skull into the brain, or exposure to explosive blast (Dixon, 2017). TBI produces mechanical injury within seconds to neurons, glia, and vessels. This primary injury rapidly triggers a secondary injury that evolves for weeks to months (Dixon, 2017). Both primary and secondary injury damages both gray and white matter. Within minutes after primary injury, neurons lose the ability to control ion homeostasis, which results in accumulation of intracellular calcium, cell depolarization, excitotoxic release of glutamate and additional disruptions of ionic gradients (Weber, 2012;Guerriero et al., 2015). Impaired mitochondrial function leads to energy failure; calcium accumulation and elevated reactive oxygen species are additional early events in secondary injury (Bains and Hall, 2012;Weber, 2012;Hill et al., 2017). Damage to vessels reduces cerebral blood flow resulting in hypoxia, hypoglycemia, and breakdown of the blood-brain barrier (Price et al., 2016). Inflammation rapidly follows TBI and persists for weeks to months after injury (Hinson et al., 2015). Acute inflammation is initiated by release by necrotic cells of damage associated molecular patterns (DAMPS) that activate astrocytes and microglia. Release of proinflammatory cytokines and chemokines lead to further breakdown of the blood brain barrier and recruitment of peripheral inflammatory cells. Microglia and astrocyte activation occurs rapidly in both gray and white matter; neuroinflammation may become chronic and continue to injure brain for months or years after injury. Later events in secondary injury include induction of cytogenic and vasogenic edema, increased intracerebral pressure, oxidative damage and necrotic and apoptotic cell loss (Bains and Hall, 2012;Hill et al., 2017). Early events in white matter include damage to axons, impaired transport and swelling. Damage to oligodendrocytes leads to demyelination and oligodendrocyte loss (Narayana, 2017). White matter damage evolves for weeks resulting in Wallerian axonal degeneration.
The pathophysiological events of secondary injury are highly interconnected. If dosed before, or soon after TBI, a variety of drugs with diverse modes of action (anti-oxidants, glutamate receptor antagonists, and anti-inflammatories) greatly limit the scope of secondary injury (Diaz-Arrastia et al., 2014). These drugs are effective despite targeting only one component of secondary injury. This suggests that, early after TBI, multiple pathophysiological events trigger the spread of secondary injury. Thus, early blockade of any one of these many injury mechanisms results in a substantial, long-term therapeutic effect. As secondary injury evolves, the efficacy of most drugs rapidly diminish through loss of drug targets; the intensification of secondary injury greatly diminishes the therapeutic effect of inhibiting one injury mechanism. Drugs that retain efficacy when dosed at longer intervals after injury may target pathophysiological events that initiate slowly after injury. Alternatively, drugs with good therapeutic windows have multiple targets that can still reduce secondary injury even after its intensification over time.
The importance of therapeutic time window in treating TBI is illustrated by comparing the preclinical testing of progesterone and CDP-choline with the design of Phase III clinical trials testing the same drugs. Progesterone was tested in two recent Phase III trials for TBI. The PROTECT III trial (NCT00822900) recruited patients with moderate to severe TBI (Glasgow Coma Score 4-12) within 4 h post-injury while the SYNAPSE trial (NCT01143064) recruited patients with severe TBI (Glasgow Coma Score < 7) within 7 h (Skolnick et al., 2014;Wright et al., 2014). Both trials were unable to demonstrate a therapeutic effect for progesterone. Prior to Phase III testing, numerous laboratories demonstrated a diverse set of therapeutic effects of progesterone in multiple rodent TBI models (Stein and Sayeed, 2018). Progesterone reduced glutamate release, prevented vasogenic edema, restored the blood brain barrier, improved aerobic respiration, and increased myelin and neurotrophin synthesis. Most importantly, progesterone reduced brain damage and improved multiple functional outcomes. Few drugs have had such wide preclinical testing on so many therapeutic outcomes. Virtually all of these studies, however, first dosed progesterone within 1 h or less after injury (Stein and Sayeed, 2018). Only three studies dosed progesterone between 4 and 6 h after injury and none of these studies performed a careful analysis of how the efficacy of progesterone changed after injury (Peterson et al., 2012(Peterson et al., , 2015. A first dose of progesterone 4 h after experimental TBI decreased gray matter damage, improved motor function and limited astrocyte activation (Peterson et al., 2012(Peterson et al., , 2015. A first dose at 6 h produced small improvements on expression of Nogo-A, GFAP, and GAP-43 (Liu et al., 2014). None of these studies examined multiple therapeutic time windows so it remains unknown how the efficacy of progesterone changed with increasingly longer times to first dose. A study of a first dose of progesterone 1 or 6 h post-stroke showed good retention of drug efficacy in a rat cerebral ischemia model (Yousuf et al., 2014). Little is understood, however, of how the analysis of therapeutic time window in animal models of stroke tells us whether an equivalent therapeutic window exists for TBI. The PROTECT III and SYNAPSE trials provided important information of how rapidly we could recruit and treat patients after moderate to severe TBI, however, due to the lack of appropriate preclinical testing, we do not know if progesterone retained sufficient potency to treat TBI when first dosed at 4-7 h after injury.
The Phase 3 COBRIT study tested the efficacy of CDPcholine on mild, moderate and severe TBI (Zafonte et al., 2012). Most patients (86%) received drugs within the first 24 h after injury. The COBRIT study did not show improvement in any outcome measures. Compared to progesterone, there was relatively little preclinical testing of CHP-choline. Dixon et al. showed that a first dose of CDP-choline beginning 24 h after injury produced mild improvements on beam balance and beam walk, and on acquisition of Morris water maze (Dixon et al., 1997). Two additional studies that dosed CDP-choline immediately after injury reported decreased lesion volume, increased neuroprotection, improvements in neurological tests, edema and protection of the blood brain barrier (Başkaya et al., 2000;Dempsey and Raghavendra Rao, 2003). A potential hypothesized mechanism of action of CDP-choline was to improve lipid metabolism, yet no study examined whether CDPcholine limited white matter injury. As with progesterone, there are no studies examining the efficacy of CDP-choline at different therapeutic time windows. Thus, inadequate drug potency at the time when patients were treated may have contributed to the futility of the PROTECT III, SYNAPSE, and COBRIT trials.

TBI PATHOPHYSIOLOGY IS A MAJOR DETERMINANT OF THERAPEUTIC TIME WINDOW
The speed of secondary injury after TBI results in the rapid appearance and disappearance of drug targets (Dixon, 2017). Studies of the therapeutic time windows of methyl-d-aspartate (NMDA) receptor agonists and antagonists illustrate how therapeutic time windows arise from the interaction of drugs with changes in TBI pathophysiological changes over time (Guerriero et al., 2015). Excessive glutamate release activates NMDA receptors within minutes after the onset of TBI (Guerriero et al., 2015). NMDA receptor activation produces calcium overload and activation of calcium-activated catabolic enzymes (Weber, 2012). If dosed soon after injury, NMDA antagonists prevent this calcium overload and prevent neuronal loss (Shohami and Biegon, 2013). The short therapeutic time window of NMDA receptor antagonists is the consequence of the speed of the calcium overload after TBI (Shohami and Biegon, 2013;Campos-Pires et al., 2015). Ongoing secondary injury subsequently produces a long-lasting downregulation of NMDA receptor expression. The loss of NMDA receptor function impairs synaptic plasticity and results in cognitive and memory deficits. The partial NMDA receptor agonist D-cycloserine when first dosed at 24 or 72 h post-injury improves Neurological Severity Score (NSS). A first dose of d-cycloserine at 24 h PI also improved performance of hippocampal-dependent tasks (Temple and Hamm, 1996;Adeleye et al., 2010;Sta Maria et al., 2017). A first dose of cycloserine at 24 h post-injury was also effective in rat model of pediatric TBI (Sta Maria et al., 2017). D-cycloserine improved performance on Novel Object Recognition and produced a mild improvement in acquisition, but not retention of Morris Water Maze (Sta Maria et al., 2017). Earlier dosing of d-cycloserine was ineffective at 8 or 16 h post-injury when NMDA receptors were downregulated (Adeleye et al., 2010). Thus, the different therapeutic time windows of NMDA receptor antagonists and agonists results from the differential consequences of NMDA receptor activation after TBI (Shohami and Biegon, 2013).

ARE STUDIES OF THERAPEUTIC TIME WINDOW IN ANIMAL MODELS RELEVANT TO HUMAN TBI?
Animal models of TBI have been invaluable for our understanding of TBI pathophysiology (Xiong et al., 2013). Most of the secondary injury events that occur in clinical TBI also occur in animal models. This has validated the use of animal models to find drug targets to treat TBI. Virtually all studies of therapeutic time window have used rodent TBI models (Table 1 and Supplementary Table 1). Studies of therapeutic time window in rodents not only assume similar TBI pathophysiology in animals and people, but that these pathophysiological events  occur with similar kinetics. Both humans and rodents rapidly develop edema, elevated extracellular glutamate, excitotoxicity and elevated intracellular Ca ++2 after TBI or experimental TBI (Palmer et al., 1993;Bullock et al., 1995;Vespa et al., 1998;Markgraf et al., 2001;Hutchinson, 2005;Weber, 2012). The increase in reactive oxygen species and its accompanying oxidative damage also occurs rapidly in animals and people (Bains et al., 2013;Cornelius et al., 2013). A variety of plasma biomarkers (GFAP, UCh-1, Tau, and S100β) show similar kinetics in rodent TBI models and clinical TBI (Mondello et al., 2016;Caprelli et al., 2017;Korley et al., 2018;Shahjouei et al., 2018). In both human TBI and TBI animal models, there is an acute and rapid increase in the levels of pro-inflammatory markers (Clausen et al., 2018;Huie et al., 2018). These data suggest that studies using rodent TBI model can provide important insights into the therapeutic window of a drug to treat clinical TBI. A variety of TBI models have also been developed using ferrets, rabbits, pigs or non-human primates (Dai et al., 2018). Although there are fewer studies using these animals than rodents, they show both rapid changes (apnea, hypertension, elevated intracranial pressure, intracranial bleeds) that is followed by a more delayed gray and white matter injury that is accompanied by a long-lasting inflammatory response (Dai et al., 2018;Vink, 2018). A major advantage of TBI models using larger animals is that they have gyrencephalic brains that better model the mechanical forces that injure the brain after TBI in humans. Thus, large animal studies better model the early pathophysiology of TBI than rodents. The study of TBI in large animals, however, is far more expensive than with rodents and often lack the spectrum of behavioral testing available in rodents. Studies of therapeutic time window frequently need large numbers of animals to achieve statistical significance which may make the use of large animal TBI models prohibitively expensive. The ability of non-rodent models to better model clinical TBI suggests a role to further confirm the therapeutic windows first determined in rodent models.

THERAPEUTIC TIME WINDOW AND LOSS OF DRUG EFFICACY AFTER TBI
Most drugs lose efficacy with increasing intervals between injury and the time to first treatment. The time course of this decrease in efficacy is known for relatively few drugs ( Table 1 and Supplementary Table 1). Most drugs that have been tested in animal TBI models have short therapeutic time windows. The N-type Ca +2 channel blocker ziconotide improved mitochondrial function when rats were first dosed 15 min after moderate CCI, but rapidly lost efficacy when first dosed between 1 and 10 h post-injury. These data are consistent with a rapid entry of calcium entry through N-type channels soon after injury (Weber, 2012). Mild hypothermia targets multiple biological processes that makes it an attractive therapy for TBI. Hypothermia inhibits apoptosis, reduces metabolic demand, promotes recovery of energy homeostasis, prevents oxidative damage, maintains the integrity of the blood brain barrier, reduces inflammation, and prevents blood coagulation and seizures. Zhao et al. systematically examined the efficacy of 4 h of hypothermia (32-34 • C) in rats beginning 15 min, 2 or 4 h after a moderate CCI (Zhao et al., 2017). Inducing hypothermia within 15 min post-injury produced the largest improvements in behavior (Neurological severity score, Morris water maze), histology (neuroprotection, edema, and neurogenesis) and inflammation (astrocyte activation and neutrophil infiltration). These outcomes also improved when hypothermia was initiated at either 2 or 4 h post-injury, but to a lesser extent than initiating therapy at 15 min. Later time points were not analyzed. Dosing between 3 and 12 h post-injury of methamphetamine has been tested using multiple outcome measures. Multiple behavioral outcomes (Neurological severity score, foot faults, and Morris water maze) were improved with a first dose of methamphetamine at 3 or 7.5 h dosing; 12 h dosing was less effective. As with hypothermia, later time points were not analyzed.

THERAPEUTIC TIME WINDOWS DIFFER DEPENDING UPON THE ANIMAL TBI MODEL AND ON THE OUTCOME MEASURE
Open head models of TBI include controlled cortical impact and fluid percussion injury. These models damage the brain through a craniotomy by directly striking or deforming the dura. The head is immobilized and unable to move (Johnson et al., 2015). Open head models produce a more focal contusion with much less diffuse white matter injury. Closed head models injure the brain by directly striking the head or the skull. In most of these models, the impact produces rapid head movements. Permitting head movement more closely models clinical TBI, and produces a more variable injury than models that prevent head movement (Johnson et al., 2015;Grin'kina et al., 2016). Closed head models produce a smaller focal contusion and a more diffuse white matter injury than open head models. Therapeutic time window has been largely studied using the open head models. Many animal models induce brain injury by selective activation of one element of secondary injury of TBI (i.e., excitotoxicity or oxidative stress). The relevance of therapeutic time window studies in these models to animal models of TBI is unknown. Therefore, only studies of therapeutic time window using animal models of TBI have been discussed in this review.
The therapeutic time window of few drugs have been tested in multiple animal models of TBI. Of the many animal studies showing the efficacy of minocycline (MINO) to improve therapeutic outcomes in experimental TBI, only two dosed MINO using a therapeutic time window of more than 1 h (Garrido-Mesa et al., 2013). A first dose of MINO 4 h after mild injury in a mouse blast model improved acquisition of Morris water maze and decreased levels of multiple biomarkers for glial and neuronal injury in both plasma and in brain tissue (Kovesdi et al., 2012). Later time points were not analyzed, so the therapeutic window of MINO in this blast model has not been fully characterized. In contrast, Sangobowale et al. analyzed the therapeutic time window of MINO in two models, a rat open head controlled cortical impact (CCI) model and a mouse model of closed head injury (CHI). These two models produce overlapping, yet different brain injuries (Johnson et al., 2015). When first dosed with MINO 24 h post-injury, injured rats and mice acquired Barnes maze, a task of spatial navigation (Sangobowale M.A. et al., 2018). Mice, however, did not acquire Barnes maze when MINO was first dosed 72 h after CHI (Whitney, Nikulina, and Bergold, unpublished results). It is unknown if rats could acquire Barnes maze when first dosed at 72 h post-injury. These data suggest that, using Barnes maze as an outcome measure, MINO has a similar therapeutic time window in multiple species and injury models. Sangobowale M.A. et al. (2018) also tested MINO-treated rats and mice on a second task, active place avoidance. In active place avoidance, rodents avoid a shock zone by attending to distal visual cues while ignoring proximal olfactory cures (Abdel Baki et al., 2010). Active place avoidance could be acquired when MINO was first dosed to mice 12 h after CHI or to rats first dosed 6 h after CCI. Both CCI and CHI induce demyelination of midline commissural tracts. Demyelination of midline commissural tracts prevents acquisition of the active place avoidance (Grin'kina et al., 2013). The therapeutic window for MINO was similar for acquisition of active place avoidance and the prevention of demyelination of midline commissural tracts (Sangobowale M.A. et al., 2018). Thus, outcome measures that have related pathophysiology have similar therapeutic windows. In both models, the therapeutic time window for active place avoidance is shorter than for Barnes maze since active place avoidance requires the coordinated activity of more brain regions than Barnes maze (Cimadevilla et al., 2001;Klur et al., 2009;Shinohara et al., 2012;Grin'kina et al., 2013). This suggests that therapeutic time windows differ depending upon outcome measure. Tasks with higher cognitive demand, such as active place avoidance will have a shorter therapeutic window than simpler tasks, such as Barnes maze. Studies of therapeutic time window should include multiple outcome measures. Given the known heterogeneity of clinical TBI, these observations suggest that the therapeutic window of drugs should be examined using multiple outcome measures in at least two TBI models.

COMBINING DRUGS CAN EXTEND THE THERAPEUTIC TIME WINDOW BEYOND THE WINDOW OF SINGLE DRUGS
Drug combinations can engage more targets than single drugs, and the ability to engage more targets may lengthen therapeutic time window (Margulies et al., 2016). Minocycline, N-acetylcysteine (NAC) and the minocycline plus N-acetylcysteine (MINO plus NAC) combination were examined in a rat CCI model and a mouse closed head injury model (Table 1). In the rat CCI model, the drug combination of minocycline and N-acetylcysteine was shown to have a longer therapeutic window than the individual drugs (Sangobowale M.A. et al., 2018). Rats acquired active place avoidance if first dosed with MINO at 1 h, but not at 6 h after injury. N-acetylcysteine-treated rats were unable to acquire active place avoidance whether first dosed at 1 or 6 h. Rats treated with MINO plus NAC acquired active place avoidance 12 h post-injury but not at 24 h post-injury. Similar time windows were obtained when retention of myelin was used as an outcome measure (Sangobowale M.A. et al., 2018). In the rat CCI model, the MINO plus NAC drug combination has a longer therapeutic time window than the individual drugs.

DRUGS WITH LONG THERAPEUTIC WINDOWS REVEAL ENZYMATIC AND CELLULAR PROCESSES THAT CAN BE TARGETED HOURS TO DAYS AFTER TBI
An important, yet understudied question about TBI is what brain functions can be targeted with drugs many hours to days after a TBI. This question can be answered, in part, by analyzing the mode of action of drugs with long therapeutic windows. Multiple drugs with long therapeutic windows target mitochondria, including cyclosporine, FCCP (p-trifluoromethoxyphenylhydrazone), and N-acetylcysteine (Pandya et al., 2009;Sullivan et al., 2011;Sangobowale M.A. et al., 2018). These drugs protect mitochondria in diverse ways; cyclosporine A prevents opening of the mitochondrial permeability transition pore and FCCP uncouples oxidative phosphorylation (Pandya et al., 2009;Sullivan et al., 2011). Both of these drugs lead to inhibition of Ca +2 entry into mitochondria. N-acetylcysteine scavenges reactive oxygen species and prevents mitochondrial oxidative stress (Abdel Baki et al., 2010). All three drugs improved outcome measures when first dosed 24 h after injury. These data suggest that drugs that protect mitochondria have long therapeutic windows.
Multiple drugs that inhibit poly (ADP-ribose) polymerase (PARP) also have long therapeutic windows (d' Avila et al., 2012;Stoica et al., 2014;Sangobowale M.A. et al., 2018). In TBI models, PARP is rapidly activated within 30 min after injury yet remains active for many days (LaPlaca et al., 1999). Multiple PARP inhibitors (PJ34, NO-1001, Veliparib, and minocycline) also block microglial activation when dosed up to 24 h postinjury (Table 1). This is due to necessity of PARP activation for microglial activation (d' Avila et al., 2012;Stoica et al., 2014). The effect of Veliparib was observed in both rat and pig CCI models (Irvine et al., 2017). PARP activity is also needed for astrocyte activation and PJ34 and Velinarib prevented astrocyte activation in both rats and pigs (Phulwani and Kielian, 2008;Irvine et al., 2017). PJ34 and NO-1001 improved sensory motor function, but neither PJ34 nor Veliparib improved cognitive outcomes.
Low dose methamphetamine has also shown a broad therapeutic window after TBI. Multiple behavioral and histological outcomes were improved with a first dose of methamphetamine at 3 or 7.5 h dosing. Twelve-hour dosing was less effective than earlier dosing yet the retention of efficacy at 12 h suggests that even later dosing should be tested (Rau et al., 2012(Rau et al., , 2014. Methamphetamine acts on multiple vesicular and cell surface transporters to alter neurotransmission of dopamine, norepinephrine, serotonin, and glutamate (Rau et al., 2016). These data suggest that drugs that target multiple neurotransmitter systems may have similarly favorable therapeutic windows.
Chronic neuroinflammation following TBI may also be an effective target for drugs. Byrne et al. showed that mice had chronic microglial activation months after CCI (Byrne et al., 2012). Treatment with the mGluR5 agonist, (RS)-2-chloro-5hydroxyphenylglycine, beginning 1 month after CCI decreased this neuroinflammation and improved multiple histological and behavioral outcomes. These effects could be blocked by co-administration of a mGluR5 antagonist, providing strong evidence that these effects were through mGluR5 activation.

RECOMMENDATIONS TO INCLUDE THERAPEUTIC WINDOW TESTING IN PRECLINICAL TBI DRUG STUDIES
Pharmacology, safety and potency are key features of any drug to treat TBI (Diaz-Arrastia et al., 2014). A favorable therapeutic time window is also required. Therefore, a careful assessment of therapeutic time window should be performed prior to clinical testing of a drug to treat TBI. We make the following suggestions of how to incorporate testing of therapeutic time window in TBI drug development: (1) Preclinical drug testing should examine drug efficacy with dosing beginning at progressively longer times after injury. This will analyze whether a drop-off in efficacy is steep or gradual as time to first dose increases.
(2) Preclinical studies of therapeutic time window should include multiple histological, physiological and behavioral outcomes. Drugs are likely to have different therapeutic windows depending upon the outcome measure. (3) Despite the demonstration that severe or moderate TBI can be dosed as early as 4 h post-injury, drugs with therapeutic windows that are substantially longer than 4 h will be needed to treat the greatest number of brain injury patients. (4) Patients with mild TBI often delay treatment, therefore drugs to treat mild TBI will require even longer therapeutic windows than drugs to treat moderate to severe TBI.

AUTHOR CONTRIBUTIONS
PB conceived and designed the study. MM and PB generated and organized the database. All authors wrote and revised the manuscript and approved the submitted version.

FUNDING
This work was supported by intramural funding to KW and PB from SUNY Downstate Medical Center.


extfieldsofstudy: Chemistry Medicine

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Pin1 Plays Essential Roles in NASH Development by Modulating Multiple Target Proteins

Pin1 is one of the three known prolyl-isomerase types and its hepatic expression level is markedly enhanced in the obese state. Pin1 plays critical roles in favoring the exacerbation of both lipid accumulation and fibrotic change accompanying inflammation. Indeed, Pin1-deficient mice are highly resistant to non-alcoholic steatohepatitis (NASH) development by either a high-fat diet or methionine–choline-deficient diet feeding. The processes of NASH development can basically be separated into lipid accumulation and subsequent fibrotic change with inflammation. In this review, we outline the molecular mechanisms by which increased Pin1 promotes both of these phases of NASH. The target proteins of Pin1 involved in lipid accumulation include insulin receptor substrate 1 (IRS-1), AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase 1 (ACC1), while the p60 of the NF-kB complex and transforming growth factor β (TGF-β) pathway appear to be involved in the fibrotic process accelerated by Pin1. Interestingly, Pin1 deficiency does not cause abnormalities in liver size, appearance or function. Therefore, we consider the inhibition of increased Pin1 to be a promising approach to treating NASH and preventing hepatic fibrosis.

Introduction
Non-alcoholic fatty liver disease (NAFLD) is characterized as hepatic steatosis that is not caused by factors such as significant alcohol consumption, chronic viral hepatitis and the side effects of medicines. In recent years, the number of NAFLD patients has been rising, showing trends similar to those of other diseases included in the metabolic syndrome category. In addition, approximately 10% of these NAFLD patients are categorized as suffering from nonalcoholic steatohepatitis (NASH), which is characterized 3.2. "Two-Hit Theory" Versus "Multi-Hit Theory" While the mechanism of NASH development has not been fully elucidated, the "two-hit theory has gained widespread recognition. According to this hypothesis, the "first hit" is the development of hepatic steatosis which is mediated mainly by lifestyle factors such as excessive caloric intake and inadequate physical exercise. The "second hit" is speculated to be induced by a variety of factors which lead to inflammation and fibrosis in the fatty liver. Oxidative stress, gut-derived endotoxins, some adipocytokines and free fatty acids (FFA) have been suggested as possible candidates for these factors [30,31]. However, recent findings raise the possibility that the two-hit theory is not fully applicable to the pathogenesis of NASH. This is because certain symptoms of inflammation are detectable before steatosis develops in some cases [32]. In addition, steatosis itself seems to be a favorable adaptation to excessive caloric intake, as triglyceride formation protects hepatocytes from lipotoxicity by "aggressive" lipids such as FFA which trigger reactive oxygen species (ROS) production and promote inflammation [33][34][35].
Furthermore, many factors including insulin resistance, dysregulation of FFA and adipokine secretion from adipose tissue and dysregulation of gut-derived signals via the alteration of gut microbiota and permeability appear to promote both steatosis and inflammation in parallel rather than sequentially. Based on these concepts, the "multiple parallel hit" theory appears to be more applicable than the "second hit" theory as an explanation for the pathogenesis of NASH [22,35].
Pin1 expression in NASH model mouse livers was shown to be dramatically increased by either a methionine-choline-deficient diet (MCDD) or a high-fat diet (HFD). Importantly, Pin1 deficiency markedly suppressed the development of hepatic steatosis, inflammation and fibrosis observed in NASH mouse livers. Bone marrow transplantation experiments revealed the presence of Pin1 in both hematopoietic and non-hematopoietic cells to be essential for inflammation and lipid accumulation, respectively, with both contributing to NASH development [36]. Also, as described below in Figure 1, Pin1 has been shown to be involved in lipid metabolism, inflammation and fibrosis in the liver. Physiological mechanisms of hepatic fibrosis development in NASH. Hepatic inflammation is enhanced by the influx of inflammatory substances from adipose tissue and the gut due to excessive calorie intake and a high-fat diet. Hepatic immune cells produce cytokines such as the tumor necrosis factor (TNF)α and activate quiescent HSCs, enhance the proliferation or survival of HSCs and cause the accumulation of lipid droplets and impairment of insulin sensitivity in hepatocytes. Activated HSCs induce hepatic fibrosis through the release of fibrotic factors. HSCs: hepatic stellate cells, PAMPs: Pathogen-associated molecular patterns. LPS: lipopolysaccharide. CXCL1: C-X-C Motif Chemokine Ligand 1. αSMA: α-smooth muscle actin. TGF-β: transforming growth factor β.
Taken together, these findings indicate upregulated hepatic Pin1 expressions to be essential for NASH development, making contributions to both inflammation and lipid accumulation, with Pin1 deletion or inhibition markedly mitigating NASH symptoms according to the "multi-hit" theory.

Role of Pin1 in the Pathogenesis of Hepatic Steatosis
Hepatic steatosis results from an imbalance between lipogenesis and fatty acid oxidation [30]. As described above, hepatic Pin1 expressions are increased by either an HFD or MCDD, and hepatic steatosis does not occur in Pin1 knockout (KO) mice [37]. These findings indicate Pin1 in the liver to be essential for the development of steatosis.
First, insulin signaling plays critical roles in lipogenesis, by inducing the expressions of two rate-limited enzymes, acetyl CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN) [38,39]. Pin1 reportedly enhances insulin signaling via its association with insulin receptor substrate 1 (IRS-1) and Akt [37,40]. Pin1 binds to IRS-1 and upregulates its tyrosine phosphorylation without altering IRS-1 protein levels. Indeed, Pin1 null mice were revealed to have impaired insulin sensitivity [37]. Moreover, Pin1 raises the level of Akt phosphorylation at Ser473 through the stabilization of the total Akt protein amount [39].
A recent report also clarified that Pin1 directly interacts with ACC1 and increases its protein stability [41]. Indeed, while Pin1 KO or knockdown markedly reduces the half-life of the ACC1 protein, Pin1 overexpression increases it. Similarly, the protein expression level of FASN is also increased by Pin1 without changing its mRNA level [42].
Third, Pin1 reportedly associates with the CBS domain in the γ subunit and reduces AMPKα subunit phosphorylation [43]. The reduction in AMPKα subunit phosphorylation mediated by Pin1 is likely due to the protective effect exerted by AMP or ADP, against dephosphorylation by protein phosphatase 2C (PP2C), being abolished. As a result, Pin1 negatively regulates AMPK activity [43]. AMPK, known as a major controller of lipid metabolism, directly phosphorylates ACC1 at the Ser79 site, and decreases both its activity and the productions of malonyl-CoA [44,45]. The reduction of malonyl-CoA indirectly upregulates fatty acid oxidation by abrogating the suppression of carnitine palmitoyltransferase 1 (CPT-1) [46,47]. In other words, AMPK inhibits lipogenesis and enhances fatty acid oxidation. Consequently, AMPK activation strongly suppresses lipid accumulation in the affected tissues.
Collectively, these observations indicate that Pin1 favors enhanced lipogenesis, functioning via at least three mechanisms; through an insulin-signaling-dependent association with IRS-1 and Akt, as well as exerting AMPK-dependent and direct actions on lipid enzymes such as ACC1 and FASN, as seen in Figure 2. The functions of Pin1 in the molecular mechanism of lipogenesis in adipocytes. Either an HFD or MCDD increases Pin1 expressions in the liver and adipocytes. In adipocytes and hepatocytes, Pin1 enhances lipogenesis through an insulin-signaling-dependent mechanism by associating with IRS-1 and Akt, exerting AMPK-dependent and direct actions on lipid enzymes such as ACC1 and FASN. In the energy consumption pathway, Pin1 downregulates the expressions of thermogenic genes, such as UCP-1, and reduces O2 consumption. Pin1 also enhances adipocyte differentiation by activating transcription of PPARγ protein in adipocytes. IRS-1: insulin receptor substrate 1. ACC: acetyl CoA carboxylase. PRDM16: PR domain containing 16. UCP-1: Uncoupling protein 1. PPARγ: peroxisome proliferator-activated receptor γ.

Essential Role of Adipose Pin1 in Obesity and NASH Development
Obesity is one of the risk factors for a fatty liver, though not all cases are obese. Recent reports have revealed that excessive lipid accumulation leads to adipocyte fibrosis [48]. Fibrotic adipocytes lose the ability to store triglycerides and ectopic fat accumulates in the affected livers. Accordingly, the amelioration of obesity rescues these livers from excessive fat storage.
Insulin resistance with hyperinsulinemia are known to further promote obesity. Pin1 controls glucose metabolism through insulin signaling, AMPK and transcription of the enzymes regulating gluconeogenesis [38]. Indeed, Pin1 null mice exhibit impairments of both glucose and insulin tolerance [49]. In addition, Pin1 is a key player in the release of insulin, as Pin1 deletion in islets alleviates insulin release in response to high glucose [50]. In contrast, adipocyte-specific Pin1 knockout (KO) mice have lower body weights, with liver triglyceride and macrophage infiltration of epididymal white adipose tissue when fed a HFD [37].
Interestingly, adipocyte-specific Pin1 KO mice show an alleviation of both HFD-induced obesity and fatty liver development, indicating the amelioration of obesity to be linked to the prevention of NASH. Pin1 enhances adipocyte differentiation by activating the transcription of peroxisome proliferator-activated receptor γ (PPARγ) protein in adipocytes without affecting PPARγ protein levels [51]. The study also revealed PPARγ expression levels to be upregulated during adipogenesis and that Pin1 knockdown impairs adipogenesis.

Essential Role of Adipose Pin1 in Obesity and NASH Development
Obesity is one of the risk factors for a fatty liver, though not all cases are obese. Recent reports have revealed that excessive lipid accumulation leads to adipocyte fibrosis [48]. Fibrotic adipocytes lose the ability to store triglycerides and ectopic fat accumulates in the affected livers. Accordingly, the amelioration of obesity rescues these livers from excessive fat storage.
Insulin resistance with hyperinsulinemia are known to further promote obesity. Pin1 controls glucose metabolism through insulin signaling, AMPK and transcription of the enzymes regulating gluconeogenesis [38]. Indeed, Pin1 null mice exhibit impairments of both glucose and insulin tolerance [49]. In addition, Pin1 is a key player in the release of insulin, as Pin1 deletion in islets alleviates insulin release in response to high glucose [50]. In contrast, adipocyte-specific Pin1 knockout (KO) mice have lower body weights, with liver triglyceride and macrophage infiltration of epididymal white adipose tissue when fed a HFD [37].
Interestingly, adipocyte-specific Pin1 KO mice show an alleviation of both HFD-induced obesity and fatty liver development, indicating the amelioration of obesity to be linked to the prevention of NASH. Pin1 enhances adipocyte differentiation by activating the transcription of peroxisome proliferator-activated receptor γ (PPARγ) protein in adipocytes without affecting PPARγ protein levels [51]. The study also revealed PPARγ expression levels to be upregulated during adipogenesis and that Pin1 knockdown impairs adipogenesis.
A recent investigation found that Pin1 is involved in the regulation of non-shivering thermogenesis in both brown and beige adipocytes. Pin1 interacts with the PR domain containing 16 (PRDM16), which is involved in the expression of the uncoupling protein (UCP-1), a major contributor to thermogenic programs. PRDM16, by associating with Pin1, is rapidly degraded through the ubiquitin-proteasome system, depending on the isomerase activity of Pin1. Indeed, Pin1 deletion in adipocytes elevates the expression of thermogenic genes and promotes O2 consumption [37].
Therefore, it has now become clear that not only hepatic but also adipose Pin1 is a major player in obesity development, contributing to the development of NAFLD or NASH, as shown in Figure 2.

Pin1 Promotes the Generation of ROS by Associating with NADPH Oxidase
Excessive free fatty acids (FFA) activate oxygen consumption in mitochondria. In the respiratory chain, which is activated in the presence of excessive FFA, complexes I and II are the main sources of mitochondrial ROS [52]. FFA present in excess also activates NADPH oxidase via protein kinase C (PKC) and produces ROS [53]. The NADPH oxidase (Nox) consists of six subcomponents (p47phox, p67phox, p40phox, Rac2, p22phox and gp91phox), in addition to Nox2 itself [54,55]. Although the mechanism of Nox2 activation is complex, it is essential that p47phox, phosphorylated by stimuli, be transferred to the membrane and then associate with p22phox.
In the Nrf2 pathway, Pin1 inhibits Nrf2 and PIP4K, which synthesize PtdInsP2 (4, 5) and remove PtdIns5P by phosphorylating it. A study on vascular smooth muscle cells showed that Pin1 overexpression increases the ubiquitination of Nrf2 and inhibits its nuclear translocation [64]. Another study showed that Pin1 and PIP4K co-localize in nuclear speckles and block the actions of PtdIns5P, which is the only known activator of Nrf2, by phosphorylating it [65].

Pin1 Enhances Inflammation
Inflammation and fibrotic change contribute to progression from NAFLD to NASH. Expressions of inflammatory cytokines are largely regulated by the pathways of the two transcription factors, nuclear factor-kB (NF-kB) and activator protein 1 (AP-1) [52,53,56]. Pin1 enhances both of these transcriptional pathways, as described below.
The family of inducible transcription factors NF-κB/Rel impacts cell survival, inflammation and the process of tumorigenesis. [66,67]. There are five structurally related NF-κB family proteins, including p50, p52, RelA (p65), RelB and c-Rel [68]. NF-κB exists in heterodimer or homodimer forms and is usually localized in the cytoplasm via interactions with IκB family members [69]. The NF-κB pathway activation begins with the inducible degradation of IκBα due to site-specific phosphorylation by the multi-subunit IκB kinase complex [70]. The detachment of IκBα induces the nuclear translocation of RelA/p50 heterodimers and initiates the induction of various pro-inflammatory genes [71]. Activated p65 and P50 bound to DNA are inactivated upon moving to the cytoplasm in response to rebinding with IκB [72,73]. Ryo et al. reported that Pin1-specific binding of p65 to the pThr254-Pro motif inhibits p65 from binding to IκBα, thereby enhancing p65 nuclear localization. Moreover, Pin1 exerts effects on p65 protein stability, since Pin1 deficiency reduces p65 protein levels. Accordingly, Pin1 regulates NF-κB pathway activation by controlling both the translocation and the protein stability of p65 [74,75]. Another study also found that Pin1 interacts with c-Rel and thereby promotes both nuclear translocation and transforming activity [76].
In the AP-1 signaling pathway, AP-1 transcriptional activity is regulated by c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) group [77]. JNK activity is upregulated by phosphorylation of Thr and Tyr residues and these phosphorylations are a response to a variety of stressors such as oxidative stress or inflammatory cytokines [78,79]. Pin1 activates this pathway by interacting with phosphorylated JNK1 [78]. The bindings of Pin1 and JNK1 induce the interaction of JNK1 to c-Jun and activating transcription factor 2 (ATF2), which are downstream members of the AP-1 family. Pin1 also activates the AP-1 signaling pathway via upregulation of p70S6K activity [80] (Figure 3). localization. Moreover, Pin1 exerts effects on p65 protein stability, since Pin1 deficiency reduces p65 protein levels. Accordingly, Pin1 regulates NF-κB pathway activation by controlling both the translocation and the protein stability of p65 [74,75]. Another study also found that Pin1 interacts with c-Rel and thereby promotes both nuclear translocation and transforming activity [76].
In the AP-1 signaling pathway, AP-1 transcriptional activity is regulated by c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) group [77]. JNK activity is upregulated by phosphorylation of Thr and Tyr residues and these phosphorylations are a response to a variety of stressors such as oxidative stress or inflammatory cytokines [78,79]. Pin1 activates this pathway by interacting with phosphorylated JNK1 [78]. The bindings of Pin1 and JNK1 induce the interaction of JNK1 to c-Jun and activating transcription factor 2 (ATF2), which are downstream members of the AP-1 family. Pin1 also activates the AP-1 signaling pathway via upregulation of p70S6K activity [80] (Figure 3).

Pin1 Activates the Pathways Leading to Fibrosis
Yılmaz and colleagues raised the possibility that Pin1 concentrations in serum may well be exploited as markers for the detection of NASH and for determining advanced fibrotic scores for this disease [81]. The serum Pin1 levels were found to correlate with histopathological features in patients with NASH and to be independent predictors of advanced liver fibrosis. Furthermore, treatment with the Pin1 inhibitor juglone reportedly ameliorates drug-induced liver fibrosis [26]. The study that obtained this finding showed the Pin1 expressions in fibrotic livers to be upregulated, and that Pin1 regulates the expression of transforming growth factor-β (TGF-β)1, a fibrogenesis regulator, and the

Pin1 Activates the Pathways Leading to Fibrosis
Yılmaz and colleagues raised the possibility that Pin1 concentrations in serum may well be exploited as markers for the detection of NASH and for determining advanced fibrotic scores for this disease [81]. The serum Pin1 levels were found to correlate with histopathological features in patients with NASH and to be independent predictors of advanced liver fibrosis. Furthermore, treatment with the Pin1 inhibitor juglone reportedly ameliorates drug-induced liver fibrosis [26]. The study that obtained this finding showed the Pin1 expressions in fibrotic livers to be upregulated, and that Pin1 regulates the expression of transforming growth factor-β (TGF-β)1, a fibrogenesis regulator, and the phosphorylation of Smad2/3, a key regulator of the fibrogenesis signaling pathway, in hepatic stellate cells (HSCs).
Numerous studies have confirmed the importance of the TGF-β-SMAD signaling pathway during the process of hepatic fibrosis. TGF-β is a ubiquitous factor which reportedly promotes fibrosis in various tissues [82,83]. TGF-β signaling occurs via both canonical Smad and non-Smad pathways [84]. TGF-β receptor I (TGF-βRI) phosphorylates the intracellular mediator receptor-regulated Smads (R-Smads), Smad2 and Smad3 [84][85][86]. Phosphorylated R-Smad is a dimer and the common mediator Smad (Co-Smad), also known as Smad4, is another member of the Smad family, and forms heterodimeric complexes in the cytosol [84,87]. The Smad complex accumulates in the nucleus and cooperates with other transcription factors to control gene transcription [84]. These Smad complex activities are negatively regulated by Smad6 and Smad7 which constitute a third class of Smad proteins, referred to as inhibitory Smad (I-Smad) [84].
Pin1 reportedly activates the TGF-Smad pathway and is thereby involved in fibrotic change [38,41,88,89]. The translational activity of TGF-β1 mRNA is regulated by various proteins. AU-rich element RNA-binding protein 1(AUF-1) binds to TGF-β1 mRNA and promotes its rapid exosome-mediated degradation [90]. Pin1 leads to the isomerization of hyper-phosphorylated AUF1 isoforms, and thereby reduces AUF1 binding to TGF-β1 mRNA and suppresses exosome-mediated mRNA decay [91]. Another study showed that Pin1 interacts with SUMOylated promyelocytic leukemia protein (PML), which acts as a nuclear translocation promoter and a TGFβ1 transcriptional regulator, increasing TGFβ1 mRNA expression and also, enhancing the TGFβ1-mediated Smad2/3 signaling pathway [92]. Pin1, as a part of the Smad pathway, interacts with both Smad2 and Smad3, thereby enhancing their phosphorylation and the resulting transcriptional activity [40,88,89].
In addition, Pin1-mediated isomerization of the inhibitory Smad6 MH2 domain may alter Smad6 functions such as nuclear localization signals and nuclear export signals. This isomerization increases phosphorylation, nuclear localization, and gene activation of Smad3 in response to TGFβ1 treatments and activates the TGF-β-Smad signaling pathway [93]. Several prior studies have confirmed that Pin1 knockdown or treatment with Pin1 inhibitors suppresses Smad phosphorylation and the expressions of fibrotic genes induced by the TGF-Smad pathway [38,89].
Signal transducers and activators of transcription (STATs) family pathways are also important for cytokine signaling and effects [94]. Leptin and IL-6 activate the STAT3 signaling pathway and increase HSC collagen mRNA expression [95]. Recent studies have shown that the complex of STAT3 and JunB can directly control activation of the COL1A2 enhancer [96,97]. In addition, STAT3 activates the TGF-Smad pathway. A study of dermal fibroblasts showed that activation of STAT3 by IL-6 results in TGFβ activation [98].
Stat3 transcriptional activity is regulated by Tyr-phosphorylation followed by dimerization and translocation from the cytosol to the nucleus. A breast cancer cell study demonstrated that Pin1 enhances STAT3 transcriptional activity by interacting with Ser727-phosphorylated STAT3 [99]. This study also revealed that Pin1 promotes STAT3-mediated epithelial-mesenchymal transition, thereby inducing liver fibrosis.
These studies showed Pin1 to be a key regulator of fibrosis and to have potential as a pharmacologic target for treating liver fibrosis, as seen in Figure 4.

Conclusions
Pin1 has been shown to regulate a variety of signal transduction pathways. In particular, Pin1 activates the pathways involved in not only lipid accumulation but also, inflammation and fibrosis. On the other hand, the results of analyses using Pin1 KO mice strongly suggest that Pin1 deficiency produces no abnormalities in liver size, formation or functions. Therefore, Pin1 inhibition is a potentially promising therapeutic method for NASH/NAFLD. In addition, it is clear that Pin1 inhibition has a marked effect on the suppression of carcinogenesis, and may exert a strong effect against hepatocellular carcinoma development. In addition, the serum Pin1 concentration is currently being studied for clinical application as a diagnostic marker for NASH/NAFLD. In conclusion, Pin1 plays a major role in all steps of the NASH process, including fat accumulation, fibrosis, and, perhaps ultimately, cancer development. However, the application of a Pin1 inhibitor, as NASH/NAFLD therapy, has not been adequately studied. Juglone (5-hydroxynaphthoquinone), epigallocatechin-3gallate, cycloheptapeptidyl inhibitor, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are known Pin1 inhibitors, but only juglone has been examined in NASH/NAFLD studies [41,100]. Since these inhibitors are not specific for Pin1, the development of a potent and highly selective Pin1 inhibitor, with minimal or no side-effects, for therapeutic use is eagerly awaited.

Conclusions
Pin1 has been shown to regulate a variety of signal transduction pathways. In particular, Pin1 activates the pathways involved in not only lipid accumulation but also, inflammation and fibrosis. On the other hand, the results of analyses using Pin1 KO mice strongly suggest that Pin1 deficiency produces no abnormalities in liver size, formation or functions. Therefore, Pin1 inhibition is a potentially promising therapeutic method for NASH/NAFLD. In addition, it is clear that Pin1 inhibition has a marked effect on the suppression of carcinogenesis, and may exert a strong effect against hepatocellular carcinoma development. In addition, the serum Pin1 concentration is currently being studied for clinical application as a diagnostic marker for NASH/NAFLD. In conclusion, Pin1 plays a major role in all steps of the NASH process, including fat accumulation, fibrosis, and, perhaps ultimately, cancer development. However, the application of a Pin1 inhibitor, as NASH/NAFLD therapy, has not been adequately studied. Juglone (5-hydroxynaphthoquinone), epigallocatechin-3-gallate, cycloheptapeptidyl inhibitor, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are known Pin1 inhibitors, but only juglone has been examined in NASH/NAFLD studies [41,100]. Since these inhibitors are not specific for Pin1, the development of a potent and highly selective Pin1 inhibitor, with minimal or no side-effects, for therapeutic use is eagerly awaited.

Conflicts of Interest:
The author declares no conflict of interest.


s2fieldsofstudy: Biology Chemistry

text:
The Organogermanium Compound THGP Suppresses Melanin Synthesis via Complex Formation with L-DOPA on Mushroom Tyrosinase and in B16 4A5 Melanoma Cells

The organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) has various biological activities. We previously reported that THGP forms a complex with cis-diol structures. L-3,4-Dihydroxyphenylalanine (L-DOPA), a precursor of melanin, contains a cis-diol structure in its catechol skeleton, and excessive melanin production causes skin darkening and staining. Thus, the cosmetic field is investigating substances that suppress melanin production. In this study, we investigated whether THGP inhibits melanin synthesis via the formation of a complex with L-DOPA using mushroom tyrosinase and B16 4A5 melanoma cells. The ability of THGP to interact with L-DOPA was analyzed by 1H-NMR, and the influence of THGP and/or kojic acid on melanin synthesis was investigated. We also examined the effect of THGP on cytotoxicity, tyrosinase activity, and gene expression and found that THGP interacted with L-DOPA, a precursor of melanin with a cis-diol structure. The results also showed that THGP inhibited melanin synthesis, exerted a synergistic effect with kojic acid, and did not affect tyrosinase activity or gene expression. These results suggest that THGP is a useful substrate that functions as an inhibitor of melanogenesis and that its effect is enhanced by combination with kojic acid.

However, the mechanism underlying the physiological effects remains unclear. In recent years, studies have shown that THGP interacts with a cis-diol structure and suggested that this interaction is involved in the physiological effects of THGP [13,14]. The identified biological substances with a cis-diol structure include catecholamine, ATP, and adenosine. L-3,4-Dihydroxyphenylalanine (L-DOPA)

THGP and L-DOPA Form a Complex via a Cis-Diol Structure
We evaluated the ability of L-DOPA to form a complex with THGP or 3-trimethylgermylpropanoic (TMGP), which contains a methyl group in place of the hydroxyl group on THGP, by 1 H-NMR (see Figure 1A). The signals at 1.5 and 2.5 ppm originated from THGP, and the L-DOPA signals are shown at 3.1, 3.9, and 6.9 ppm. The mixture of THGP and L-DOPA produced a spectrum that differed from the characteristic spectra of THGP and L-DOPA alone ( Figure 1B). The mixture of these substances yielded novel signals at 1.8 and 2.6 ppm, which were not found on the L-DOPA or THGP spectra and were thus derived from the complexed compounds. However, the mixture of THGP and L-DOPA also showed the individual spectra of THGP and L-DOPA, which suggests that THGP does not form a complete complex with L-DOPA. In contrast, after the mixing of TMGP (signals at 0.1, 1.0, and 2.2 ppm) with L-DOPA ( Figure 1C), only the signals from the individual compounds were observed. In summary, the data suggest that THGP forms a complex with L-DOPA through the hydroxyl group of THGP ( Figure 1D). mixture of these substances yielded novel signals at 1.8 and 2.6 ppm, which were not found on the L-DOPA or THGP spectra and were thus derived from the complexed compounds. However, the mixture of THGP and L-DOPA also showed the individual spectra of THGP and L-DOPA, which suggests that THGP does not form a complete complex with L-DOPA. In contrast, after the mixing of TMGP (signals at 0.1, 1.0, and 2.2 ppm) with L-DOPA ( Figure 1C), only the signals from the individual compounds were observed. In summary, the data suggest that THGP forms a complex with L-DOPA through the hydroxyl group of THGP ( Figure 1D).

THGP Inhibits Melanin Synthesis through a Reaction Mediated by Mushroom-Derived Tyrosinase
We confirmed that the mixing of L-DOPA and mushroom tyrosinase synthesized melanin. The resulting melanin solutions without added organogermanium (left) or with added THGP (center) or TMGP (right) are displayed in Figure 2A. The melanin content of the control (without added organogermanium) is shown as a percentage in Figure 2B and compared with the control condition. THGP significantly suppressed melanin synthesis (p < 0.01) at a concentration of at least 1000 µM (Figure 2A,B). THGP also inhibited melanin synthesis in a dose-dependent manner (93% inhibition with a concentration of 1000 µM to 71% inhibition with a concentration of 5000 µM), whereas TMGP did not inhibit melanin synthesis. This result suggests that the hydroxyl group on THGP is important for melanin synthesis, and THGP might thus inhibit the synthesis of melanin by complexing with L-DOPA via the hydroxyl group of THGP. Kojic acid also inhibited melanin synthesis in a dose-dependent manner ( Figure 2C).
The synergistic inhibitory effects between THGP and kojic acid are shown in Figure 2D. The theoretical value for the combination of THGP and kojic acid was considered the standard (with a value of 1), and the synergistic effect of THGP was calculated in comparison to this standard. For example, 5000 µM THGP alone resulted in a melanin content of 67.8% compared with the control, and 1000 µM kojic acid alone yielded an inhibition rate of 51.9%. The theoretical value was found to equal 35.2%, which corresponds to the multiplication of 67.8% by 51.9%. However, because the experimentally measured inhibition rate was 12%, the synergistic effect was 2.93-fold higher than the theoretical value. This synergistic effect was due to the interaction of THGP with the substrate and the interaction of kojic acid with the enzyme. value of 1), and the synergistic effect of THGP was calculated in comparison to this standard. For example, 5000 μM THGP alone resulted in a melanin content of 67.8% compared with the control, and 1000 μM kojic acid alone yielded an inhibition rate of 51.9%. The theoretical value was found to equal 35.2%, which corresponds to the multiplication of 67.8% by 51.9%. However, because the experimentally measured inhibition rate was 12%, the synergistic effect was 2.93-fold higher than the theoretical value. This synergistic effect was due to the interaction of THGP with the substrate and the interaction of kojic acid with the enzyme. The results were statistically analyzed using Dunnett's method, and 0 μM THGP was used as the The results were statistically analyzed using Dunnett's method, and 0 µM THGP was used as the control. Circles: 0 µM THGP; filled circles: 1000 µM THGP; triangles: 2500 µM THGP; squares: 5000 µM THGP. The data are presented as the means ± SDs from six independent experiments. ** p < 0.01 vs. the control.

THGP Inhibits Melanin Production by the B16 4A5 Melanoma Cell Line
The effect of THGP or kojic acid on B16 4A5 melanoma cell proliferation was evaluated through the MTS assay. The results showed that 5000 µM THGP did not affect cell proliferation, whereas 5000 µM kojic acid showed cytotoxicity (80% proliferation; Figure 3). This result suggests that THGP is less cytotoxic and safer than kojic acid.

THGP Inhibits Melanin Production by the B16 4A5 Melanoma Cell Line
The effect of THGP or kojic acid on B16 4A5 melanoma cell proliferation was evaluated through the MTS assay. The results showed that 5000 μM THGP did not affect cell proliferation, whereas 5000 μM kojic acid showed cytotoxicity (80% proliferation; Figure 3). This result suggests that THGP is less cytotoxic and safer than kojic acid. We examined the effect of THGP or kojic acid on melanin synthesis by B16 4A5 melanoma cells. The melanin contents in the extracellular solution are shown in Figure 4A, and the intracellular melanin contents are shown in Figure 4B. The values in both figures are displayed as percentages The results were statistically analyzed by Dunnett's method, and 0 µM THGP was used as the control. The results are presented as the means ± SDs from eight independent experiments. ** p < 0.01 vs. the control.
We examined the effect of THGP or kojic acid on melanin synthesis by B16 4A5 melanoma cells. The melanin contents in the extracellular solution are shown in Figure 4A, and the intracellular melanin contents are shown in Figure 4B. The values in both figures are displayed as percentages relative to the melanin content in the untreated control cells. The amount of extracellular melanin after being treated with 50, 500, and 5000 µM THGP decreased to approximately 60%, 40%, and 30% of the control levels, respectively ( Figure 4A). In addition, 500 µM THGP suppressed melanin production by 62%, whereas 500 µM kojic acid exerted an inhibitory effect of 55%. These results suggest that the inhibitory effect of THGP on melanin production was as high as that of kojic acid at the same concentration (500 µM). However, treatment with 5000 µM THGP increased the intracellular melanin content by approximately 1.8-fold compared with the control ( Figure 4B). This figure does not show the ratio of the extracellular to intracellular melanin contents, but the extracellular melanin content was approximately 5-fold higher than the intracellular melanin content. This result suggests that the total amount of intracellular and extracellular melanin was suppressed by THGP. Cells treated with 6 mM THGP for 48 h were observed under an optical microscope, and their morphologies are shown in Figure 4C. The cellular accumulation of melanin clearly increased after THGP treatment. These results suggest that the THGP and-L-DOPA complex inhibited not only the synthesis but also the release of melanin. Moreover, the extracellular melanin content of cells treated with the combination of THGP and kojic acid is shown in Figure 4D. The results indicate that the extracellular melanin level was synergistically suppressed by the combination of THGP and kojic acid. In particular, melanin production was suppressed by approximately 90% by the combination of 500 µM THGP with 50 µM kojic acid.

THGP Does Not Affect Tyrosinase Activity or the Expression of Melanin Synthesis-Related Genes
The influence of THGP treatment on the tyrosinase activity of B16 4A5 melanoma cells was examined. The activities observed after different THGP treatments are shown in Figure 5A, and the mRNA expression levels of genes related to melanin production in B16 4A5 cells are shown in Figure  5B. The results show that THGP did not affect tyrosinase activity ( Figure 5A), and the expression levels of tyrosinase, Trp-1, Trp-2, and MITF, which are the melanin production-related genes, were The suppressive effect of the combination of THGP and kojic acid on melanin synthesis was evaluated. The results were statistically analyzed using Dunnett's method, and 0 µM THGP served as the control. The data are presented as the means ± SDs from six independent experiments. * p < 0.05 and ** p < 0.01 vs. the control.

THGP Does Not Affect Tyrosinase Activity or the Expression of Melanin Synthesis-Related Genes
The influence of THGP treatment on the tyrosinase activity of B16 4A5 melanoma cells was examined. The activities observed after different THGP treatments are shown in Figure 5A, and the mRNA expression levels of genes related to melanin production in B16 4A5 cells are shown in Figure 5B. The results show that THGP did not affect tyrosinase activity ( Figure 5A), and the expression levels of tyrosinase, Trp-1, Trp-2, and MITF, which are the melanin production-related genes, were also not impacted by treatment with THGP alone or in combination with L-DOPA ( Figure 5B,C). These results suggest that THGP does not influence the tyrosinase activity or gene expression to inhibit the synthesis of melanin by forming a complex with L-DOPA.
4A5 cells were treated with THGP or kojic acid for 72 h, and the (A) extracellular and (B) intracellular melanin amounts were then evaluated. (C) Optical microscopy images of cells treated with THGP (6000 μM). The arrow means melanin accumulated in large amount in B16 4A5 and the circle means the enlarged location in the figure below. (D) The suppressive effect of the combination of THGP and kojic acid on melanin synthesis was evaluated. The results were statistically analyzed using Dunnett's method, and 0 μM THGP served as the control. The data are presented as the means ± SDs from six independent experiments. * p < 0.05 and ** p < 0.01 vs. the control.

THGP Does Not Affect Tyrosinase Activity or the Expression of Melanin Synthesis-Related Genes
The influence of THGP treatment on the tyrosinase activity of B16 4A5 melanoma cells was examined. The activities observed after different THGP treatments are shown in Figure 5A, and the mRNA expression levels of genes related to melanin production in B16 4A5 cells are shown in Figure  5B. The results show that THGP did not affect tyrosinase activity ( Figure 5A), and the expression levels of tyrosinase, Trp-1, Trp-2, and MITF, which are the melanin production-related genes, were also not impacted by treatment with THGP alone or in combination with L-DOPA ( Figure 5B,C). These results suggest that THGP does not influence the tyrosinase activity or gene expression to inhibit the synthesis of melanin by forming a complex with L-DOPA.

THGP Suppresses Melanin Production in α-MSH-or L-DOPA-Induced Melanogenesis
Because melanogenesis is stimulated by α-MSH, we evaluated the influence of THGP treatment in the presence of α-MSH in the culture media on melanin synthesis and the extracellular melanin content, and the results are shown in Figure 6A. The amount of extracellular melanin increased after treatment with α-MSH (from approximately 70% to 100%, i.e., 1.4-fold increase), and treatment with THGP at concentrations above 50 μM reduced the extracellular melanin levels compared with those obtained with α-MSH treatment ( Figure 6B). In particular, the addition of THGP at high concentrations (500 and 5000 μM) completely suppressed melanogenesis. The extracellular melanin content after the addition of the L-DOPA substrate is shown in Figure 6B. Compared with the control condition, the addition of L-DOPA markedly accelerated melanogenesis in melanoma cells by approximately 10-fold. In addition, the cells treated with L-DOPA, THGP, and kojic acid exhibited decreased melanin contents compared with the control cells. THGP (at concentrations of 5, 50, 500, and 5000 μM) inhibited melanogenesis in a dose-dependent manner (to levels that were 90%, 85%, 60%, and less than 10% of the control levels, respectively). At most high concentrations (5000 μM), THGP decreased the melanin level to the same level found in the untreated group (without L-DOPA), but the effect of kojic acid treatment was lower (approximately 90% to 85%) than that obtained with THGP treatment (Figure 6B). This result might reflect the different interactions of THGP and kojic acid: THGP complexes with L-DOPA, whereas kojic acid inhibits tyrosinase activity.

THGP Suppresses Melanin Production in α-MSHor L-DOPA-Induced Melanogenesis
Because melanogenesis is stimulated by α-MSH, we evaluated the influence of THGP treatment in the presence of α-MSH in the culture media on melanin synthesis and the extracellular melanin content, and the results are shown in Figure 6A. The amount of extracellular melanin increased after treatment with α-MSH (from approximately 70% to 100%, i.e., 1.4-fold increase), and treatment with THGP at concentrations above 50 µM reduced the extracellular melanin levels compared with those obtained with α-MSH treatment ( Figure 6B). In particular, the addition of THGP at high concentrations (500 and 5000 µM) completely suppressed melanogenesis. The extracellular melanin content after the addition of the L-DOPA substrate is shown in Figure 6B. Compared with the control condition, the addition of L-DOPA markedly accelerated melanogenesis in melanoma cells by approximately 10-fold. In addition, the cells treated with L-DOPA, THGP, and kojic acid exhibited decreased melanin contents compared with the control cells. THGP (at concentrations of 5, 50, 500, and 5000 µM) inhibited melanogenesis in a dose-dependent manner (to levels that were 90%, 85%, 60%, and less than 10% of the control levels, respectively). At most high concentrations (5000 µM), THGP decreased the melanin level to the same level found in the untreated group (without L-DOPA), but the effect of kojic acid treatment was lower (approximately 90% to 85%) than that obtained with THGP treatment ( Figure 6B). This result might reflect the different interactions of THGP and kojic acid: THGP complexes with L-DOPA, whereas kojic acid inhibits tyrosinase activity.
content after the addition of the L-DOPA substrate is shown in Figure 6B. Compared with the control condition, the addition of L-DOPA markedly accelerated melanogenesis in melanoma cells by approximately 10-fold. In addition, the cells treated with L-DOPA, THGP, and kojic acid exhibited decreased melanin contents compared with the control cells. THGP (at concentrations of 5, 50, 500, and 5000 μM) inhibited melanogenesis in a dose-dependent manner (to levels that were 90%, 85%, 60%, and less than 10% of the control levels, respectively). At most high concentrations (5000 μM), THGP decreased the melanin level to the same level found in the untreated group (without L-DOPA), but the effect of kojic acid treatment was lower (approximately 90% to 85%) than that obtained with THGP treatment ( Figure 6B). This result might reflect the different interactions of THGP and kojic acid: THGP complexes with L-DOPA, whereas kojic acid inhibits tyrosinase activity.

Discussion
In this study, we evaluated the ability of THGP to suppress melanogenesis. We first attempted to confirm the formation of a complex between THGP and L-DOPA as an early substrate in melanin synthesis. In 2015, the ability to complex with a substance containing a cis-diol structure was reported to be a chemical property of THGP [31]. Moreover, a previous study provided the first confirmation that THGP functions physiologically in vitro via the formation of a complex with cis-diol compounds [13]. Specifically, THGP suppresses the influx of Ca 2+ by interacting with adrenaline and ATP, which have cis-diol structures. Other studies have demonstrated that THGP interacts with adenosine, D-glucose, and D-fructose [14,32]. These results suggest that the physiological activities of THGP are mediated by binding to cis-diol structures. Therefore, we considered whether THGP could form a complex with L-DOPA, which would affect melanogenesis, and our 1 H-NMR analysis clarified that THGP can also form a complex with L-DOPA, which is a precursor substance in melanin synthesis. As shown in Figure 1, the presence of THGP yielded signals of a complexed compound, whereas TMGP did not yield any signals of the complex. These findings show that THGP can interact with L-DOPA through dehydration between the two molecules. A previous crystal structure analysis revealed that THGP forms a complex with noradrenaline, which contains the same catecholamines as L-DOPA, by dehydration condensation via the diol group on catechol. During the formation of a complex of THGP with diol, the propanoic group originating from THGP forms a lactone ring and a coordinate bond with the vacant d-orbital of germanium, resulting in the formation of a pentacoordinate germanium (V) atom. Moreover, we also confirmed that THGP does not form a complex with tyrosine (phenol derivative of L-DOPA). Therefore, these data indicate that the interaction between THGP and L-DOPA requires both Ge-OH and vicinal diol and might be achieved via dehydration between the two molecules. Therefore, we subsequently performed a study using mushroom tyrosinase and found that the synthesis of melanin from L-DOPA was inhibited by the addition of THGP. Additionally, THGP inhibited the tyrosinase-mediated reaction that produced melanin, and this inhibitory effect was not observed with TMGP ( Figure 2). This result suggests that the inhibition of melanin synthesis by THGP was caused by complex formation with L-DOPA. Subsequently, the combination of THGP with kojic acid, which is reportedly a tyrosinase activity inhibitor, exerted a synergistic effect (Figure 4). It can be inferred that THGP and kojic acid suppress melanin synthesis via different mechanisms of action because THGP acts on the substrate as described above and kojic acid acts on the enzyme. In addition to kojic acid, α-arbutin, licorice flavonoid, and similar substances are used as whitening components [28,29,33]; therefore, these substances might exert similar synergistic effects with THGP against melanogenesis. Because these substances act on enzymes such as tyrosinase, TRP-1, and TRP-2, the inhibition of melanin synthesis can likely be enhanced by the combined use of THGP and these substances. These whitening agents have some known problematic side effects. Specifically, it has been reported that rhododenol caused vitiligo in Japan in 2014 and that rhododenol caused white spot damage on the skin by inhibiting enzymes involved in melanin production, such as tyrosinase, and inducing cell damage [34]. Hydroquinone has been used in the field of medicine and cosmetics due to its whitening effect, but its use is prohibited in many European countries due to its carcinogenic activity [35][36][37]. Therefore, the development of a safe cosmetic product with a whitening effect is needed [38]. This study demonstrates that THGP does not influence tyrosinase activity or the expression of genes involved in melanogenesis ( Figure 5). In addition, previous studies have demonstrated that THGP exerts antitumor and antioxidant effects; thus, the side effects of THGP are likely limited [39][40][41][42]. Furthermore, because THGP did not affect cell proliferation at concentrations up to 5000 µM, THGP is considered safe up to this concentration ( Figure 3).
In the cellular experiment, the extracellular melanin content was effectively decreased by the addition of THGP, but the amount of melanin that accumulated in the THGP-treated cells was greater than the control cells. It can therefore be considered that THGP has the effect of accumulating melanin in cells and suppressing the release of melanosome in addition to the inhibitory effect of melanin production by complex formation with L-DOPA. The mechanism, however, is not clear. Melanosomes mature in melanocytes and are released extracellularly, but THGP may inhibit melanosome maturation or melanosome release. It is necessary to clarify this mechanism in the future. However, as THGP did not affect the tyrosinase activity or the expression of melanin production-related genes, it may be involved in the melanin release from melanocytes and delivery to keratinocytes. In skin pigmentation, melanocytes are present in the spinous layer and deliver melanin to keratinocytes. Then, keratinocytes containing the melanin accepted from melanocytes progressively turn over in the epidermis. As a result, spots and darkening appear on the skin [43]. THGP not only suppresses melanin production but may also induce a whitening effect by inhibiting melanin transport. The inhibitory effects of the single use of THGP are shown in Figure 2, Figure 4, and Figure 6, and the inhibitory effects against the enzymatic reaction shown in Figure 2 are lower than those found in the other cell-based studies shown in Figures 4 and 6. We believe that this difference originates from the suppressive effect of THGP on the release of melanin from melanocytes. Further investigation into this transport mechanism induced by the addition of THGP is strongly recommended. Moreover, as shown in Figure 6B, an additional study of L-DOPA revealed a mechanistic difference between THGP and kojic acid. The results of this study suggest that THGP is a novel substrate that suppresses the production of melanin and can potentially be applied in the cosmetic field. Figure 7 shows a schematic of the effect of THGP on melanin production. In this study, we demonstrated that THGP forms a complex with L-DOPA by 1 H-NMR. Furthermore, a test using mushroom tyrosinase proved that THGP exerts an inhibitory effect on melanin synthesis from L-DOPA. In addition, TMGP, which has a methyl group in place of the hydroxyl group present in THGP, did not exert an inhibitory effect on melanin synthesis, and this result suggests that THGP inhibits melanogenesis via complex formation with cis-diol structures. In an experiment using B16 4A5 melanoma cells, THGP also inhibited melanin synthesis. Furthermore, THGP did not affect tyrosinase activity or the expression of melanin production-related factors. These results suggest that THGP affects substrates rather than enzymes involved in melanin metabolism. Finally, kojic acid enhanced the inhibition of melanogenesis when administered in combination with THGP. In this study, tyrosinase enzyme and mouse melanoma cells were used, and similar results thus may not be obtained with human cells. Therefore, the inhibitory effects on cytotoxicity and melanin production need to be assessed in future studies using normal human melanocytes. tyrosinase activity or the expression of melanin production-related factors. These results suggest that THGP affects substrates rather than enzymes involved in melanin metabolism. Finally, kojic acid enhanced the inhibition of melanogenesis when administered in combination with THGP. In this study, tyrosinase enzyme and mouse melanoma cells were used, and similar results thus may not be obtained with human cells. Therefore, the inhibitory effects on cytotoxicity and melanin production need to be assessed in future studies using normal human melanocytes. Figure 7. Mechanism underlying the inhibitory effect of the combination of THGP and kojic acid on melanin synthesis. Compared with the control condition, THGP administration inhibits the amount of extracellular melanin but slightly increases the amount of intracellular melanin. In addition, THGP does not affect tyrosinase activity or the expression of melanin production-related genes. Moreover, THGP shows a synergetic effect with kojic acid. "up arrow" means up-regulated, "down arrow" Figure 7. Mechanism underlying the inhibitory effect of the combination of THGP and kojic acid on melanin synthesis. Compared with the control condition, THGP administration inhibits the amount of extracellular melanin but slightly increases the amount of intracellular melanin. In addition, THGP does not affect tyrosinase activity or the expression of melanin production-related genes. Moreover, THGP shows a synergetic effect with kojic acid. "up arrow" means up-regulated, "down arrow" means down-regulated, "grey arrow" means reaction path about melanin syhthesis and "T arrow" means inhibitory effects.

Reagents
The THGP used in this study was an aqueous hydrolysis solution of poly-trans-[(2-carboxyethylgerma) sesquioxane] synthesized at the Hakodate manufacturing plant of Asai Germanium Research Institute Co., Ltd. (Kawasaki, Japan). TMGP was synthesized at Asai Germanium Research Institute Co., Ltd. THGP was dissolved in water to a concentration of 500 mM, and the pH was adjusted to 7.05. This stock was diluted 100-fold to the working concentration of 5 mM using media.

1 H-NMR
The 1 H-NMR spectrum of each sample was measured using a Mercury Plus 300 MHz instrument (Agilent Technologies Inc., Santa Clara, CA, USA). The concentration and pH of each sample were adjusted to 5 mM and 7.05, respectively, and the samples were mixed at a molar ratio of 1:1. All solvents were prepared using heavy water. The light water (HOD: 4.80 ppm) remaining in the heavy water solvent was used as the standard for the assessment of chemical shifts. The measurement temperature was 25 • C, and the number of integrations was 16. Deuterium oxide (Kanto Chemical Co., Inc., Tokyo, Japan) and 40% sodium deuteroxide (NaOD; D, 99.5%) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were used for the NMR measurements.

MTS Assay
The cells were seeded at 5 × 10 3 cells/well in a 96-well plate. After 24 h, 0 to 5000 µM THGP or 0 to 500 µM kojic acid was added. The cells were treated for 48 h, and an MTS assay was then performed using a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay kit (Promega Corp., Madison, WI, USA) according to the manufacturer's recommended protocol. The absorbance at 495 nm was measured, and the results are presented as percentages relative to the nontreated group (designated 100%).

Measurement of the Melanin Content in Cells
The cells were seeded at 1 × 10 5 cells/well on a 24-well plate, and after 24 h of incubation, THGP and/or kojic acid were added. In the combination tests, 0 to 500 µM THGP and 0 to 50 µM kojic acid were added. After 72 h of treatment, the supernatant was collected in 96-well plates, and the cells were dissolved by incubation at 60 • C for 20 min with 1 M NaOH (FUJIFILM Wako Pure Chemical Corporation). The absorbances of the cell lysate and supernatant were then measured at 405 nm. The results are presented as percentages relative to the nontreated group (designated 100%).

Measurement of Tyrosinase Activity
The cells were plated at a density of 1 × 10 6 cells/well in a 60 mm dish. After 24 h, 0 to 5000 µM THGP was added, and after 48 h of treatment, the proteins were extracted from the cells using 0.1% Triton-X (Nacalai Tesque, Inc., Kyoto, Japan) in PBS (−). The cell lysate was frozen at −80 • C for 30 min and then thawed on ice. After centrifugation at 20,000× g for 20 min, the supernatant was collected. A Bradford assay (Bio-Rad Laboratories Inc., Hercules, CA, USA) was performed to quantify the protein concentration. The extracted protein sample (8 µg), including the enzymatic solution and 5 mM L-DOPA, was mixed and measured after incubation at 37 • C for 3 h. The absorbance at 405 nm was measured, and the results are presented as percentages relative to the nontreated group (designated 100%).

Quantitative Polymerase Chain Reaction (PCR)
The cells were seeded at 2 × 10 5 cells/well in a six-well plate, and after 24 h of incubation, THGP (0-5000 µM) with or without 100 µM L-DOPA was added. After at least 24 h of treatment, total RNA was extracted using Isogen (Nippon Gene, Inc., Tokyo, Japan) according to the manufacturer's recommended protocol. The extracted RNA was reverse transcribed by SuperScript III (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) with 1 µg of template. The PCRs consisted of 40 cycles of 95 • C for 5 sec and 60 • C for 30 sec and were performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio, Inc., Otsu, Japan) and a LightCycler 96 (Roche Diagnostics GmbH, Mannheim, Germany). The primers used in the PCR analysis are shown in Table 1, and β-actin was used as an internal control. The cells were seeded at 1 × 10 5 cells/well in a 24-well plate. After 24 h, 1 µM α-MSH (Peptide Institute Inc., Osaka, Japan) was added, and after 24 h, 0 to 5000 µM THGP and 0 to 500 µM kojic acid were added. After 72 h of treatment, the extracellular melanin contents were measured using the above-described measurement method (Section 4.6). The results are presented as percentages relative to the α-MSH group (designated 100%).

Measurement of the Melanin Content in Cells Treated With L-DOPA
The cells were seeded at 1 × 10 5 cells/well in a 24-well plate. After 24 h, 0 to 5000 µM THGP, 0 to 500 µM kojic acid, and 250 µM L-DOPA were added, and after 72 h of treatment, the extracellular melanin contents were measured using the above-described measurement method (Section 4.6). The results are presented as percentages relative to the L-DOPA group (designated 100%).

Statistical Analysis
Dunnett's method was used for all evaluations.