To study the 7,8-DHF’s PK profiles, we have performed a panel of in vitro ADMET assays. We found that 7,8-DHF is stable in liver microsomal assay but labile in hepatocytes, indicating that 7,8-DHF might be readily subjected to secondary modification-conjugation. Caco-2 permeability assay, parallel artificial membrane permeability assay (PAMPA) and PAMPA-BBB assays demonstrate that 7,8-DHF possess reasonable absorption rate and is able to penetrate BBB [50]. MDR1-MDCKII permeability assay also indicates that 7,8-DHF is a weak P-glycoprotein (Pgp) substrate (unpublished data). hERG assay reveals that 7,8-DHF exhibits IC 50 >25 μM. CYP inhibition and induction assays reveal that 7,8-DHF has no time-dependent inhibition nor induction on major CYP enzymes. Concur with this result, 12 months chronic treatment or high dose acute treatment with 7,8-DHF suggests that 7,8-DHF is non-toxic to the rodents.

In vivo PK study shows that plasma 7,8-DHF concentration peaks at 10 min with 70 ng/ml, and brain 7,8-DHF also climaxes at 10 min with concentration of 50 ng/g of brain. 7,8-DHF in plasma can still be detected after 8 h (5 ng/ml) after administration. In contrast, only 7 ng/g of 7,8-DHF can be found in brain at 4 h and it is below the quantitative limit at 6 h [50]. In vivo metabolism study shows that 7,8-DHF is subjected to glucuronidation, sulfation and methylation [33]. Among these modifications, glucuronidation and sulfation are mainly responsible for the in vivo clearance of the flavonoids. Indeed, O-methylated metabolites including 7-methoxy-8-hydroxy-flavone (7M8H-flavone) and 7-hydroxy-8-methoxy-flavone (7H8M-flavone) can be detected in both the plasma and brain samples after oral administration of 7,8-DHF. Interestingly, these mono-methylated metabolites are still active in triggering TrkB activation in primary neurons and mouse brain [50].

Catechol containing compounds usually have short in vivo half-life and are prone to be cleared in the circulation system after oxidation, glucoronidation, sulfation or methylation. For instance, Apomorphine is a catechol-containing non-narcotic morphine derivative that acts as a potent dopaminergic agonist. Its metabolism occurs through several enzymatic pathways, including N-demethylation, sulfation, glucuronidation, and catechol-O-methyltransferase as well as by nonenzymatic oxidation [51]. L-DOPA is the mainstay of Parkinson’s disease (PD) therapy; this drug is usually administered orally, but it is extensively metabolized in the gastrointestinal tract, so that relatively little arrives in the bloodstream as intact L-DOPA. To minimize the conversion to dopamine outside the central nervous system, L-DOPA is usually given in combination with inhibitors of amino acid decarboxylase and COMT (catechol methyltransferase) [52]. Our preliminary in vivo PK study revealed that 7,8-DHF has t 1/2 more than 2 h in mouse circulation after oral administration [50]. Conceivably, glucuronidation, sulfation and methylation pathways may explain the relative short half-life of 7,8-DHF and its synthetic derivatives.

To improve the poor PK profiles intrinsic to catechol-containing molecules, we synthesized numerous prodrugs by modifying 7,8-dihydroxy groups with esters, carbamates or phosphates to improve the oral bioavailability and brain exposure of 7,8-DHF. Currently, an optimal prodrug R7 has been found with favorable in vitro ADMET (absorption, distribution, metabolism, excretion and toxicity) characteristics. R7 exhibits approximately 18 % oral bioavailability with C max of 1554.9 ng/ml, T max of 0.28 h and T 1/2 for PO of 2.32 h. Noticeably, 7,8-DHF plasma concentration released from R7 (PO, 50 mg/kg) is much higher than orally administrating the same dose of parent 7,8-DHF. The oral bioavailability is increased from 4.6 % (parental 7,8-DHF) to 84.2 % (R7). Accordingly, the brain exposure for 7,8-DHF is significantly increased by R7 than the parent compound upon oral administration of comparable dosage (unpublished data). TrkB and its downstream p-Akt/p-MAPK signalings are potently activated upon oral administration of R7, which is tightly correlating with 7,8-DHF concentrations in the animal brain. R7-provoked TrkB activation also fits well with the in vivo PK data, underscoring that the released 7,8-DHF from R7 prodrug triggers a long-lasting TrkB signalings in the mouse brain. This prodrug is now under preclinical IND-enabling study for the indication of Alzheimer’s disease.

7,8-DHF displays robust therapeutic efficacy toward Alzheimer’s disease

There is mounting evidence that 7,8-DHF mimics the physiological activities of BDNF and exhibits promising therapeutic efficacy toward various neurological diseases including Parkinson’s disease (PD) [26], Huntington’s disease (HD) [27], ALS (Amyotrophic lateral sclerosis) [53, 54], Alzheimer’s disease (AD) [55–58], Posttraumatic Stress Disorder (PTSD) [59], and Rett Syndrome [60]. Moreover, 7,8-DHF displays therapeutic effect toward axon regeneration [61], and spiral ganglion degeneration [48]. Noticeably, it also demonstrates therapeutic activities in mental diseases like depression [33, 56, 62, 63]. Here, we focus on discussing its effects in treating AD, the leading cause of dementia worldwide, which is characterized by the accumulation of the β-amyloid peptide (Aβ) within the brain along with deposition of hyperphosphorylated and cleaved microtubule-associated protein Tau. It is suggested that reductions of BDNF content or TrkB inactivation may play a role in the pathogenesis of AD. Indeed, BDNF expression is reduced in the brain of AD patients and delivery of BDNF gene has been shown as a novel potential therapeutic in diverse models related to AD [64]. BDNF also displays a protective role against AD pathogenesis by increasing learning and memory of demented animals [65]. Thus, these preclinical evidence strongly supports that BDNF might be useful as a therapeutic agent for treating AD.

Reduced acetylcholine neurotransmission due to loss of neurons in the basal forebrain and depletion of choline acetyltransferase are observed in AD pathology. Currently, there are two types of medication to treat AD: cholinesterase inhibitors and NMDA antagonist. However, these drugs can only delay the inevitable symptomatic progression of the disease without eliminating the main neuropathological hallmarks of the disease (i.e. formation of senile plaques and neurofibrillary tangles) nor rescuing the neuronal loss. 7,8-DHF potently stimulates hippocampal progenitor neurogenesis. For instance, oral administration of 7,8-DHF (5 mg/kg) in wild-type C57BL/6 J mice for a few weeks strongly induces neurogenesis [46]. Intraperitoneal administration of 7,8-DHF also elicits robust neurogenesis in depressive vulnerable or non-vulnerable rat [62]. This neurotrophic effect by 7,8-DHF has also been observed in APP/PS1 AD mouse model [66]. Devi and Ohno showed that 7,8-DHF rescued memory deficits in transgenic mice that co-express five familial Alzheimer’s disease mutations (5XFAD) during the spontaneous alternation Y-maze task. In addition, 7,8-DHF restores deficient TrkB signaling in 5XFAD mice without affecting endogenous BDNF levels. While 5XFAD mice exhibit elevations in the β-secretase enzyme (BACE1) that initiates amyloid-β (Aβ) generation, as observed in sporadic AD, 7,8-DHF blocks BACE1 elevations and lowers the levels of the β-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Aβ40, and Aβ42 in the brains of these mice. Most strikingly, they demonstrated that BACE1 expression can be decreased by 7,8-DHF administration in wild-type mice, suggesting that BDNF-TrkB signaling is also important for downregulating baseline levels of BACE1. Hence, this study supports that TrkB activation with systemic 7,8-DHF administration can ameliorate AD-associated memory deficits, attributable to reductions in BACE1 expression and β-amyloidogenesis [55]. Nevertheless, the authors employed a subchronic paradigm (10 days intraperitonial injection) in aged 5X FAD mice (12–15 months old mice). Since 5X FAD mice develop amyloid plaques at 2 months old and exhibit cognitive defects at 5 months of age, we employed a different treatment strategy: feeding the mice at 2-months-old till 5-months-old and monitored the cognitive activity in Morris Water maze. In addition, we examined the amyloid plaque deposit, synapse formation and long-term potentiation (LTP) at the end of the treatment. Our data showed that 7,8-DHF protects primary neurons from Aβ-induced cell death and promotes dendrite branching and synaptogenesis. Chronic oral administration of 7,8-DHF activates TrkB signaling and prevents Aβ deposition in 5XFAD mice [56]. In alignment with these findings, 7,8-DHF significantly increases spine density and reduces synaptic and neuronal loss in Cam/Tet-DTA, an inducible model of severe neuronal loss in hippocampus and cortex, and demonstrates substantial improvements in spatial memory in the lesioned mice [58]. These results strongly suggest that 7,8-DHF represents a novel oral bioactive therapeutic agent for treating AD.

7,8-DHF inhibits obesity through activating muscular TrkB

Obesity is a metabolic disorder with increasing prevalence worldwide. According to the World Health Organization (WHO), more than 39 % (~1.9 billion) of adults are overweight. Of these, over 600 million (~13 %) are obese in 2014. These numbers have been doubled since 1980. Therefore, developing effective pharmacotherapy to control excess body weight gain is a hot research direction nowadays.

In addition to the neurotrophic activities, BDNF/TrkB signaling also plays a critical role in food intake and body weight control. In rodents, pharmacological treatments with BDNF induce a reduction of food intake, whereas genetic models with reduced BDNF/TrkB signaling display hyperphagia and obesity [67, 68]. Recent evidence indicates that BDNF acts as an energy metabolism regulator in both CNS and peripheral organs. It has been reported that BDNF levels are low in obesity or patients with type 2 diabetes [68, 69]. BDNF is expressed in non-neurogenic tissues, including skeletal muscle, and exercise increases BDNF levels in brain, plasma and skeletal muscle. Pederson et al. reported that BDNF increased phosphorylation of AMP-activated protein kinase (AMPK) and acetyl coenzyme A carboxylase (ACC) and enhanced fatty oxidation both in vitro and ex vivo. These data points to the fact that BDNF is a contraction-inducible protein in skeletal muscle that is capable of enhancing lipid oxidation via activation of AMPK. Thus, BDNF appears to be an active player in both neurobiology and peripheral metabolism [70].

Because BDNF has anti-obesity activity by suppressing food intake, we thus initiated a test to see if 7,8-DHF can be used to prevent the development of obesity. We investigated the effect of 7,8-DHF (drinking the dissolved 7,8-DHF in water) on mouse body weight gain under chow diet or high-fat diet (HFD) feeding for 6 months [49]. To our surprise, 7,8-DHF consumption does not suppress food intake, which is in contrast to BDNF administration. Nevertheless, we found that 7,8-DHF significantly decreases the body weight gain in both chow diet and HFD paradigms, with more striking effect on HFD. The white adipocyte tissue (WAT) mass is significantly decreased about 20–30 % in 7,8-DHF-treated HFD group. Indirect calorimetry study showed that 7,8-DHF treatment decreases RER (respiratory exchange ratio), favoring the usage of lipid as the main fuel. Our study also reveals the mechanism of the anti-obesity actions by 7,8-DHF. By performing experiments in animal model and cell culture (C2C12) system, we identified that 7,8-DHF mainly acts on the muscle TrkB receptors to induce uncoupling protein 1 (UCP1) expression and activates AMP-activated protein kinase (AMPK). As a result, the energy expenditure and lipid oxidation in 7,8-DHF activated muscle cells are increased, leading to the lean phenotype observed. Unexpectedly, this anti-obesity effect is predominantly associated with female mice but not male mice, presumably due to estrogen content of the animals. Mice with 7,8-DHF treatment also exhibit improved blood insulin concentration, lower blood glucose level and increased insulin sensitivity in tissues such as liver, fat and muscles, suggesting 7,8-DHF is effective in alleviating the obesity-induced diabetes as well. These exciting findings identify a new function of BDNF/TrkB signaling in the skeletal muscle that the cascade controls cellular energy expenditure, which also provides the pre-clinical evidence that 7,8-DHF administration is an effective means to suppress body weight gain during energy surplus.