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, 115 (3), 578-583

The Prodrug of 7,8-dihydroxyflavone Development and Therapeutic Efficacy for Treating Alzheimer's Disease

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The Prodrug of 7,8-dihydroxyflavone Development and Therapeutic Efficacy for Treating Alzheimer's Disease

Chun Chen et al. Proc Natl Acad Sci U S A.

Abstract

The BDNF mimetic compound 7,8-dihydroxyflavone (7,8-DHF), a potent small molecular TrkB agonist, displays prominent therapeutic efficacy against Alzheimer's disease (AD). However, 7,8-DHF has only modest oral bioavailability and a moderate pharmacokinetic (PK) profile. To alleviate these preclinical obstacles, we used a prodrug strategy for elevating 7,8-DHF oral bioavailability and brain exposure, and found that the optimal prodrug R13 has favorable properties and dose-dependently reverses the cognitive defects in an AD mouse model. We synthesized a large number of 7,8-DHF derivatives via ester or carbamate group modification on the catechol ring in the parent compound. Using in vitro absorption, distribution, metabolism, and excretion assays, combined with in vivo PK studies, we identified a prodrug, R13, that prominently up-regulates 7,8-DHF PK profiles. Chronic oral administration of R13 activated TrkB signaling and prevented Aβ deposition in 5XFAD AD mice, inhibiting the pathological cleavage of APP and Tau by AEP. Moreover, R13 inhibited the loss of hippocampal synapses and ameliorated memory deficits in a dose-dependent manner. These results suggest that the prodrug R13 is an optimal therapeutic agent for treating AD.

Keywords: 7,8-dihydroxyflavone; Alzheimer’s disease; TrkB; pharmacokinetics; prodrug.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) R13: 7,8-DHF prodrug chemical structure. (B) In vitro absorption, distribution, metabolism, and excretion screening strategy for prodrugs.
Fig. 2.
Fig. 2.
7,8-DHF concentrations in brain and plasma after oral administration of R13. (A and B) Twenty-four male CD1 mice were given 78 mg/kg of R13 (equal to 7,8-DHF 50 mg/kg), which was dissolved in DMSO and resuspended in 5% DMSO/95% methylcellulose (0.5%, wt/vol). At indicated time points, three mice/group were killed, and serum and brain samples were collected. 7,8-DHF was quantitatively analyzed by LC-MS/MS. Even at 8 h after oral administration, >19 ng/mL of 7,8-DHF was detected in the plasma. 7,8-DHF >8 ng/g was detected in mouse brains at 2 h and persisted for >4 h. (C) Six 5XFAD mice were given 7.25 mg/kg, 21.8 mg/kg, and 72.5 mg/kg of R13, and another six 5XFAD mice were given 7,8-DHF at 5 mg/kg, 15 mg/kg, and 50 mg/kg. After 4 h, two mice/group were killed, and serum and brain samples were collected. 7,8-DHF was quantitatively analyzed by LC-MS/MS. (D) R13 hydrolysis route. T1 is the major intermediate released from R13 in pH 1.2–7.4 buffer, mimicking the pH change in the transition from stomach to intestine. (E) Nine mice were given R13 at doses of 21.8, 43.6, and 72.5 mg/kg. After 4 h of administration, three mice/group were killed, and serum and brains were collected. 7,8-DHF and T1 were quantitatively analyzed by LC-MS/MS.
Fig. 3.
Fig. 3.
R13 elicits TrkB and downstream signaling activation in 5XFAD mice. (A) R13 activates TrkB signaling cascade in the hippocampus of 5XFAD mice in a dose-dependent manner. R13 was dissolved in pure DMSO, then suspended in 0.5% methylcellulose at final concentration of 5% DMSO/0.5% methylcellulose. The suspension was orally administrated to 2 mo old 5XFAD mice (7.25, 21.8 or 43.6 mg/kg/d) consecutively for 3 mo, and the brain lysates were prepared. The p-TrkB and its downstream signals were monitored by immunoblotting, and the ratio of p-TrkB/TrkB, p-Akt/Akt and p-ERK/ERK were quantitatively analyzed. Data are shown as mean ± SEM. *P < 0.01. (B) IHC staining of p-TrkB in 5XFAD brain sections. Here 2-mo-old 5XFAD mice were fed with R13 or vehicle consecutively for 3 mo. The phosphorylation of TrkB in dentate gyrus was detected by IHC with anti–p-TrkB 816 antibodies. (Scale bar: 50 μm.) Quantification of p-TrkB+ neurons in the dentate gyrus. Note that R13 treatment elicited the phosphorylation of TrkB in 5XFAD mice. Data are shown as mean ± SEM. *P < 0.01.
Fig. 4.
Fig. 4.
R13 prevents the synaptic loss in hippocampal CA1 area of 5XFAD mice. (A) R13 reversed the synaptic loss in 5XFAD mice. The dendritic spines from apical dendritic layer of the CA1 region were analyzed by Golgi staining. (Scale bar: 5 μm.) (B) Quantitative analysis of spine density. The decreased spine density in 5XFAD mice was reversed by R13 in a dose-dependent manner. n = 6 in each group. *P < 0.01. (C) Quantitative analysis of the synaptic density in vehicle- and R13-treated 5XFAD mice. 5XFAD mice show decreased synaptic density, which was reversed by R13. Data are shown as mean ± SEM. *P < 0.01. (D) Representative EM image of the synaptic structures. Red stars indicate the synapses. (Scale bar: 1 μm.) (E) Immunoblotting analysis of synaptic markers in brain homogenates from mice treated with vehicle or R13. R13 treatment increased the expression of synaptic markers in 5XFAD mice. (F) LTP of field excitatory postsynaptic potentials (fEPSPs) was induced by 3XTBS (theta-burst stimulation) (four pulses at 100 Hz, repeated three times with 200-ms intervals). The traces shown are representative fEPSPs recorded at time points 1 (vehicle-treated 5XFAD mice), 2, 3, and 4 (R13-treated 5XFAD mice). The magnitude of LTP in 5XFAD mice is significantly lower in vehicle-treated transgenic mice, and R13 treatment reversed the LTP impairment. n = 5 in each group. Data are presented as mean ± SEM. *P < 0.05, vehicle-treated vs. R13-treated mice.
Fig. 5.
Fig. 5.
R13 alleviates Aβ deposition and reduces the concentrations of total Aβ, improving the spatial learning and memory of 5XFAD mice. (A) IHC analysis of Aβ deposits in 5XFAD mice. (Scale bar: 100 μm.) (B and C) Quantitative analysis of amyloid plaques. Amyloid deposition in 5XFAD mice was significantly decreased by orally administrated R13 at all doses. *P < 0.01. (D and E) Aβ42 and Aβ40 ELISA. R13 significantly reduced the concentrations of Aβ40, but not of Aβ42, in mouse brain at all doses. (FH) R13 improves cognitive functions in 5XFAD mice. 5XFAD mice (n = 8–10/group) orally administered control vehicle or different doses of R13 were trained in the water maze over 5 d. Shown are mean ± SEM latency to mount the escape platform (F), the AUC of latency (G), and the percentage of time spent in the target quadrant (H). *P < 0.05 compared with vehicle-treated 5XFAD mice.
Fig. 6.
Fig. 6.
R13 alleviates inflammation, inhibits AEP activation, and reduces the concentrations of AEP-derived APP fragments and Tau fragments. (A) Western blot showing the processing of APP and Tau by AEP. R13 significantly inhibited AEP activation by the reduction of AEP cleavage formation, which attenuated Tau and APP cleavage. (B) R13 repressed AEP expression in 5XFAD mice. IHC analysis showing the presence of AEP-positive cells in hippocampus and cortex of both vehicle-treated and R13-treated mice. (Scale bar: 50 μm.) (C and D) Quantitative analysis of AEP-positive cells. AEP expression in 5XFAD mice was significantly decreased by orally administered R13 at all doses. *P < 0.01. (E) AEP enzymatic activity analysis. Data are presented as mean ± SEM; n = 5, one-way ANOVA. *P < 0.01 compared with vehicle-treated mouse brains. (F) Proinflammatory cytokines, such as IL-1β, IL-6, and TNFα, were determined by ELISA. Chronic R13 treatment significantly decreased the IL-1β, IL-6, and TNFα production in mouse brains compared with age-related vehicle-treated mice (n = 5).

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