Compound AD110 Acts as Therapeutic Management for Alzheimer's Disease and Stroke in Mouse and Rat Models

Article abstract of DOI:10.1021/acschemneuro.9b00651

Anti-inflammatory therapy may be an effective therapeutic intervention for neurological diseases, such as Alzheimer's disease (AD) and stroke. As an important anti-inflammatory cytokine, interleukin-10 (IL-10) inhibits proinflammatory responses of both innate and adaptive immune cells. We tested the hypothesis that drug-induced promotion of IL-10 expression is effective in improving cognitive abilities and neurologic outcomes of AD and stroke. An orally small molecule AD110 was synthesized and subjected to in vitro and in vivo analyses. We found that AD110 enhanced IL-10 release in lipopolysaccharide (LPS)-activated BV2 microglial cells. Y-Maze and Morris water maze tests showed improved cognitive abilities in AD mice treated with AD110. Moreover, AD110 attenuated cerebral ischemic injury in a transient middle cerebral artery occlusion (tMCAO) rat model. This study not only provides a promising lead compound with IL-10-promoting activity, but also supports the hypothesis that promoting IL-10 expression is a potential therapeutic strategy for AD and stroke.

Full text of DOI:10.1021/acschemneuro.9b00651

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Article
Compound AD110 acts as therapeutic management for
Alzheimer’s disease and stroke in mouse and rat models
Ping Sun, Wei Zhou, Hu Yue, cheng zhang, yitao ou, Zhongjin Yang, and Wenhui Hu
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00651 • Publication Date (Web): 27 Feb 2020
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Compound AD110 acts as therapeutic management for Alzheimer’s disease
and stroke in mouse and rat models
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Ping Sun†,#, Wei Zhou‡,#, Hu Yue†, Cheng Zhang†, Yitao Ou†, Zhongjin Yang†,*, Wenhui Hu†,*
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†
Key Laboratory of Molecular Target & Clinical Pharmacology and State Key Laboratory of
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Respiratory Disease, School of Pharmaceutical Sciences & the Fifth Affiliated Hospital,
Guangzhou Medical University, Guangzhou, Guangdong, 511436, China
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School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology,
Guangzhou, Guangdong, 510006, China
# These authors contributed equally to this work
* Corresponding Author
Zhongjin Yang: yzj23@163.com
Wenhui Hu: huwenhui@gzhmu.edu.cn
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ABSTRACT:
Anti-inflammatory therapy may be an effective therapeutic intervention for
neurological diseases, such as Alzheimer’s disease (AD) and stroke. As an
important anti-inflammatory cytokine, Interleukin-10 (IL-10) inhibits
proinflammatory responses of both innate and adaptive immune cells. We
tested the hypothesis that drug-induced promotion of IL-10 expression is
effective in improving cognitive abilities and neurologic outcomes of AD and
stroke. An orally small molecule AD110 was synthesized and subjected to in
vitro and in vivo analyses. We found that AD110 enhanced IL-10 release in
lipopolysaccharide (LPS)-activated BV2 microglial cells. Y maze and Morris
water maze tests showed improved cognitive abilities in AD mice treated with
AD110. Moreover, AD110 attenuated cerebral ischemic injury in a transient
middle cerebral artery occlusion (tMCAO) rat model. This study not only
provides a promising lead compound with IL-10-promoting activity, but also
supports the hypothesis that promoting IL-10 expression is a potential
therapeutic strategy for AD and stroke.
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Keywords: Alzheimer’s disease, anti-inflammatory cytokine, AD110,
interleukin-10, Neuroinflammation, stroke
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INTRODUCTION
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Uncontrolled neuroinflammation, a hallmark of neurological disorders such as
Alzheimer’s disease (AD) and stroke, may lead to neural dysfunction and
aggravate disease progression1. The primary cells involved in the inflammatory
response of the brain are microglial cells, which are resident macrophage-like
cells of the central nervous system (CNS) that release various cytokines in
response to an external stimulus2. Microglia can be classified into two broad
subpopulations: M1 and M2 phenotypes3. The M1 phenotype is associated
with proinflammatory cytokines; when activated, M1 phenotype microglia
produce a wide range of proinflammatory cytokines and neurotoxic mediators,
including interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNFα), and nitric
oxide (NO)4, 5. These accumulated neurotoxic substances act directly on
neurons to cause cell death6, 7. Conversely, the M2 phenotype is associated
with anti-inflammatory responses; activated M2 phenotype microglia release
anti-inflammatory cytokines and mediators that promote tissue repair8-11, such
as IL-4, IL-10, IL-13, IL-33, IGF-1, and transforming growth factor-β (TGF-β)3.
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Preclinical data indicate that anti-inflammatory therapy may be an effective
therapeutic intervention for AD and stroke12-14, although effective therapies
are still being developed. One such strategy is to modulate immune cell
function by inhibiting proinflammatory cytokine release and enhancing
anti-inflammatory cytokine expression12. IL-10 is an important
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immunoregulatory cytokine that inhibits proinflammatory responses of both
innate and adaptive immune cells15. Pre-treatment with IL-10 decreases
nuclear translocation of the p50 and p65 subunits of NF-κB and production of
IL-6, TNF-α, IL-12, and NO16, 17. Furthermore, IL-10 exerts neuroprotective
effects, recovers neurite outgrowth, and promotes neuronal synapse
formation18, 19. Lower IL-10 level is associated with increased stroke risk20, and
postischemic gene transfer of IL-10 into the lateral ventricle attenuates brain
infarction and hippocampal damages in a rat model21. Combarros et al. found
that IL-10-1082A/G (rs1800896) polymorphism contributed to chronic
inflammation in elderly people and increased the risk of AD22. In addition, IL-10
significantly reduces astro- and microgliosis, enhances neurogenesis, and
improves spatial cognitive dysfunction in an APP/PS1 AD mouse model23.
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In this study, we evaluated whether the use of anti-neuroinflammatory
compounds to upregulate IL-10 expression was a potential strategy for
improving cognitive abilities and neurologic outcomes of AD and stroke. We
investigated
the
anti-neuroinflammatory
effects
of
AD110,
a
quinazoline-4-carboxamide scaffold (Figure 1), by enhancing IL-10 release in
lipopolysaccharide (LPS)-stimulated BV2 microglial cells. In vivo, we assessed
the effect of AD110 treatment on spatial learning and memory in AD mice via Y
maze test (YMT) and Morris water maze test (MWMT). Besides, we used a
transient middle cerebral artery occlusion (tMCAO) rat model to study the
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effect of AD110 on cerebral ischemic injury. The pharmacokinetic properties of
AD110 were also studied.
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RESULTS
Design and Synthesis of AD110
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Neuroinflammation is a prominent feature of the AD brain and plays a
continuous role in modulating AD progression. Minozac24, a pyridazine scaffold,
presented potential anti-neuroinflammatory effect upon in vitro and in vivo
evaluation. Based on Minozac, we designed two small molecules GIBH-130 and
AD110 (Figure 1) with a novel scaffold, using scaffold-hopping, bioisosterism
and fragment-based drug design strategy25. GIBH-130 was reported to have
good inhibition activities against pro-inflammatory cytokines25. Herein, we
investigated whether AD110 could achieve the anti-neuroinflammatory effects
in LPS-stimulated BV2 microglial cells by enhancing anti-inflammatory cytokine
IL-10.
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The synthetic scheme for the production of AD110 is shown in Figure 2. A
cyano group was introduced into the position 4 of compound 1
(4-chloro-2-phenylquinazoline) by nucleophilic substitution reaction with
potassium cyanide (KCN) at 80°C. The acid 3 (2-phenylquinazoline-4-carboxylic
acid) was obtained by hydrolysis of 2 with lithium hydroxide in methanol and
H2O. Treatment of the acid 3 with oxalyl chloride, followed by condensation
with 1-(4-pyridyl)piperazine ，resulted in the synthesis of the desired amide
AD11026.
AD110 enhances IL-10 release and CD206 expression in BV2 cells
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We detected cytokine release in the supernatant and lysate of LPS-treated BV2
cells. As shown in Figure 3A, AD110 strongly enhanced IL-10 levels in the
supernatant (ANOVA: p < 0.001; post hoc analysis: 1 nM: 74.4%, p < 0.05; 10
nM: 135.0%, p < 0.01; 100 nM: 128.4%, p < 0.01; and 1 μM: 149.1%, p < 0.01) ,
while GIBH130 significantly improved IL-10 expression only at 10 μM (ANOVA:
p < 0.001; 10 μM: 133.9%, p < 0.01). Concentrations of proinflammatory
cytokines TNFα, IL-6, and KC in cell supernatant and IL-1β in lysate, however
were not significantly affected by AD110 treatment (p > 0.05; Figure 3B-E).
IFN-γ and IL-12p70 were detected below or close to background levels in our
study (data not shown). The immunohistochemistry study suggested AD110
enhanced CD206 expression, a marker for the M2 phenotype of microglia,
as shown in Figure 4. The findings indicate that AD110 provided
considerable anti-neuroinflammatory capacity in LPS-stimulated BV-2
cells.
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Locomotor activity and anxiety in AD mice are unaffected by AD110
treatment
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Open field test to evaluate the locomotor activity after AD110
administration showed that treatment with either AD110 or donepezil
had no effect on spontaneous locomotor activity (p > 0.05; Figure 5B).
And the total distance traveled did not significantly differ between the
Aβ1-42-injected mice and the sham-operated group (Figure 5B). The
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findings suggest that AD110 did not influence the locomotor activity and
anxiety of AD mice.
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Spatial learning and memory in AD mice are improved by AD110
treatment (Y maze test)
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Using YMT, we found that both AD110 and donepezil administration
significantly improved the behavioral performance of the Aβ1-42-injected
mice compared to that of the vehicle-administered mice (donepezil, p <
0.05; AD110, p < 0.05; Figure 6A), as indicated by increased percent
alternation. We did not detect a significant difference in the total number
of arm entries (Figure 6B), excluding hyperactivity as a possible
confounding factor for cognitive improvement27, 28. It demonstrates that
AD110 have a positive effect comparable to the vehicle-administered
mice.
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Spatial learning and memory in AD mice are improved by AD110
treatment (Morris water maze test)
In MWMT, on Days 2 and 3 of training, escape latency (Day 2, p < 0.01;
Day 3, p < 0.05, Figure 7A) and swimming distance (Day 2, p < 0.01; Day 3,
p < 0.01, Figure 7B) of Aβ1-42-injected mice were significantly longer than
that of the sham-operated mice, but daily oral administration of
donepezil (Day 2, p < 0.05 for both escape latency and swimming distance;
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Figure 7A,B) or AD110 (Day 2, p < 0.01 and Day 3, p < 0.05 for escape
latency; Day 2 and Day 3, p < 0.01 for swimming distance; Figure 7A,B)
reversed this impairment. In the MWMT probe test, the Aβ1-42-injected
mice showed a reduced percentage of distance and time spent in the
target quadrant as well as fewer platform crossings compared to the
sham-operated group (Figure 7C-E). The mice treated with AD110 showed
significantly better performance in the percentage of path length in the
target quadrant (donepezil, p < 0.01; AD110, p < 0.05; Figure 7C), time
spent in the target quadrant (donepezil, p < 0.01; AD110, p < 0.05; Figure
7D), as well as the number of crossings through the platform region
(donepezil, no difference; AD110, p < 0.01; Figure 7E). Taken together,
the findings suggest that AD110 prevented Aβ-induced cognitive
impairments as assessed by MWMT.
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AD110 attenuates cerebral ischemic injury in rats
The study results showed that both Edaravone and AD110 significantly
ameliorated cerebral ischemic injury in rats (p < 0.05; Figure 8; Figure S5).
Compared to the control group, which had an infarct volume of 40 ± 9%,
rats treated with AD110 and Edaravone had reduced infarct volume by
37.5% and 42.5%, respectively (Figure 8A). The functional assessment
revealed a significant improvement in Bederson score in AD110-treated
rats (1.87 ± 0.8) and Edaravone-treated rats (1.93 ± 0.82) compared to
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vehicle-treated rats (2.53±0.52) (Figure 8B). Therefore, AD110 exerted
considerable neuroprotective effects against cerebral ischemia injury.
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In vivo rat pharmacokinetics
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The pharmacokinetic parameters of AD110 determined after po (25
mg/kg) and iv (5 mg/kg) administration in rats was summarized in Table 1.
Mean maximal concentrations of 1.207 mg/L (po) and 1.343 mg/L (iv)
were achieved. Good bioavailability (F = 75.5%, po) and moderate
biological half-life (t1/2 = 4.68 h, po; t1/2 = 4.35 h, iv) of AD110 provided
adequate concentration and durability in the systemic circulation. The
results show AD110 possessed good pharmacokinetic profiles.
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DISCUSSION
The key findings of this study are that the novel small molecule AD110
enhances anti-inflammatory cytokine IL-10 expression, improves spatial
memory deficits in Aβ-injected AD mice model and attenuates cerebral
ischemic injury in tMCAO stroke rat model. The findings are consistent
with the concept of microglial activation as a therapeutic strategy in both
AD and stroke models25, 29-33
.
AD110 dramatically increases IL-10 expression, even at nanomolar
concentrations. IL-10 is one of the classic cytokines released by M2
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phenotype of activated microglia34, 35. Several compounds have been
identified to regulate microglial M1 and M2 polarization. Other
substances that exhibit neuroprotective properties such as resveratrol,
withaferin A, folic acid, and paeoniflorin also increase IL-10 expression,
suggesting a possible shift in microglial phenotypes from M1 to M2, i.e.,
pro-inflammatory to anti-inflammatory state36-39. Though most of these
compounds downregulate proinflammatory M1 cytokines, however we
found that AD110 did not affect the expression of IL-1β, IL-6, TNFα, or KC.
Our data imply that AD110 may specifically promote M2 polarization of
microglia.
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We found that treatment with AD110 induced positive effects in the
treatment of stroke and AD animals. These effects may be due to
anti-neuroinflammatory properties of AD110 and upregulation of IL-10
expression. IL-10 controls inflammation by regulating inflammatory
pathways and reducing cytokine receptor expression and activation40. In
vitro and in vivo models of ischemic stroke have convincingly
demonstrated IL-10-mediated neuroprotection directly and indirectly41.
IL-10-deficient (IL-10−/−) C57BL/6 mice exhibit 30% larger brain infarct
volumes than wild type (WT) mice 24ꢀh post stroke42. In this model,
IL-10−/− mice and WT mice respond similarly within the first 6ꢀh after
ischemia, but IL-10−/− mice are found to have greater neuroinflammation
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on day 443. Overexpression of IL-10 led to a 40% reduction in brain infarct
volumes 4ꢀdays after permanent MCAO44. In our study, treatment of
AD110 could effectively reduce infarct volume by 37.5%.
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The exact role of inflammation in the pathology of AD remains unclear.
Although IL-10 is undoubtedly an important anti-inflammatory cytokine,
the specific effects are not well understood. AD patients have been found
to have increased IL-1β and IL-10 levels, whereas IL-1β production is 7-10
times greater than IL-10 production45, 46. This suggests that a probable
imbalance in proinflammatory and anti-inflammatory activities exists in
AD. Elevated IL-10 expression may play a potential role in a compensation
mechanism. Preclinical studies have shown that upregulation of IL-10
significantly improved spatial cognitive dysfunction in APP/PS1 mice23,
and IL-10−/− mice developed cognitive deficits more frequently than WT
mice during peripheral infection47. However, Chakrabarty et al. found that
IL-10 overexpression weakened the microglial phagocytosis of soluble Aβ and
exacerbated Aβ-induced cognitive impairment in TgCRND8 mice and Tg2576
mouse model48. They promoted IL-10 expression before the formation of
typical AD pathology48. In our study, AD110 was administrated after Aβ
injection, so their study results cannot be compared directly with our results. It
is also possible the source of the discrepant results could be attributed to the
use of different kind of AD models.
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No inflammatory factors were detected during or after the animal tests, which
should be considered as a limitation of this study. In this study, we were not
able to verify that whether the in vivo effectiveness of AD110 is dependent on
its anti-neuroinflammatory capacity. Mechanism research of AD110 will be
performed in the future, including pro-inflammatory/anti-inflammatory
cytokines detection in the brain of animal models.
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CONCLUSIONS
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Overall, AD110 may reduce neuroinflammation through upregulation of
IL-10. Our results suggest that treatment with AD110 improved cognition
in the AD animal model and attenuated cerebral ischemic injury in the
stroke rat model. This study not only indicates that promoting IL-10
expression is a potential therapeutic strategy for AD and stroke but also
provides a promising lead compound, which has efficacy comparable to
that of donepezil and edaravone, for further discovery and development
of oral drugs for AD and stroke.
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METHODS
Synthesis of AD110
Details corresponding to each of the above steps in the synthetic scheme
are described below. Unless otherwise mentioned, all reactions were carried
out under anhydrous conditions in a nitrogen atmosphere with dry solvents.
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Reactions were monitored by thin-layer chromatography carried out on 0.25
mm Tsingdao silica gel plates (60F-254). Visualization was achieved using UV
light. Tsingdao silica gel (200 – 300 mesh) was used for flash column
chromatography. Reagents of the highest commercial quality were purchased
and used without further purification. Nuclear magnetic resonance (NMR)
spectra were recorded with a spectrometer (1H, 400 MHz; 13C, 100 MHz) and
referenced to the peak for solvents—CDCl3 or DMSO-d6. Data were reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m=
multiplet), coupling constants, and integration.
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Compound 3: 2-phenylquinazoline-4-carboxylic acid
A solution of compound 1 (10.0 g, 41.5 mmol), potassium cyanide (3.01 g,
46.1 mmol) and sodium p-toluenesulfonate (4.84 g, 24.9 mmol) in DMF (100
mL) was heated at 80°C for 6 h. The reaction mixture was cooled at room
temperature, followed by addition of water (400 mL) and extraction with ethyl
ether (3 × 150 mL). The combined organic layers were washed with water (200
mL) and saturated brine (200 mL) and concentrated to give an oily crude
product 2 (10.6 g).
A solution of LiOH (3.83 g, 91.3 mmol) in H2O (40 mL) and MeOH (40 mL)
was added to the above crude product 2. The mixture was stirred for 2 h at
room temperature. Ethyl acetate (100 mL × 2) was added to the mixture to
extract impurities. The water phase was subsequently adjusted to pH 3 with
hydrochloric acid (1.0 M). The precipitating white solid was filtered, washed
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with water (50 mL × 2), and dried under vacuum at 40°C for 24 h to obtain the
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product 3, 2-phenylquinazoline-4-carboxylic acid (6.8 g, 65.5% yield). H NMR
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(400 MHz, DMSO-d6) δ 8.57 – 8.49 (m, 2H), 8.36 (d, J = 8.4 Hz, 1H), 8.11 (d, J =
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8.0 Hz, 1H), 8.04 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.75 (ddd, J = 8.2, 6.8, 1.3 Hz,
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1H), 7.58–7.51 (m, 3H); C NMR (100 MHz, DMSO-d6) δ 166.9, 160.1, 159.6,
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151.9, 137.3, 135.8, 131.7, 129.4, 129.3 (C-3ʹ,5ʹ on phenyl), 129.2, 128.7
(C-3ʹ,5ʹ on phenyl), 126.5, 119.8; HRMS (ESI) calcd for C15H11N2O2 (M+H)+
251.0815, found 251.0814.
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AD110: (2-phenylquinazolin-4-yl)(4-(pyridin-4-yl)piperazin-1-yl)methanone
Over a period of 30 min, oxalyl chloride (3.5 mL, 41.3 mmol) was slowly
dropped into a solution of 3 (6.8 g, 27.2 mmol) in anhydrous dichloromethane
(150 mL) and N, N-dimethylformamide (0.05 mL). The mixture was stirred for
12 h at room temperature. The solvent and excess oxalyl chloride were
removed under vacuum at 35 °C to give the acid chloride 4.
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A solution of 1-(pyridin-4-yl)piperazine (5.3 g, 32.6 mmol) and
triethylamine (3.8 mL, 27.2 mmol) in anhydrous dichloromethane (150 mL) was
slowiy added into a solution of acid chloride 4 in anhydrous dichloromethane
(150 mL) at 0°C . The resulting mixture was stirred in an ice-water bath for 3 h,
followed by addition of water (100 mL), and was subsequently extracted with
dichloromethane (3 × 150 mL). The combined organic layers were washed with
saturated brine.Afterwards, thet were dried over Na2SO4 and concentrated to
give an oily crude product, which was purified on a silica gel column to obtain
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the desired AD110 (8.1 g, 75.3% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 8.64 – 8.51 (m, 2H), 8.29 (m, 2H), 8.12 (d, J = 8.5 Hz, 1H), 8.05 – 7.98
(m, 1H), 7.92 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.60 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H),
7.54 – 7.46 (m, 3H), 6.65 (m, 2H), 4.09 (m, 2H), 3.62 – 3.54 (m, 2H), 3.50 (m,
3
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2H), 3.34 (m, 2H); C NMR (100 MHz, CDCl3) δ 165.1, 162.3, 160.0, 154.6,
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152.0, 150.5 (C-2ʺ, 6ʺ on pyridyl), 137.4, 134.8, 131.1, 129.4, 128.8 (C-3ʹ,5ʹ on
phenyl), 128.7 (C-2ʹ, 6ʹ on phenyl), 128.2, 125.6, 120.4, 108.8 (C-3ʺ,5ʺ on
pyridyl), 46.7, 46.2, 46.1, 41.4; HRMS (ESI) calcd for C24H22N5O (M+H)+
396.1819, found 396.1817.
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In vitro study and cytokine detection
BV2 cells were cultured in Dulbecco's modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA,
USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 μg/mL
streptomycin (Invitrogen, Carlsbad, CA, USA). The serum-containing medium
was removed, and the cells were treated either with or without AD110 at four
concentrations for 24 h in parallel with 0.1 µg/mL LPS (Sigma, MO, USA) in
serum-free Opti-MEM medium (Invitrogen, Carlsbad, CA, USA). Appropriate
controls, including a vehicle control and LPS only stimulated cells, were
included in the analysis. Cell supernatants or lysates were collected and
subjected to Mesoscale Discovery Mouse Proinflammatory 7-Plex
immunosorbent assay (IL-1β, IL-10, IL-6, keratinocyte-derived cytokine [KC],
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IFN-γ, IL-12p70, and TNFα).
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Immunohistochemistry
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For BV2 cells staining, the cells were fixed with 4% PFA (dissolved in PBS) at
room temperature for 20 min and then blocked for another 30 min at room
temperature first. Then, cells were incubated with goat anti-CD206 (Abcam,
MA, USA; 1:200) overnight at 4 °C before they were were incubated with
donkey anti-goat IgGs conjugated with Alexa 568 (Life science, Carlsbad, CA,
USA; 1:800) together with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Images
were taken using the Leica DM4000 microscope (Leica, Wetzlar, Germany). The
mean fluorescence intensity of the CD206 signal (n = 30 per group) was
measured and analyzed using ImageJ software.
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AD mouse treatment
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All the animal studies complied with the ARRIVE guidelines, and all
experiments were approved by Animal Research Committee of Guangzhou
Medical University. The male mice (n = 8-10 per group) were anesthetized
using 350 mg per kg body weight of chloral hydrate intraperitoneal injection
and then restrained onto a stereotaxic apparatus (Narishige scientific
instrument lab, Japan). The bregma coordinates used for injection were: −1.0
mm lateral, −0.3 mm posterior, and −2.5 mm below. Each mouse received a
single intracerebroventricular injection of either 3 μL of aggregated Aβ1-42
solution (410 pmol, Sigma-Aldrich, St. Louis, MO, USA) or 3 μL of physiological
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saline (sham-operated group). The saline−diluted Aβ1-42 was incubated for
aggregation at 37°C for 120 h before injection. Aβ1-42-injected mice received
oral AD110 (2.5 mg/kg, diluted in CMC-Na solution), donepezil hydrochloride
(0.65 mg/kg), or CMC-Na solution (control). The sham-operated group received
an oral administration of CMC-Na solution. The behavioral tests began at Day 7
after the Aβ1-42 injection and were performed sequentially (Figure 5a).
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Open field test
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The open field test was conducted in a sound-attenuated room. After each trial,
the box was wiped with 96% ethanol to remove odor cues. A camera
connected to the computer was used to record animal activities. On the first
day of training, each mouse was submitted to a habituation session by placing
it in the empty open field for 5 min. There were three habituation training
sessions for each mouse with a 4 h interval. 24 h after the last training sessions,
each mouse was submitted to a 5-min trial. The circle had a diameter of 15 cm
diameter, half of which was considered as middle zone. The locomotor activity
of the mice was recorded as total distance covered within 5 min.
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Y maze test
YMT was conducted on Day 8 after Aβ1-42 injection. Each mouse was placed at
the end of the start arm and allowed to move freely through the maze without
reinforcers during an 8 min session. The sequence of arm entries was manually
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recorded. A mouse was considered to have entered an arm when all four paws
were positioned in the arm runway. The percent of alternation and the total
number of arm entries were measured. An alternation was defined as entries
into all three arms on consecutive trials. The percent of alternation was
defined as the [total number of alternations / (total number of arm entries - 2)]
× 100%.
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Morris water maze test
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MWMT was performed according to previous reports29. Hidden platform
training was carried out from Day 9 to Day 12 after Aβ1-42 injection with three
spatial training sessions per day. 24 h after the last training session, a probe
test was conducted on Day 13 in the same spatial training pool with the
platform removed. All mice started the probe test from the opposite quadrant
of the target quadrant. The dependent variables were percentage of path
length or swimming time in target quadrant and the crossing time.
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Transient middle cerebral artery occlusion
Procedures for transient middle cerebral artery occlusion (tMCAO) were
performed as previously described with minor alterations49. Adult male
Sprague-Dawley rats (n = 15 per group) were anesthetized with 5% isoflurane
in oxygen/nitrogen (N2O/O2 [30/70]) and a mixture of 2% isoflurane and
N2O/O2 (30/70) during the course of surgery. The left common carotid artery,
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internal carotid artery, and external carotid artery were exposed surgically. A
monofilament nylon suture (Beijing Sunbio Biotech Co Ltd) with a rounded tip
was introduced into the internal carotid artery through the external carotid
artery stump and gently advanced to occlude the middle cerebral artery (MCA).
After 90 min of MCAO, animals were reperfused before euthanization for an
additional 24 h by withdrawing the suture. Rats were treated with Edaravone
(6 mg/kg) or AD110 (2.5 mg/kg) at the time of beginning of reperfusion by
cauda vein. After the animals regained consciousness, neurological deficiencies
were evaluated by Bederson’s method by two blinded independent observers.
Neurological findings were scored on a four-point scale, with a score of “0” for
no neurological deficits, a score of “1” for any amount of consistent forelimb
flexion and no other abnormality, a score of “2” for consistently reduced
resistance to a lateral push toward the paretic side, and a score of “3” for
consistently circling toward the paretic side. After euthanasia, the brains from
all study animals were removed and dissected. Five pieces of 2-mm-thick serial
coronal sections were obtained from each brain. Brain sections were then
stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) at 37°C for 10
min and then fixed in 10% formalin at room temperature overnight. White
areas indicated infarcted regions and pink areas indicated normal undamaged
regions. After fixation, these brain sections were scanned using a flat-bed
scanner. Each side of the 5 sections was scanned and analyzed, to obtain a
total of 10 images per brain. From each section, only the damaged side of the
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hemispheres was analyzed. The images were then quantified with ImageJ
software (NIH, Bethesda, MD, USA).
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Pharmacokinetic analysis
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Pharmacokinetic analysis was conducted on male Sprague-Dawley rats (n = 4
per group; body weight of 180-220 g). After an acclimatization period of 1
week, AD110 was administered at a dose of 25 mg/kg (po) and 5 mg/kg (iv).
Blood samples were obtained at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h
and stored at −20°C until processed for quantification. For bioanalysis, high
performance liquid chromatography-mass spectrometry (HPLC-MS) method
was used to determine the concentration of AD110 in the blood samples, using
propranolol hydrochloride as an internal standard.
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Statistical analyses
Data are expressed as mean ± standard deviation (SD) or standard error of the
mean (SEM). We carried out statistical analyses using non-parametric tests
such as the Kruskal-Wallis test or one-way ANOVA (SPSS 16.0 software). Post
hoc group-wise comparisons were conducted using Dunn’s multiple
comparison test. p-values of < 0.05 were considered significant.
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Corresponding Author
Email:
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Zhongjin Yang: yzj23@163.com
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Wenhui Hu: huwenhui@gzhmu.edu.cn
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Author Contributions
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WH designed the research studies. PS, WZ, HY, CZ, YO, ZY performed the
experiments. PS, ZY drafted the manuscript with the assistance of the other
authors. All authors have given approval to the final version of the manuscript.
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Funding
This work is supported by Natural Science Foundation of Guangdong Province,
China (Project No. 2017A030310171), National Natural Science Foundation
(Project No. 81872743; Project No. 81803364) and Start-up grant from
Guangzhou Medical University.
Notes
The authors declare that they have no competing interests.
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Abbreviations
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Aβ: β-amyloid; AD: Alzheimer’s disease; CNS: Central Nervous System; icv:
Intracerebroventricular; IL-10: interleukin 10; iv: intravenous; KC:
Keratinocyte-derived cytokine; LPS: lipopolysaccharide; MWMT: Morris water
maze test; NO: nitric oxide; PDEs: phosphodiesterases; PK: pharmacokinetic;
po: per oral; tMCAO: middle cerebral artery occlusion；TNF-α: tumor necrosis
factor alpha; TTC: 2,3,5-triphenyltetrazolium chloride; WT: Wild type; YMT: Y
maze test
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Supporting Information
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Figure S1 1H NMR of compound 3.
Figure S2 13C NMR of compound 3.
Figure S3 1H NMR of AD110.
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Figure S4 13C NMR of AD110.
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Figure S5 AD110 attenuated cerebral ischemic injury in rats. Representative images of infarct volume
measurements in vehicle, Edaravone and AD110 treated rats subjected to transient middle cerebral
artery occlusion (tMCAO) for 90 min followed by 24 h reperfusion.
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Table 1. Key Pharmacokinetic Parameters of AD110 after a Single Dose in
Rats
compd route Dose
Cmax
Tmax
T1/2
[h]
Vz/F
CLz/F
F[%]
AUC0-∞[μg· h/L
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[mg/kg] [mg/L] [h]
[L/kg]
[L/h/kg]
]
AD110 iv
(n=4)
5
1.343
1.207
4.35
10.218 1.698
3038.261
po
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3.5
4.68
14.68
2.218
11470.016
75
(n=4)
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Data are mean values. Abbreviations: iv, intravenous; po, per oral; Cmax,
peak plasma concentration of a drug after administration; Tmax, time to
reach Cmax; T1/2, elimination half-life; Vz/F, apparent volume of
distribution; CLz/F, total plasma clearance; AUC, area under the
concentration−time curve; F, bioavailability.
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Figure legends
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Figure 1. The structures of AD110 and GIBH-130.
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Figure 2. Synthetic scheme for synthesis of AD110.
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Reactions, conditions and yields: a) KCN, sodium p-toluenesulfonate, DMF,
80°C, 6 h; b) LiOH, H2O, r.t, 2 h; c) oxalyl chloride, DMF, CH2Cl2, 12 h; d)
1-(4-Pyridyl)piperazine, CH2Cl2, TEA, 0°C.
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Figure 3. AD110 enhanced anti-inflammatory cytokine IL-10 release in
BV2 cells. BV2 cells were treated with or without AD110 at four
concentrations for 24 h in serum-free Opti-MEM medium in parallel with 0.1
µg/mL LPS. AD110 treatment strongly enhanced anti-inflammatory cytokine
IL-10 levels in the supernatant (SN) in a concentration-dependent manner
(A). Pro-inflammatory cytokine TNFα (B), IL-6 (C), and KC (D) in SN and
IL-1β (E) in cell lysate were not significantly affected by AD110 treatment.
Data are presented as means ± SD. *p < 0.05, **p < 0.01.
Figure 4. AD110 enhanced CD206 expression in BV2 cells. BV2 cells were
treated with or without AD110 at 1 μM for 24 h in serum-free Opti-MEM
medium in parallel with 0.1 µg/mL LPS, and then stained with anti-CD206 (A, B).
Scale bar represents 50 µm. The mean fluorescence intensity of the CD206
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