Stereoselective glucuronidation metabolism, pharmacokinetics, anti-amnesic pharmacodynamics, and toxic properties of vasicine enantiomers in vitro and in vivo

Article abstract of DOI:10.1016/j.ejps.2018.07.058

Vasicine (VAS) is a potential natural cholinesterase inhibitor for treatment of Alzheimer's disease. Due to one chiral centre (C-3) presenting in molecule, VAS has two enantiomers, d-vasicine (d-VAS) and l-vasicine (l-VAS). The study was undertaken to investigate the stereoselective glucuronidation metabolism, pharmacokinetics, anti-amnesic effect and acute toxicity of VAS enantiomers. In results, the glucuronidation metabolic rate of l-VAS was faster than d-VAS in human liver microsomes and isoenzymes tests, and it was proved that the UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B15 were the major metabolic enzymes for glucuronidation of l-VAS, while only UGT1A9 for d-VAS, which take responsibility of the significantly less metabolic affinity of d-VAS than l-VAS in HLM and rhUGT1A9. The plasma exposure of d-VAS in rats was 1.3-fold and 1.6-fold higher than that of l-VAS after intravenous and oral administration of d-VAS and l-VAS, respectively. And the plasma exposure of the major glucuronidation metabolite d-VASG was one of tenth of l-VASG or more less, no matter by intravenous or oral administration. Both d-VAS and l-VAS were exhibited promising acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, and the BChE inhibitory activity of d-VAS with IC₅₀ of 0.03 ± 0.001 μM was significantly stronger than that of l-VAS with IC₅₀ of 0.98 ± 0.19 μM. The molecular docking results indicated that d-VAS and l-VAS could bind to the catalytic active site (CAS position) either of human AChE and BChE, and the BChE combing ability of d-VAS (the score of GBI/WAS dG ?7.398) was stronger than that of l-VAS (the score of GBI/WAS dG ?7.135). Both d-VAS and l-VAS could improving the learning and memory on scopolamine-induced memory deficits in mice. The content of acetylcholine (ACh) after oral administration d-VAS increased more than that of l-VAS in mice cortex, through inhibiting cholinesterase (ChE) and increasing choline acetyltransferase (ChAT). In addition, the LD₅₀ value of d-VAS (282.51 mg·kg^?1) was slight lower than l-VAS (319.75 mg·kg^?1). These results indicated that VAS enantiomers displayed significantly stereoselective metabolic, pharmacokinetics, anti-amnesic effect and toxic properties in vitro and in vivo. The d-VAS might be the dominant configuration for treating Alzheimer's disease.

Full text of DOI:10.1016/j.ejps.2018.07.058

Accepted Manuscript
Stereoselective glucuronidation metabolism, pharmacokinetics,
anti-amnesic pharmacodynamics, and toxic properties of vasicine
enantiomers in vitro and in vivo
Yudan Zhu, Wei Liu, Shenglan Qi, Hanxue Wang, Yuwen Wang,
Gang Deng, Yunpeng Zhang, Shuping Li, Chao Ma, Yongli Wang,
Xuemei Cheng, Changhong Wang
PII:
DOI:
Reference:
S0928-0987(18)30362-2
doi:10.1016/j.ejps.2018.07.058
PHASCI 4634
To appear in:
European Journal of Pharmaceutical Sciences
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Revised date:
Accepted date:
19 February 2018
24 July 2018
30 July 2018
Please cite this article as: Yudan Zhu, Wei Liu, Shenglan Qi, Hanxue Wang, Yuwen
Wang, Gang Deng, Yunpeng Zhang, Shuping Li, Chao Ma, Yongli Wang, Xuemei Cheng,
Changhong Wang , Stereoselective glucuronidation metabolism, pharmacokinetics, anti-
amnesic pharmacodynamics, and toxic properties of vasicine enantiomers in vitro and in
vivo. Phasci (2018), doi:10.1016/j.ejps.2018.07.058
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ACCEPTED MANUSCRIPT
Stereoselective
Glucuronidation
Metabolism,
Pharmacokinetics,
Anti-Amnesic
Pharmacodynamics, and Toxic Properties of Vasicine Enantiomers in vitro and in vivo
a
a
a
a
Yudan Zhu a, 1, Wei Liu a, 1, Shenglan Qi , Hanxue Wang , Yuwen Wang , Gang Deng , Yunpeng
a
Zhang a, Shuping Li , Chao Ma a, Yongli Wang a, b, Xuemei Cheng a,b, Changhong Wang a, b
*
aInstitute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine; The MOE
Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New
Resources and Quality Evaluation of Chinese Medicines, 1200 Cailun Rood,Shanghai 201203,China
bShanghai R&D Centre for Standardization of Chinese Medicines, 1200 Cailun Rood, Shanghai
201203,China
*Correspondence to: Professor Changhong Wang,Institute of Chinese Materia Medica, Shanghai
University of Traditional Chinese Medicine, Shanghai 201203, China. Tel: 086-021-51322511, Fax:
086-021-51322519, E-mail: wchcxm@shutcm.edu.cn; wchcxm@hotmail.com (C.H. Wang)
1Yudan Zhu and Wei Liu contributed equally to this work.
Keywords: Vasicine enantiomers; Anti-amnesic; Glucuronidation; Pharmacokinetics; Stereoselective
1

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Abstract
Vasicine (VAS) is a potential natural cholinesterase inhibitor for treatment of Alzheimer’s disease.
Due to one chiral centre (C-3) presenting in molecule, VAS has two enantiomers, d-vasicine (d-VAS)
and l-vasicine (l-VAS). The study was undertaken to investigate the stereoselective glucuronidation
metabolism, pharmacokinetics, anti-amnesic effect and acute toxicity of VAS enantiomers. In results,
the glucuronidation metabolic rate of l-VAS was faster than d-VAS in human liver microsomes and
isoenzymes tests, and it was proved that the UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B15
were the major metabolic enzymes for glucuronidation of l-VAS, while only UGT1A9 for d-VAS,
which take responsibility of the significantly less metabolic affinity of d-VAS than l-VAS in HLM
and rhUGT1A9. The plasma exposure of d-VAS in rats was 1.3-fold and 1.6-fold higher than that of
l-VAS after intravenous and oral administration of d-VAS and l-VAS, respectively. And the plasma
exposure of the major glucuronidation metabolite d-VASG was one of tenth of l-VASG or more less, no
matter by intravenous or oral administration. Both d-VAS and l-VAS were exhibited promising
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activit ies, and the BChE
inhibitory activity of d-VAS with IC50 of 0.03 ± 0.001 μM was significantly stronger than that of l-VAS
with IC50 of 0.98 ± 0.19 μM. The molecular docking results indicated that d-VAS and l-VAS could bind
to the catalytic active site (CAS position) either of human AChE and BChE, and the BChE combing
ability of d-VAS (the score of GBI/WAS dG -7.398) was stronger than that of l-VAS (the score of
GBI/WAS dG -7.135). Both d-VAS and l-VAS could improving the learning and memory on
scopolamine-induced memory deficits in mice. The content of acetylcholine (ACh) after oral
administration d-VAS increased more than that of l-VAS in mice cortex, through inhibiting
cholinesterase (ChE) and increasing choline acetyltransferase (ChAT). In addition, the LD50 value of
d-VAS (282.51 mg ·kg–1) was slight lower than l-VAS (319.75 mg·kg–1). These results indicated that
VAS enantiomers displayed significantly stereoselective metabolic, pharmacokinetics, anti-amnesic
effect and toxic properties in vitro and in vivo. The d-VAS might be the dominant configuration for
treating Alzheimer’s disease.
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1. Introduction
Chiral drugs exhibit distinct biochemical and pharmacological behaviors in the body, and the
preclinical evaluation of an enantiomer must be carried out to develop chiral drugs containing at least
one chiral centre and produce 2n-1 pairs of enantiomers (Shen et al., 2013). Widely used chiral drugs,
such as amlodipine (antihypertensive), methylphenidate (stimulant) and salbutamol (antihasthma), play
important roles in the treatment of some human diseases (Maddi et al., 2010; Bartl et al., 2017;
Jacobson et al., 2017). The physiochemical and biochemical properties of racemic mixtures and
individual stereoisomers might be different, and the affinity of enantiomers with target molecules
(enzymes, receptors and transporters) might also be different, it would result in dissimilar
pharmacodynamics and pharmacokinetics. In some cases, one enantiomer may have a desired
beneficial effect, whereas others may produce deleterious side-effects or toxicity (Smith, 2009). Thus,
the U.S. Food and Drug Administration (FDA), China State Food and Drug Administration (SFDA)
and some departments in other countries have agreed that the pharmacological, pharmacokinetic and
toxicological evaluation of individual enantiomer of each developing chiral drug must be quantified
(FDA, 1992; SFDA, 2007).
Vasicine (VAS), a pyrrolo [2,1-b] quinazoline type alkaloid, has a chiral centre in C-3 and two
enantiomers, namely d-vasicine (d-VAS) and l-vasicine (l-VAS) (the chemical structures were shown in
Fig. S1) (Joshi et al., 1996). VAS was first isolated in 1924 from Adhatoda vasica (Acanthaceae), and
most of the work on this molecule was conducted between the 1960s and 1990s. VAS has diverse
pharmacologic actions, such as bronchodilatory (Mahindroo et al., 2005), antitussive (Liu et al., 2015a),
abortifacient (Chandokhe, 1987), antimicrobial (Pa and Mathew, 2012), anti-inflammatory and
antioxidant effects (Srinivasarao et al., 2006). In recent years, as a potential natural cholinesterase (ChE)
inhibitor, VAS has exhibited promising anticholinesterase activity in preclinical models and has been
studied in the development of treatments for Alzheimer's disease (AD) (Ali et al., 2016; Li et al., 2017;
Liu et al., 2016; Zhao et al., 2013). Liu and colleagues suggested that VAS showed strong inhibition of
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities with IC50 of 3.24 ± 0.08 μM
and 0.1 ± 0.0 µM, respectively (Liu et al., 2014). Moreover, the in vivo pharmacokinetic study of VAS
was found that the oral absolute bioavailability (F) of VAS was approximately 54.56% ± 7.33% with a
short half-life of 1.5 h in rats (Liu et al., 2015b). VAS could be metabolized and converted into
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vasicinone (VAO), 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-b-D-glucuronide (VASG),
1,2,3,9-tetrahydropyrrolo
[2,1-b]
quinazolin-3-ylhydrogen
sulfate
(VASS)
and
9-oxo-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-b-D-glucuronide (VAOG) in rat in vivo (Liu et al.,
2015c). Notably, the exposure level of VAS and its glucuronidation metabolite (VASG) in rat plasma is
comparable, whether by oral or intravenous administration (Liu et al., 2015b; Liu et al., 2015c), and
VAS and VASG were cumulatively excreted 29.9 ± 3.78% and 41.0 ± 6.92% from urine and feces
within 72 hours after oral administration 45 mg/kg of Rac-VAS, respectively (Liu et al., 2016),
implying that the glucuronidation of VAS is one of the most important metabolic pathway in rats. In
addition, most metabolites maintain potential inhibitory activity against AChE and BChE, although it is
weaker than that of VAS. These results implied that VAS undergoes a metabolic inactivation process in
vivo with respect to cholinesterase inhibitory activity (Liu et al., 2015c). Although many studies have
been conducted on VAS, the stereoselective properties of VAS enantiomers with respect to metabolism,
pharmacokinetics, pharmacodynamics and toxicity were rarely studied.
The aim of the present study is to investigate the stereoselective metabolism, pharmacokinetics,
anti-amnesic effects, and acute toxicity of d-VAS and l-VAS and provide valuable scientific research
data for the discovery of novel anti-amnesic drugs from VAS enantiomers. The stereoselective
pharmacokinetics and glucuronidation metabolic diversities of d-VAS and l-VAS were carried out in
rats and in human liver microsomes (HLMs), pre-clinical animal liver microsomes and
UDP-glucuronosyltransferase (UGT) isoenzymes. The in vitro anticholinesterase activity assay and
molecular docking assay were performed and scopolamine-induced memory deficit mouse model were
used to evaluate the stereoselective anti-amnesic effects of d-VAS and l-VAS. The toxic properties of
VAS enantiomers were evaluated by acute toxicity tests.
2. Materials and methods
2.1. Chemicals and reagents
Racemic VAS (rac-VAS) and VAO were isolated from Peganum harmala L., and racemic VASS,
VAOS, and VASG (purity> 98%) were obtained from rat urine samples after the oral administration of
VAS in our laboratory (Liu et al., 2015b). They were identified using NMR (Bruker NMR AV 400,
Bruker Bio Spin GmbH, Rheinstetten, Germany) and mass spectrometry data with purity of more than
98% and assayed through peak area normalization via HPLC. Pseudoephedrine hydrochloride (PSH)
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was provided by the National Institute for the Control of Pharmaceutical and Biological Products
(Beijing, China). Choline acetyltransferase (ChAT) assay kit was purchased from Nanjing Jiancheng
Bioengineering Institute (No. A079-1, Nanjing, China). BChE from equine serum, AChE from
Electrophorus electricus, chlormequat chloride, butyrocholine chloride (BCh), acetylcholine chloride
(ACh), choline chloride (Ch), alamethicin, glucose 6-phosphate, glucose-6-phosphate dehydrogenase,
nicotinamide adenine dinucleotide phosphate (NADPH), uridine diphosphate glucuronic acid
(UDPGA), tris base, fluconazole, phenylbutazone, hecogenin, niflumic acid, and diclofenac were
obtained from Sigma Aldrich Co. (St. Louis, MO, USA). Recombinant human UGT1A1, 1A3, 1A4,
1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17 (5 mg ·mL–1) and pooled HLM (20 mg·mL–1, 32
donor pool; equal number of males and females) were purchased from BD Biosciences (San Jose, CA,
USA). The liver microsomes of rats, mice, pigs, guinea pigs, dogs, monkeys, rabbits, bulls, sheep and
camels were prepared using the reported method in our laboratory (Li et al., 2016). MCI Gel CHP 20P
was purchased from Mitsubishi Chemical Corporation (Tokyo, Japan). Silica Gel C18 (MB 100-40/75)
was purchased from Fuji Silysia Chemical Ltd. (Kasugai, Japan). HPLC grade acetonitrile and
methanol were purchased from Fisher Scientific Co. (Santa Clara, USA). Formic acid of HPLC grade
was purchased from Tedia Co. (Fairfield, USA). HPLC grade water was obtained by a Milli-Q
Academic System (Millipore, Billerica, MA). The ACQUITY UPLCTM HSS T3 column (100 mm × 2.1
mm, 1.8 μm) was purchased from Waters Corp. (MA, USA). All other reagents and solvents were of
analytical grade.
2.2. Animals
Male C57BL/6 mice aged 6 weeks and guinea pigs (body weight 200–250 g) were obtained from the
Animal Research Centre of Shanghai University of Traditional Chinese Medicine (Shanghai,
China).Male and female Kunming mice (body weight, 18–22 g) and Sprague-Dawley rats (body weight,
180–200 g) were obtained from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). All the
animals were raised under controlled conditions at a temperature of 25 ± 2°C and 12 h light–dark cycle
with free access to food and water for at least 7 days before the experiment. All the experiments
performed on the animals were in accordance with the P.R. China legislation on the use and care of
laboratory animals and approved by the Animal Care and Use Committee of Shanghai University of
Traditional Chinese Medicine (Approval Number: ACSHU-2011-G115).
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2.3. In vitro metabolic diversity of rac-VAS, d-VAS and l-VAS in human livermicrosomes
2.3.1. Elimination of rac-VAS and its enantiomersin human liver microsomes
The elimination rates of rac-VAS and its enantiomers in HLM were determined. The entire
incubation volume was 100 μL and contained HLM (0.5 mg protein·mL-1) and alamethicin (50 μg·mg-1
protein) in 100 mM Tris-HCl buffer (pH 7.4). The solution with HLM and alamethicin was incubated 5
min at 37°C with shaking in advance. Subsequently, 10 mM glucose 6-phosphate, 4 mM magnesium
chloride, 1 unit·mL-1of glucose-6-phosphate dehydrogenase and 1 μM rac-VAS, d-VAS or l-VAS were
added in the incubation systemsuccessively. The incubation reaction was initiated by the addition of 1
mM NADP+ and 5 mM UDPGA (Nishimuta et al., 2011). The reaction process was terminated with
500 μL of ice-cold acetonitrile containing IS after incubation for 0, 10, 20, 40, 60, and 80 min,
respectively. The mixture was centrifuged at 12000 × g for 10 min, and 540 μL of supernatant was
evaporated to dryness by a gentle streamof nitrogen (37°C). The residue was dissolved by 90 μL of 5%
methanol and centrifuged at 12000 × g for 10 min. The supernatant (5 μL) was injected for
UPLC-MS/MS analysis. Data was processed through the GraphPad Prism 5.0, the concentration of the
prototype VAS before the incubation start (0 min) is considered as 100%, and the remaining prototype
concentration remaining at the remaining time points is converted to the remaining %. Then, the natural
logarithm and incubation time of all remaining quantities were made into regression curves, and the
slope K was obtained. The intrinsic clearance (CLint) of rac-VAS and its enantiomers were estimated
from the in vitro half-life of substrate depletion (t1/2 = -0.693/k), incubation volume (V) and
concentration of microsomal proteins in the incubation mixture (P) according to the following equation:
CLint =(0.693/t1/2) × (V/P) (Obach et al., 1997).
2.3.2. Formation rates of glucuronide metabolites in pooled HLMs and pre-clinical animal liver
microsomes
The formation rates of glucuronide metabolites in pooled HLM and pre-clinical liver microsomes
(MoLM, monkey liver microsomes; CLM, camel liver microsomes; BLM, bull liver microsomes; SLM,
sheep liver microsomes; DLM, dog liver microsomes; GLM, guinea pig liver microsomes; RLM, rat
liver microsomes; MLM, mouse liver microsomes; PLM, pig liver microsomes; and RaLM, rabbit liver
microsomes) were evaluated. The entire incubation volume was 100 μL and contained pooled HLM or
pre-clinical animal liver microsomes (0.5 mg protein·mL-1) and alamethicin (50 μg·mg-1 protein) in 100
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mM Tris-HCl buffer (pH 7.4). The solution with HLM or pre-clinical liver microsomes and alamethicin
were incubated 5 min at 37°C with shaking in advance. Subsequently, 10 mM glucose 6-phosphate, 4
mM magnesium chloride, 1 unit·mL-1of glucose-6-phosphate dehydrogenase and 0.5 mM rac-VAS,
d-VAS or l-VAS were added in the incubation system successively. Finally, the incubation reaction
was initiated by the addition of 1 mM NADP+ and 5 mM UDPGA (Nishimuta et al., 2011). The 100 μL
mixed solution was incubated for 60 min at 37°C, 500 μL of ice-cold acetonitrile mixed with 5 ng·mL-1
IS (PSH) was added to immediately terminate the reactions. The mixture was then centrifuged at 12000
× g for 10 min, and 540 μL of the supernatant was evaporated to dryness by a gentle streamof nitrogen
(37°C). The residue was dissolved in 90 μL of 5% methanol and centrifuged at 12000 × g for 10 min,
and 5 μL of the supernatant was injected for UPLC-MS/MS analysis. Control samples without NADP+,
UDPGA or substrates were prepared. Each of incubation reaction was performed in three replicates.
2.3.3. Effect of UGT inhibitorson production rates of glucuronide metabolites in pooled HLM
In the effect of UGT inhibitors assay, rac-VAS and its enantiomers were incubated with pooled
HLM (0.5 mg protein·mL-1) and specific chemical inhibitors in the UDPGA-regenerating system. The
incubation system (100 μL) contained the pooled HLM (0.5 mg protein·mL-1), alamethicin (50 μg·mg-1
protein), rac-VAS or its enantiomers (250 μM), 4 mM magnesium chloride, Tris-HCl buffer (pH 7.4)
and 5 mM UDPGA. The mixture was incubated at 37°C for 60 min, and the reaction was terminated by
the addition of 500 μL of ice-cold acetonitrile containing IS (5 ng·mL-1 PSH). The selective inhibitors
used were atazanavir (UGT1A1, IC50 2.4 μM) (Zhang et al., 2005), hecogenin (UGT1A4, IC50 1.5 μM)
(Uchaipichat et al., 2006) and fluconazole (UGT2B7, IC50 2.5 μM) (Raungrut et al., 2010) at individual
concentrations of 1, 10 and100 μM; niflumic acid (UGT1A9, IC50 25 μM) (Miners et al., 2011) at
concentrations of 10, 100 and 500 μM; phenylbutazone (UGT1As, IC50 500 μM) (Ballard et al., 2016)
and diclofenac (UGT2B15, IC50 500 μM) (Uchaipichat et al., 2004) at concentrations of 100, 500 and
1000 μM. The other operations were similar to the formation rates of glucuronide metabolites in pooled
HLM. The control samples without inhibitors were prepared. Each of incubation was performed in
three replicates. The percent inhibition was calculated as the formation rate of VASG in the inhibited
samples (Vinhibited) relative to the formation rate of VASG in the control samples (Vcontrol) according to
the following equation: Percent inhibition=(1-Vinhibited/Vcontrol)× 100% (n=3).
2.3.4. Identification of rhUGT isoforms in human recombinant UGT systems
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In the human recombinant UGTs assay, rac-VAS and its enantiomers were incubated with 12 human
rhUGT enzymes (rhUGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 2B4, 2B7, 2B10, 2B15 and 2B17).
The incubation system(100 μL) contained the rhUGT enzymes (0.5 mg·mL-1), alamethicin (50 μg·mg-1
protein), rac-VAS or its enantiomers (250 μM), 4 mM magnesium chloride, Tris-HCl buffer (pH 7.4)
and 5 mM UDPGA. The mixture was incubated at 37°C for 60 min, and the reaction was terminated by
the addition of 500 μL of ice-cold acetonitrile containing IS (5 ng·mL-1 PSH). The other operations
were similar to the formation rates of glucuronide metabolites in pooled HLM. The control samples
without human UGTs or substrates were prepared. Each of incubation reaction was performed in three
replicates.
2.3.5. Glucuronidation kineticsin human recombinant UGT1A9 and UGT2B15 systems
The standard incubation mixtures contained d-VAS and l-VAS (15–250 μM) or rac-VAS (30–500
μM) and HLM (0.5 mg of protein·mL-1) or rhUGT1A9 (0.5 mg of protein·mL-1) and rhUGT2B15 (0.5
mg of protein·mL-1). The procedures for the incubation were performed according to the protocols used
in the recombinant human UGT enzymes. Each of incubation reaction was performed in three
replicates. The supernatants were analysed by UPLC-MS/MS. The glucuronidation metabolite
(d-VASG and l-VASG) formation rates of the VAS enantiomers were plotted against the
concentrations of d-VAS and l-VAS to obtain the Michaelis-Menten constant (Km) and maximum
velocity (Vmax) values of the reactions (V = [Vmax1 × S/(Km1+ S)]+ [ Vmax2 × S/(Km2 + S)]), [S] is the
substrate concentration. This procedure was performed on Prism software (GraphPad Software Inc.,
San Diego, CA). All the results were expressed as mean ± standard error.
2.4. In vivo stereoselective pharmacokineticsofd-VAS and l-VAS in rats
2.4.1. Dose administration and pharmacokinetics
Thirty-two Sprague-Dawley rats were equally divided into four groups, two were intravenous dosage
groups (1 mg·kg-1 of d-VAS and l-VAS, respectively) and two were oral dosage groups (7.5 mg·kg-1 of
d-VAS and l-VAS, respectively). Approximately 0.25 mL of venous blood was collected from the
angular vein of each rat at the following time points: 0, 2, 5, 15, 30, 45, 60, 120, 240, 480 and 720 min
after intravenously and orally administration. The blood samples were centrifuged at 5000 × g at 4°C
for 10 min to separate the plasma, which was then stored at –20°C until UPLC/MS/MS analysis.
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2.4.2. Preparation of plasma samples and UPLC-MS/MS analysis
Aliquots of 100 μL of plasma samples were added with 50 μL of standard work solutions of PSH (IS)
and 250 μL of acetonitrile in tubes to precipitate the protein. The mixture was vortex-mixed for 1 min
and then centrifuged at 12000 × g for 10 min. Aliquots of 320 μL of the supernatant were transferred to
another clean tube and dried by a gentle stream of nitrogen (37°C). Initial mobile phase (80 μL) was
then added, and the mixture was vortexed for 1 min. An aliquot of 5 μL was injected into the
UPLC-MS/MS system with a ACQUITY UPLCTM HSS T3 column (2.1 mm × 100 mm, 1.8 mm, MA,
USA) for analysis according to a previous protocol (Liu et al., 2015c) with minor modifications and
partial verification for the VASG enantiomer assay (see Supplementary Table S1).
2.5. Anticholinesterase activity ofd-VAS and l-VAS in vitro
The anticholinesterase activities of l-VAS, d-VAS, and 11 mixture samples with different ratios of
l-VAS and d-VAS (l-VAS/d-VAS: 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10) were performed
as previously described, in which the ACh and BCh were used as substrates to determine the inhibitory
activities against AChE and BChE, respectively (Liu et al., 2014). All the test mixtures were dissolved
in 0.2% DMSO to obtain 20 mM solutions. Thereafter, a 100 µL incubation system containing 10 µL
of the test compound solution and 40 µL of the enzyme solutions (with final concentrations: of 0.0035
unit·mL−1 for AChE, or 0.008 unit·mL−1 for BChE) were mixed and pre-incubated for 15 min at 25°C.
Then, 50 µL of the substrate solution (final concentrations 5.50 µM for ACh or 7.15 µM for BCh) was
added, and the resulting mixture was incubated for 20 min at 25°C. The reaction was terminated
immediately by adding 300 µL of ice-cold acetonitrile solution (0°C), which was dissolved with 1.899
µM IS (chlormequat chloride). The mixed solution was then centrifuged (15000 × g, 10 min), and the
supernatant was used for the UPLC-MS/MS analysis. The IC50 values of the VAS mixtures on AChE
and BChE were calculated using the Prism software (GraphPad Software Inc., San Diego, CA, USA).
2.6. Moleculardocking of d-VAS and l-VAS with human AChE and BChE
For the comparison between d-VAS and l-VAS with human AChE and BChE (hAChE and hBChE),
in respect to interactions and bonds, antagonist-bound human ChE [Protein Data Bank code: 4BDT]
was selected as the template of hAChE and hBChE (Nachon et al., 2013). The conformations of VAS
enantiomers were searched in the MMFF94X force field of MOE. The best model generated and sorted
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by GBVI/WSA dG score was energy minimized, and hydrogen ions were added using Protonate 3D in
MOE. The final model was subjected to binding site analysis and docking study.
2.7. Anti-amnesic effect of rac-VAS, d-VAS, and l-VAS on scopolamine-induced memory deficitsin mice
2.7.1. Morris watermaze test of rac-VAS, d-VAS, and l-VAS
The detailed procedure of Morris water maze (MWM) test was conducted according to a previously
described method (He et al., 2015). A total of 48 male C57BL/6 mice were randomly divided into six
groups (eight mice per group), namely vehicle-treated group, scopolamine-treated group (1 mg·kg−1),
donepezil-treated group (3 mg·kg−1), d-VAS-treated group (15 mg·kg−1), l-VAS-treated group (15
mg·kg−1), and rac-VAS-treated group (15 mg·kg−1). Donepezil, rac-VAS, d-VAS and l-VAS were
administered by oral gavage 1 week prior to the behavioural testing. During the tests, donepezil,
rac-VAS, d-VAS, and l-VAS were administered 1 h before the trial, and scopolamine was
intraperitoneally (i.p.) injected 30 min after.
2.7.2. Collection of brain tissues
After the end of the MWM test, all mice were anaesthetized and decapitated, and their brains were
removed rapidly on ice. The entire cerebral cortex and hippocampus of each brain tissue were isolated
on ice and then immediately snap-frozen by liquid nitrogen and stored at −80°C until further analysis.
2.7.3. Analysis of AChE activity and ACh content levelsin brain tissues
Some portions of the cerebral cortices and hippocampi were homogenised in 20 mM PBS (pH 7.6,
1:10 w/v) on ice, and the homogenates were centrifuged at 12000 × g for 15 min at 4°C. The
supernatants were used to measure the activity of AChE through a validated UPLC-MS/MS method
(Liu et al., 2014). Briefly, a mixture of 30 µL of ACh (200 µM) and 30 µL of supernatant was added in
a 1.5 mL Eppendorf tube, and the reaction was terminated after co-incubation for 5 min and 10 min at
25°C by the addition of 180 µL of cold acetonitrile (0°C), respectively. Each sample was prepared in
triplicate. After being centrifuged at 12000 × g for 10 min, the Ch content in the supernatant of the
reaction mixture was measured by UPLC-MS/MS (Liu et al., 2014). The activity of AChE was
calculated using the following equation:
The activity of AChE (unit·g−1) = K × (Mean C10 min–Mean C5 min)/∆T.
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where K represents the dilution factor of the sample, Mean C10 min represents the mean content of Ch in
triplicate samples co-incubated for 10 min, Mean C5 min represents the mean content of Ch in triplicate
samples co-incubated for 5 min and ∆T represents the time difference to terminate reaction.
The remaining supernatants of the cerebral cortices and hippocampi were detected by the validated
UPLC-MS/MS method (Liu et al., 2014) for the determination of the ACh and Ch contents.
2.7.4. Analysis of choline acetyltransferase activity in brain tissues
The levels of ChAT in the cerebral cortex and hippocampus homogenates were measured with
commercially available assay kits in accordance with the manufacturer’s protocols. The levels of ChAT
in the cerebral cortex and hippocampus tissues were normalizedand expressed as unit·g-1 tissue.
2.7.5. Western blot analysis
The hippocampus and cortex sections of mice were homogenised on ice with CellLyticTMMT
mammalian tissue lysis reagent (Sigma, C3228), which contains protease and phosphatase inhibitor
cocktails (Sigma, P3840). The supernatant was separated fromthe homogenate, which was centrifuged
at 12000 ×g for 10 min at 4°C. Protein concentration was quantified by BCA assay. Thereafter, 20 μg
of protein from each sample was electrophoresed by 10% SDS-PAGE and transferred to PVDF
membrane (Millipore, Temecula, CA, USA). The membrane was incubated with blocking solution (5%
BSA) for 1 h at room temperature and then incubated overnight with primary antibodies against AChE
(Millipore, Temecula, CA, USA), ChAT and GAPDH (Abcam, Cambridge, UK) at 4°C. The membrane
was then washed five times with PBS containing 0.1% Tween 20 (PBST), incubated with horse radish
peroxidase-conjugated secondary antibodies for 1 h at room temperature, washed five times with PBST
and visualised with ECL prime kit (GE Healthcare, NA, UK; He et al., 2015).
2.8. Acute toxicity test for d-VAS and l-VAS in mice
In the acute toxicity study, Kunming mice were divided into 12 groups (five males and five females
for each group) after an appropriate pre-experiment. The mice underwent fasting for 12 h with water ad
libitum before treatment. l-VAS was orally administered at doses of 219, 258, 303, 357, 420 and 494
mg·kg-1, and d-VAS was orally administered at doses of 186, 219, 258, 303, 357 and 420 mg·kg-1,
respectively. The general behaviours of the mice were observed continuously for 1 h after the treatment,
intermittently for 4 h and then over 24 h. The mice were further observed for up to 14 days after
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treatment to search for any sign of toxicity and death. The median lethal dose (LD50) values and 95%
confidence limits of d-VAS and l-VAS were calculated by Bliss method.
2.9. Isolation and purification of d-VAS and l-VAS
The VAS enantiomers were separated by using a CHIRALPAK AY-H column (0.46 cm × 15 cm,
Daicel Chemical Industries, Ltd., Tokyo, Japan) with a flow-rate of 0.5 mL·min−1 and UV detection at
210 nm with ethanol/diethanol amine ration of 100/0.1 (v/v). The HPLC chromatogram was shown in
Supplementary Fig. S2. Fraction solutions of 6.129 min and 9.297 min were collected and concentrated
in vacuo to yield residues. The purities of d-VAS and l-VAS were determined to be more than 98%
through the normalization of the peak areas detected by HPLC-DAD (Fig. S2B and S2C). The fraction
solution of 6.129 min was confirmed as l-VAS by the specific rotation value of −43.67 (c=0.1 mg·mL−1,
ethanol) after setting the temperature at 20°C. The relatively specific rotation value of the fraction
solution of 9.297 min was +41.83 (c=0.1 mg·mL−1, ethanol), which was confirmed as d-VAS.
2.10. Isolation and purification of glucuronide metabolitesd-VASG and l-VASG
Rac-VAS was administered to guinea pigs (n=10) by gavage in a dose of 100 mg·kg−1, and
approximately 0.44 L of urine samples from the guinea pigs were subjected to MCI Gel CHP 20P
column chromatography (4.5 × 60 cm; 1000 mL). The column was then eluted with a gradient system
of methanol–water (0:100; 5:95; 10:90; 20:80; 30:70; 50:50; 100:0). The elution profiles of the
metabolites were obtained from 10% and 20% methanol fractions. Both fractions were concentrated in
vacuo to yield residues, which were then dissolved in a small amount of water and purified by
semi-preparative HPLC.
Semi-preparative HPLC was performed with a ZORBAX SB-C18 column (9.4 mm × 25 cm, 5 μm;
Agilent, USA) at 30°C in an LC3000 liquid chromatograph (Beijing Tong Heng Innovation
Technology Co., Beijing, China). The detection wavelength was set at 280 nm. The mobile phase
consisted of methanol (A) and 0.1% aqueous formic acid (B). The mixture of VASG enantiomers (138
mg) was obtained using a gradient elution of 0 min to 10 min (5% to 20% A, with) at a flow rate of 3
mL·min–1. The purity of the mixture of VASG enantiomers detected through HPLC-DAD and
UPLC-MS/MS was more than 98% after area normalization. The separation and preparation of
d-VASG and l-VASG were carried out on a Spursil C18 column (9.4 mm × 25 cm, 5μm; Dikma
Limited, CA, USA). Finally, pure d-VASG and l-VASG were prepared.
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2.11. Statistical analysis
All data were analysed on SPSS 18.0 and expressed as mean ± standard deviation (SD) unless
otherwise indicated. The escape latency and escape rate data in MWM test were analysed using
two-way analysis of variance (ANOVA) with repeated measures. The other behavioural data and the
biomarker changes in vivo and in vitro were analysed by one-way ANOVA followed by Tukey's Post
Hoc Test. The pharmacokinetic parameters were also expressed as the mean ± standard deviation. The
plasma concentration versus time curves were plotted and all the pharmacokinetic data were processed
using the noncompartmental pharmacokinetics data analysis software program PK solutions 2™
(Summit Research Services, USA). The following pharmacokinetic parameters of quantitative
compounds were calculated: absorption rate constant (ka), absorption half-life (t1/2ka), distribution rate
constant (kd), distribution half-life (t1/2kd), elimination rate constant (ke), elimination half-life (t1/2ke),
apparent volume of distribution (Vd/F), apparent clearance rate (CL/F), and mean residence time (MRT).
The maximum peak concentration (Cmax), the time of maximum plasma concentration (Tmax) and area
under the plasma concentration versus time curve from zero to time t (AUC(0-t)) were obtained directly
from the observed concentration versus time data. The area under the plasma concentration versus time
curve from zero to infinity (AUC(0-∞)) was calculated by means of the trapezoidal rule with
extrapolation to infinity with a terminal ke. For all statistical tests, the value of p < 0.05 was regarded as
significant.
3. Results
3.1. Metabolic diversity of VAS enantiomersin vitro
3.1.1. Elimination rates of rac-VAS, d-VAS and l-VAS in HLM
The elimination rates of rac-VAS, d-VAS and l-VAS were determined in HLM in vitro. The results
were shown in Supplementary Fig. S3. After incubation of 2 h, the residues of rac-VAS, d-VAS, and
l-VAS were 68.2 ± 8.0%, 72.4 ± 10.4%, and 44.7 ± 10.5%, with t1/2 of 227.5±3.8, 294.1±28.2, and
131.4 ± 18.3 min, and the CLint of 61.2±1.3, 48.4 ± 6.2, and 118.2 ± 14.3 μL·min-1·mg-1 protein,
respectively, indicating that the elimination rate of l-VAS was significantly faster than those of d-VAS
(p < 0.01).
3.1.2. Formation rates of metabolitesin pooled HLM and pre-clinical livermicrosomes
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The glucuronidation metabolites d-VASG and l-VASG were detected in the HLM after incubation
with 500 μM rac-VAS, l-VAS and d-VAS (Supplementary Fig. S4). The rate of the d-VASG production
(peak area, 37933.5 ± 2814.99) was significantly lower than that of l-VASG (peak area, 201368.5 ±
4331.03) in HLM after incubation with rac-VAS. The amount of d-VASG production (peak area, 6824
± 354.97) accounted for 3.21% of the amount of l-VASG production (peak area: 212721.5 ± 9594.73)
in HLM after incubation with the l-VAS (Fig. S4B). However, the area responses of d-VASG and
l-VASG were 27404 ± 1890.80 and 3906.5 ± 228.40, respectively, when the substrate was d-VAS (Fig.
S4C). Although both l-VASG and d-VASG could be detected in the d-VAS group, but the l-VASG
production only accounted 14.2% of d-VASG. These data implied that l-VAS was prone to be
metabolized into l-VASG, and d-VAS was metabolized into d-VASG, but the rate of l-VASG
production was significantly faster than that of d-VASG in HLMs with the same substrate
concentration (p < 0.01). These results indicated that stereoselective metabolism occurred between
d-VAS and l-VAS, and l-VAS was more prone to be metabolized by glucuronidation in HLM.
The glucuronidation metabolites d-VASG and l-VASG were evaluated in the pre-clinical liver
microsomes after incubation with 500 μM rac-VAS, l-VAS and d-VAS (Supplementary Fig. S5). The
rates of the d-VASG and l-VASG production in some pre-clinical liver microsomes (MoLM, BLM,
SLM, DLM, GLM, RLM, MLM, PLM, and RaLM) were significantly faster than in HLM after
incubation with 500 μM rac-VAS, l-VAS or d-VAS. It is worth noting that the rates of the d-VASG and
l-VASG production in GLM was the fastest among other pre-clinical liver microsomes, and the
production d-VASG and l-VASG in GLM were also comparable after incubation with rac-VAS. The
urinary excretion of d-VASG and l-VASG in guinea pigs were also comparable after performing oral
administration of rac-VAS on rats (data was not shown). It benefited from the result that the standard
substances of d-VASG and l-VASG were isolated and obtained from urine of guinea pigs after oral
administration of rac-VAS. In additional, it was found that the rate of the l-VASG production was faster
than d-VASG in various pre-clinical liver microsomes, except for GLM. All of these results indicated
that the glucuronidation metabolism of VAS or its enantiomers has certain difference in species.
3.1.3. Effect of UGT inhibitorson production ratesin pooled HLM
The inhibitory reaction activities for the VASG enantiomer formation caused by niflumic acid
(UGT1A9 inhibitor) and diclofenac (UGT2B15 inhibitor) were below 35% in the rac-VAS and VAS
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enantiomers, as shown in Fig. 1, no matter the substrate was rac-VAS or VAS enantiomers. These
results indicated that UGT1A9 and UGT2B15 were the major enzymes responsible for the
glucuronidations of l-VAS and d-VAS.
3.1.4. Identification of UGT isoforms in recombinant human UGT systems
The results of recombinant human UGTs (rhUGTs) assay and specific inhibitory studies were
displayed in Fig. 2. They showed that rhUGT1A1, rhUGT1A3, rhUGT1A7, rhUGT1A8, rhUGT1A9,
rhUGT2B4, rhUGT2B7, rhUGT2B15 and rhUGT2B17 were involved in the metabolism of rac-VAS
and l-VAS to l-VASG, and the most active among them were rhUGT1A9 and rhUGT2B15 (Fig. 2A
and 2B). rhUGT1A1, rhUGT1A3, rhUGT1A9, rhUGT2B4 and rhUGT2B7 involved in catalysing the
metabolism of d-VAS to d-VASG, and rhUGT1A9 was the most active (Fig. 2C). Different metabolic
enzymes could possibly induce stereoselective metabolic differences between d-VAS and l-VAS.
3.1.5 Enzyme kinetics studies on the formation of glucuronidation metabolites in HLM and rhUGT
isoenzymes
The best fit of data shown in Fig. 3 and Fig. 4 was obtained using the double Michaelis-Menten
equation for enzyme kinetics. The Eadie-Hofstee transformations revealed evidence of enzyme kinetics
in HLM, as well as in rhUGT1A9 and rhUGT2B15. The Vmax of the metabolic conversion of d-VAS to
d-VASG (0.35 ± 0.09 pmol·min-1·mg-1 protein) was much lower than that of l-VAS to l-VASG (8.77 ±
0.72 pmol·min-1·mg-1 protein) in HLM (Table S2). Similarly, the Vmax of metabolic conversion of
d-VAS to d-VASG (0.84 ± 0.08 pmol·min-1·mg-1 protein) was much lower than that of l-VAS to
l-VASG (1.72 ± 0.52 pmol·min-1·mg-1 protein) in rhUGT1A9 (shown in Table 1; p < 0.05). Moreover,
the CL of the metabolism of l-VAS to l-VASG (0.015 ± 0.001 L·min-1·mg-1 protein, p < 0.001) was
significantly higher than that of d-VAS (0.003 ± 0.001 L·min-1·mg-1protein, p < 0.001) in HLM. These
data illustrated that l-VAS is tend to be metabolized into l-VASG. Furthermore, d-VASG formation was
inadequate to calculate the kinetic parameters in rhUGT2B15.
3.2. In vivo stereoselective pharmacokinetic ofd-VAS and l-VAS in rats
The plasma concentration time curves of d-VAS and l-VAS in rats after intravenously and orally
administration of d-VAS and l-VAS were shown in Fig. 5. The similar profiles were obtained for both
d-VAS and l-VAS after intravenously and orally administration. To compare the difference of
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pharmacokinetics between d-VAS and l-VAS and their main metabolites in the plasma of rats, the
pharmacokinetics parameters after intravenously and orally administration of d-VAS and l-VAS were
calculated and summarized in Table 2 and Table 3, respectively. It could be seen from Fig. 5A and 5B,
similar profiles were obtained for both d-VAS and l-VAS after intravenously and orally administration.
Tmax could be used to define the time of maximum plasma concentration after orally administration,
which is similar for d-VAS and l-VAS. However, differences observed in Cmax and AUC values between
d-VAS and l-VAS after intravenously and orally administration. The AUC(0-∞) and Cmax after the
intravenously administration of d-VAS were nearly 1.4-fold and 1.2-fold higher than that of l-VAS,
respectively (Table 2). The AUC(0-∞) and Cmax after the oral administration of d-VAS were nearly
1.6-fold and 1.7-fold higher than that of l-VAS, respectively (Table 3).
The absolute bioavailability (F) of d-VAS and l-VAS were 80.6% and 65.7%, respectively, implying
that the first pass effect of d-VAS was lower than that of l-VAS. Furthermore, the t1/2Ke and MRT after
the intravenously administration of d-VAS were nearly 2.6-fold and 2.2-fold longer than that of l-VAS,
the t1/2Ke after the orally administration of d-VAS was nearly 1.3-fold longer than that of l-VAS, and
d-VAS had significantly slower elimination rates in plasma after oral administration (p < 0.05; Table 2)
and intravenous administration (p < 0.001; Table 3). After intravenously and orally administration of
l-VAS, the plasma exposure (AUC(0-∞)) of l-VASG was more 5.2-fold and 46.3-fold higher than that of
d-VASG (Fig. 5C and 5D). Similarly, the l-VASG and d-VASG enantiomers were both detected in the
plasma when d-VAS was intravenously and orally administered (Fig. 5E and 5F), but the plasma
exposure of l-VASG was also 1.2-fold and 1.2-fold higher than that of d-VASG (Tables 2 and 3).
3.3. Inhibitory activity of d-VAS and l-VAS on AChE and BChE in vitro
The IC50 values of the technical mixtures of two stereoisomers with different proportions of AChE
and BChE were compared. As indicated in Fig. 6, both d-VAS and l-VAS showed potent inhibitory
activities against AChE and BChE, but no significant difference was observed between d-VAS (IC50:
1.73 ± 0.21 μM) and l-VAS (IC50: 1.49 ± 0.03 μM) when the values were plotted against AChE (Fig. 6).
On the contrary, significant differences between the IC50 values of d-VAS and l-VAS when the values
were plotted against BChE. The IC50 values decreased as l-VAS/d-VAS ratio decreased. With respect
to the inhibition of BChE inhibitory activity, the IC50 value of d-VAS (0.03 ± 0.00 μM) was 30 times
higher than that of l-VAS with IC50 of 0.98 ± 0.19 μM (Fig. 6).
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3.4. Moleculardocking of d-VAS and l-VAS with human AChE and BChE
The molecular binding abilities of d-VAS and l-VAS with hAChE and hBChE were illustrated in Fig.
7. The binding pocket and ligand binding of d-VAS were similar with that of l-VAS when hAChE and
hBChE were used for the structural composite. Both d-VAS and l-VAS could bind to the catalytic
active site (CAS) and subsequently inhibit the activities of compounds (Changeux, 1996; Rosenberry et
al., 2008). In our study, d-VAS and l-VAS ranked first and thus were selected for subsequent analyses.
AChE molecular docking results revealed that a hydrogen bond could be generated between Trp439
and one hydrogen atom in the benzene ring in d-VAS. A strong π-π hydrophobic interactions could be
observed (Fig. 7A and 7B), and a hydrogen atom in the hydroxyl group of l-VAS formed a hydrogen
bond with H2O (Fig. 7C and 7D). In the BChE molecular dock, a weak hydrogen–benzene interaction
was formed by the hydrogen on the C11 of d-VAS. The other hydrogen atom on the same position
formed a hydrogen bond with Glu197. An oxygen atom in the hydroxyl group formed a hydrogen bond
with Gly116, and the pyridine ring of d-VAS was characterized by a strong π–π hydrophobic
interaction between Trp82 and the benzene ring of d-VAS (Fig. 7E and 7F). The π–π hydrophobic
interaction and hydrogen bond of l-VAS with Glu197 were the same as those observed in d-VAS.
However, a hydrogen bond between hydrogen on C11 and His438 was present, and the
hydrogen–benzene interaction was absent in l-VAS (Fig. 7G and 7H).
In summary, the ability of VAS enantiomers to combine with AChE was slightly weaker than that
with BChE because of the weaker affinity of d- and l-VAS with AChE (the score of GBI/WAS dG:
–6.902 and –6.898) compared with BChE and the stronger combing ability of d-VAS to combine with
hBChE (the score of GBI/WAS dG: -7.398) compared with l-VAS (the score of GBI/WAS dG: -7.135).
These results were in agreement with the in vitro AChE and BChE inhibition assay described above,
where inhibitory activity of d-VAS against BChE was strongerthan that against l-VAS.
3.5. VAS enantiomersattenuated cognitive impairmentsinduced by scopolamine in mice
To explore whether rac-VAS, d-VAS and l-VAS prevent acute memory impairment, we measured
their anti-amnesic effects in scopolamine-induced and memory-deficient mice by the MWM tasks. In
the hidden platform–swimming trials, the scopolamine-treated mice showed markedly impaired
memory in their path length on the third, fourth and fifth testing days , respectively (Fig. 8A, 11.71 ±
5.56 m vs. 5.80 ± 2.09 m, p < 0.01; 14.99 ± 9.06 m vs. 4.98 ± 3.56 m, p < 0.05 and 15.67 ± 5.04 m vs.
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3.37 ± 1.99 m, p < 0.001), and the escape latency on the fourth and fifth testing days, respectively (Fig.
8B, 39.25 ± 20.12 s vs. 25.13 ± 16.50 s, p < 0.01; 47.00 ± 15.20 s vs. 21.43 ± 17.73 s, p < 0.05). By
contrast, both rac-VAS and d-VAS could effectively shorten the path length on the third testing day
(6.76 ± 4.48 m vs. 11.71 ± 5.56 m, p < 0.05; 6.62 ± 2.48 m vs. 11.71 ± 5.56 m, P < 0.05), as well as
with the l-VAS and donepezil (8.40 ± 2.78 m vs. 14.99 ± 9.06 m, p < 0.05; 8.45 ± 4.85 m vs. 14.99 ±
9.06 m, p < 0.05) on the fourth testing day (Fig. 8A). Moreover, on the fourth testing day, rac-VAS,
d-VAS, l-VAS and donepezil could effectively shorten the escape latency of scopolamine-treated mice
(25.37 ± 22.99 s vs. 39.25 ± 20.12 s, p < 0.05; 23.00 ± 9.90 s vs. 39.25 ± 20.12 s, p < 0.05; 26.43 ±
11.82 s vs. 39.25 ± 20.12 s,p < 0.05 and 22.71 ± 10.84 s vs. 39.25 ± 20.12 s, p < 0.05; Fig. 8B).
One day after the last hidden platform-swimming test, the spatial probe test was performed to
determine whether mice could memorise the location of the platform. The spatial memory of
scopolamine-induced mice was significantly damaged in contrast to those in the control group. The
mice passed less through the target zone where the hidden platform was placed 24 h before (Fig. 8C
and 8D, p < 0.001). Conversely, mice treated with rac-VAS, d-VAS and l-VAS at 15 mg·kg-1 and
donepezil at 3 mg·kg-1 crossed the site more often than those by treated with scopolamine alone (p <
0.05).
3.6. d-VAS increased ACh content by moderating the activitiesof AChE and ChAT
The ACh and Ch contents detected by UPLC MS/MS in the cerebral cortices were shown in Fig. 9A
and 9B. The ACh contents in the scopolamine-treated mice were significantly lower (p < 0.05) than
those in the control. By contrast to the scopolamine-treated mice, the ACh contents in mice treated with
d-VAS showed significant increase (p < 0.05), and became higher than those in mice treated with
l-VAS. No difference was observed on the Ch contents among the treatment group, scopolamine group,
and donepezil group, by comparing to that in control group.
In order to explore how VAS enantiomers affecting on ACh content in vivo, the activities of AChE in
the cerebral cortex and hippocampus, and the activities of ChAT in the cerebral cortex of
scopolamine-induced mice were measured. Long-term d-VAS and l-VAS treatment for 15 days could
significantly inhibit AChE activity (Fig. 9C and Fig. 9D) and decrease the protein expression of AChE
in the cerebral cortex and hippocampus (Fig. 9F, Fig. 9G, Fig. 9I, and Fig. 9J), especially for d-VAS (p
< 0.01 and p < 0.05, respectively). On the contrary, the activity of ChAT was enhanced in the cerebral
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cortex (Fig. 9E) and ChAT protein expression was increased in the cerebral cortex and hippocampus
after treatment with rac-VAS, d-VAS and l-VAS. But, treatments with d-VAS and l-VAS did not
produce significant difference on the activity and expression of ChAT (Fig. 9F, Fig. 9H, Fig. 9I, and Fig.
9K).
3.7. Acute toxicity of d-VAS and l-VAS in mice
The results of acute toxicity tests of d-VAS and l-VAS in mice were shown in Supplementary Table
S3. The LD50 values of d-VAS and l-VAS after oral administration into mice were 282.51 mg·kg–1 (95%
confidence limits 250.23 - 315.76 mg·kg–1) and 319.75 mg·kg–1 (95% confidence limits 271.85 -
363.39 mg·kg–1) by Bliss method, respectively. These finding indicated a slight lower LD50 value of
d-VAS than l-VAS in mice, but there were no significant differences.
4. Discussion
On chiral drugs, the FDA has regulated the study of the pharmacokinetics, pharmacodynamics and
toxicology properties of pure isomers (FDA, 1992). For example, clausenamide (Clau) is an active
compound originally extracted from the traditional Chinese herbal medicine, Clausena lansium, which
has four chiral centres. The Clau eutomer (-)-Clau and the distomer (+) has been demonstrated to
improve cognitive function in normal physiology and pathological conditions (Chu et al., 2016). In
addition, bambuterol is a long-acting β adrenoceptor agonist and cholinesterase inhibitor used in the
treatment of asthma, it is administered as a racemic mixture, and the recent study has found
R-bambuterol showing a stronger inhibitory effect than S-bambuterol in BChE stereoselective
inhibition (Pistolozzi et al., 2015). Similarly, the present study focused on the stereoselectivity of VAS
enantiomers on improvement of learning and memory abilities, and aimed to find out which of d-VAS
and l-VAS is the dominant enantiomer.
AD is a common neurodegenerative disease, and AChE and BChE are the two enzymes involved in
breaking down the neurotransmitter ACh. Many researchers consider ChE inhibitors, such as
physostigmine and galantamine, to be selective for AChE (Mukherje et al., 2007). These inhibitors
could prolong the availability of acetylcholine. Some studies revealed that AChE predominates in the
healthy brain, with BChE considered to play a minor role in regulating brain ACh levels. However,
BChE activity progressively increases in patients with AD, while AChE activity remains unchanged or
declines (Lam et al., 2009; Greig et al., 2002; 2005). Thus, AD treatment has been focused on BChE
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inhibition rather than on AChE in recent years (Giacobini, 2004; Greig et al., 2002), and BChE has
become as a new therapeutic target and diagnostic marker in AD treatment. Huperzine A, a
quinolizidine alkaloid from Huperzia serrata, was found to reversibly inhibit AChE (IC50=47 nM) and
BChE (IC50=30 nM) (Giacobini, 2004), showing potential use in the treatment of AD at an advance
stage. The in vitro anticholinesterase activity assays of VAS enatiomers indicated that d-VAS has a
stronger inhibitory activity against BChE than l-VAS. The inhibitory activity of d-VAS against BChE
(IC50= 0.029 ± 0.001 μM) was similar to that of huperzine A.
As anti-cholinergic drug, scopolamine impairs cholinergic system activity by acting on muscarinic
receptor and increase AChE activity. Therefore, the scopolamine-induced memory deficits model has
been widely used in screening for candidate drugs with cognitive improvement (Gutierres et al., 2014;
Liu et al., 2017). As a common cholinesterase inhibitor, donepezil has been used to improve cognition
and behavior of AD patients in clinic. In the present study anti-amnesic effects were measured in
scopolamine-induced and memory-deficient mice through MWM tasks to determine whether rac-VAS,
d-VAS and l-VAS prevent acute memory impairment. The results indicated that rac-VAS, d-VAS and
l-VAS administration could significantly improve spatial learning and memory in cognitively impaired
mice. The ACh content in the mouse cortices after d-VAS oral administration increased, and the activity
of ChE in the mouse cortices and hippocampi after d-VAS oral administration decreased. However, the
behavioral test disclosed few differences between VAS enantiomers and rac-VAS. The reason may be
multifaceted. Dysfunction of cholinergic system, neuroinflammation, oxidative stress and amyloid
plaque deposition were the early events in the development and progression of AD (Hamodat et al.,
2017; Kanamaru et al., 2015; Dong et al., 2004). Intraperitoneal injection of scopolamine not only
could cause memory impairment and cholinergic system dysfunction but also could trigger ROS and
inflammation production in the brain (Tao et al., 2014; Xian et al., 2015; Guo et al., 2016). Thus,
d-VAS and l-VAS not only could improve the learning and memory of cognitively impaired mice by
regulating cholinergic nervous systembut also acting on otherroutes.
The metabolism of rac-VAS was systematically investigated in vitro and in vivo in our previous
study, and the glucuronidation of VAS was one of the most important metabolic pathways (Liu et al.,
2015b; Liu et al., 2015c). In the present study, the stereoselective glucuronidation metabolism of VAS
enantiomers was investigated. In order to obtain quantitative experimental results, the reference
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standard substances of d-VASG and l-VASG were isolated and purified. The in vitro phase II
glucuronidation metabolite of rac-VAS was incubated in the liver microsomes of 11 mammalian
species (human, monkey, camel, bull, sheep, dog, guinea pig, rat, mouse, pig and rabbit;
Supplementary Fig. S5A). The production rates of d-VASG and l-VASG significantly vary among
different mammalian liver microsomes, and the highest production rates were observed in guinea pigs
liver microsomes, implying that guinea pigs were the most suitable species for preparing the d-VASG
and l-VASG metabolites. The urinary excretion of d-VASG and l-VASG in guinea pigs were also
comparable after performing oral administration of rac-VAS on rats (data was not shown). Thus, the
d-VASG and l-VASG from the urine of guinea pigs were isolated and purified after performing oral
administration of 100 mg·kg-1 of rac-VAS. When the substrate was d-VAS or l-VAS, the diversity of
glucuronidation metabolite in 11 mammalian liver microsomes was prominent (Fig. S5B and S5C).
Given the significant differences among the production rates of d-VASG and l-VASG was observed in
different liver microsomes, it could provide an available animal models to prepare the desired
metabolites, and it could provide a very useful strategy for the study of othersimilar drugs.
The glucuronidation metabolism of rac-VAS, d-VAS and l-VAS were detected in the HLM
(Supplementary Fig. S5). The amount of d-VASG and l-VASG production showed a significant
difference in the rac-VAS group. The amount of l-VASG was significantly higher than that of d-VASG,
indicating that the rate of l-VASG production was significantly faster than that of d-VASG in HLM.
Subsequent studies on d-VAS and l-VAS in HLM have further confirmed this conclusion. l-VAS could
be metabolized into l-VASG and d-VASG, and d-VAS could also be metabolized into l-VASG and
d-VASG in HLM (Supplementary Fig. S4). Similar results have been found in the pharmacokinetic
study of VAS enantiomers in rats (Table 2 and Table 3). However, on the UGT isoforms assay, d-VAS
and l-VAS could be only metabolized into d-VASG and l-VASG, respectively (Fig. 2). The complex
and variable metabolic enzymes, a large number of enterobacteria and a variety of endogenous
substances in liver microsomes and mice, may be the reasons for the conversion configuration of
l-VASG and d-VASG (Zhang et al., 1994; Reist et al., 1998; Ma et al., 2005). However, the conversion
configuration between l-VAS and d-VAS has not been found in HLM in vitro (Supplementary Fig. S6),
and in rats plasma by a pilot test in vivo (Supplementary Fig. S7).
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In addition, the results of rhUGTs assay indicated that both UGT1A9 and UGT2B15 participated in
catalysing l-VAS to l-VASG (Fig. 2B), and only UGT1A9 was the main enzymes involved in
metabolism of d-VAS to d-VASG. Enzyme kinetics studies indicated that the Vm of rhUGT1A9 and
rhUGT2B15 for metabolizing l-VAS to l-VASG were 1.72 ± 0.52 pmol·min-1·mg-1 protein and 2.01 ±
0.00 pmol·min-1·mg-1 protein, respectively. And the Vm of rhUGT1A9 for the metabolic conversion of
d-VAS to d-VASG was 0.84 ± 0.08 pmol·min-1·mg-1 protein. It indicated that not only different rhUGT
isoforms could metabolize VAS enantiomers but the metabolic capacity of a given UGT isoform could
induce the stereoselective metabolic differences between d-VAS and l-VAS.
The results of in vivo pharmacokinetics study disclosed that the plasma exposure of d-VAS was
1.3-fold and 1.6-fold higher than that of l-VAS and the major glucuronidation metabolite d-VASG was
approximately one-tenth of l-VASG in rats after intravenous and oral administration of d-VAS and
l-VAS, respectively. In lights of the exposure difference, the different metabolic enzymes responsible
for VAS enantiomers, such as UGT1A9 and UGT2B15, might play a key role in pharmacokinetics
processes ofd-VAS and l-VAS.
In a previous in vitro study, the AChE and BChE inhibitory activities of VAS and its major
metabolites were investigated, the results indicated that most metabolites maintain potential inhibitory
activity against AChE and BChE but is weaker than that of the prototype VAS, implying that VAS
undergoes metabolic inactivation process in vivo with respect to cholinesterase inhibitory activity (Liu
et al., 2015b; Liu et al., 2015c). In the present study, the plasma exposure level of d-VAS was higher
than in l-VAS after intravenous or oral administration of the same dose of d-VAS or l-VAS in rats. In
addition, d-VAS displayed a stronger BChE in vitro inhibitory activity than that of l-VAS at same
concentration. The activity of AChE in the mouse cortex and hippocampus was extensively decreased
more after oral administration of d-VAS than that of l-VAS, implying that excessive cholinesterase
inhibition was more likely to occur in high doses of d-VAS in acute toxicity test.
The evaluation of the stereoselective toxicity of different enantiomers is extremely important.
Despite all this, published studies on the toxicity of VAS to mammals were rare. It was reported that the
LD50 values after oral administration of VAS in mice and rats were 290 and 640 mg·kg–1 (Claeson et al.,
2000). In the present study, the LD50 values of d-VAS and l-VAS after oral administration in mice were
282.51 mg·kg–1 (95% confidence limits 250.23 - 315.76 mg·kg–1) and 319.75 mg·kg–1 (95% confidence
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limits 271.85 - 363.39 mg·kg–1) by the Bliss method, respectively. Thus, d-VAS and l-VAS could
experience the same acute toxicity with similar LD50 values.
Species difference exists in physiology, anatomy, and metabolic enzymes expression/activity
(Fujiwara, et al., 2018) among different species of animals and human. There are great pressures for
extrapolating data obtained from animals to clinical trial and toxicity of new drugs candidate.
In conclusion, it was firstly confirmed that VAS enantiomers exhibit significantly stereoselective
anti-amnesic effects, metabolic, and pharmacokinetic properties in vitro and in vivo. Based on these
proofs, it could be believed that d-VAS might be a dominant configuration suitable as a drug candidate
for AD treatment. Of course, there is still much more challenging researches to be done in the future,
such as clinic pharmacokinetics, pharmacodynamics, and safety evaluation, etc.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China and Xinjiang Uygur
Autonomous Region of China (No. U1130303), the National Nature Science Foundation of China (No.
81173119), the Key Project of Ministry of Science and Technology of China (2012ZX09103201-051)
awarded to Professor Changhong Wang for financial support of this study. Yudan Zhu and Wei Liu
were contributed equally to this work.
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