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Introduction
Alzheimer disease (AD) is a progressive
neurodegener-ative disorder associated with a global impairment of higher
mental function, and presenting an impairment of memory as
the cardinal symptom[1]. Histopathological hallmarks of the
disease are the extracellular deposition of amyloid
b-peptide (Ab) in senile plaques, the appearance of intracellular
neurofibrillary tangles (NFT), a loss of cholinergic neurons, and
extensive synaptic changes in the cerebral cortex,
hippo-campus and other areas of brain essential for cognitive
functions. The key symptoms of AD are primarily caused by
cholinergic dysfunction. A significant correlation has been
found between a decrease in cortical cholinergic activity and
the deterioration of mental test scores in patients with
AD[2]. Based on the cholinergic hypothesis of AD, cholinergic
enhancement strategies have been at the forefront of efforts to
pharmacologically palliate the cognitive impairments. Among
the various therapeutic approaches investigated to enhance
cholinergic transmission, cholinesterase inhibitors (ChEI) are
the first group of compounds that have shown some
pro-mise in the treatment of AD. The most significant
therapeutic effect of ChEI in AD treatment is to stabilize cognitive
function at steady level during at least a 1 year period in
approximately 50% of patients. Most clinical studies show
that in a certain percentage of AD patients (approximately
20%) cognitive function can be stabilized for a period of up
to 24 months. In addition, the AD patients who do not
respond to therapy with one ChEI can be switched to a second
one with a 50% rate of success. To date, four ChEI, tacrine,
donepezil, galanthamine and rivastigmine have been
approved by the US Food and Drug Administration for the
treatment of AD, and several new ChEI are being
studied[3-6]. However, the clinical usefulness of ChEI has been limited by
their short half-lives and excessive side effects caused by
activation of peripheral cholinergic systems, as well as
hepatotoxicity, which is the most frequent and important
side effect of tacrine therapy[7-9]. To obtain better
therapeutic benefit in the treatment of AD, the search for a
long-acting ChEI that exerts minimal clinical side effects is still
ongoing[5].(-)-Huperzine A (HupA), a novel
Lycopodium alkaloid, is isolated from the Chinese medicinal herb
Huperzia serrata (Qian Ceng Ta; Figure 1). The herb has been used in China
for centuries in the treatment of such conditions as
contu-sions, strains, swelling, and schizophrenia. HupA, a
compound that is chemically unique in comparison with other
agents under study for AD, is a reversible, potent, and
selective acetylcholinesterase (AChE) inhibitor. Its potency and duration of AChE inhibition rival those of tacrine,
galantha-mine, donepezil, and
rivastigmine[10-13]. HupA has been found
to improve cognitive deficits in a broad range of animal
models. The phase IV clinical trials conducted in China have
demonstrated that HupA induces significant improvement
in the memory of elderly people and patients with AD and
vascular dementia (VD) without any notable side
effects[14-17]. In this paper the pharmacological properties,
pharmacokine-tics, and toxicology of HupA, in addition to the clinical trials
so far conducted using this agent, are reviewed.
Effects on cholinesterase activity and
inhibi-tion mechanism
The cholinesterase inhibition by HupA has been
evaluated in vitro and in vivo, using a spectrophotometric
me-thod[18] with minor modifications. For assay of AChE or
butyrylcholinesterase (BuChE) activity, a reaction mixture
of 4 mL containing acetylthiocholine iodide (0.3 mmol/L) or
butyrylthiocholine iodide (0.4 mmol/L), 1 mL sodium
phosphate buffer (0.1 mmol/L), the test compound (0.1-0.5 mL),
and enzyme (0.1-0.2 mL) was incubated at 37 °C for 8 min.
In vitro and in vivo comparison studies with respect to
AChE inhibition showed that the potency of HupA was
similar or superior to the inhibitors currently being used in AD
treatment (Table 1)[11-13,19-21]. Based on the 50% inhibitory
concentration (IC50), HupA was more potent than tacrine,
physostigmine, galanthamine, and rivastigmine with respect
to inhibition of AChE activity, whereas HupA was the least
potent BuChE inhibitor among the inhibitors
tested[11,21-23]. The IC50 ratio of HupA for BuChE:AChE was much greater
than those of the other 4 inhibitors. HupA exerted inhibitory
effects on AChE from different sources to a similar extent
but, interestingly, was a weaker inhibitor of human serum
BuChE relative to BuChE from other sources. Studies using
8 and 4 cholinesterase isoenzymes, which were extracted
from mouse and dog plasma, respectively, showed that HupA
had a more selective inhibition on AChE isoenzymes, but
had little or no inhibition on
BuChE[24]. The better reversible effect of
(±)-HupA on AChE was also demonstrated in
studies with porcine intrinsic cardiac neurons, which express both
AChE and BuChE[25].
The apparent inhibition constants
(Ki value) for AChE are in the nanomole range, which indicates that these
inhibitors have high affinity for the enzyme. However, the doses
of donepezil and tacrine used orally are much higher than
that of HupA[26], which might be explained by their low
bioavailability and/or by rapid metabolism.
AChE exists in multiple molecular forms that can be distinguished by their subunit associations and hydrodynamic
properties[27,28]. In mammalian brain, the bulk of AChE
occurs as a tetrameric, G4 form (10S) together with much smaller
amounts of a monomeric, G1 (4S)
form[29,30]. There is evidence that AChE inhibitors do not inhibit all forms equally
well. Studies from our laboratory showed that HupA
preferentially inhibited tetrameric AChE (G4 form), whereas tacrine
and rivastigmine preferentially inhibited monomeric AChE
(G1 form). Donepezil showed pronounced selectivity for G1
AChE in striatum and hippocampus, but not in cortex.
Physostigmine showed no form-selectivity in any brain region.
In cortex, the most potent inhibitors of G4 AChE were HupA
and donepezil. The potent inhibitors of cortical G1 AChE
were donepezil and tacrine. In hippocampus, HupA and
physostigmine were the most potent inhibitors of G4 AChE,
whereas donepezil and tacrine were the most potent against
G1 AChE. In striatum, HupA and donepezil were the most
potent against G4 AChE, and again donepezil was the most
potent against G1 (Table 2). It is well known that
approximately 60%-90% of G4 AChE is ectocellular, and is the major
form for metabolizing ACh. The G4 AChE is the
physiologically relevant form at cholinergic synapses, and its
inhibition would be expected to prolong the action of ACh. The
results mentioned above suggest that the use of AChE
inhibitors in the treatment of AD must consider both
form-specific and region-specific characteristics of AChE
inhibition[12].
Significant inhibition of AChE activity was demonstrated
in the cortex, hippocampus, striatum, medial septum, medulla
oblongata, cerebellum, and hypothalamus of rats that were
killed 30 min following the administration of HupA at several
dose levels compared with the saline
control[26,31,32]. There was a clearly dose-dependent inhibition of AChE in the brain
region by HupA. In contrast to the inhibition of AChE
activity in vitro, the relative inhibitory potency of oral HupA on
cortex AChE was found to be approximately 24- and
180-fold, on an equimolar basis, that of donepezil and tacrine,
respectively[19]. Correlated to the dosage of AChE inhibition,
however, only donepezil and tacrine produced significant
BuChE inhibition in serum[19]. Tacrine was a more potent
inhibitor of serum BuChE than that of brain AChE. The
inhibitory potency of HupA on AChE differs from that of
donepezil and tacrine following different routes of
administration. HupA exerted an almost similar anti-ChE
efficacy in rats following oral and ip administration, whereas
tacrine ip produced a greater inhibition both on brain AChE
and serum BuChE. At doses of 0.03 µmol/L (8 µg) and 0.06
mmol/L (16 µg), HupA significantly inhibited brain AChE
activity 30 min after intraventricular injection, which was less potent than donepezil, but more potent than
tacrine[26]. These findings indicate that HupA, in contrast to donepezil and
tacrine, has higher oral bioavailability and better
penetrability through the blood-brain barrier.
AChE inhibition in rat whole brain reached a maximum at
60 min and was maintained for 360 min following oral
admini-stration of HupA, at a dose of 1.5 µmol/kg (3.6 mg/kg). Peak
inhibition in cortex and serum was observed at 30-60 min,
and inhibition exceeding 10% in the cortex was maintained
between 15-240 min. The BuChE activity recovered to the
control level at 360 min after administration of HupA, whereas
20% and 46% inhibition still existed for donepezil and tacrine,
respectively[19]. The rapid decrease of AChE and BuChE
activity seen in red blood cells and plasma, respectively,
with HupA correlates with the short-lasting and mainly
peripheral side effects[31]. Repeated oral doses (once daily for
8 d and 30 d) of HupA produced no significant difference in
AChE inhibition as compared to a single dose, indicating
that no tolerance to HupA
developed[19,33].
The enantiomers of HupA differ greatly in their ability to
inhibit AChE. At equivalent doses, (+)-HupA was much
weaker than (-)-HupA in inhibiting AChE in NG108-15 cells[34]. Careful measurement of AChE inhibition in cell-free
systems revealed that (-)-HupA was 49-fold more potent
than (+)-HupA as shown both by IC50 values and
Ki (Table 3). The natural isomer was also more selective for AChE, as
shown by the 9-fold higher ratio of BuChE
IC50 versus AChE IC50. Similar differences in anti-cholinesterase potency were
seen when changes in ChE activity in whole brain, cortex
and serum were compared 30 min after oral administration of
(-)-HupA and (+)-HupA. Although both enantiomers of
HupA produced a dose-dependent inhibition of ChE, (-)-HupA was approximately 50-fold more potent than (+)-HupA[34]. This difference in activity measured between
the 2 enantiomers may be partially ascribed to the fact that
the H-bond between the ethylidene methyl of (-)-HupA and
His440 is absent in the (+)-HupA
complex[35,36].
The mechanisms by which HupA inhibits AChE have
been extensively studied by using
kinetics[11,23,37], computer-aided docking
studies[36,38] and X-ray crystallography
approaches[35]. The Lineweaver-Burke plot representation of
the inhibition of rat erythrocyte membrane AChE by HupA
indicates a mixed competitive type of inhibition, because the
intersection of the lines occurs in the second quadrant (Figure
2)[11,22,23]. A similar type of inhibition was found in porcine
brain immobilized AChE[23]. Rat erythrocyte membrane AChE
activity did not exhibit progressive decrease with prolonged
incubation with HupA in vitro, and the AChE activity
recovered to 94% of the control after being washed 5 times, indicating that the inhibitory action of HupA was reversible and
different from that of isoflurophate
(DFP)[11,23].
Immense effort has been directed towards gaining
insights into the binding between HupA and AChE since 1991,
when the 3-D structure of the native TcAChE was
determined by using both X-ray crystallography and molecular
modeling. The 2.5 Å resolved crystal structure of a
Torpedo AChE-HupA complex demonstrated the "ingenious design"
of the natural alkaloid[39] to bind more tightly and specifically
to the enzyme than do other known AChE inhibitors such as
tacrine and edrophonium. Furthermore, the refined
structure clearly identifies the principal protein-ligand interactions
responsible for the efficacy of the inhibitor upon binding to
AChE[35]. The principal interactions include: (i) direct and
strong hydrogen bonds between the carbonyl group of
HupA and the hydroxy oxygen of Tyr130 (located at the
peripheral site of the enzyme), as well as between the thylidene
methyl group and the main-chain oxygen of His440 (a
modality of the catalytic triad); (ii) indirect hydrogen bonds,
mediated by 1 or 2 water molecules, between HupA and residues
of the enzyme that constitute the active center (eg the ring
nitrogen of HupA is hydrogen-bonded to carboxylic oxygen
of Glu199, whereas the -NH3+ group is bonded to hydroxyl
oxygen of Tyr 121); (iii) the cation-p interaction induced
upon binding between the -NH3+
group of HupA and the aromatic rings of Trp84 and Phe 330 at the choline site (it
should be noted that other reversible AChE inhibitors such
as tacrine and edrophonium bind to the same
site)[40]; and (iv) a large number of hydrophobic interactions that are
established between a carbon atom of HupA and the various
oxygen, nitrogen, or carbon atoms of the amino acid
residues comprising the enzyme.
Computer-assisted docking studies and the resolution
of high-resolution crystal structure data for the AChE-HupA
complex provide a valuable platform for the rationalization of
the higher selectivity of the inhibitor, as well as the distinct
thermodynamic stability of the complex. For example, HupA
can form an extra hydrogen bond with Tyr337 within the
choline site that exists only in the mammalian homologue of
AChE, but not in Torpedo enzymes and
BuChE[41,42]. This particular interaction may be largely responsible for the much
stronger inhibitory effect of HupA on mammalian AChE than
that on the other 2 enzymes. In addition, the peptide flip
between Gly117 and Gly118 (induced only by the binding of
HupA) may explain why HupA possesses a longer residence
time than other commonly used anticholinesterase
agents[37].
Dvir et al reported that the oxyanion holes of AChE
composed of Gly117, Gly118, and Gly119 were disrupted by HupA.
The carbonyl oxygens of HupA appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between
Gly117 and Gly118 to undergo a peptide
flip[36]. The new conformation is stabilized by Gly117O making H-bonds with
Gly119N and Ala201N, the other 2 functional elements of the
3-pronged oxyanion hole characteristic of
AChE[36]. It has been suggested that the peptide flip itself is responsible for
the low on-rates observed for AChE inhibition by
(-)-HupA[35]. Its stabilization may contribute to the low rates of
dissociation observed[37].
Effects on cholinergic parameters
To study the effect of HupA on cholinergic transmission
at mouse neuromuscular junctions in vitro, isolated mouse
phrenic nerve-hemidiaphragm preparations were used with
a conventional intracellular recording technique. HupA at a
concentration of 1 µmol/L increased the amplitude,
time-to-peak, and half-life of miniature end-plate potentials (MEPP)
of muscle fiber[43]. HupA had no effect on resting membrane
potentials, the mean quantal content of end-plate potentials
and the frequency of MEPP of muscle fiber, indicating that
the effects of HupA may not be mediated through
presynaptic or postsynaptic mechanisms. In contrast to donepezil
and tacrine, neither the appearance of giant MEPP nor slow
MEPP was changed by HupA, ruling out the possibility of
non-specific promoting effects on terminal ACh
release[44]. In a study of toad paravertebral ganglia (PVG) using
intracellular recording techniques[45], HupA, at concentrations of
0.3, or 1 µmol/L, did not change membrane potential or input
resistance, but increased the rate of orthodromic action
potential evoked by preganglionic stimulation, in contrast to
physostigmine[46] and
tacrine[47]. HupA increased exogenous ACh- but not carbachol-induced depolarization, indicating
that the facilitating effect of HupA on ACh transmission is
mainly mediated by its anti-AChE activity.
Neuronal nicotinic acetylcholine receptors (nAChR) are
involved in cognition and may play a role in AD. Studies on
nAChR in rat hippocampal CA1 interneurons in slices using
patch-clamp techniques showed that HupA had no
significant effect on either the amplitude or kinetics
of a7 nAChR activated by ACh, but slowed the rate of recovery from
desensitization through an indirect mechanism. For
non- a7 receptors, HupA significantly increased the amplitude and
decay phase for responses induced by ACh (but not carbachol), also through an indirect mechanism. The results
suggested that AChEI were likely to be important regulators
of cholinergic signaling in the
hippocampus[48].
In isolated rat phrenic nerve-diaphragm preparation,
HupA significantly increased the amplitude of muscle con
traction induced by stimulating the nerve. The anticurare
effect of HupA was found to be much more potent than that
of neostigmine in anesthetized rat sciatic-tibialis preparation.
The salivation induced by HupA was less potent than that
induced by neostigmine[49].
HupA produced an altered electroencephalography (EEG)
result in conscious rabbits, which showed a decrease in lower
frequency components and the total EEG power in the
cortical area, and the dominant frequency changed from delta
rhythm to theta rhythm in the hippocampus. These effects
are cholinergic in nature and can be reversed by
scopolamine or atropine[49-51].
The acetylcholine potentiating action of HupA has been
observed in the frog rectus abdominus muscle, rat phrenic
nerve diaphragm preparation, guinea pig ileum, and human
iris sphincter muscle. HupA has greater acetylcholine
potentiating activity on vertebrate muscles than does
physostigmine[49,52].
In the hippocampus, high-affinity choline transport was
reduced by 28% after multiple ip doses of 0.5 mg/kg
HupA[33]. Because the effect of HupA was completely reversible with
time and not mediated through a direct interaction with the
transporter, this effect was probably mediated through
regulatory control of high-affinity choline transport in response
to ACh increases following ChE inhibition rather than by
directly acting on the transporter.
Studies on the displacement of [3H]QNB- and
[3H](-)nicotine-specific binding showed that HupA had little direct
effect on cholinergic receptors compared with tacrine and
heptylphysostigmine[31,53]. The concentration required to
display 20% specific binding was 20 µmol/L for
[3H](-)nicotine and 160 µmol/L for
[3H]QNB, indicating that lower concentrations of HupA have a stronger displacing effect on
[3H](-)nicotine- than on [3H]QNB-specific binding. A
stronger effect of a low dose HupA on central nicotinic receptors
may constitute an additional therapeutic advantage in the
treatment of AD. The low level of ACh synthesis in the
cortex of a patient with AD may maintain presynaptic
nicotinic receptors in an active state with no
desensitization[54]. In such a state, nicotinic receptors may become more
sensitive to stimulation by HupA.
HupA at concentrations of 1-100 µmol/L tested
in vitro did not affect the electrically evoked release of
[3H]ACh from rat cortical
slices[31], which contrasted with the decreased
release seen with tacrine, physostigmine, and
metrifonate[55]. This finding suggests that HupA might not have exerted
direct action on the cholinergic presynaptic receptors
controlling ACh release.
Effects on brain neurotransmitters
Compared with other inhibitors being used in the therapy
of AD, HupA produced a more prolonged increase of ACh
levels than did tacrine, donepezil, rivastigmine,
physostig-mine, or metrifonate after systemic
administration[13,31,32,53,56]. There is considerable regional variation in the increase of
ACh levels after HupA administration, with maximal increase
seen in frontal and parietal cortex, and smaller increases in
the striatum and cerebellum[31]. Considering that ACh level
is particularly low in the cerebral cortex of patients with
AD[57], this particular regional specificity produced by HupA may
constitute a therapeutic advantage. A positive correlation
was seen between ACh levels and AChE activity in the
frontal cortex and whole
brain[13,31,32,53]. Studies using
microdialy-sis techniques in conscious, freely moving rats showed that
HupA dose-dependently elevated the level of ACh in cortex
and hippocampus. The time course of cortical AChE
inhibition with HupA mirrored the increase of ACh at the same
doses[13]. This result is consistent with the idea that the
increase in extracellular ACh was due primarily to the
inhibition of cortical AChE. In molar terms, HupA was 8- and
2-fold more potent than donepezil and rivastigmine,
respec-tively, in increasing cortical ACh levels, with a
longer-lasting effect (Figure 3)[13]. Tolerance or accumulation of
increasing ACh level was not formed after multiple
administration of HupA (Liang YQ et al, unpublished data). HupA did
not alter choline levels[32] or the activity of choline
acetyltransferase (ChAT) in any region of the rat brain
assayed[32,33], suggesting that the increase of ACh levels by
HupA was not likely to be mediated through an increase in
the rate of ACh synthesis.
Brain norepinephrine (NE) and dopamine (DA) levels
increased significantly following either systemic
administration of HupA or local administration of HupA through
microdialysis, but 5-HT level was not
affected[56]. HupA produced an 11- and 2-fold more potent increase in the DA level
of the middle prefrontal cortex than donepezil and
rivastigmine, respectively. The increasing effect of HupA
on DA level was more potent than that on NE level (Liang
YQ et al, unpublished data). Oxotremorine (a muscarinic
agonist) and mecamylamine (a nicotinic antagonist)
completely blocked the increasing effects of HupA on DA and
NE levels, suggesting that ACh regulation by presynaptic
ACh muscarinic receptors or nicotinic receptors accounts
for the effect of HupA on DA and NE. These effects may be
involved in the memory improvement effected by HupA
(Liang YQ et al, unpublished data).
adult rhesus monkeys or those occurring naturally in aged
monkeys using a delayed-response
task[66]. HupA (0.01-0.1 mg/kg, im) improved the memory deficits induced by
scopolamine in young adult monkeys. In aged monkeys, HupA
(0.001-0.01 mg/kg, im) significantly increased choice
accuracy in delayed response performance (Figure 4). The
beneficial effects of HupA were long lasting. Monkeys retained
improved performance for approximately 24 h after a single
injection of HupA. Given that the NE and DA levels
decreased significantly with aging in
monkeys[75], the effect of HupA on memory may be associated with increased levels
of NE and DA. HupA (0.01-0.1 mg/kg, im) also produced
significant improvement on reserpine- (a catecholamine
depleting agent) and yohimbine- (an a2-adrenoceptor
antago-nist) induced memory impairments in monkeys
(Figure 5). These effects are primarily due to the increase of NE level by
HupA, which might stimulate the a2-adrenoceptor in the prefrontal cortex to improve the delayed response
performance[67].
Deposition of b-amyloid protein is considered a crucial
event in initiating the neuritic and neuronal degeneration in
AD, which mainly affects the areas involved in cognitive
function, such as cortices, some limbic structures, and the
forebrain nuclei projecting to those areas. Repeated icv
infusion of b-amyloid protein-(1-40) induced marked amnesic
effects along with signs of neurodegeneration and
extracellular amyloid deposits throughout the frontoparietal cortex
and hippocampus, which indicated that b-amyloid protein
deposition in the brain was related to cognitive impairment,
hypofunction of cholinergic neurons and neuronal death.
Daily intraperitoneal administration of HupA for 12
consecutive days produced significant reversals of the
b-amyloid-induced deficit in learning a water maze task (Figure 6).
Treatment with HupA also attenuated the neuronal degeneration
induced by Ab1-40 in the cortex and hippocampus,
indicating that neuroprotection is involved to some extent in the
favorable effect of HupA on Ab-induced memory
deficits[76].
It is well-documented that
N-methyl-D-aspartate (NMDA)-receptor activation mediates the generation of
long-term potentiation (LTP), a cellular process that underlies
learning and memory[77,78]. There is evidence that the
suppressive action of Ab on LTP in both CA1 and dentate gyrus
operates via an NMDA receptor-independent pathway that
involves cholinergic terminals in the hippocampus. It is of
some interest that HupA (1.0 µmol/L) was found to enhance
LTP, but at a much lower dose (0.1 µmol/L) largely blocked
the suppressive effects of Ab on LTP
induction[79,80], which might involve the mechanism of HupA against
Ab-induced cognitive deficits.
Enhancing effects on cognition
HupA has been found to be an effective cognition
enhancer in a number of different animal species.
Enhancement of learning and memory performance was demonstrated
in passive footshock avoidance[58-61], water maze escape task[63,64], and spatial discrimination in a radial arm
maze[10,65], as well as in delayed response performance in
monkeys[66,67]. Beneficial effects were seen not only in intact adult rodents,
aged rodents[58,64] and
monkeys[66], but also in rodents cogni-tively impaired by
scopolamine[10,31,50,60-63,68],
AF64A[26,69],
electroshock[61,68],
cycloheximide[61],
NaNO2[61],
CO2[58,60], and
D-galactose[70]. Inverted U-shaped dose-response curves
typical of cognition enhancers were found with HupA. The
duration of improvement induced by oral HupA on learning
and memory retention processes were longer than those
induced by physostigmine, galanthamine, and tacrine,
respectively[71]. HupA has a higher efficacy than tacrine and
donepezil given orally[19]. The improvement effected by
HupA was more pronounced in working memory than in
reference memory[10], which may benefit AD patients because
the cognitive deficits in memory of recent events are more
severe in AD. In aged rats, HupA could significantly reduce
the latent period of finding the platform and increased the
time in the probe quadrant in the Morris water maze
performance[64]. In addition, HupA improved cognition in
cholinergically lesioned rats[58,61] and the spatial working memory
deficit induced by lesions in the nucleus basalis
magnocellu-laris[72].
HupA reversed memory deficits induced by bilateral
injection of scopolamine and muscimol (a
GABAA agonist) into hyperstriatum ventrale in chicks. The improvement was
observed from 30 min to 90 min, but not at 10 min after
training, indicating that HupA participated in the
modulation of intermediate-term memory and long-term memory
formation in a passive avoidance task. This finding suggests
that HupA improved memory formation not only by acting
as a highly potent inhibitor of AChE, but also by
antagonizing the effects mediated through the
GABAA receptor[62]. It is well known that deficiencies in ACh and GABA content
have been observed in the cortical regions of AD
brains[73]. Anatomical evidence also suggests an important interaction
between GABA and cholinergic neurons in the septum and
hippocampus[74]. Thus, a drug that is able to enhance
synaptic ACh and also antagonize the
GABAA receptor could be ideal for treatment of AD.
To extend the antiamnesic effect of HupA to non-human
primates, HupA was evaluated for its ability to reverse the
deficits in spatial memory produced by scopolamine in young
Apart from AD, the most common dementia in the elderly
is vascular dementia (VD). This disorder, like AD, presents
as a clinical syndrome of intellectual decline produced by
ischemia, hypoxia, or hemorrhagic brain lesion. Rats with
permanent bilateral ligation of the common carotid arteries
exhibit learning and memory impairments and neuronal
damage resembling those in VD. In these rats, daily oral
administration of HupA produced a significant improvement of the
deficits in learning the water maze task, beginning 28 d after
ischemia, along with approximately 33%-40% inhibition of
AChE activity in the cortex and
hippocampus[81]. Similar cognitive improvement of HupA was observed in a gerbil
model of transient global ischemia[82]. The protective effects
of HupA against hypoxic-ischemic (HI) brain injury were also
found in neonatal rats. Unilateral HI brain injury was
produced by ligation of the left common carotid artery followed
by 1 h hypoxia with 7.7% oxygen in 7-d-old rat pups. After 5
weeks, HI brain injury in rat pups resulted in working memory
impairments as shown by increased escape latency in a
water maze and reduced time spent in the target quadrant. Rats
treated with HupA at doses of 0.05 or 0.1 mg/kg ip for 5
weeks after HI injury performed better than saline-treated HI
rats, and neuronal damage in the ipsilateral hemisphere was
attenuated (Figure 7). These findings suggest that HupA
might be beneficial in the treatment of HI encephalopathy in
adults and neonates[83].
Neuroprotective effects
Several neurodegenerative disorders such as AD,
cerebral ischemia-reperfusion injuries and head injuries are
thought to be related to changes in oxidative metabolism.
Increased oxidative stress, resulting from free radical
damage to cellular function, can be involved in the events
leading to AD, and is also connected with lesions called tangles
and plaques. Plaques are caused by the deposition of
Ab and are observed in the brains of AD
patients[2,84-86]. Studies show that oxygen radicals initiate amyloid build-up, leading
to neurodegeneration[85,87]. HupA has been found to protect
against H2O2- and Ab-induced cell lesion, decrease the level
of lipid peroxidation, and increase antioxidant enzyme
activities in rat PC12 and NG108-15 cell lines and primary cultured
cortical neurons (Figure 8)[34,88-91]. Following 6 h exposure of
the cells to H2O2 (200 µmol/L) or 48 h exposure to
Ab25-35 (1 µmol/L), a marked reduction in cell survival, activity of
glutathione peroxidase (GSH-Px) and catalase (CAT), as well as
increased production of reactive oxygen species (ROS) and
malondial-dehyde (MDA) were observed. Pretreatment of
the cells with HupA (0.1-10 µmol/L) 2 h before
H2O2 or Ab exposure significantly elevated cell survival. HupA reversed H2O2- and Ab-induced decreases in GSH-Px and CAT activity,
as well as causing increases in the production of ROS, MDA
and superoxide dismutase (SOD).
Oxygen-glucose deprivation (OGD) for 30 min caused
death in more than 50% of rat pheochromocytoma PC12 cells,
along with major changes in morphology and biochemistry,
including elevated levels of lipid peroxide, SOD activity and
lactate concentration. Cells pretreated for 2 h with HupA (0.1, 1, or 10 µmol/L), however, had increased survival and
reduced biochemical and morphologic signs of toxicity.
HupA protected PC12 cells against OGD-induced toxicity
most likely by alleviating disturbances of oxidative and
energy metabolism[92].
In rat studies, intracerebroventricular infusion of
b-amyloid1-40 (800 pmol×3) induced significant morphological
injury and decreases in cortical ChAT activity. Daily ip
administration of HupA for 12 consecutive days attenuated the
loss of ChAT activity in the cerebral cortex and the neuronal
degeneration induced by
b-amyloid1-40[76].
MDA level and manganese-SOD (Mn-SOD) activity in
hippocampus, cerebral cortex, and serum of aged male rats
were 2.3-2.8 times and 1.8-2.8 times greater, respectively,
than those of adult male rats. HupA (0.05 mg/kg, ig)
markedly lowered the levels of MDA and the activities of
Mn-SOD in aged male rats following 7-14 consecutive days of
daily administrations[93]. A reduction of oxygen free radicals
in the plasma and erythrocytes was also demonstrated in a
clinical study[16].
In chronic cerebral hypoperfused rats, HupA restored
the decrease in ChAT activity in the hippocampus, improved
neuronal morphological damage, and restored SOD and lipid
peroxides activities, as well as lactate and glucose
concentrations to their normal
levels[81]. Similar protective effects
of HupA were observed in the studies of transient global
ischemia in gerbils[82]. The protective effect of HupA on HI
brain injury has also been found in neonatal
rats[81]. A unilateral HI brain injury was produced by the ligation of the left
common carotid artery (CCA) followed by 1 h hypoxia with
7.7% oxygen in 7-d-old rat pups. After 5 weeks, the HI brain
injury in rat pups caused damage in the ipsilateral striatum,
cortex and hippocampus, as well as a marked reduction in
CA1 neuron density. These neuropathologic signs were
attenuated by the administration of HupA at a dose of 0.1
mg/kg (Figure 9). These results raise the possibility that
HupA may be potentially useful in treating HI
encephalopathy in neonates.
In studies carried out to examine the stereoselectivity of
the cellular protective effect induced by (-)-HupA, it was
found that (-) and (+) HupA exerted similar potency in pro
tecting against the cellular toxicity induced by
Ab25-35. This result contrasted with the stereoselectivity of cholinesterase
inhibition in vitro and in vivo (Figure
10)[34]. The ability of HupA to inhibit the catalyzing activity is not parallel to its
neuroprotective effect, implying that the cytoprotective
effect of the 2 enantiomeric forms of HupA might relate to the
non-catalytic action of AChE.
It was recently reported that HupA exerted a
neuroprotec-tive effect via modulating the intracellular
Ca2+ ([Ca2+]i) level
together with the mRNA transcription of calmodulin (CaM)
and calmodulin-dependent protein kinase II (CaMPK II) in
hippocampal neurons[94]. Mice given repeated CCA
ligation-reperfusion treatment showed a marked increase in
[Ca2+]i, and a decrease in CaM and CaMPK II mRNA levels in
hippocampal neurons. Daily oral administration of HupA
(0.05 mg/kg) for 30 consecutive days significantly reversed
the shift in [Ca2+]i, CaM and CaMPK II mRNA levels induced
by ischemia.
The findings mentioned above indicate that HupA has
protective effects against free radical-, ischemia- and
Ab-induced cell toxicity, which might be beneficial in the
treatment of patients with AD and VD.
Apoptosis is the process by which neurons die during
normal development and is also a feature of chronic and
acute neurodegenerative diseases and
stroke[95]. In accordance with previous reports, studies from our lab
demonstrate typical apoptotic changes when neuron-like cells are
exposed to stressors such as
H2O2[96], Ab
peptides[91], oxygen-glucose deprivation
(OGD)[97], serum
deprivation[98], the protein kinase C (PKC) inhibitor
staurosporine[99], and global
ischemia[82]. These changes include DNA laddering
(Figure 11), cell shrinkage, generation of nuclear apoptotic bodies,
terminal deoxyribonucleo-tidyl transferase-mediated
dUTP-digoxigenin nick end-labeling (TUNEL) positive staining
(Figure 12), chromatin condensation (Figure 13), and other
classic hallmarks of apoptosis (Figure
14)[91,96,97,99]. Such
abnormalities are markedly relieved by HupA.
Administration of HupA (0.1 or 0.2 mg/kg, ip, per day) for 12
consecutive days conferred substantial neuroprotection on rats that received icv injections of b-amyloid1-40
(800 pmol×3): the number of apoptotic-like neurons were markedly
reduced[76]. In primary cultured neurons, preincubation with HupA at
concentrations higher than 0.01 µmol/L led to a large
dose-dependent attenuation of cell toxicity induced by
Ab25-35[91]. Moreover, HupA (1 µmol/L) caused large reductions in the
amounts of subdiploid DNA detected in a flow cytometry
assay and weakened the ladder pattern on agarose gel
electrophoresis, which is typically seen after exposure to
Ab (Figure 11)[91]. The inhibition of ROS formation may involve the anti-apoptotic actions of HupA[91].
The cellular commitment to apoptosis is regulated by
the Bcl-2 family of proteins. High levels of Bcl-2 expression
will inhibit apoptosis. In contrast, an increased expression
of P53 and Bax is associated with the initiation of
apoptosis[100]. Treatment with HupA attenuated
H2O2-, Ab- and OGD-induced overexpression of mRNA and protein levels for c-jun,
Bax and P53, and downregulated that of Bcl-2 to normal
levels (Figure 15)[76,96,97].
In the mitochondrial-mediated cell death pathway, a key
step is transient opening of the mitochondrial permeability
transition (MPT), involving a non-specific increase in the
permeability of the inner mitochondrial
membrane[101,102]. In this process, cytochrome c moves from the intermembrane
space into the cytoplasm[103], where it binds to Apaf-1 (the
molecular core of apoptosome, which executes
mitochondria-dependent apoptosis). In the presence of dATP, this
complex polymerizes into an oligomer known as the apoptosome. The apoptosome activates the protease,
caspase-9, which in turn activates caspase-3. The cascade
of proteolytic reactions also activates DNase, which leads
to cell death[104]. Our recent results showed that PC12 cells,
when pre-incubated with HupA at concentrations above 0.01 µmol/L, were markedly protected against apoptosis
induced by Ab, with a significant reduction in mitochondrial
swelling and improved mitochondrial membrane potentials
(Gao X et al, unpublished data).
A series of studies were carried out in our laboratory that
focused on caspase activation in primary cultures of rat
cortical neurons subjected to a variety of stresses.
Measurements of caspase-3-like fluorogenic cleavage demonstrated
that HupA (1 µmol/L) attenuated the Ab25-35-induced
increase in caspase-3 activity at 6, 12, 24, and 48
h[91]. Western blot analyses confirmed these results at the protein level.
HupA also inhibited caspase-3 activation in models of
apoptosis induced by serum deprivation and staurosporine
treatment. The apoptosis induced by serum deprivation for
24 h was accompanied by enhanced caspase-3 activity and a
release of mitochondrial cytochrome c into the
cytosol[98]. HupA (0.1-10 µmol/L) improved neurons survival, inhibiting
the rise in caspase-3 activity and protein expression (Figure
16)[98]. Likewise, cell survival was greatly enhanced when HupA
(0.1-100 µmol/L) was introduced 2 h before 24 h exposure to
0.5 µmol/L staurosporine. Staurosporine-induced DNA
fragmen-tation, upregulation of the pro-apoptotic gene
bax, downregulation of the antiapoptotic gene
bcl-2, and decrease in caspase-3 proenzyme protein level were all attenuated by
HupA at dose of 1 µmol/L[99].
A potassium channel with delayed rectifier characteristics may play an important role in Ab-mediated
toxicity[105]. The upregulation of an outward
K+ current known as
Ik mediates several forms of neuronal apoptosis and might
specifically contribute to the pathogenesis of Ab-induced neuronal
death. Exposure to 20 µmol/L Ab25-35 or Ab1-42 is known to
enhance the apoptosis-related current,
IK[106]. Expression of
wild-type PS-1 or PS-2 increases outward
K+ current densities in HEK293 cells relative to untransfected or
mock-transfected cells[107]. These data raise the intriguing possibility
that manipulations aimed at reducing outward
K+ current may provide an approach to reducing neuronal
degeneration in patients with AD.
HupA reversibly inhibited the fast transient potassium
current (IA) in CA1 pyramidal neurons acutely dissociated
from rat hippocampus. The effect was voltage-independent
and insensitive to atropine. HupA slowed down the decay
of IA and its recovery from inactivation and showed no effect
on steady-state inactivation, but hyperpolarized the
activation curve of IA by 6 mV, suggesting that HupA may act as a
blocker at the external mouth of the A
channel[108]. In addition, HupA inhibited another important outward
K+ current, the sustained potassium current
(IK) in a voltage-dependent manner in acutely dissociated rat hippocampal neurons. The
effect was insensitive to atropine. HupA hyperpolarized the
activation curve of the current by 16 mV, and markedly
prolonged the decay time constant
t2[109]. Because outward K+ current has proven very important in apoptosis induction,
the reversible inhibitory effects of HupA on
IA and IK might
contribute to its anti-apoptotic effect.
In light of these findings, we can conclude that HupA, in
addition to being a potent, highly specific and reversible
inhibitor of AChE, possesses the ability to protect neurons
against cytotoxicity and apoptosis induced by
H2O2, Ab, OGD, ischemia, serum deprivation and staurosporine. The
protective and anti-apoptotic actions of HupA may involve
inhibiting the production or the effects of ROS, improving
energy metabolism, regulating apoptosis-related gene
expression, protecting mitochondrial function, as well as
modulating intracellular Ca2+ concentrations and inhibiting
outward K+ currents. The neuroprotective effect of HupA is
not correlated with its AChE inhibitory activity. These
findings suggest that the therapeutic effects of HupA may be
exerted via a multi-target mechanism.
Protection of HupA against glutamate-induced
cytotoxicity
Glutamate is the main excitatory neurotransmitter in the
central nervous system (CNS), with important roles in neu
rotransmission and functional plasticity. Excitatory amino
acid neurotransmitters are also involved in CNS pathology.
The deleterious effects of overstimulation with excitatory
amino acids have been implicated in a variety of acute and
chronic neurodegenerative disorders, including ischemic
brain damage, AD and neuronal cell
death[110-115]. Glutamate-mediated overactivation of receptors induces excessive
Ca2+ influx, which results in elevated intracellular
Ca2+ concentrations[116,117] with serious consequences such as necrosis and
apoptosis[118]. Blockade of glutamate receptors prevents
most of the Ca2+ influx and neuronal cell death induced by
glutamate exposure[119,120].
It has been reported that HupA protects against
gluta-mate-induced toxicity. HupA (100 µmol/L) was found to
decrease neuronal cell death caused by a toxic level of glutamate.
In those experiments, HupA reduced glutamate-induced
calcium mobilization but did not affect the increase in
intracellular free calcium induced by exposure to high KCl or a
calcium activator Bay-K-8644[121]. HupA dose-dependently
inhibited the NMDA-induced toxicity in primary neuronal cells,
most likely by blocking NMDA ion channels and inhibiting
the subsequent Ca2+ mobilization at or near the
phencyclidine (PCP) and MK-801 ligand
sites[122]. HupA reversibly inhibited NMDA-induced current in acutely dissociated rat
hippocampal pyramidal neurons and blocked specific
[3H]MK-801 binding in synaptic membranes from rat cerebral
cortex[123]. Of all AChE inhibitors tested, HupA is the most powerful
both in protecting mature neurons and in blocking the
binding of [3H]MK-801. The effect was non-competitive, and
showed neither "voltage-dependency" nor
"use-dependency"[124]. HupA acts as a non-competitive antagonist of
the NMDA receptors, via a competitive interaction with one
of the polyamine binding sites[125]. It is interesting that
natural (-)-HupA and synthetic (+)-HupA reduced the binding of
[3H]MK-801 with similar
potency[126], indicating that HupA inhibits NMDA receptors in rat cerebral cortex without
stereoselectivity. This result is in dramatic contrast with the
stereoselective inhibition of acetylcholinesterase.
Neuronal cell death caused by overstimulation of
gluta-mate receptors has been proposed as the final common
pathway for a variety of neurodegenerative diseases, including
AD. The ability of HupA to attenuate glutamate-mediated
neurotoxicity may be one additional reason for considering
this agent as a potential therapeutic for dementia and as a
means of slowing or halting the pathogenesis of AD at an
early stage[122].
Effects on secretory amyloid precursor protein
and protein kinase C-a
Ab is a self-aggregating 39-43-amino-acid peptide that
originates from a larger polypeptide that is known as the
Alzheimer¡¯s amyloid precursor protein (APP). Alternate
pathways for APP processing have been described: the
non-amyloidogenic secretory pathway, which releases a soluble
ectodomain (APPs) and prevents Ab
formation[127], and the endosomal-lysosomal pathway, which produces
amyloido-genic products[128]. The amyloid hypothesis of
AD[129,130], which is focused on the potential toxic role of an excessive
production of Ab, suggests that the aberrant metabolism of
APP is a central pa, thogenetic mechanism for the disease.
Several factors can affect the secretory
non-amyloido-genic pathway of APP. For example, the stimulation of
phospholipase C (PLC)-coupled receptors, such as muscarinic
m1 and m3, has been shown to potentiate the secretion of
APPs in cell cultures. These effects are probably mediated
mainly by PKC[131]. It has also been reported that several
AChEI affect APP processing in addition to the catalytic
function of AChE[6,132,133].
HupA can alter the disturbance of PKC and APPs
induced by Ab in both rats and the HEK293sw cell
line[134]. The levels of APPs and PKCa were significantly decreased
after infusion with Ab1-40 in rats. HupA caused a marked
reduction of these changes (Figure 17), but had no
significant effects on APPs or PKCa in normal rats. In HEK293sw
cells, HupA increased the levels of APPs and PKCa, but had
no effect on the levels of PKCd and PKCe. These findings
suggest that HupA may reduce APP processing through
upregulating PKC, especially PKCa levels.
In an attempt to clarify the receptor mechanisms involved
in such effects, we found that HEK293wt cells treated with
scopolamine, a nonselective muscarinic antagonist, partly
blocked the HupA-induced increase in the levels of APPs
and PKC. In contrast, the nicotinic antagonist mecamylamine
had no effect, suggesting that stimulation of the
non-amyloidogenic secretory pathway of APPs metabolism by
AChE inhibition is probably accomplished via an effect on
the muscarinic receptors/PKC cascade (Yan H et
al, unpublished data). Because PKC is a key enzyme in signal
transduc-tion, and because APPs itself has neuroprotective effects,
modulating the levels of these 2 proteins by HupA may be
beneficial in AD therapy.
Effects on nerve growth factor
Nerve growth factor (NGF) is a member of the neurotrophin
family that promotes the survival and outgrowth of central
cholinergic neurons[135]. The decrease in trophic support for
the neurons in the aging brain is associated with neuronal
death and appearance of neurodegenerative disorders such
as AD[136]. Accumulated data suggest that cholinergic
mechanisms are involved in the regulation of NGF synthesis and
release, and some AChE inhibitors are known to exert
NGF-like activities by potentiating the neuritogenic effect of NGF[137]. HupA has been shown to increase neurite
outgrowth from undifferentiated PC12 cells (Figure
18) and to enhance the expression and secretion of NGF, as well as
increase p75NTR mRNA in primary astrocytes
(Figure 19). These effects suggest the possibility that HupA increases
the NGF-induced enhancement of neuron survival and
function improvement, which is helpful in the rescue of injured
neurons in neuro-degenerative disease. In addition to
inhibiting AChE activity, AChE mRNA expression and protein
levels were significantly upregulated after treatment with
HupA. Accumulating evidence indicates that AChE may
influence neurite outgrowth through a non-catalytic
mechanism[138]. Hence, the effect of HupA on neurite outgrowth
may be associated with the level of AChE
expression[139].
Neurotrophic factors such as NGF not only promote the
survival of responsive neurons but also protect them from
oxidative injury. Our study with SH-SY5Ycells used
H2O2 to generate an oxidative stress sufficient to cause cell loss along
with a substantial decrease in the mRNA and protein levels
of NGF, neurotrophin receptor p75
(p75NTR) and tyrosine kinase A (TrkA). HupA not only reduced the overt signs of
cytotoxicity, but also preserved the expression of NGF and
its receptors (Figure 20). These neuroprotective effects of
HupA on H2O2-induced cytotoxicity were blocked by the
TrkA phosphorylation inhibitor K252a, and were antagonized
by the mitogen-activated protein (MAP) /extracellular
signal-regulated kinase (ERK) inhibitor PD98059 (Figure
21). These findings indicate that the NGF and TrkA receptor
mediate key events required for the neuroprotective actions
of HupA. Among the downstream signaling events
triggered by the action of NGF at the TrkA receptor, activation
of the MAP/ERK kinase pathway may be particularly
important for the ability of HupA to protect SH-SY5Y cells against
oxidative stress[140].
Pharmacokinetics
The pharmacokinetics of HupA have been studied in rodents and healthy human volunteers. HupA is absorbed
rapidly, distributed widely in the body, and eliminated at a
moderate rate (Table 4). The levels of HupA in the blood
following iv or po administration of
[3H]HupA in rats declined in 2 phases, namely the distribution phase and
elimination phase. The oral bioavailability was 96.9% in mice,
with the highest radioactivities in the kidney and liver. The
majority of the radioactivity was excreted in the urine 24 h
after iv administration of [3H]HupA. Only 2.4% was
recovered from the feces. Paper chromatograms of rat urine
revealed that [3H]HupA was excreted partially as prototype
and its metabolite[141]. Autoradiographic studies in mice
showed that HupA was present in all regions of the brain,
but was particularly concentrated in the frontoparietal cortex,
striatal cortex, hippocampus, and nucleus accumbens after
iv injection[32].
The plasma concentration-time curves of HupA in dogs
were determined by using the liquid chromatography- mass
spectrum-mass spectrum (LC-MS-MS) method after the last
intramuscular injection (10 µg/kg per day for 15 d) of a
sustained-release formulation, and the mean
Cmax was 0.36±0.08 ng/mL, which occurred at 48.0±24.5 h. The mean plasma
elimination half-life was 54.8±5.6 h, and the mean area under
the plasma concentration versus time curve was 92.6±4.5 ng·
h/mL[142]. A pharmacokinetic study using HupA transdermal
patches in 6 beagle dogs showed that the HupA patches
were able to deliver sustained or controlled drug release
in vivo. Following application of the first patch (4 mg per 20
cm2), HupA concentrations in serum increased for
approximately 12-24 h, reaching an average maximum concentration
of 3.4 ng/mL. Thereafter, blood concentrations were
maintained up to 84 h during the period in which the patches were
worn[143].
In young healthy volunteers, HupA levels in plasma were
determined by reverse phase high performance liquid
chromatography (HPLC) by using a spectrophotometric detector.
The time course of plasma concentration conformed to a
one-compartment open model with first-order absorption
following oral administration of 0.99 mg HupA. HupA was
rapidly absorbed and widely distributed in
vivo[144]. The half-life of HupA was at least 4-17 times longer than that of
tacrine or physostigmine[145]. A pharmacokinetic
comparison between young and elderly human volunteers treated
with 0.225 mg HupA was recently conducted. The areas
under the blood level-time curve
(AUC0®36) was 75.0 and 97.2 nmol/L·h in young and elderly volunteers, respectively. The
mean maximum blood concentrations
(Cmax) were 9.1 and 6.8 nmol/L, respectively, and the elimination half-lives
(T1/2) of HupA were 10.0 and 13.0 h, respectively. Thus the mean
residence times (MRT0-72) were 10.3 and 12.2 h, respectively
(Chen Y et al, unpublished data). The elimination of HupA
in the elderly volunteers was slower than that in the young
volunteers. HupA was released in a slow and prolonged manner after oral administration.
To identify which cytochrome P450 (CYP) isoenzymes
are involved in the metabolism of HupA, an in
vitro study was performed with rat liver microsomes, and
immunoinhibi-tion and chemical inhibition methods. HupA metabolism was
analyzed with HPLC and expressed as the HupA
disappearance rate. The results showed that 76.2% of HupA
metabolism was inhibited by a CYP1A2 antibody and 17.8% by a
CYP3A1/2 antibody. The inhibitory effects produced by
CYP2C11 and 2E1 antibodies were minor. The CYP1A2
substrate phenacetin produced an inhibitory effect of 70.3%.
These data suggest that HupA metabolism in rat liver
microsomes is mediated primarily by CYP1A2, with a probable
secondary contribution by CYP3A1/2. CYP2C11 and 2E1
are probably not involved in HupA
metabolism[146].
To predict possible drug interactions and confirm the
safety of HupA as a medication, the effects of HupA on the
activity and expression of CYP were examined. Liver
microsomes and total mRNA were prepared from rats treated
orally with 0, 0.1, 1, or 2 mg/kg HupA for 2 weeks.
Phenobarbital, 3-methylcholanthrene (3-MC), ethanol, and
dexamethasone were used as positive controls. No change
in isoenzyme expression or catalytic activity was found in
rats treated with 0.1 mg/kg HupA, but CYP1A2 activity and
levels of CYP1A2 protein and mRNA were increased when
treated with HupA at doses of 1 and 2 mg/kg, although they
were minor when contrasted with 3-MC. HupA produced no
effects on CYP2C11, CYP2B1/2, 2E1, or 3A. These results
indicate that the activity and expression of liver CYP
isoenzymes are not affected in rats treated with pharmacological
doses of HupA, but may elicit a slight inductive response in
CYP1A2 at a toxicological dose. The CYP1A2 induction
produced by HupA is related to transcription
enhancement[147].
Toxicology and detoxification
A series of studies have been conducted to evaluate the
toxicity of HupA in mice, rats, rabbits, and dogs.
Dose-response curves for salivation indicated that HupA was less
potent than other ChE inhibitors[49]. The characteristic
symptoms of cholinergic hyperactivity were less severe for HupA
in rats compared with donepezil and tacrine; fasciculation or
other cholinergic signs were not found with oral HupA at a
dose of 0.48 mg[19]. The
LD50 doses of HupA were 4.6 mg
(po), 3.0 mg (sc), 1.8 mg (ip) and 0.63 mg (iv) in mice.
Atropine exerted a significant antagonistic effect on the toxicity
induced by HupA. Studies to evaluate subacute toxicity
have been conducted in rats, rabbits, and dogs, in which no
histopathological changes were found in liver, kidney, heart,
lung, or brain in rats (1.5 mg/kg, po) or dogs (0.6 mg/kg, im)
after administration of HupA for 180 d. No teratogenic effect
was detected in mice (0.019-0.38 mg/kg, ip) or rabbits
(0.02-0.2 mg/kg, im) after the administration of HupA.
To examine the acute effects of HupA on rat liver, changes
in liver coefficient, serum biochemistry, and histopathology
were detected after a single dose. The cytotoxicity of HupA
was also assessed by determining extracellular and
intracellular amounts of lactate dehydrogenase in cultured
hepato-cytes. Similar to tacrine, HupA raised the liver coefficient
and increased serum levels of aspartate aminotransferase
and alanine aminotransferase. Unlike tacrine, however, acute
administration of HupA did not induce histopathological
changes in the liver. That atropine redressed the effects of
HupA on the liver indicates that the acute effects of HupA
on rat liver are not related to
hepatotoxicity[148].
A study in mice also demonstrated that HupA did not
perturb respiration at a dose inhibiting 40% of AChE, and at
a lethal dose, did not affect any other enzyme important for
respiration[149].
HupA has been tested as a prophylactic drug against
poisoning with soman and other nerve gases with
reasonable outcomes[150]. It works by protecting cortical AChE from
soman inhibition and preventing subsequent seizures.
Several studies have reported that acute administration
of HupA protects rodents against organophosphate (OP)
intoxication without the typical cholinergic
side-effects[150-152]. Similarly, in primates (Chinese rhesus monkeys), HupA by
itself has been shown to protect animals against the toxic
signs and lethality induced by the injection of 1.3
LD50 of soman[151]. When compared with pyridostigmine
(PYR)[153,154], the cumulative dose of soman needed to produce
convulsions and epileptic activity was 1.5-fold greater in animals
that received HupA compared with the group of primates
pretreated with PYR. HupA also selectively inhibited red
cell AChE activity , whereas PYR also inhibited plasma BuChE.
This result was confirmed in a study with
guinea-pigs[155]. PYR cannot protect against seizures and subsequent
neuropathology induced by OP agents, because it does not
penetrate into the brain. HupA, when combined with atropine
methyl nitrate or without any supporting therapy acting on
the CNS (atropine sulfate or benzodiazepine), prevented
lethality and manifested anticonvulsant and central
neuropro-tective properties[155]. The superior protection offered by
HupA appears to be related both to the selectivity of HupA
for red cell AChE, which preserves the scavenger capacity
of plasma BuChE for OP agents, and to its protective effect
on cerebral AChE[154]. HupA is more stable than the
carbamates used as pretreatment for OP poisoning. These prophy
lactic effects make HupA a potential protective agent against
OP intoxication.
Clinical trials
The efficacy and safety of HupA in AD patients have
been evaluated by clinical trials in China. In an early
double-blinded study conducted in 100 elderly patients with 17
probable cases of AD, HupA (0.03 mg, im) produced a significant
improvement in all rating scores as evaluated by Buschke
Selective Reminding performance[156]. A more
comprehensive clinical study was conducted in 819 patients who met
the AD criteria of National Institute for Communicative
Disorders and Stroke-Alzheimer¡¯s Disease and Related
Disorders Association (NINCDS-ADRDA) and Diagnostic and
Statistic Manual of Mental Disorders-Third Edition-Revised
(DSM-III-R) at 39 mental hospitals in China. After treatment
with HupA at a dose of 0.03-0.4 mg/d, patients showed
improvement in their memory, cognitive skills, and ability in
their daily life. No severe side effects were
found[14-16,157-170]. The results from a 12-week, double-blinded, randomized and
placebo-controlled nationwide clinical trial with 202 patients
with the diagnosis of possible or probable AD confirmed the
efficacy of HupA in improving the results of cognitive tasks.
In this study, their cognitive functions [measured with
Mini-mental State Examination Scale (MMSE) and Alzheimer¡¯s
Disease Assessment Scale-Cognitive Subscale (ADAS-Cog)], non-cognitive functions (measured with mood and
behavior-ADAS-non-Cog) and activity of daily living (ADL)
were all improved significantly at week 6, and further
improved at week 12 after patients had been treated with HupA
at a dose of 0.1-0.2 mg, bid[17]. The results of a clinical trial
with HupA carried out over a longer time period were
reported by Jiang et al, and showed that HupA at a dose of
0.15 mg twice per day significantly improved the cognition of
33 AD patients at 12, 24, and 48 weeks, with no statistical
difference among the 3 time points[164].
Combined therapy with HupA plus other medicine or
mental training also showed favorable clinical results. In a
placebo-controlled clinical trial with 24 AD patients and 35
VD patients, subjects showed significant favorable
differences in MMSE and ADL scores after treated with HupA (0.15 mg, twice per day) plus nicergoline (20 mg, twice per
day), HupA plus nicergoline and conjugated estrogen, or
HupA alone; combined treatment was better than HupA alone[171]. The superiority of combined therapy over HupA
alone was also suggested by the marked improvement in
MMSE, clinical dementia rating (CDR) and ADL scores from
a clinical trial, in which 30 female AD patients were treated
with 2 mg nilestriol once every fortnight, and 0.1 mg HupA
twice per day for 24 weeks[172]. In a group of 43 patients with
mild to moderate AD, American Association of Mental
Deficiency (AAMD) and ADL scores were significantly improved
after treatment with HupA (0.1 mg, twice per day) combined
with training in daily life activities for 8
weeks[173]. After 22 patients with AD and 38 with VD who were treated for 8
weeks with HupA (0.1 mg, twice per day) complemented with
a mental stimulation program consisting of reminiscence,
reality orientation and remotivation, Hasegawa dementia
scale (HDS), CDR and ADL scores were significantly
improved[174]. These results suggest that combined treatment
using HupA might be a reasonable means in the clinical
therapy.
The therapeutic effects of HupA on VD were evaluated
early in 1991 by conducting a randomized, matched and
double-blinded study involving 56 patients with multi-infarct dementia (MID)[175]. Using a self-controlled design,
marked improvement in memory deficits were observed after
0.15-0.45 mg HupA given orally for 4
weeks[176,177]. Patients with AD (23 cases) or VD (41 cases) were treated with HupA
for 8 weeks, and memory deficiency and recognition decline
was markedly improved in both AD and
VD[178]. Yin et al reported that 39 patients who met the DSM-R criteria for mild
to moderate vascular dementia, showed a significant increase
in AAMD, MMSE and ADL scores after treated with 0.1 mg
HupA twice per day for 8 weeks[179]. A similar result was
reported in a trial involving 20
patients[180]. Moreover, HupA has been shown to be more effective than pyritinol in the
treatment of MID[181]. Wang et
al reported that the CDR and ADL scores of VD patients who met the DSM-IV criteria
were markedly improved in MMSE after treatment for 6 months
with HupA[20]. HupA also showed efficacy in improving
cognitive deficiency in endemic
cretins[182], and urinary incontinence in patients with cerebral
stroke[183].
A survey involving 50 middle-aged and elderly patients
with different degrees of dysmnesia demonstrated the
efficacy of HupA in improving verbal recall, retention and
retrieval in patients with mild and moderate dysmnesia. Values
of total recall, long-term retrieval, long-term storage,
consistent long-term retrieval and unreminded recall were markedly
increased when patients were treated with 0.1 mg HupA orally
twice daily for 2 weeks[184]. In a study with conducted in
children who had language delay and other developmental
conditions, treatment with 0.05 mg HupA twice daily for more
than 3 months improved language delay by a total of
67.56%[185].
Zhang et al reported that HupA could improve memory
function and neurotransmission in patients with mild and
moderate traumatic brain injury. Thirty patients were treated
with traditional therapeutic (0.8 g piracetan and 20 mg
nimodipine, twice per day, combined with function
convalescence training), and other 30 patients were treated with 0.1 mg HupA bid besides traditional therapeutic. Both
groups showed significant improvement in memory and
cognition after both 1 and 3 months, and the improvement in
memory and cognition in patients treated with HupA was
more dramatic than the improvement in patients treated with
traditional therapeutics[186].
It is well documented that people with schizophrenia have
neurocognitive impairments across multiple domains,
including impairments in motor functioning, various aspects of
attentional abilities, executive functions and memory
functioning. Ma et al have recently studied the effect of
HupA on memory disorders in schizophrenic patients. Sixty
patients with schizophrenia, who were at the rehabilitation
stage, were divided into groups that received HupA or a
placebo, and 30 non-schizophrenic people were used as
controls. After 12 weeks of treatment with 0.2-0.4 mg HupA,
memory functions were significantly
improved[187]. Similar results were also reported by Fang
et al[188] and
Yang[189].
HupA has also shown some efficacy in ameliorating
sleeping. It was reported that alternating HupA treatment
with clonazepam treatment in the day and night, respectively,
markedly extends sleeping duration, modifies the rhythm of
sleep, and improves the quality of sleeping in patients with
chronic insomnia. With this treatment regime, the dosage of
clonazepam can be decreased
gradually[190].
Several clinical studies have shown that HupA is
effective for the treatment of benign senescent forgetfulness
(BSF)[156,175,191-193], in addition to AD and VD. Seventy four
percent of patients treated with HupA (0.15 mg, bid) for 4
weeks showed improvement in memory quotient (MQ) and
Wechsler memory scale (WMS)
scores[15,194-197]. The improving effect of HupA was studied in 34 pairs of junior high
school students who had complained of memory inadequacy.
HupA at a dose of 0.1 mg, twice per day for 4 weeks,
increased the scores for "accumulation", "recognition",
"reproduction", "association", "tactual memory", and
"number of recitation" factors, but not the "understanding"
factor[170].
In the early clinical studies using HupA for the treatment
of myasthenia gravis (MG), it was found that 99% of 128
patients with MG had their clinical manifestations controlled
or improved with HupA treatment. The duration of action of
HupA lasted for 7±6 h, and the side effects were minimal
compared with neostigmine[198]. Xia
et al reported similar improving effects in 63 MG patients treated with im HupA at
a dose of 0.2 mg twice per day[199].
Summary
AD is a multi-causal and multi-factorial progressive
neurodegenerative disease with complicated pathogenesis,
thus it is likely that multiple drugs or drugs with
poly-pharmacological activities will be the best therapeutic approaches
to address the varied pathological aspects of the disease.
Based on the characteristic cholinergic deficits in AD, AChE
inhibitors are still the drugs of choice for the symptomatic
therapy of AD. As a potent and reversible AChE inhibitor,
HupA has attracted attention because, relative to other
well-known AChE inhibitors, it has greater potency, higher
selectivity with respect to its AChE inhibitory effect, and marked
memory-enhancing efficacy in a broad range of animal
models of cognitive impairment, and in patients with AD, VD,
and other cognitive problems. Interestingly, new data from
our lab show that HupA, besides inhibiting the hydrolysis
of synaptic ACh, has neuroprotection, APP metabolism
modulation and NGF-like neurotrophic activities, indicating that
the non-cholinergic effects of HupA could play important
roles for the treatment of neurodegenerative disease through
interfering with the key factors of the disease. These
encouraging preclinical and clinical findings suggest that HupA
is a promising candidate for the treatment of
neurodegenera-tive diseases such as AD and VD, and is very likely to exert
its therapeutic effects via a multi-target mechanism, which
therefore provides us with a large amount of exciting
research to carry out.
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