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Ibuprofen and Glutathione Conjugate as a Potential Therapeutic Agent for Treating Alzheimer's Disease.

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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
139
Full Paper
Ibuprofen and Glutathione Conjugate as a Potential
Therapeutic Agent for Treating Alzheimer’s Disease
Francesco Pinnen1, Piera Sozio1, Ivana Cacciatore1, Catia Cornacchia1, Adriano Mollica1,
Antonio Iannitelli1, Eleonora D’Aurizio1, Amelia Cataldi2, Susi Zara2, Cinzia Nasuti3, and
Antonio Di Stefano1
1
Dipartimento di Scienze del Farmaco, Università ‘‘G. D’Annunzio’’, Chieti, Italy
Dipartimento di Biomorfologia, Università ‘‘G. D’Annunzio’’, Chieti, Italy
3
Dipartimento di Medicina Sperimentale e Sanità Pubblica, Università di Camerino, Camerino (MC), Italy
2
Non-steroidal anti-inflammatory drugs (NSAIDs) and antioxidant therapy might protect against
the development of Alzheimer’s disease (AD). In the present work, we synthesized a molecular
combination of glutathione (GSH) and ibuprofen (IBU) via an amide bond and investigated its
potential for targeted delivery of the parent drugs to neurons, where cellular oxidative stress and
inflammation are related to AD. Evaluation of its physicochemical and in-vitro antioxidant properties
indicated that compound 1 exhibits good stability toward human plasma enzymatic activity, and, like
GSH, displays in-vitro free radical scavenging activity in a time and concentration-dependent manner.
The new compound was also assessed by infusion in a rat model for Alzheimer’s disease for
its potential to antagonize the deleterious structural and cognitive effects of b-amyloid(1-40). In
behavioral tests of long-term spatial memory, animals treated with codrug 1 performed significantly
better than those treated with b-amyloid (Ab) peptide. Histochemical findings confirmed the
behavioral data, revealing that Ab protein was less expressed in cerebral cortex treated with 1
than that treated with IBU. Taken together, the present findings suggest that conjugate 1
treatment may protect against the oxidative stress generated by reactive oxygen species (ROS) and
the cognitive dysfunction induced by intracerebroventricular (i.c.v.) infusion of Ab(1-40) in rats, and
thus that codrug 1 could prove useful as a tool for controlling AD induced cerebral amyloid deposits
and behavioral deterioration.
Keywords: Alzheimer Disease / Codrug / Glutathione / Ibuprofen
Received: July 13, 2010; Revised: September 23, 2010; Accepted: September 24, 2010
DOI 10.1002/ardp.201000209
Introduction
Alzheimer’s disease (AD), characterized by neuronal degeneration, is the most frequent form of senile dementia among
people age 65 and older [1]. Although the etiology of the
disease is still unclear, many factors are thought to play
important roles in its pathogenesis, among them, oxidative
stress, abnormal protein accumulation, excessive metal ions,
and reduced acetylcholine (Ach) levels [2]. Early in the pathogenesis of AD, there is progressive deposition of b-amyloid
Correspondence: Antonio Di Stefano, Dipartimento di Scienze del
Farmaco, Università ‘‘G. D’Annunzio’’, Via dei Vestini 31, 66100 Chieti, Italy.
E-mail: adistefano@unich.it
Fax: þ39-871-3555267
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(Ab) peptide in hippocampal and cerebral cortical regions
accompanied by the presence of neurofibrillary tangles
(NTFs) and senile plaques. NTFs are principally formed by
aggregates of hyperphosphorylated microtubular Tau
protein, whereas the senile plaques are complex extracellular
lesions in which an Ab-containing core is surrounded by
reactive microglia, fibrillary astrocytes, interleukins, and
dystrophic neuritis [3, 4]. AD brain tissues are also characterized by neuroinflammatory changes that, together with the
Ab deposition, might elevate levels of reactive oxygen species
(ROS), accompanied by simultaneous induction of glutathione depletion, the key factor determining vulnerability
to oxidant attack [5, 6].
Epidemiological studies suggest a reduced prevalence of
AD among users of non-steroidal anti-inflammatory drugs
140
F. Pinnen et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
(NSAIDs); in particular, long term NSAID therapy delayed the
onset and progression of the disease and slowed the rate of
cognitive impairment [7, 8]. In-vitro studies have demonstrated that ibuprofen (IBU), meclofenac, ketoprofen, diclofenac, and acetyl salicylic acid dose-dependently inhibited
formation of Ab fibrils in fresh Ab(1-40) and Ab(1-42), and also
limited their extension [9]. Among the tested NSAIDs, IBU
seems the most promising agent, acting by allosteric modulation of g-secretase, the enzyme responsible for the final
proteolytic cleavage of amyloid precursor protein (APP),
which liberates Ab(1-42) peptide [10]. Furthermore, these
NSAIDs exhibit a concentration range of activity where
Ab(1-42) production is greatly reduced without inhibition
of Notch receptors, the key factor of mechanism-based side
effects associated with g-secretase inhibitor treatment [11].
While the etiology of AD remains unclear, accumulating
evidence indicates that oxidative stress and mitochondrial
dysfunction play an important role [12, 13]; it is thought that
Ab, in concert with metal ions, may cause oxidative stress,
which that in turn causes lipid and protein peroxidation, free
radical formation, and neurodegeneration [14]. Prompted by
these findings, researchers have explored whether exogenous
antioxidants in food and supplements can prevent or
delay AD onset, and found beneficial effects of combined
administration of antioxidant vitamin E and vitamin C
supplements on the incidence of AD in an elderly population
[15, 16]; one study has proposed enhancement of the body’s
own antioxidant and free radical scavenger systems to
counter the oxidative stress related to AD brain [14]. One
of these endogenous systems in the brain is associated with
glutathione (GSH), the most abundant intracellular non
protein thiol, which is able to detoxify various oxidants by
directly free radical scavenging or acting as a coenzyme in
GSH-peroxidase-catalyzed reactions [17]. GSH (g-glutamyl–
S
O
TFA . H2N
COOMe
SBut
H
N
N
H
cysteinyl–glycine), a water-soluble endogenous tri-peptide
present in mammalian cells in concentrations up to
10 mM, is the key functional element in the cysteinyl moiety
that provides the reactive thiol group. Within the cell, GSH is
kept in its thiol-reduced form (>98%) by glutathione disulfide
(GSSG) reductase, an NADPH-dependent enzyme; additional
amounts of GSH are present as GSSG and as glutathione
conjugates (GS-R). Maintaining optimal GSH/GSSG ratios in
the cell is critical to survival, since GSH is one of the primary
endogenous antioxidant defence systems in the brain, removing hydrogen- and lipid-peroxides [18]. Since GSH concentrations decline with age and in some age-related diseases,
and may be involved in AD pathology in humans, it can be
hoped that repletion of systemic GSH might prove promising
for the management of AD [19].
In previous works, GSH codrugs were synthesized and
evaluated as antiparkinson agents with antioxidant properties. These GSH transporter targeted conjugates had three
components: the carrier (GSH), the active drug, and a suitable
linker for conjugation of the carrier with the drug molecule
[20, 21]. To design cleavable conjugates, a linker containing
an ester or amide bond is preferable, because it can be cleaved
by plasma esterases or peptidases to release the individual
drugs that then act independently; thus, cleavable conjugates can be seen as unique molecular entities with notable
synergistic advantages over drug cocktails at the moment of
administration. Inspired by these facts, in this work we
sought to enhance the brain availability of GSH and IBU
by synthesizing conjugate 1 (Scheme 1), that is, covalently
joining GSH and IBU, as a carrier to deliver the drugs to the
central nervous system (CNS) (Fig. 1). Our compound might
permit targeted delivery of IBU and GSH directly to neurons,
where cellular stress and inflammatory processes are
associated with AD. We evaluated the physicochemical and
a
H
N
COOMe
O
O
S
O
COOMe
N
H
SBut
H
N
COOMe
O
3
2
b
O
SH
O
H
N
COOMe
N
H
H
N
COOMe
O
1
Scheme 1. Synthesis of codrug 1. Reagents and conditions: (a) IBU, DCC, TEA, dry DCM, 2 h, rt; (b) (n-Bu)3P, n-PrOH/H2O (2:1),
1.5 h, rt, pH 8.5.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
COOMe
O
Figure 1. Chemical structure of codrug 1.
biological properties of codrug 1 and investigated its pharmacological in-vivo behavior; in particular, its potential effects in
the Ab(1-40) infused AD rat model; immunohistochemical
analysis was also employed to assess the effects of codrug 1 on
Ab(1-40) plaque formation [22].
Results and discussion
Lipophilicity is an essential feature for predicting a molecule’s penetration through the blood brain barrier (BBB).
We assessed this property using reverse-phase chromatographic retention times (RT) and calculating the octanol/
water partition coefficients (logP) in n-octanol/phosphate buffer of pH 7.4; the logK0 value was determined using a mixture
of acetonitrile and water as eluant [23]. These values are listed
in Table 1. The lipophilicity of compound 1 was also calculated using the ACD logP software package, version 4.55
(Advanced Chemistry Development Inc., Toronto, Canada)
(Table 1). It is generally accepted that good absorption of
an orally administered drug can be obtained when the logP
value is more than 1.35, and the aqueous solubility is >10 mg/mL.
Factors such as absorption, excretion, and penetration of the
CNS may also be related to the logP value of a drug. In general,
assuming passive transport through biological membranes,
a logP of 2 0.7 can be suitable for optimum CNS
penetration [24]. The values obtained for our compound
satisfy the requirements for g.i. absorption and for CNS
penetration.
Stability studies of the new compounds were performed at
378C in isotonic sodium phosphate buffer (pH 7.4), in
Simulated Gastric Fluid (SGF, pH 1.3) and in rat and human
plasma, as previously described [25]. The disappearance of
codrug 1 was monitored by the HPLC UV-DAD method and
the pseudo-first-order hydrolysis rate constants (Kobs) for
hydrolysis were calculated [26]. In buffer solutions, the
new compound was stable in all media (t½ > 16 h),
while in human plasma, a slow bioconversion to IBU was
Table 1. Physicochemical properties of codrug 1.
logP
a
2.70 ( 0.20)
clogP
3.02 ( 0.72)
logK
a
3.30 ( 0.14)
Solubility
in watera (mg/mL)
10.3 ( 0.9)
a Values are means of three experiments, standard deviation is
given in parentheses.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
141
observed (t½ > 45 min), indicating good resistance to
peripheral enzymatic degradation (pseudo-first order
kinetics, Kobs ¼ 0.015 minS1). The extracts were analyzed
using a previous proposed LC/MS/MS method and all MS
experiments were performed using electrospray ionization
(ESI) mode; we were only able to measure the relative amount
of parent drug IBU (data not shown) [27, 28].
The rapid DPPH-HPLC method and the DMSO competition
test were used to evaluate the free radical scavenging activity
of our compound. The scavenging of the DPPH (1,1-diphenyl2-picrylhydrazyl) radical is a simple model reaction providing
information about the relative ability of antioxidant compounds to scavenge free radicals, whereas the DMSO competition test is useful for evaluating the hydroxyl radical
scavenging activity. GSH and cysteine were used as reference
standards [29, 30]. The DPPH formation, observed at 517 nm,
decreases stoichiometrically with respect to the number of
electrons taken up. Since the decrease in the peak depends on
the radical scavenging activity of tested compounds and the
time of incubation, the absorbance was recorded at concentrations between 50 and 300 mM and at different incubation
times (up to 120 h). In our experiments IBU did not interact
with DPPH radical; instead, the radical scavenging activity of
100
% scavenging
COOMe
N
H
H
N
80
60
Cys
40
codrug 1
20
GSH
0
25
50
150
300
conc. (µM)
Figure 2. % DPPH radical scavenging activity after 20 min
incubation.
100
% scavenging
O
SH
O
H
N
Ibuprofen and Glutathione Conjugate
80
60
Cys
40
codrug 1
GSH
20
0
0
50
100
150
Time (h)
Figure 3. % DPPH radical scavenging activity over time of
incubation.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
80
70
vehicle
Aβ
Aβ + IBU
Aβ + GSH
Aβ + 1
60
Latency (s)
treatment, the neuroprotective effects of drugs on spatial
and memory learning in the AD rat model were assessed.
In Figure 4, the results showed that although performance
of each group improved significantly across days of trials,
learning was significantly slower in Ab, Ab þ IBU,
Ab þ GSH, and Ab þ 1-treated groups compared with the
control group. However, on the second day of training,
Ab þ IBU, Ab þ GSH, and Ab þ 1-treated groups were
quicker to find the platform than the Ab-treated group
(P < 0.05). There were no significant differences among the
groups during days 4 and 5 of training (P > 0.05). Despite
slower learning in the IBU, GSH and 1-treated groups, performance at the probe tests given 24 h (day 6) and one week
(day 12) after learning showed that spatial memory was not
significantly impaired in these tasks (Fig. 5a and 5b). On the
contrary, the Ab-treated group showed higher impairment of
long-term memory on day 12 compared with all groups
(P < 0.05).
To be sure that the difference observed in water maze
performance among groups was not due to locomotor
activity alterations rather than to differences in spatial memory, we assessed ambulatory, rearing and grooming episodes
of all groups by open field task as previously described [32].
No differences of locomotor activities among groups were
measured (data not shown). To assess the potential activity of
compound 1 in AD therapy, hematoxylin-eosin and AgNO3
stainings together with immunohistochemical analysis of
Ab(1-40) were performed, determining the plaque placement
and formation of Ab aggregates [33]. Hematoxylin-eosin and
AgNO3 stainings in the control sample indicated well-organized cell arrangement within the cerebral cortex, visible
50
40
*∆
30
∆
20
∆
10
0
1
2
3
4
5
Day of training
Figure 4. Effects of drug administration on spatial reference memory in a Morris water maze. Performance during acquisition was
expressed as the mean (SE) latency to find a submerged platform
from control, Ab, Ab þ IBU, Ab þ GSH, and Ab þ 1 groups during
five consecutive days of training (4 trials per day). P < 0.05 compared with Ab group; ~ P < 0.05 compared to control group.
compound 1 and GSH and cysteine increased with concentration (Fig. 2) and with incubation time (Fig. 3); these results
were also confirmed by DMSO competition tests (data not
shown) [31].
In this study, we also investigated whether codrug 1 could
improve learning and memory impairment in an infused AD
rat model. Ab(1-40) was continuously infused into the lateral
ventricle of rats for 28 days and contemporaneously IBU,
GSH, 1 or vehicle were injected s.c. One month after
Time spent in the target quadrant (s)
45
40
35
30
25
20
15
10
5
0
Day 6
b)
Time spent in the target quadrant (s)
vehicle
Aβ
Aβ + IBU
Aβ + GSH
Aβ + 1
a)
45
40
vehicle
Aβ
Aβ + IBU
Aβ + GSH
Aβ + 1
35
*∆
*∆
*∆
30
25
*
20
15
10
5
0
Day 12
Figure 5. Effects of drug administration on spatial reference memory in a Morris water maze. Performance during probe trial was expressed
as the mean (SE) of time spent in the target quadrant where the platform had been during five consecutive days of training from control, Ab,
Ab þ IBU, Ab þ GSH, and Ab þ 1 groups on day 6 (a) and on day 12 (b). P < 0.05 compared with Ab group; ~ P < 0.05 compared to
control group.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
nerve connections, and many astrocytes, while cerebral
cortices injected with Ab showed cell disorganization (data
not shown). Immunohistochemical analysis of Ab(1-40)
protein disclosed many different sized plaques within capillary vessels in the cerebral cortex of Ab(1-40) and/or drug
vehicle injected rats (Fig. 6a and 6b). Ab-injected cerebral
cortices treated with IBU, GSH or compound 1 showed few
plaques within capillary vessels and, in particular, Ab(1-40)
protein was less expressed in codrug 1 treated than in IBU
and GSH treated cerebral cortex. Apoptosis is a fundamental
biological process that serves many important functions in
the developing and adult organism [34]. However, the aberrant induction of apoptosis can have direct consequences,
particularly in irreplaceable cells such as neurons. Activation
Ibuprofen and Glutathione Conjugate
143
of pro-apoptotic pathways appears to contribute to AD and
other important neurological diseases. Because inhibitors of
apoptosis can prevent cell death even in the continued presence of the apoptosis-inducing trigger, these pathways are
attractive targets for therapeutic intervention [35]. To assess
the anti-apoptotic capacity of compound 1, we performed a
TUNEL (terminal transferase-mediated dUTP-biotin nick end
labeling) analysis, a technique that specifically detects apoptotic cells by utilizing terminal transferase to incorporate
biotinylated nucleotides into the fragmented DNA of apoptotic cells; the results are shown in Fig. 7a and 7b; codrug 1,
combining antioxidant, anti-inflammatory and neuroprotective activities, showed an anti-apoptotic action compared to
IBU and GSH alone.
Figure 6. (A) Immunohistochemical detection of Ab(1-40) in rat brain in different experimental conditions. Magnification 20. (a): Control;
(b): Ab injected cerebral cortex; (c): Ab injected cerebral cortex þ GSH; (d): Ab injected cerebral cortex þ IBU; (e): Ab injected cerebral
cortex þ 1; (f): Negative control. Arrows indicate Ab plaques. (B) Densitometric analysis of Ab (1-40) positive area, expressed as % SD,
assessed by direct visual counting of three fields for each of five slides per each of 5 samples at 40 magnification by MetaMorph Software
System. Data are the mean SD of three different consistent experiments.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
Figure 7. (A) TUNEL analysis of rat brain in different experimental conditions. The presence of DNA fragmentation has been quantified by
direct visual counting of brown counterstained nuclei. Magnification: 40. (a): Control; (b): Ab injected cerebral cortex; (c): Ab injected
cerebral cortex þ GSH; (d): Ab injected cerebral cortex þ IBU; (e): Ab injected cerebral cortex þ 1; (f): Negative control. Arrows indicate
positive nuclei. (B) Graphical representation of TUNEL analysis. Five slides were examined per sample. Apoptotic cells were counted out of a
total of 100 cells. Percentage values represented in the graph are means SD. n ¼ 3 for all groups
In summary, this report describes the synthesis of
codrug 1 as a carrier for delivery of GSH and IBU to
the CNS and evaluates this conjugate as a potential neuroprotective agent in AD therapy. Our findings showed
that compound 1 has good stability toward human
plasma enzymatic activity, displaying free radical scavenging effects in an in-vitro test; we also examined the
effects of codrug 1 on chronic treatment following
intracerebroventricular (i.c.v.) infusion of Ab(1-40)
protein. In behavioral tests of long-term spatial memory,
animals treated with codrug 1 performed significantly
better on day 12 of the probe than those treated with
Ab(1-40). Taken together, these results indicate that codrug
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1 may have potential as a neuroprotective agent for AD
therapy.
Our histochemical findings confirmed the behavioral data,
revealing that Ab protein was less expressed in cerebral
cortex treated with 1 than that treated with IBU. These findings suggest that conjugate 1 treatment may protect against
the oxidative stress generated by ROS and the cognitive
dysfunction induced by i.c.v. infusion of Ab(1-40) in rats,
and thus that codrug 1 could prove useful as a tool for
controlling AD induced cerebral amyloid deposits and behavioral deterioration.
It is hoped that the increased bioavailability potentially
afforded by coadministration of GSH and IBU may offer a
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
Ibuprofen and Glutathione Conjugate
145
Experimental
Yield: 65%; Rf ¼ 0.47, CHCl3/MeOH (95:5); 1H-NMR (CDCl3) d: 0.83
(6H, d, 2 CH3), 1.26 (9H, s, 3 CH3), 1.42 (3H, d, CH3), 1.78–1.98 (2H, m,
CH2), 2.14–2.19 (2H, m, CH2), 2.21–2.28 (1H, t, CH), 2.40–2.43 (2H, d,
CH2), 3.03 (2H, d, CH2), 3.61 (1H, s, CH), 3.65 (6H, 2 s, CH3), 3.88–4.10
(2H, m, CH2), 4.58 (1H, m, CH), 4.78 (1H, m, CH), 6.41 (1H, d,
J ¼ 7.51 Hz, NH), 6.47 (1H, t, J ¼ 5.71 Hz, NH), 6.97 (1H, d,
J ¼ 8.41 Hz, NH), 7.18–7.23 (4H, dd, Ar); 13C-NMR (CDCl3) d: 18.67
and 18.69 (2 CH3), 22.60 (CH3), 28.37 (CH2), 30.06 (3 CH3), 30.40
(CH2), 32.42 (CH2), 41.50 (CH2), 41.75 (CH2), 45.23 (CH), 46.62 (CH),
48.56 (C), 51.93 (CH), 52.61 (CH), 52.72 and 53.05 (2 OMe), 127.47–
140.99 (6 Ar), 170.27, 170.70, 172.58, 172.63, and 175.17 (5 CO).
General
Ibu-Glu[Cys-Gly-OMe]-OMe (1)
promising therapeutic strategy for AD. The combination of
the anti-apoptotic and neuroprotective functions of compound 1 derived from the IBU and GSH moieties may be of
therapeutic benefit not only for treatments of AD, but also for
other neurodegenerative disease in which apoptotic death is
a component of neuronal degeneration, such as amyotrophic
lateral sclerosis, Parkinson’s and Huntington’s diseases.
Microanalyses were performed on a 1106 Carlo Erba CHN analyzer (Carlo Erba, Milan, Italy), with results within 0.4% of the
calculated values.
Codrug 1 was characterized by 1H- and 13C-NMR, and their
purities (>95%) were quantified by HPLC. Analytical HPLC
measurements were run on a Waters 600 HPLC pump, equipped
with a Waters 2996 photodiode array detector, a 20-mL Rheodyne
injector and a computer integrating apparatus. The column was a
Waters Symmetry RP-C18 column (4.6 mm 150 mm, 5 mm), the
mobile phase was a mixture of water/methanol (25:75) at a flow
rate of 1 mL/min, and the UV-detector was set at a length of
220 nm. 1H- and 13C-NMR spectra were recorded on a Varian
VXR 300 MHz spectrometer (Varian Inc., Palo Alto, CA, USA).
Chemical shifts are reported in parts per million (d) downfield
from the internal standard tetramethylsilane (Me4Si). The identity
of new compound was confirmed by elemental analysis, and NMR
data; homogeneity was confirmed by TLC on silica gel Merck 60
F254 (Merck, Germany). Solutions were routinely dried over anhydrous sodium sulphate prior to evaporation. Chromatographic
purifications were performed by Merck 60 70–230 mesh ASTM
silica gel column. IBU was purchased from Sigma (St. Louis, MO,
USA). H-Glu-[Cys(SBut)-Gly-OMe]-OMe trifluoroacetate (2) was
synthesized as previously reported [21]. All other chemicals used
were of the highest purity commercially available.
A solution of 3 (2.94 mmol) in a mixture of n-PrOH/H2O (2:1)
(30 mL) was brought to pH 8.5 with 25% aqueous NH3 and flushed
with nitrogen. After 30 min tri-n-butylphosphine (3.53 mmol) was
added and the stoppered flask stirred at room temperature. After
1 h the reaction mixture was concentrated and subjected to
column chromatography on silica gel using CHCl3/MeOH (9:1)
as eluant to afford the corresponding reduced compound 1.
Yield: 45%; Rf ¼ 0.64, CHCl3/MeOH (9:1); 1H-NMR (CDCl3) d:
0.82 (6H, d, 2 CH3), 1.42 (3H, d, CH3), 1.72–1.96 (2H, m, CH2),
2.09–2.19 (2H, m, CH2), 2.21–2.31 (1H, t, CH), 2.39–2.41 (2H, d,
CH2), 2.71–3.01 (2H, m, CH2), 3.61 (1H, s, CH), 3.63 (6H, 2 s, CH3),
3.91–4.14 (2H, m, CH2), 4.55 (1H, m, CH), 4.63 (1H, m, CH), 6.41
(1H, d, J ¼ 7.51 Hz, NH), 6.54 (1H, t, J ¼ 5.71 Hz, NH), 6.79 (1H, d,
J ¼ 8.41 Hz, NH), 7.08–7.28 (4H, dd, Ar); 13C-NMR (CDCl3) d: 18.48
and 18.68 (2 CH3), 22.59 (CH3), 26.58 (CH2), 28.07 (CH2), 30.39
(CH2), 31.90 (CH2), 41.39 (CH2), 45.18 (CH), 46.54 (CH), 43.07 (CH2),
52.20 (CH), 52.62 (2 OMe), 52.93 (CH), 127.43–141.07 (Ar), 170.35,
170.53, 172.41, 172.88, and 175.06 (5 CO).
Aqueous solubility
Compound 1 (50 mg) was placed in a microtube containing 1 mL
of deionized water and was shaken at 258C for 1 h to ensure the
solubility equilibrium. After centrifugation, a 20-mL portion of
the supernatant was analyzed by HPLC [36].
Chemistry
Codrug 1 was synthesized as outlined in Scheme 1 employing
solution phase procedures by elongation of the suitably protected GSH peptide chain in the N-direction with ibuprofen.
Deprotection of the cysteine -SH to give the desired codrug 1
was obtained when the corresponding protected precursor was
treated for 1.5 h at room temperature with a small excess (1.2
equiv.) of tri-n-butylphosphine in a water/n-propanol solution,
made slightly alkaline (pH 8.5) by aqueous ammonia. 1H- and 13CNMR spectra are relative to the prevalent diastereomer.
Ibu-Glu[Cys(Sbut)-Gly-OMe]-OMe (3)
To a stirred solution of tripeptide H-Glu-[Cys(SBut)-Gly-OMe]-OMe
trifluoroacetate (2) (4.55 mmol) in dry CH2Cl2 (13 mL) were
added TEA (4.55 mmol) and IBU (4.55 mmol) in dry CH2Cl2
(10 mL) followed by portionwise addition of a solution of DCC
(4.55 mmol) in dry CH2Cl2 (10 mL). After 2 h at room temperature, the reaction mixture was filtered and the resulting solution
was evaporated under vacuum. The residue was taken up in
CHCl3 and the organic layer washed with 1 N KHSO4, saturated
aqueous NaHCO3 and brine. The residue obtained after drying
and evaporation was chromatographed on silica gel using CHCl3/
MeOH (95:5) as eluant to give 3.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Lipophilicity
clogP
The calculated clogP was determined using ACD LogP software
package, version 4.55 (Advanced Chemistry Development Inc.,
Toronto, Canada).
logP
Octanol/water partition coefficients were determined by placing
approximately 5 mg of compound 1 in 1 mL of anhydrous
n-octanol, shaking vigorously for about 2 min and filtering.
An equal volume of phosphate buffer pH 7.4 was added and
the mixture was equilibrated by repeated inversions of up to
200 times for 5 min and then allowed to stand for 30 min for the
phases to fully separate. Thereafter the respective phases were
analyzed by HPLC.
logK0
Oil/water partition coefficients can be estimated using reversephase chromatographic RT due to the good relationship between
logP and log capacity factor (logK) values determined using
octadecyl silica columns [37].
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Kinetics of chemical hydrolysis
A 0.02 M hydrochloridric acid buffer of pH 1.3 as non-enzymatic
simulated gastric fluid (SGF) and 0.02 M phosphate buffer of pH
7.4 were used in this study [37].
Kinetics of enzymatic hydrolysis
Plasma from rats and human was obtained by centrifugation of
blood samples containing 0.3% citric acid at 3000 g for 15–
20 min. Plasma fractions (4 mL) were diluted with 0.02 M phosphate buffer (pH 7.4) to give a final volume of 5 mL (80% plasma).
Incubations were performed at 37 0.58C using a shaking water
bath as previously described [37].
In vitro antioxidant activity
DPPH radical scavenging activity
The DPPH radical scavenging activity was estimated according to
the method previously reported [37].
DMSO competition for hydroxyl radicals
The hydroxyl radicals were generated by mixing the Fe3þ/
ascorbic acid system, as previously described by Andreadoua
et al. with some modifications [38]. The reaction mixture contained EDTA (0.1 M), Fe3þ/EDTA (167 mM), DMSO (12.5 M) in
phosphate buffer (50 mM, pH 7.4), the tested compounds (0.04
and 0.1 M) and ascorbic acid (20 mM). After 30 min of incubation
(378C) the reaction was stopped with CCl3COOH (17% w/v) and
ethyl 3-oxobutanoate was added to detect formaldehyde by
HPLC–diode-array system [39].
In vivo studies
Animals
Male Wistar rats (n ¼ 40) (Harlan, UD, Italy) that weighed 200–225 g
at the beginning of the experiments were used. The animals were
individually housed in a room on a 12 h light/dark cycle (lights off at
7:00 a.m.) at constant temperature (20–228C) and humidity (45–55%).
Rats were offered food pellets (4RF; Mucedola, Settimo Milanese,
Italy) and tap water ad libitum and were handled once a day for 5 min
during the first week after arrival. Each animal was weighted weekly
throughout the experimental period. All procedures were conducted
in adherence to the European Community Council Directive for Care
and Use of Laboratory Animals.
Surgical procedure
After anesthesia with 10 mg/kg of a mixture of zolazepam and
tiletamine (Zoletil 100, Italmed, Italy) by intraperitoneal injection,
a stainless steel cannula was implanted in the animal’s lateral
cerebroventricle using a stereotaxic instrument at the following
coordinates, in mm with reference to the bregma: Anteroposterior
(AP), 1.0; lateral (L), 1.8. Coordinates were taken from Paxinos and
Watson and adjusted for the body weight of the animals [40]. The
cannula, connected by a Ø 0.8 mm capillary tube to an osmotic
pump (Alzet, model 2004, Charles River, Italy), containing either
Ab peptide(1-40) solution or the vehicle alone, was quickly placed
subcutaneously under the dorsal skin of the rat. The outlet of the
pump was implanted in the left ventricle 3.5 mm dorsoventral to
cranium and attached to the skull with screws and dental cement.
The content of the mini-osmotic pump (4.6 Ab nmol/rat) at a final
volume of 200 mL was delivered for 28 days at a flow of 0.25 mL/h,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
according to the manufacturer. All animals were housed individually and received food and water ad libitum.
Drug preparation and animal treatments
To fill osmotic pump, the Ab(1-40) solution and vehicle solution
(vehicle in pump) were prepared on the same day of i.c.v. pump
implantation. Ab(1-40) was dissolved in a vehicle solution comprising 35% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid
(TFA) to achieve a final concentration of 4.6 nmol/200 mL. IBU, GSH
and 1 were solubilized in sterile saline containing 20% (v/v) DMSO
and were daily administered subcutaneously for 28 days at a dose
of 23 mmol/kg. A vehicle solution, prepared with sterile saline
containing 20% (v/v) DMSO, was also administered subcutaneously
for 28 days at a dose volume of 250 mL/kg as IBU, GSH, and 1.
The animals were randomly divided into 5 groups (n ¼ 8 rats):
(i) Control group, (ii) Ab group, (iii) Ab þ IBU, (iv) Ab þ GSH, and
(v) Ab þ 1 group. The first group received vehicle continuously
infused into the lateral ventricle by osmotic pump for 28 days and
contemporaneously vehicle was injected daily s.c. The second,
third and fourth groups received Ab(1-40) continuously infused
into the lateral ventricle of rats for 28 days and contemporaneously vehicle, IBU, GSH or 1 were injected daily s.c., respectively.
Water maze test
To assess spatial learning and memory, rats were trained in a
standard Morris water maze task one month after treatment with
IBU, GSH and codrug 1 in order to assess the effect on the impairment of learning abilities in Ab-infused AD rat model [41]. This
paradigm tested the animal’s ability to learn and remember the
spatial location of a platform submerged 1 cm below the surface
of the water in a circular pool 1.5 m in diameter (maintained at
208C). The water was rendered opaque by the addition of nontoxic
white paint. The pool was placed in the center of a large room with
numerous extramaze cues (e.g., circles, squares, triangles) on the
walls so that the rats can use these visual cues as a means of
navigating in the maze. Four points around the edge of the pool
were designated as north (N), south (S), east (E) and west (W) which
allowed the apparatus to be divided into four corresponding
quadrants (i.e., NE, SW, NW, and SE). Rats were introduced into
the water maze facing the wall at N, S, E, or W, and swam until
they found the hidden platform placed in the center of the NE
quadrant or until 90 s elapsed, at which time they were placed on
the platform, where they remained for 30 s. The starting point
was randomly distributed across the four quadrants. Training
consisted of 4 trials per day for five consecutive days and the
intertrial interval was 30 s. On day 6 (i.e., 24 h following the last
hidden platform trial) a probe trial was conducted in which the
platform was removed from the pool to measure the time spent in
the target quadrant where the platform was located during training. This assessment provided a estimate of the strength and
accuracy of the memory of the previous platform location. On
day 12 (i.e., 6 days post-training) a second probe trial was conducted in which the platform was removed. This assessment
provided an estimate of the long-term spatial memory.
Histological examination
After the completation of the behavioral experiment rats were
sacrified with anhydride carboxide and their whole brains were
removed. For western blot analysis, they were immediately frozen in 2-methylbutane and stored at –808C, whereas they were
dehydrated for immunohistochemical and histological analysis.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 139–148
Light microscopy
Brain samples were fixed in phosphate-buffered formalin
solution, dehydrated through ascending alcohols (ethanol 708,
ethanol 908, absolute ethanol) and xylene and then paraffin
embedded. The samples were then de-waxed (xylene and
alcohol progressively lower concentrations) and processed for
hematoxylin-eosin staining, Bielschowsky staining according to
manufacturer’s instructions (Bio-Optica, Milan, Italy) and for
immunohistochemical analysis [42].
Immunohistochemistry
In order to detect Ab(1-40), immunohistochemistry performed
using an UltraVision LP Detection System HRP Polymer &
DAB Plus Chromogen kit (Thermo Fisher Scientific, CA, USA)
and processed according to data sheet. Sections (5 mm)
were incubated in the presence of mouse anti-human Ab(1-40)
monoclonal antibody and then in presence of HRP
conjugated secondary antibody. Peroxidase was developed using
diaminobenzidin chromogen (DAB). Nuclei were hematoxylin
counterstained. Negative controls were performed omitting the
primary antibody. Samples were then observed with a light microscope (Leica, Heidelberg, Germany) equipped with a Coolsnap
video camera for computerized images (RS Photometrics,
Tucson, AZ).
TUNEL analysis
TUNEL analysis, which allows to identify DNA strand breaks,
yielded during apoptosis, was performed according to manufacturer’s explanations (Calbiochem, Darmstadt, Germany).
Computerized morphometry measurements and image
analysis
After digitizing the images deriving from immunohistochemistry
stained sections, Metamorph Software System (Universal Imaging
Corporation, Molecular Device Corporation, PA, USA) (Crysel
Instruments, Rome, Italy) was used to evaluate Ab(1-40) expression. Image analysis of protein expression was performed through
the quantification of threshold area for immunohistochemical
brown colors per field of light microscope observation.
Metamorph assessments were logged to Microsoft Excel and processed for Standard Deviations and Histograms.
Statistical analysis
Results are expressed as means S.E. For intergroup differences,
the data were analyzed by one-way ANOVA followed by NewmanKeul test post hoc comparison.
The authors would like to thank Professor Adriano Antonucci for helpful
discussions and Sheila Beatty for linguistic revision of the manuscript.
Financial support from Ministero dell’Istruzione, dell’Università e della
Ricerca (MIUR) is gratefully acknowledged.
The authors have declared no conflict of interest.
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