In vitro and in vivo investigation of dexibuprofen derivatives for CNS delivery

Article abstract of DOI:10.1038/aps.2011.144

Aim:Dexibuprofen, the S(+)-isomer of ibuprofen, is an effective therapeutic agent for the treatment of neurodegenerative disorders. However, its clinical use is hampered by a limited brain distribution. The aim of this study was to design and synthesize brain-targeting dexibuprofen prodrugs and to evaluate their brain-targeting efficiency using biodistribution and pharmacokinetic analysis.Methods:In vitro stability, biodistribution and pharmacokinetic studies were performed on male Sprague-Dawley rats. The concentrations of dexibuprofen in biosamples, including the plasma, brain, heart, liver, spleen, lung, and kidney, were measured using high pressure lipid chromatography (HPLC). The pharmacokinetic parameters of the drug in the plasma and tissues were calculated using obtained data and statistics.Results:Five dexibuprofen prodrugs that were modified to contain ethanolamine-related structures were designed and synthesized. Their chemical structures were confirmed using 1 H NMR, 13 C NMR, IR, and HRMS. In the biodistribution study, 10min after intravenous administration of dexibuprofen (11.70 mg/kg) and its prodrugs (the dose of each compound was equivalent to 11.70 mg/kg of dexibuprofen) in male Sprague-Dawley rats, the dexibuprofen concentrations in the brain and plasma were measured. The C brain /C plasma ratios of prodrugs 1, 2, 3, 4, and 5 were 17.0-, 15.7-, 7.88-, 9.31-, and 3.42-fold higher than that of dexibuprofen, respectively (P<0.01). Thus, each of the prodrugs exhibited a significantly enhanced brain distribution when compared with dexibuprofen. In the pharmacokinetic study, prodrug 1 exhibited a brain-targeting index of 11.19 {DTI=(AUC brain /AUC plasma) 1 /(AUC brain /AUC plasma) dexibuprofen }.Conclusion:The ethanolamine-related structures may play an important role in transport across the brain blood barrier.

Full text of DOI:10.1038/aps.2011.144

Acta Pharmacologica Sinica (2012) 33: 279–288
© 2012 CPS and SIMM All rights reserved 1671-4083/12 $32.00
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Original Article
In vitro and in vivo investigation of dexibuprofen
derivatives for CNS delivery
*
Xuan ZHANG, Xing LIU, Tao GONG, Xun SUN, Zhi-rong ZHANG
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041,
China
Aim: Dexibuprofen, the S(+)-isomer of ibuprofen, is an effective therapeutic agent for the treatment of neurodegenerative disorders.
However, its clinical use is hampered by a limited brain distribution. The aim of this study was to design and synthesize brain-targeting
dexibuprofen prodrugs and to evaluate their brain-targeting efficiency using biodistribution and pharmacokinetic analysis.
Methods: In vitro stability, biodistribution and pharmacokinetic studies were performed on male Sprague-Dawley rats. The concen-
trations of dexibuprofen in biosamples, including the plasma, brain, heart, liver, spleen, lung, and kidney, were measured using high
pressure lipid chromatography (HPLC). The pharmacokinetic parameters of the drug in the plasma and tissues were calculated using
obtained data and statistics.
Results: Five dexibuprofen prodrugs that were modified to contain ethanolamine-related structures were designed and synthesized.
Their chemical structures were confirmed using 1HNMR, 13C NMR, IR, and HRMS. In the biodistribution study, 10 min after intravenous
administration of dexibuprofen (11.70 mg/kg) and its prodrugs (the dose of each compound was equivalent to 11.70 mg/kg of dexibu-
profen) in male Sprague-Dawley rats, the dexibuprofen concentrations in the brain and plasma were measured. The Cbrain/Cplasma ratios
of prodrugs 1, 2, 3, 4, and 5 were 17.0-, 15.7-, 7.88-, 9.31-, and 3.42-fold higher than that of dexibuprofen, respectively (P<0.01).
Thus, each of the prodrugs exhibited a significantly enhanced brain distribution when compared with dexibuprofen. In the pharmacoki-
netic study, prodrug 1 exhibited a brain-targeting index of 11.19 {DTI=(AUCbrain/AUCplasma)1/(AUCbrain/AUCplasma)dexibuprofen}.
Conclusion: The ethanolamine-related structures may play an important role in transport across the brain blood barrier.
Keywords: dexibuprofen; ethanolamine; prodrugs; brain targeting
Acta Pharmacologica Sinica (2012) 33: 279–288; doi: 10.1038/aps.2011.144
Therefore, it is critical to enhance the brain distribution of
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs), includ- NSAIDs to achieve therapeutic concentrations at normal
ing ibuprofen, naproxen and flubiprofen, are widely used as doses.
neuroprotective agents in the treatment of neurodegenerative
Over 90% of therapeutic agents are excluded from CNS by
disorders[1-3]. However, the clinical application of NSAIDs is the blood brain barrier (BBB), which is composed of the tightly
impeded by limited brain accessibility. Several in vivo animal connected endothelial cells of brain capillaries and the sur-
studies have shown that the concentrations of ibuprofen or rounding astrocytes and pericytes[10, 11]. Typically, molecules
flurbiprofen in the brain achieved at normal plasma concentra- that are capable of crossing the BBB by passive diffusion are
tions are only 0.9–1.5 µmol/L[4–6]. It has also been found that small (Mr<500 Da), lipophilic and uncharged at physiological
most NSAIDs, including naproxen, ibuprofen, flurbiprofen, pH[12, 13]. Various facilitated or energy-dependent transport-
ketoprofen, and indomethacin, exhibit very limited distribu- ers and receptors are highly expressed at the BBB; examples
tion in the CNS, with steady state brain/plasma concentration of such transporters include members of the organic cation
ratios of 0.01– 0.05[7]. Several factors account for the low CNS transporter (OCT) families, organic anion transporters (OATs)
distribution of NSAIDs, including high plasma protein bind- and multidrug resistance proteins (MRPs)[14, 15]. A number of
ing rates, limited passive transport and efflux transport[8, 9]
.
studies aimed at enhancing the distribution of therapeutics in
the brain based on carrier- and receptor-mediated transport
systems have been performed[16–18]. Due to the negative charge
carried by the BBB, which expresses acetylneuraminic acid
on the capillary luminal membrane and heparin sulfate on
*
To whom correspondence should be addressed.
E-mail zrzzl@vip.sina.com
Received 2011-06-15 Accepted 2011-09-30

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the basal lamina, compounds carrying positive charges have
Materials and methods
also been designed to cross through adsorption-mediated Materials
transport[19, 20]
.
Dexibuprofen (99.8% purity) was purchased from the Fourth
Although several NSAID prodrugs have been synthesized Pharmaceutical Company of Suzhou (Suzhou, China). Ace-
to enhance the anti-inflammation activity or reduce the gas- tonitrile (HPLC grade) was purchased from Kemiou (Tianjin,
trointestinal toxicity of their parent compounds, few stud- China). Water was prepared using an Aquapro water purifi-
ies have investigated the targeted delivery of NSAIDs to the cation system (Chongqing, China). All of the other chemicals
CNS[21, 22]. In a previous study, a series of NSAID prodrugs and reagents used in this study were of analytical grade and
were synthesized to enhance transport to the brain. The car- obtained commercially. TLC (thin-layer chromatograpy)
boxylic groups of NSAIDs were coupled with a 1,4-dihydro- (silica gel GF254) was used to detect spots using UV radia-
1-methylpyridine-3-carboxylate moiety[23]. The results of this tion. The purification of intermediates and final synthesized
study revealed that all of the prodrugs were more lipophilic compounds was achieved by flash chromatography on silica
than their corresponding parent compounds, suggesting that gel. 1H-NMR and 13C-NMR analyses were performed using
prodrugs may exhibit better brain penetration. In another an AMX-400 Bruker Spectrometer. Infrared (IR) spectra were
study, six ibuprofen prodrugs containing a proline moiety obtained using a Perkin-Elmer 983 Infrared Spectrophotome-
or related structures were synthesized and evaluated[24]. The ter (USA). MS spectroscopy was recorded on a Waters Q-TLF
results revealed that the synthesized prodrugs possessed anti- Premier mass spectrometer (USA) utilizing electrospray
inflammatory properties; furthermore, three of these com- ionization (ESI). Optical rotations were determined using a
pounds inhibited lipoxygenase.
Perkin–Elmer 341 polarimeter.
In this study, the S(+)-isomer of ibuprofen, (2S)-2-(4-
isobutylphenyl)propionic acid (dexibuprofen), was used as a Chemistry
model drug because it exhibits better anti-inflammatory effects The synthesis routes of the dexibuprofen derivatives (1, 2, 3, 4,
and fewer side effects than ibuprofen. Ethanolamine and its and 5) are outlined in Scheme 1. Dexibuprofen chloride was
related structures were chosen as modifications to dexibu- prepared as described by Song et al[27]. In brief, dexibuprofen
profen in the preparation of prodrugs. Although some etha- was treated with thionyl chloride (SOCl2) at reflux for 4 h.
nolamine derivatives of NSAIDs were synthesized to improve The reaction mixture was concentrated under reduced pres-
the anti-inflammatory activity of parent compounds and sure. The resulting dexibuprofen chloride was coupled with
decrease gastrointestinal toxicity[25, 26], an investigation of their the hydroxyl group of ethanolamine-related structures via
potential as brain targeting agents has not yet been performed. an ester bond. To generate N,N-disubstituted ethanolamine,
Ethanolamine is a commonly found small molecule. Etha- esterification was performed in a one-step reaction by mixing
nolamine and its related structures are not only present in commercially available N,N-dimethylaminoethanol or N,N-di-
endogenous substances including phospholipids (eg, neu- ethylaminoethanol with dexibuprofen chloride in anhydrous
rotransmitters) but also in various CNS-accessible therapeu- dichloromethane (CH2Cl2) in the presence of triethylamine
tics, such as meclofenoxate, cetirizine and procaine. Con- (TEA) to give target prodrug 1 or 2 in high yield[26]. For amino-
taining an amino group, ethanolamine-related structures are ethanol or 2-(methylamino)ethanol, which has a free H atom
positively charged under physiological conditions, which may at the amino group, the amino group was protected by di-
allow them to interact with the negatively charged BBB. More- tertbutyldicarbonate in the first step. The obtained intermedi-
over, prodrugs that are modified by ethanolamine-related ate, either 3a or 4a, was then coupled with dexibuprofen chlo-
structures may act as substrates of the organic cation or cho- ride in the presence of TEA to give either prodrug 3b or 4b.
line transporters that are located at the BBB. After being trans- Prodrug 3b or 4b was then deprotected by HCl in ethyl acetate
ported across the BBB, the prodrugs can be further hydrolyzed to yield prodrug 3 or 4, respectively. In the preparation of
by esterases in the brain to release dexibuprofen.
N-acetyl ethanolamine dexibuprofen ester, N-acetyletha-
In this study, a series of dexibuprofen prodrugs modified by nolamine was synthesized in the first step. Ethanolamine was
ethanolamine-related structures were designed and synthe- dissolved in dioxane with added acetyl chloride in the pres-
sized. The active carboxyl group of dexibuprofen was coupled ence of sodium hydride (NaOH) to yield prodrug 5a. Prodrug
with ethanolamine via an ester bond to give prodrug 4. The 5a was then condensed with dexibuprofen chloride to yield
amino group of ethanolamine was substituted by one methyl final compound 5.
group, two methyl groups, two ethyl groups or one acetyl
group to yield prodrugs 3, 1, 2, and 5, respectively. The stabil- Animals
ity of these prodrugs in the plasma and brain homogenates of Male Sprague-Dawley rats (200–230 g) were provided by the
Sprague-Dawley rats was tested. Biodistribution and phar- Laboratory Animal Center of Sichuan University (Chengdu,
macokinetic studies of dexibuprofen and the prodrugs were China). All rats were maintained under standard conditions
performed in male Sprague-Dawley rats to evaluate the brain- with access to food and water ad libitum. All animal study
targeting efficiency of the prodrugs. The influence of their procedures were approved by the Sichuan University Animal
chemical structures on their transport efficiency is discussed.
Ethical Experimentation Committee according to the require-
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Scheme 1. Synthesis of dexibuprofen derivatives modified
by ethanolamine related structures (1, 2, 3, 4, 5). Reagents
and conditions: (a) CH2Cl2, TEA; (b) (Boc)2O, NaOH; (c)
dexibuprofen chloride, TEA; (d) EtOAc, HCl, RT; (e) CH3COCl,
NaH, dioxane; (f) dexibuprofen chloride, TEA.
ments of the National Act on the use of experimental animals stability of the prodrugs. These values were obtained using
(China).
the formula t1/2=0.693/K. The K values were calculated from
the slope rate of the profile, which was obtained from a plot
of time versus the natural logarithm of the remaining prodrug
In vitro stability
To determine whether ester prodrugs of dexibuprofen were in the medium. The remaining prodrug concentration was
hydrolyzed into free dexibuprofen in rat plasma and brain determined by subtracting the released dexibuprofen concen-
homogenates, we incubated prodrugs 1, 2, and 3 with the pre- tration from the initial concentration of added prodrug in the
pared cultures at 37°C. At a specific time point, aliquots of rat medium.
plasma or brain homogenate were collected and processed.
The released dexibuprofen concentrations were determined Processing of biosamples
using high pressure liquid chromatography (HPLC) with a UV In an EP tube, an aliquot of 200 μL plasma or 500 μL tissue
detector.
homogenates was mixed with naproxen solution in methanol
Rat plasma was prepared immediately before each experi- (1.266 μg/mL, 150 μL), which was used as an internal stan-
ment. Briefly, blood was obtained from the ocular artery and dard. A total of 1 or 2 mL of methanol was then added to the
placed in a heparinized EP tube prior to centrifugation at plasma and tissue homogenates, respectively. The mixtures
1110×g for 5 min. The resulting plasma was incubated with were then vortexed for 10 min, followed by centrifugation at
prodrugs without dilution. Rat brains were removed and 1665×g for 10 min. The supernatant was then transferred to
homogenated with 0.9% sterile saline (g/mL) at a ratio of 1:2 an EP tube and dried at 35°C under air flow. The residue was
immediately before use.
then ultrasonically redissolved in 150 μL of methanol for 5
Prodrugs 1, 2, and 3 (2 mg each) were dissolved in a mixed min. After centrifugation at 10010×g for 10 min, 20 μL of the
solvent solution (2 mL) of ethanol, 0.9% saline and propylene obtained supernatant was injected into an HPLC system to
glycol at a ratio of 1:2:2. The resulting solution (0.2 mL) was determine the dexibuprofen concentration.
then added in the plasma or brain homogenates (9.8 mL),
resulting in a final concentration of 20 μg/mL. The mixtures Chromatographic conditions
were incubated at 37 °C. Samples were prepared at prede- An HPLC-based method was developed to determine the con-
termined time points (plasma: 0, 5, 15, 20, 25, 30, and 40 min; centration of dexibuprofen in biosamples including plasma,
brain homogenates: 0, 0.5, 1, 2, 3, 4, 6, and 12 h). Aliquots of brain, heart, liver, spleen, lung and kidney. The HPLC system
plasma (200 μL) or brain homogenate (500 μL) were with- (Waters, USA) was equipped with a Waters Delta 600 pump, a
drawn to determine the concentration of released dexibupro- Waters 2487 Dual λ absorbance detector, a Waters 600 control-
fen. Naproxen solution in methanol (1.266 μg/mL, 150 μL) ler, a model 100 column heater (CBL photoelectron technol-
was added immediately. A total of 1 mL or 2 mL methanol ogy) and a 20-μL injector loop. The Kromasil C-18 column
was then added to the plasma or brain homogenates, respec- (150 mm×4.6 mm, ODS, 5 μm) was protected by a Phenom-
tively, to prevent further hydrolysis of the prodrugs. The bio- enex guard column (4 mm×3 mm, ODS). The column was
sample processing method and chromatographic conditions eluted using a mobile-phase system containing acetonitrile/
are described in the following sections.
water (49/51), with 0.2% (v/v) triethylamine added. Phos-
Hydrolysis half-life measurements were used to evaluate the phoric acid was used to adjust the pH to 2.5. The mobile
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phase was then filtered and degassed by sonication before artery and sacrificed by cervical dislocation. Blood samples
use. The flow rate used was 1.0 mL/min, and the diode-array were collected and placed into heparinized EP tubes. Imme-
detector was operated at 220 nm.
diately following collection, the samples were centrifuged at
Naproxen was used as an internal standard. An HPLC 1110×g for 5 min. The plasma was then stored at -20°C until
assay for determining the concentration of dexibuprofen in assays were performed. Tissue samples, including samples
different tissues was developed, including protocols for gen- from the brain, heart, liver, spleen, lung, and kidney, were
erating calibration curves and determining intra- and inter- removed and flushed with saline. The tissue samples were
day precision and extraction recoveries. Under the optimized then homogenized using 0.9% saline (g/mL) at a ratio of
HPLC conditions, the retention times of naproxen and dexi- 1:2. The homogenates were stored at -20°C until assays were
buprofen were 5.5 and 13.83 min, respectively. These values performed. Aliquots of 200 μL plasma or 500 μL tissue homo-
were well separated from endogenous substances and prod- genates were processed, and the released dexibuprofen was
rugs 1, 2, 3, 4, and 5, which had retention times of 2.730, 3.595, measured as described above.
2.265, 2.730, and 12.710 min, respectively (Figure 1).
Data analysis
Biodistribution and pharmacokinetic studies of dexibuprofen and The pharmacokinetic parameters of dexibuprofen and the
its prodrugs in rats
prodrugs were described as a two-compartment open model.
To further evaluate the brain targeting efficiency of the prod- The pharmacokinetic parameters in the plasma and tissues
rugs, a biodistribution and pharmacokinetic study was per- were calculated using Data and Statistics software pakage
formed in vivo. Male Sprague-Dawley rats (200–230 g) were (DAS, Shanghai, China). The area under the curve (AUC), the
provided by the Laboratory Animal Center of Sichuan Uni- maximal concentration (Cmax) and the mean residence time
versity. Prodrugs and dexibuprofen were injected through (MRT0–t) for the plasma, brain, heart, liver, spleen, lung and
the caudal vein. For each sampling time point, five non-fasted kidney were calculated separately. Statistical evaluation was
male rats were treated with a single dose of either dexibu- performed using analysis of variance followed by a t-test. P
profen (11.70 mg/kg) or prodrug 1 (15.73 mg/kg), 2 (17.34 values less than 0.01 and 0.05 were considered significant. The
mg/kg), 3 (14.94 mg/kg), 4 (14.15 mg/kg), or 5 (16.53 mg/kg). data shown represent mean values±SD.
Each prodrug dose was equivalent to 11.70 mg/kg of dexi-
The relative uptake efficiency (RE), concentration efficiency
buprofen. Prior to administration, the compounds were dis- (CE) and drug targeting index (DTI) were calculated to evalu-
solved in a mixed solvent containing a 1:2:2 ratio of ethanol, ate the brain targeting efficiency of the prodrugs. The ratio of
0.9% saline and propylene glycol, to a final concentration of 60 the dexibuprofen concentration in the brain compared to that
mmol/L. In the biodistribution study, the rats were sacrificed in the plasma {(Cbrain/Cplasma
)dexibuprofen} was also used to evalu-
10 min after the injection of either dexibuprofen or prodrugs 1, ate brain targeting efficiency. The values of RE, CE, and DTI
2, 3, 4, or 5. For the pharmacokinetic investigations of dexibu- are defined as follows:
profen and prodrug 1, the rats were sacrificed at 5, 10, 15, 30, RE=(AUC)prodrug/(AUC)dexibuprofen
45, 60, 120, and 240 min post-dosing.
CE=(Cmax prodrug/(Cmax dexibuprofen and
At a specific time point, the rats were bled from the ocular DTI=(AUCbrain/AUCplasma
prodrug/(AUCbrain/AUCplasma
,
)
)
)
)
dexibuprofen
Figure 1. HPLC chromatograms of
dexibuprofen and prodrugs 1, 2, 3,
4, and 5. Compound was dissolved
in methanol with concentration of 5
μg/mL.
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Results
In vitro stability
The in vitro stability studies revealed that all of the tested dexi-
buprofen derivatives released dexibuprofen when incubated
with rat plasma and brain homogenates (Table 1). These
result suggest that compounds 1, 2, and 3 were rapidly hydro-
lyzed in rat plasma. The half-lives of compounds 1 and 2 were
less than 5 min. The half-life of prodrug 3 was 11.63±1.04
min. These results imply that prodrugs 1, 2, and 3, which all
contain ester bonds, may be substrates of specific esterases in
the plasma and are efficiently hydrolyzed. Investigation of
the stability of the prodrugs in rat brain homogenates showed
that compounds 1, 2, and 3 were cleaved by enzymes at differ-
ent rates. The hydrolysis half-lives of compounds 1, 2, and 3
were 0.273±0.009, 1.150±0.061, and 3.712±0.174 h, respectively.
The results suggested that prodrug 3 was more stable in brain
homogenates than either compound 1 or 2, which was consis-
tent with the findings for the plasma.
Figure 2. Dexibuprofen concentrations (μg/g) in the plasma and brain
10 min after intravenous administration of dexibuprofen (11.70 mg/kg)
or prodrugs 1, 2, 3, 4, 5 (each compound was equivalent to 11.70 mg/kg
of dexibuprofen) in rats. Each point represents the mean±SD of five
experiments. cP<0.01 vs dexibuprofen.
Table 1. Hydrolysis half-life (t1/2) of prodrugs 1, 2, 3 in rat plasma or brain
homogenate at 37ºC (n=3).
t1/2
Compound
Plasma
Brain homogenate
1
2
3
<5.0 min
<5.0 min
11.63±1.04 min
0.273±0.009 h
1.150±0.061 h
3.712±0.174 h
Figure 3. Comparison of (Cbrain/Cplasma
)
ratios of dexibuprofen and
dexibuprofen
prodrugs. Cbrain, Cplasma: dexibuprofen concentration (μg/g) in the brain or
plasma 10 min after intravenous administration of dexibuprofen (11.70
mg/kg) or prodrugs 1, 2, 3, 4, 5 (each compound was equivalent to 11.70
mg/kg of dexibuprofen) in rats. Each point represents the mean±SD of
five experiments. cP<0.01 vs dexibuprofen.
Biodistribution and pharmacokinetic study of dexibuprofen and
prodrugs in rats
To evaluate the brain-targeting efficiency of prodrugs 1,
2, 3, 4, and 5 (Scheme 1), a biodistribution study was per- the highest distribution efficiency in the brain.
formed. We determined the concentrations of dexibuprofen
We further investigated the biodistribution and pharmacoki-
in the plasma, brain, heart, liver, spleen, lung and kidney 10 netic parameters of dexibuprofen and prodrug 1 in Sprague-
min after intravenous administration of dexibuprofen (11.70 Dawley rats. The concentrations of dexibuprofen after the
mg/kg) or prodrugs (at doses equivalent to 11.70 mg/kg of systemic administration of dexibuprofen (11.70 mg/kg) and
dexibuprofen). The concentration of dexibuprofen in the brain prodrug 1 (15.73 mg/kg, a dose equivalent to 11.70 mg/kg
versus that in the plasma (Cbrain/Cplasma
)dexibuprofen was used to of dexibuprofen) were determined and statistically analyzed.
evaluate the brain-targeting efficiency of the compounds. The The dexibuprofen concentration profile versus time is shown
concentrations of dexibuprofen in the brain and plasma 10 in Figure 4. The Cbrain/Cplasma ratio versus time is shown in Fig-
min after the injection of dexibuprofen or prodrug 1, 2, 3, 4, or ure 5. The pharmacokinetic parameters are reported in Table
5 are shown in Figure 2 (n=5). Prodrugs 1, 2, and 3 exhibited 2. An evaluation of the targeting efficiency of prodrug 1 is
enhanced drug concentrations in the brain in comparison with shown in Table 3.
dexibuprofen (P<0.01). The (Cbrain/Cplasma
)
ratios 10
The results revealed that 5 min after the administration of
dexibuprofen
min after administration of dexibuprofen or prodrug 1, 2, 3, dexibuprofen, the concentration in the plasma was initially
4, or 5 were 0.037±0.011, 0.626±0.091, 0.576±0.119, 0.290±0.114, very high, with a Cplasma of 64.44±14.00 μg/g (Figure 4). How-
0.342±0.132, and 0.126±0.026, respectively (Figure 3; n=5). The ever, at the same time point, the concentration of dexibuprofen
(Cbrain/Cplasma)dexibuprofen ratios of prodrugs 1, 2, 3, 4, and 5 were in the brain was only 2.81±1.16 μg/g. At different time points,
17.0-, 15.7-, 7.88-, 9.31-, and 3.42-fold higher, respectively, the Cbrain/Cplasma ratios were consistently low after the adminis-
than that of dexibuprofen. Thus, the prodrugs exhibit good tration of dexibuprofen, never exceeding 0.05 (Figure 5). The
brain-targeting efficiency (P<0.01), with a relative efficiency of AUCbrain/AUCplasma ratio was only 0.025. These results indi-
1>2>4>3>5. Among prodrugs (2S)-2-(4-isobutylphenyl) pro- cated that the BBB was able to strongly prevent the entry of
pionic acid 2-dimethylaminoethyl ester (prodrug 1), exhibited dexibuprofen into the brain. This finding is in agreement with
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Table 2. Pharmacokinetic parameters (calculating from the measured dexibuprofen concentrations) in rat plasma and tissues after intravenous
administration of dexibuprofen (11.70 mg/kg) or prodrug 1 (15.73 mg/kg, equivalent to 11.70 mg/kg of dexibuprofen). Each point represents the
mean±SD of five experiments. bP<0.05, cP<0.01 vs dexibuprofen.
Dexibuprofen
Cmax (μg/g)
Prodrug 1
Cmax (μg/g)
Tissue
AUC0→t (μg/g·h)
MRT0–t (h)
AUC0→t (μg/g·h)
MRT0–t (h)
Plasma
Brain
Heart
Liver
Spleen
Lung
Kidney
44.79±7.73
1.10±0.41
8.11±1.39
14.03±3.91
4.01±0.62
10.60±2.10
6.34±1.10
64.44±14.00
2.81±1.16
0.789±0.046
0.274±0.023
0.767±0.158
0.961±0.086
1.128±0.357
1.220±0.072
0.700±0.077
28.70±2.35b
7.89±1.23c
6.66±1.04
12.98±5.12
4.12±1.12
22.07±4.84c
7.58±1.34
29.74±2.97c
21.79±3.37c
10.46±1.78
10.34±1.49b
6.24±3.15
29.03±4.41c
11.59±2.68
0.900±0.119
0.237±0.034
0.733±0.167
1.052±0.16
0.591±0.096b
0.853±0.037c
0.592±0.043
11.99±3.70
16.94±3.25
5.60±1.27
13.19±2.56
9.43±2.72
Figure 5. (Cbrain/Cplasma)dexibuprofen ratios of dexibuprofen or prodrug 1 versus
time (h). Cbrain, Cplasma: dexibuprofen concentration (μg/g) in the brain or
plasma after intravenous administration of dexibuprofen (11.70 mg/kg) or
prodrug 1 (15.73 mg/kg, equivalent to 11.70 mg/kg of dexibuprofen) in
rats. Each point represents the mean±SD of five experiments. bP<0.05,
cP<0.01 vs dexibuprofen.
a previous study that revealed that most NSAIDs exhibited
very limited distribution in the CNS, with steady state brain/
plasma concentration ratios of 0.01–0.05[7].
Figure 4. Dexibuprofen concentrations in the plasma and brain (μg/g) vs
time (h) after intravenous administration of dexibuprofen (11.70 mg/kg)
and prodrug 1 (15.73 mg/kg, equivalent to 11.70 mg/kg of dexibuprofen)
in rats. Each point represents the mean±SD of five experiments.
bP<0.05, cP<0.01 vs dexibuprofen.
Compared with the administration of free dexibuprofen, the
concentration of dexibuprofen in the plasma after adminis-
tration of prodrug 1 was moderate. The concentration in the
plasma was 29.74±2.97 μg/g 5 min after the administration
of prodrug 1 (Figure 4), which was lower than that of dexibu-
profen (P<0.01). With moderate concentrations in the plasma,
prodrug 1 exhibited a high concentration of dexibuprofen
(21.79±3.37 μg/g) in the brain 5 min after administration
Table 3. Targeting efficiency of Prodrug 1 compared with dexibuprofen
after intravenous administration of dexibuprofen (11.70 mg/kg) and 1
(15.73 mg/kg, equivalent to 11.70 mg/kg of dexibuprofen) in male rats
(Figure 4). The (Cbrain/Cplasma
)
ratio of prodrug 1 was
dexibuprofen
0.829 at 5 min, much higher than that of dexibuprofen, which
was only 0.044 (Figure 5) (P<0.01). These results revealed
almost no barrier effect of the BBB in response to prodrug 1.
However, the high concentration of dexibuprofen observed
decreased rapidly, with an MRT of 0.237±0.034 h, showing an
active exclusion effect of the BBB toward dexibuprofen.
The AUC0–t values after administration of dexibuprofen
and prodrug 1 were 1.10±0.41 and 7.89±1.23 μg/g·h in the
brain, respectively. The Cmax values after the administration
(mean±SD, n=5). aRE=(AUC)1/(AUC)dexibuprofen
.
bCE=(Cmax)1/(Cmax
)
.
dexibuprofen
cDTI=(AUCtissue /AUCplasma)1/(AUCtissue/AUCplasma
)
.
dexibuprofen
Brain
Heart
Liver
Spleen
Lung
Kidney
REa
CEb
DTIc
7.17
7.75
11.19
0.82
0.87
1.28
0.93
0.61
1.44
1.4
1.11
2.18
1.24
2.20
1.94
1.49
1.23
2.32
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of dexibuprofen and prodrug 1 were 2.81±1.16 and 21.79±3.37 prodrugs are positively charged under physiological condi-
μg/g in the brain, respectively (Table 2). The relative effi- tions, which may allow them to interact with the negatively
ciency (RE=AUC1/AUCdexibuprofen) was 7.17, and the (Cmax)1/ charged BBB.
(Cmax
)
ratio was 7.75 in the brain (Table 3). The drug
Among the prodrugs tested, prodrug 1, which was prepared
by coupling N,N-dimethylaminoethanol and dexibuprofen
dexibuprofen} was 11.19. Taken together, these data via an ester bond, exhibited the highest targeting efficiency.
dexibuprofen
targeting index {DTI=(AUCbrain/AUCplasma)1/(AUCbrain
AUCplasma
/
)
indicated a remarkable brain targeting efficiency of prodrug Because prodrug 1 was rapidly hydrolyzed in the rat plasma
1 with a marked enhancement of brain distribution, accom- with a half-life less than 5 min, its high distribution in the
panied by a decreased plasma distribution. This finding sup- brain indicated remarkable brain-transport efficiency.
ports the use of such compounds as therapeutic agents, per-
During the synthesis of prodrugs 1, 2, 3, 4, and 5, the active
mitting enhanced therapeutic concentrations in the brain with carboxyl group of dexibuprofen was coupled with etha-
decreased peripheral side effects.
nolamine-related structures via an ester bond. The present
study reveals that although prodrugs have similar structures,
their stability in media and brain-transport efficiency were
Discussion
In this study, ethanolamine and its related structures were quite different. It is interesting to note that many CNS-acces-
coupled with dexibuprofen to generate potential brain-target- sible therapeutics, such as meclofenoxate, cetirizine and pro-
ing agents. The in vitro stability and in vivo biodistribution of caine, contain N,N-dimethylaminoethanol structures. Because
these compounds were investigated and biopharmacokinetic the structure of N,N-dimethylaminoethanol is very similar to
studies were performed to evaluate their brain-targeting effi- choline, we hypothesize that prodrug 1 might be a substrate
ciency. The results obtained were compared with that of free of the choline transporters located at the BBB. After entering
dexibuprofen. The influence of their chemical structures on the brain, prodrug 1 was hydrolyzed by esterases, resulting in
the transport efficiency was also discussed.
In the stability study, the results showed a rapid hydrolysis rug 2 also exhibited a high transport efficiency. The (Cbrain
of prodrugs 1, 2, and 3 in the plasma (Table 1). Prodrugs 1, 2, Cplasma
ratio of prodrug 2 was 15.7-fold higher than
the release of dexibuprofen. In addition to prodrug 1, prod-
/
)
dexibuprofen
and 3, which each contain ester bonds, were likely degraded that of free dexibuprofen 10 min after administration (Figure
by specific esterases in the plasma. Because N,N-dimethylam- 3, P<0.01). This result implies that the small alkyl groups
inoethanol, N,N-diethylaminoethanol and 2-(methylamino) disubstituted onto the amino group of ethanolamine seem to
ethanol have structures that are similar to that of choline, facilitate the recognition and transport of these compounds by
prodrugs 1, 2, and 3 could be substrates of plasma esterases, specific transporters at the BBB.
such as cholinesterase. These findings were in agreement
with those of a previous study that showed compounds with of prodrug 1, the (Cbrain/Cplasma
Our results revealed that at 5 min after the administration
ratio reached levels as
)
dexibuprofen
structures similar to that of prodrug 1 undergoing very fast high as 0.829 (Figure 5). Subsequently, the high concentration
hydrolysis in 80% human plasma, with half-lives less than 5 of dexibuprofen in the brain decreased rapidly. Because dexi-
min[28]. The hydrolysis profiles of the prodrugs in rat brain buprofen is negatively charged at physiological pH, prevent-
homogenates revealed that prodrug 3 was more stable than ing it from passively penetrating through the BBB, the rapid
either prodrug 1 or 2, suggesting that prodrugs 1 and 2 are efflux of dexibuprofen was probably facilitated by efflux trans-
better substrates of specific esterases in the brain than prodrug porters located at the BBB. A sustained-release drug delivery
3 and underwent hydrolysis with high efficiency to release system for prodrug 1 designed to maintain a steady concentra-
dexibuprofen.
tion of dexibuprofen in the CNS will be investigated in future
In our preliminary investigations, we investigated the in studies. A more stable ester bond could also be designed to
vitro stability of prodrugs 4 and 5. The hydrolysis profiles of allow prodrugs to be slowly hydrolyzed by brain esterases
prodrugs 4 and 5 revealed that they were not stable and did after entering the brain.
not follow the linear kinetic equation. Therefore, we have only
presented in vitro stability data relating to prodrugs 1, 2, and
3.
Conclusions
In this study, we designed and synthesized a series of dexi-
In our biodistribution studies, each prodrug exhibited an buprofen derivatives that were modified with ethanolamine-
enhancement relative to dexibuprofen in brain-targeting effi- related structures. This is the first time that ethanolamine-
ciency (Figure 3, P<0.01). As observed from the results of our related structures have been utilized in the modification of
HPLC studies, the lipophilicity of prodrugs 1, 2, 3, 4, and 5 compounds for brain-targeting delivery. The distribution
are not increased when compared with dexibuprofen (Figure and pharmacokinetic studies revealed that all of the prepared
1); thus, the observed enhancement of brain distribution is derivatives exhibited enhanced brain distribution com-
probably due to movement by specific transporters located at pared with dexibuprofen (P<0.01). These results imply that
the BBB. Because ethanolamine-related structures are similar ethanolamine-related structures may play an important role
to choline and small molecular cations, the high transport in transport across the BBB. The mechanism responsible for
efficiency of prodrugs is probably facilitated by the action of this enhancement will likely be further elucidated in the near
organic cations or choline transporters at the BBB. In addition, future.
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Zhang X et al
286
mL). Di-tert-butyldicarbonate (1.90 g, 8.6 mmol) was then
(2S)-2-(4-isobutylphenyl)propionic acid 2-dimethylaminoethyl added. The mixture was then stirred at room temperature for
ester (1)
5 h. The mixture was concentrated under reduced pressure,
Experimental
N,N-dimethylaminoethanol (1.30 g, 14.6 mmol) and TEA (5 and the residue was then subjected to silica gel chromatogra-
mL, 36 mmol) were dissolved in anhydrous CH2Cl2 (15 mL). phy (dichloromethane/methanol, 20:1) to yield prodrug 3a as
Dexibuprofen chloride (2.24 g, 10.0 mmol) was added drop- a colorless oil (0.86 g, 71.1%).
wise under an ice bath. The reaction mixture was stirred con-
1H-NMR (CDCl3, 400 MHz): δ 3.733 (s, 2H, HOCH2CH2),
tinuously at room temperature for 4 h. The mixture was then 3.382 (s, 2H, NCH2CH2), 2.907 (s, 3H, CH2NCH3), 1.452 [s, 9H,
extracted with CH2Cl2. The organic layer was combined and COOC(CH3)3].
sequentially washed with sodium bicarbonate solution (aq)
and water. It was then dried over anhydrous sodium sulfate (2S)-2-(4-isobutylphenyl)propionic acid 2-(N-tert-butoxycarbonyl-
and filtered. The resulting filtrate was concentrated under N-methylamino) ethyl ester (3b)
reduced pressure. The residue was then subjected to silica gel Prodrug 3a (0.70 g, 4.1 mmol) was dissolved in CH2Cl2 (10 mL)
chromatography (dichloromethane/methanol, 20:1) to yield with added TEA (1 mL, 7.2 mmol). Dexibuprofen chloride
prodrug 1 as a colorless oil (2.32 g, 83.7%).
(1.10 g, 4.9 mmol) was added dropwise under an ice bath. The
mixture was stirred at room temperature for 4 h. The mixture
[α]20D+36.3° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.178 (d, 2H, J=8.0 Hz, 2’-H, 6’-H), was concentrated, and the residue was subjected to silica gel
7.059 (d, 2H, J=8.0 Hz, 3’-H, 5’-H), 4.191–4.107 (m, 2H, CH2CH2OCO), chromatography (petroleum ether/ethyl acetate, 7:1) to yield
3.660–3.720 (m, 1H, COCHCH3), 2.503 (t, 2H, J=11.2 Hz, NCH2CH2), prodrug 3b as a colorless oil (1.16 g, 78.5%).
2.418 [d, 2H, J=7.2 Hz, CH2CH(CH3)2], 2.182 [s, 6H, N(CH3)2], 1.850–
1.783 [m, 1H, CH2CH(CH3)2], 1.459 (d, 3H, J=6.8 Hz, COCHCH3), 0.870
[d, 6H, J=6.4 Hz, CH2CH(CH3)2].
[α]20D+16.0° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.186 (d, 2H, J=8.0 Hz,
2’-H, 6’-H), 7.086 (d, 2H, J=8.0 Hz, 3’-H, 5’-H), 4.165 (s, 2H,
IR (KBr cm-1): 3089, 2955, 2869, 2821, 1735, 1513, 1460, 1160, CH2CH2OCO), 3.657–3.750 (m, 1H, COCHCH3), 3.368 (m,
1067.
2H, NCH2CH2), 2.674–2.722 (m, 3H, CH2NCH3), 2.438 [d, 2H,
J=7.2 Hz, CH2CH(CH3)2], 1.799–1.875 [m, 1H, CH2CH(CH3)2],
1.482–1.515 (m, 3H, COCHCH3), 1.437 [s, 9H, COOC(CH3)3],
HRMS for [M+H]+: m/z calculated 278.2120; found 278.2115.
(2S)-2-(4-isobutylphenyl)propionic acid 2-diethylaminoethyl ester 0.879–0.901 [m, 6H, J=6.8 Hz, CH2CH(CH3)2].
(2)
N,N-diethylaminoethanol (0.88 g, 7.51 mmol) and TEA (2 mL, (2S)-2-(4-isobutylphenyl)propionic acid 2-methylaminoethyl ester
14.4 mmol) were dissolved in anhydrous CH2Cl2 (10 mL). (3)
Dexibuprofen chloride (1.12 g, 5.0 mmol) was added dropwise Concentrated hydrochloric acid (0.1 mL) was added to a solu-
under an ice bath. The reaction mixture was stirred continu- tion of compound 3b (1.0 g, 2.7 mmol) in ethyl acetate (10 mL).
ously at room temperature for 2.5 h. The mixture was then The mixture was stirred at room temperature for 14 h. The
extracted with CH2Cl2. The organic layer was combined and solution was then concentrated, and the residue was subjected
sequentially washed with sodium bicarbonate solution (aq) to silica gel chromatography (dichloromethane/methanol,
and water. It was then dried over anhydrous sodium sulfate 10:1) to yield prodrug 3 as a white solid (0.48 g, 57.2%).
and filtered. The resulting filtrate was concentrated under
reduced pressure. The residue was subjected to silica gel
chromatography (dichloromethane/methanol, 20:1) to yield
prodrug 2 as a yellow-tinted oil (1.10 g, 72.4%).
mp 103–105°C.
[α]20D+9.0° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.203 (d, 2H, J=8.0 Hz, 2’-H,
6’-H), 7.085 (d, 2H, J=8.0 Hz, 3’-H, 5’-H), 4.508–4.549 (m, 1H,
CH2CH2OCO), 4.317–4.341 (m, 1H, CH2CH2OCO), 3.847–3.898
[α]20D+39.4° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.197 (d, 2H, J=8.0 Hz, 2’-H, (m, 1H, COCHCH3), 3.053–3.146 (m, 2H, NCH2CH2), 2.517
6’-H), 7.083 (d, 2H, J=8.0 Hz, 3’-H, 5’-H), 4.145 (t, 2H, J=6.0 Hz, (s, 3H, CH2NCH3), 2.433 [d, 2H, J=7.2 Hz, CH2CH(CH3)2],
CH2CH2OCO), 3.695 (q, 1H, J=7.2 Hz, COCHCH3), 2.675 (t, 1.796–1.863 [m, 1H, CH2CH(CH3)2], 1.508 (d, 3H, J=6.8 Hz,
2H, J=6.0 Hz, NCH2CH2), 2.505 [q, 4H, J=7.2 Hz, N(CH2CH3)2], COCHCH3), 0.886 [d, 6H, J=6.8 Hz, CH2CH(CH3)2].
2.440 [d, 2H, J=6.8 Hz, CH2CH(CH3)2], 1.786–1.888 [m,
1H, CH2CH(CH3)2], 1.482 (d, 3H, J=6.8 Hz, COCHCH3), 1073.
0.976 [t, 6H, J=7.2 Hz, (CH2CH3)2], 0.891 [d, 6H, J=6.8 Hz,
CH2CH(CH3)2].
IR (KBr cm-1): 3391, 2955, 2869, 2845, 1735, 1510, 1464, 1163,
HRMS for [M+H]+: m/z calcd 264.1964; found 2764.1960.
IR (KBr cm-1): 2967, 2872, 2808, 1736, 1513, 1461, 1380, 1164, N-tert-butoxycarbonyl-ethanolamine (4a)
1069.
Ethanolamine (0.60 g, 10.0 mmol) and sodium hydroxide
HRMS for [M+H]+: m/z calcd 306.2433; found 306.2436.
(0.40 g, 10.0 mmol) were dissolved in water (10 mL) and diox-
ane (10 mL). Di-tert-butyldicarbonate (2.7 g, 12.0 mmol) was
then added, and the mixture was stirred at room temperature
N-tert-butoxycarbonyl-2-methylaminoethanol (3a)
2-(methylamino)ethanol (0.50 g, 7.0 mmol) and sodium for 3.5 h. The mixture was concentrated, and the residue was
hydroxide (0.28 g, 7.0 mmol) were dissolved in methanol (10 subjected to silica gel chromatography (petroleum ether/
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Zhang X et al
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ethyl acetate, 1:1) to yield prodrug 4a as a colorless oil (1.54 g,
95.1%).
1H-NMR (CDCl3, 400 MHz): δ 1.990 (s, 3H, CH3CO), 3.326–
3.390 (m, 2H, CH2NCO), 3.682 (t, 2H, CH2O)
1H-NMR (CDCl3, 400 MHz): δ 3.650 (t, 2H, J=5.2 Hz,
HOCH2CH2), 3.241 (t, 2H, J=5.2 Hz, NCH2CH2), 1.415 [s, 9H, (2S)-2-(4-isobutylphenyl)propionic acid 2-(acetylamino)ethyl ester
COOC(CH3)3].
(5)
Compound 5a (0.5 g, 4.85 mmol) and TEA (2 mL, 14.4 mmol)
(2S)-2-(4-isobutylphenyl)propionic acid 2-(N-tert-butoxycarbonyl- was dissolved in anhydrous tetrahydrofuran (20 mL). Dexi-
amino) ethyl ester (4b)
buprofen chloride (1.31 g, 5.84 mmol) was added dropwise
Compound 4a (0.7 g, 4.3 mmol) was dissolved in CH2Cl2 (10 under an ice bath. The reaction mixture was then stirred
mL) with added TEA (1 mL, 7.2 mmol). Dexibuprofen chlo- continuously at room temperature for 2 h. The mixture was
ride (1.44 g, 6.45 mmol) was added dropwise under an ice concentrated, and the residue was subjected to silica gel chro-
bath. The mixture was stirred at room temperature for 3.5 h. matography (petroleum ether/ethyl acetate, 1:1) to yield com-
The mixture was then concentrated, and the residue was sub- pound 5 as a colorless oil (0.94 g, 66.2%).
jected to silica gel chromatography (petroleum ether/ethyl
acetate, 10:1) to yield prodrug 4b as a colorless oil (1.38 g,
92.1%).
[α]20D+17.8° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.111 (d, 2H, J=8.0 Hz,
3’-H,5’-H), 7.200 (d, 2H, J=8.4 Hz, 2’-H, 6’-H), 4.174–4.228
(m, 1H, CH2O), 4.056–4.109 (m, 1H, CH2O), 3.685–3.737 (m,
[α]20D+23.1° (c 1, CH3OH).
1H-NMR (CDCl3, 400 MHz): δ 7.190 (d, 2H, J=7.6 Hz, 1H, COCHCH3), 3.397–3.436 (m, 2H, CH2NCO), 2.444 [d,
2’-H, 6’-H), 7.088 (d, 2H, J=7.6 Hz, 3’-H, 5’-H), 4.102 (s, 2H, J=7.2 Hz, CH2CH(CH3)2], 1.826–1.872 [m, 4H, CH3CO,
2H, CH2CH2OCO), 3.672–3.725 (m, 1H, COCHCH3), 3.288 CH2CH(CH3)2], 1.492 (d, 3H, J=7.2 Hz, COCHCH3), 0.892 [d,
(s, 2H, NCH2CH2), 2.439 [d, 2H, J=7.2 Hz, CH2CH(CH3)2], 6H, J=6.4 Hz, CH2CH(CH3)2].
1.793–1.891 [m, 1H, CH2CH(CH3)2], 1.478 (d, 3H, J=6.8 Hz,
COCHCH3), 1.421 [s, 9H, COOC(CH3)3], 0.890 [d, 6H, J=6.0 Hz, 1459, 1373, 1292, 1166, 1070, 799.
IR (KBr cm-1): 3294, 3085, 2956, 2871, 1736, 1657, 1553, 1515,
CH2CH(CH3)2].
HRMS for [M+H]+: m/z calcd 292.1913; found 292.1916.
(2S)-2-(4-isobutylphenyl)propionic acid 2-aminoethyl ester (4)
Acknowledgements
Concentrated hydrochloric acid (0.1 mL) was added to a solu- This work was supported by the National Natural Science
tion of compound 4b (1.0 g, 2.8 mmol) in ethyl acetate (10 mL). Foundation of China (Grant No 81130060).
The mixture was stirred at room temperature for 14 h. The
solution was then concentrated, and the residue was subjected
to silica gel chromatography (dichloromethane/methanol, Xuan ZHANG, Zhi-rong ZHANG, Xing LIU, Tao GONG, and
Author contribution
10:1) to yield compound 4 as a white solid (0.36 g, 43.5%).
mp 117–120°C.
Xun SUN designed the research strategy; Xuan ZHANG and
Xing LIU performed the research; Xuan ZHANG and Xing
LIU analyzed the data; and Xuan ZHANG wrote the paper.
[α]20D+14.0° (c 1, CH3OH).
1H-NMR (DMSO-d, 400 MHz): δ 7.214 (d, 2H, J=8.0 Hz,
2’-H, 6’-H), 7.116 (d, 2H, J=8.0 Hz, 3’-H, 5’-H), 4.278–4.335 (m,
1H, CH2CH2OCO), 4.044–4.100 (m, 1H, CH2CH2OCO), 3.781–
3.835 (m, 1H, COCHCH3), 3.041–3.054 (m, 2H, NCH2CH2),
2.413 [d, 2H, J=7.2 Hz, CH2CH(CH3)2], 1.770–1.837 [m, 1H,
CH2CH(CH3)2], 1.408 (d, 3H, J=6.8 Hz, COCHCH3), 0.853 [d,
6H, J=6.4 Hz, CH2CH(CH3)2].
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