Brain-specific delivery of dopamine mediated by N, N-dimethyl amino group for the treatment of Parkinson's disease

Article abstract of DOI:10.1021/mp500352p

Parkinson's disease (PD) has become one of the most deadly diseases due to a lack of effective treatment. Herein, N-3,4-bis(pivaloyloxy)dopamine-3- (dimethylamino)propanamide (PDDP), a brain-specific derivative of dopamine, was designed and synthesized, which consists of a brain targeted ligand, N,N-dimethyl amino group, and two dipivaloyloxy groups for lipophilic modification. PDDP was investigated both in vitro and in vivo by comparing with L-DOPA and another derivative (BPD) without N,N-dimethyl amino group. PDDP showed a more pronounced accumulation in mouse brain microvascular endothelial cells (bEnd.3) than BPD via an active transport process. The increased cellular uptake of PDDP was proven to be mediated by putative pyrilamine cationic transporters. Following intravenous administration, the concentration of PDDP in the brain was 269.28-fold and 6.41-fold higher than that of L-DOPA and BPD at 5 min, respectively. Additionally, PDDP effectively attenuated the striatum lesion caused by 6-hydroxydopamine (6-OHDA) in rats. More importantly, PDDP presented antioxidant and antiapoptotic effects on 6-OHDA-induced toxicity in human neuroblastoma cells (SH-SY5Y). Thus, N,N-dimethyl amino group-based PDDP represents an effective and safe treatment for PD.

Full text of DOI:10.1021/mp500352p

Article
pubs.acs.org/molecularpharmaceutics
Brain-Specific Delivery of Dopamine Mediated by N,N‑Dimethyl
Amino Group for the Treatment of Parkinson’s Disease
Yanping Li, Yangyang Zhou, Bowen Qi, Tao Gong, Xun Sun, Yao Fu, and Zhirong Zhang*
Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, Sichuan University, Sichuan, People’s Republic
of China
S
* Supporting Information
ABSTRACT: Parkinson’s disease (PD) has become one of the most deadly
diseases due to a lack of effective treatment. Herein, N-3,4-bis(pivaloyloxy)-
dopamine-3-(dimethylamino)propanamide (PDDP), a brain-specific deriv-
ative of dopamine, was designed and synthesized, which consists of a brain
targeted ligand, N,N-dimethyl amino group, and two dipivaloyloxy groups
for lipophilic modification. PDDP was investigated both in vitro and in vivo
by comparing with L-DOPA and another derivative (BPD) without N,N-
dimethyl amino group. PDDP showed a more pronounced accumulation in
mouse brain microvascular endothelial cells (bEnd.3) than BPD via an active
transport process. The increased cellular uptake of PDDP was proven to be
mediated by putative pyrilamine cationic transporters. Following intravenous administration, the concentration of PDDP in the
brain was 269.28-fold and 6.41-fold higher than that of L-DOPA and BPD at 5 min, respectively. Additionally, PDDP effectively
attenuated the striatum lesion caused by 6-hydroxydopamine (6-OHDA) in rats. More importantly, PDDP presented antioxidant
and antiapoptotic effects on 6-OHDA-induced toxicity in human neuroblastoma cells (SH-SY5Y). Thus, N,N-dimethyl amino
group-based PDDP represents an effective and safe treatment for PD.
KEYWORDS: N,N-dimethyl amino group, lipophilic derivatization, dopamine, brain-targeting, Parkinson’s disease
distribution of the parent drug, but no systematic investigation
was conducted to address the therapeutic effect and side effects.
In previous works, N,N-dimethyl amino group-related
structures were proven to significantly enhance the brain-
uptake efficiency of dexibuprofen15 and naproxen.16 This small
molecular ligand offers several advantages for drug develop-
ment. Specifically, N,N-dimethyl amino group possesses a
simple and well-defined structure. Moreover, the N,N-dimethyl
amino group has a high degree of safety without the potential
toxicity associated with macromolecular carriers. Additionally,
N,N-dimethyl amino group has high efficiency of brain
targeting which would likely result in superior therapeutic
effect. To our best knowledge, no published reports are
available regarding N,N-dimethyl amino group as a brain-
targeted ligand by other groups.
Therefore, N-(3,4-dihydroxyphenethyl)-3-(dimethylamino)
propanamide (DDP) was initially designed and synthesized,
which is the conjugate of dopamine and N,N-dimethylamino
propanoic acid to improve the brain targetability of dopamine.
However, DDP, which could not be transported across the BBB
(data not shown), was shown not to be a suitable candidate for
brain targeting. Similar results were obtained with other drugs
(e.g., zidovudine) conjugated with the N,N-dimethyl amino
1. INTRODUCTION
Parkinson’s disease (PD) is a progressive neurodegenerative
disorder associated with the loss of dopamine (DA) neurons in
the nigrostriatal pathway.1 A population of approximately 5
million globally suffer from PD, and the prevalence is expected
to increase dramatically in the coming decades due to the rapid
aging of the population worldwide.2 To date, oral admin-
istration of L-DOPA has been the gold standard for PD
treatment which compensates for the dopamine deficiency.3−5
However, L-DOPA therapy is associated with a number of
problems such as poor bioavailability and peripheral effects.6,7
Therefore, developing brain-targeted drug delivery systems
would be of great significance to improve the therapeutic effects
and reduce the side effects.
The blood brain barrier (BBB), which is characterized by
tight junctions between endothelial cells, the absence of
fenestrations, and low occurrence of pinocytic activity, presents
the greatest challenge for brain drug delivery.8 To improve the
brain penetration, numerous medicinal chemistry- and
pharmaceutical technology-based strategies have been explored
and developed.9 Among them, drug-carrier conjugates that can
be specifically transported across the BBB via carrier-mediated
mechanisms have drawn attention. Recently, glucose trans-
porters (GluT1),10−12 amino acid transporters (large neutral
amino acid transporters 1, LAT1),13 and 2-phenyl- imidazopyr-
idine moiety-14 based delivery systems have been successfully
developed to specifically deliver L-DOPA or dopamine to the
brain. The above conjugates were shown to increase the brain
Received: May 13, 2014
Revised: June 23, 2014
Accepted: July 29, 2014
Published: July 29, 2014
© 2014 American Chemical Society
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a
Scheme 1. Synthetic Route of Compounds 3 and 4
a
Reagents and conditions: (a) (Boc)2 in dioxane/NaOH aqueous solution; (b) pivaloyl chloride, THF; (c) CF3COOH, CH2Cl2; (d) 3-
(dimethylamino)propanoic acid hydrochloride, EDCI/HOBt, EtN3 in dry CH2Cl2.
group, which suggested that simply conjugating the drug with
the ligand did not result in improved brain targetability of the
conjugate. Regarding the differences in physicochemical
properties between the two kinds of drugs, nonsteroidal anti-
inflammatory drugs (NSAIDs) are lipophilic drugs that have
great affinity to tissues. In contrast, dopamine and zidovudine
are water-soluble drugs that are easily ionized in the
bloodstream and rapidly metabolized.17 Thus, lipophilicity
would be an important impact factor for achieving brain
targeting. Therefore, N,N-dimethylamino group-conjugated
drug derivatives with sufficient lipophilicity appear to be
transported more specifically to the brain.
On the basis of this hypothesis, we presented a first attempt
to confirm the brain targetability of the N,N-dimethyl amino
group with improved lipophilicity. Initially, dipivaloyloxy was
introduced in the design of the conjugate to achieve the
lipophilic derivatization of the catechol system in dopamine. N-
3,4-Bis(pivaloyloxy)dopamine-3-(dimethylamino)propanamide
(PDDP) was thus synthesized and investigated in vitro and in
vivo. Moreover, PDDP showed enhanced cellular uptake in
bEnd.3 cells which was attributed to putative pyrilamine
cationic transporters. In addition, tissue distribution, brain
bioavailability, and therapeutic efficacy of PDDP were evaluated
and compared with those of L-DOPA which demonstrated that
PDDP would be an excellent candidate to achieve brain-specific
dug delivery.
principles of care and use of laboratory animals and were
approved by the experiment animal administration committee
of Sichuan University.
2.2. Cell Lines and Cell Culture. L929 (fibroblast cells),
bEnd.3 cells (immortalized mouse brain microvascular
endothelial cells), and SH-SY5Y (human neuroblastoma cells)
were purchased from the American Type Culture Collection
(Rockville, MD, U.S.A.) which are from passage 3 to 20. Cells
were cultured in DMEM with high glucose (Hyclone, U.S.A.)
supplemented with 10% FBS (Hyclone, U.S.A.), 100 IU/mL
penicillin, and 100 μg/mL streptomycin. Cells were maintained
at 37 °C in a humidified atmosphere containing 5% CO2, and
the culture medium was changed every other day.
2.3. Synthesis and Characterization of N-3,4-Bis-
(pivaloyloxy)dopamine-3-(dimethyl amino)-
propanamide (PDDP). The synthetic routes of BPD,
PDDP, and DDP are described in Scheme 1 and Scheme S1
of the Supporting Information (SI). The detailed methods are
presented in the SI.
The stability of BPD and PDDP were investigated in PBS
(pH 7.4), plasma, and brain homogenate at 37 °C for 8 h. At
predetermined time points, 100 μL of the sample was collected.
Then the samples were diluted with acetonitrile, and the
percentages of the residual BPD and PDDP were determined
by LC−MS/MS.
2.4. Sample Preparation and LC−MS/MS Analysis.
BPD and PDDP were subjected to hydrolysis before LC−MS/
MS analysis. To cell digests (0.1 mL), plasma (0.1 mL), or
tissue homogenates (0.1 mL) was added 100 μL pig liver
esterase (100 units/mL, Sigma-Aldrich, U.S.A.) and then
mixed. After 15 min hydrolysis at 37 °C, dopamine and DDP
were released from PD and PDDP, respectively, and 0.6 mL
acetonitrile was added. For L-DOPA, 0.2 or 1.0 mL of
acetonitrile was added to cell digests (0.1 mL), plasma (0.5
mL), or tissue homogenates (0.5 mL), respectively. The
mixtures were vortexed for 5 min and centrifuged at 12,000
rpm for 10 min, and then supernatants (1 μL) were analyzed by
LC−MS/MS assay.
The LC−MS/MS system consisted of an Agilent 1200 series
RRLC system, which includes an SL autosampler, degasser and
SL binary pump, and an Agilent triple-quadruple MS. LC−MS/
MS analyses were performed on a diamonsil ODS column (50
mm × 4.6 mm, 1.8 μm) with the corresponding guard column
(ODS, 5 μm). The mobile phase consisted of 80% aqueous
2. MATERIALS AND METHODS
2.1. Materials and Animals. Dopamine hydrochloride was
purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China),
L-DOPA and 6-OHDA were supplied by Sigma-Aldrich (St.
Louis, MO, U.S.A.) with purity of 99.0%, and 3-
(dimethylamino)propanoic acid hydrochloride was obtained
from Tokyo Chemical Industry CO. Ltd. (Tokyo, Japan).
Annexin V-FITC/PI apoptosis detection kit was purchased
from KeyGEN Biotech (China). Acetonitrile (HPLC grade)
was purchased from Kemiou (Tianjin, China). All the other
chemicals and reagents were of analytical grade obtained
commercially.
Sprague−Dawley rats (male; body weight: 200
20 g),
provided by the West China Experimental Animal Center of
Sichuan University (China), were maintained in a germ-free
environment and allowed free access to food and water. All
animal experiments were performed in accordance with the
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solution containing 0.1% formic acid and 20% acetonitrile (v/
v), at a flow rate of 0.3 mL/min.
valinomycin, a potassium ionophore, cells were preincubated
with 10 μM valinomycin for 10 min, and then uptake of PDDP
was measured as described above. The uptake was also
measured at acidic or alkaline pH (6.0 and 8.4, respectively).
2.7. Tissue Distribution and Brain Bioavailability in
Rats. L-DOPA, BPD, or PDDP were injected through tail vein
of the rats. For each preparation and sampling time point, five
rats were treated with a single dose of L-DOPA, BPD, and
PDDP which was 19.31, 31.43, and 41.17 mg/kg respectively,
equivalent to 18 mg/kg dopamine. The rats were sacrificed at 5,
15, 30, 60, and 120 min after injection. The blood plasma and
tissue homogenates (hearts, livers, spleens, lungs, kidneys, and
brains) were quickly collected and stored at −40 °C until assay.
For the brain bioavailability study, the experimental
procedures were kept similar to the biodistribution study. At
each predetermined time point, rats were sacrificed, and brains
were quickly collected and stored at −40 °C until assay. Tissue
concentration of drug was measured accordingly.
2.8. Therapeutic Effect on 6-OHDA-Induced PD Rat
Model. 2.8.1. Unilateral Intrastriatal Infusion of 6-OHDA. 6-
OHDA-induced PD rat model was established according to K.
Hu et al.18 Male Sprague-Dawley (SD) rats (250 20 g) were
anesthetized with chloral hydrate (400 mg/kg, i.p.), and then
placed in a stereotaxic apparatus (Anhui Zhenghua Bio
Equipment Co., Ltd., Huaibei, China) to achieve a flat skull
position. A sagittal incision was made in the scalp with sterile
blade, then the skin and inferior tissue layers covering the skull
were retracted and a small hole was drilled at the following
coordinates: lateral (L) 3 mm, antero-posterior (AP) 0.2 mm,
dorso−ventral 4.8, and 5.6 mm from the bregma point. The 6-
OHDA solution (10 μg/mL dissolved in 0.2% ascorbic acid)
was infused unilaterally through a stainless steel cannula into
the striatal region at 0.3 μL/min, 1 μL each point. Another
group of rats, which were injected with a 6-OHDA-free solution
(0.2% ascorbic acid saline) in a similar manner, served as the
sham group.
2.8.2. Experimental Design. Sixty rats were randomly
divided into the following five groups (n = 12). Sham-operated
group: rats were subjected to the surgical procedure as
described above; 6-OHDA lesioned group: in this group, rats
were injected with normal saline via caudal vein every day after
6-OHDA lesioning; 6-OHDA + L-DOPA group: rats received
L-DOPA (5 mg/kg/day) via caudal vein every day after 6-
OHDA lesioning; 6-OHDA + BPD group: rats received BPD
(8.15 mg/kg/day, equal to 5 mg/kg/day of L-DOPA) via
caudal vein every day after 6-OHDA lesioning; 6-OHDA +
PDDP group: Rats received PDDP (10.66 mg/kg/day, equal to
5 mg/kg/day of L-DOPA) via caudal vein every day after 6-
OHDA lesioning. After 21 days, all rats were sacrificed, and
brains were harvested for immunohistochemistry stain of
tyrosine hydroxylase (TH), LC−MS/MS detection of the
dopamine, and assessment of enzymatic antioxidants.
2.8.3. Detection of Striatal Dopamine. Five rats of each
group were sacrificed with each left and right striatum harvested
separately for the detection of dopamine (DA) as described
previously. Briefly, each striatum was sonicated in 0.1 M
perchloric acid, and then diluted with acetonitrile. Homoge-
nates were vortexed for 5 min and centrifuged at 12,000 rpm
for 10 min at 4 °C. The supernatants were collected and
detected with LC−MS/MS method as described above.
2.8.4. Tyrosine Hydroxylase (TH) Immunohistochemistry.
Two rats of each group were anaesthetized with chloral hydrate,
and perfused with normal saline and followed by 4%
Detection was operated on a mass spectrometer in the
positive electrospray source ion mode, and quantification was
performed using multiple reaction monitoring (MRM). MRM
of m/z 154 → 137.1 and 253 → 58.1 were adopted to qualify
dopamine and DDP. L-DOPA was monitored at m/z 198.1 →
152. The corresponding optimized collision-induced dissocia-
tion voltages of the analysis were 103 and 20 eV for the
fragmentor and collision energy, respectively. Instrumental
parameters were as follows: gas temperature of 350 °C, gas flow
of 8 mL/min, nebulizer of 30 psi, capillary of 4000 v.
2.5. Cellular Uptake Assay. 2.5.1. Time-Dependent
Cellular Uptake. The bEnd.3 cells were seeded in 6-well
culture plates (5 × 105 cells/well). On the second day, when
they had grown to confluence, cells were exposed to L-DOPA,
BPD, and PDDP, which were diluted in Hank’s buffer (122
mM NaCl, 3 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4
mM CaCl2,10 mM D-glucose, 10 mM HEPES, pH 7.4) at a final
concentration of 40 μM. Then bEnd.3 cells were incubated in
triplicate for 0.25, 0.5, 1, or 2 h, respectively. After that, cells
were washed with PBS (pH 7.4) to remove extracellular drugs.
Cells were then trypsinized, centrifuged at 3000 rpm for 5 min,
and cell pellets were washed twice with PBS. The cell pellets
were added with ultrapure water and lysed by repetitive freeze−
thawing to release the intracellular drugs. Sample processing
procedures are described in section 2.4, and the intracellular
concentrations of L-DOPA, BPD, or PDDP were measured by
LC−MS/MS. Cellular uptake was expressed as the amount
(nmol) of L-DOPA, BPD, or PDDP per 1 mg of total cellular
protein. The total protein of cell lysates was determined in
parallel wells by BCA assay (Pierce, U.S.A.).
2.5.2. Dose-Dependent Cellular Uptake. The bEnd.3 cells
were seeded in 6-well culture plates (5 × 105 cells/well). On
the second day, cells were incubated with L-DOPA, BPD, or
PDDP at the following concentrations, 10, 20, 40, and 80 μM,
respectively, for 0.5 h at 37 °C. Cells were rinsed three times
with ice-cold PBS and collected for the determination of
intracellular concentration.
2.5.3. Effect of Temperature and Energy Inhibitors on
Cellular Uptake. The bEnd.3 cells were seeded in 6-well culture
plates (5 × 105 cells/well). On the second day, cells were
exposed to L-DOPA, BPD or PDDP (40 μM) for 0.5 h at 37
°C, 4 °C, or in the presence of NaN3. Then, cells were washed
three times with ice-cold PBS and collected for the
determination of intracellular concentration.
2.5.4. Cellular Uptake on Different Cell Lines. The bEnd.3
cells and L929 cells were incubated with L-DOPA, BPD or
PDDP (40 μM) for 0.5 h at 37 °C, respectively. Then, cells
were washed three times with ice-cold PBS and collected for
the determination of intracellular concentration.
2.6. Mechanistic Study in the Brain Uptake of PDDP in
vitro. 2.6.1. Competitive Inhibition Assay. The bEnd.3 cells
were incubated with Hank’s buffer in the absence or presence
of various inhibitors. After incubation for 15 min at 37 °C,
PDDP (40 μM) was added to the cells, and then the mixture
was incubated for another 0.5 h. Then cells were rinsed three
times with ice-cold PBS and collected as the sample for each
group. The intracellular concentration of PDDP was
determined as described before.
2.6.2. The Uptake Property of PDDP in bEnd.3 Cells. In
sodium-free experiments, sodium ions were replaced with N-
methylglucamine or potassium chloride. For the study of
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paraformaldehyde in PBS. The brains were removed, kept in
4% paraformaldehyde fixative overnight and embedded in
paraffin and serially cut for TH immunohistochemistry
according to previous studies.18,19 Photomicrographs were
taken with a microscope camera (Nikon Eclipse 80i, Nikon
Instech Co. Ltd., Kawasaki, Kanagawa, Japan) at 50×
magnification.
2.8.5. Studies Related to Oxidative Stress. To assess free
radical-mediated effects following 6-OHDA lesioning and to
explore the free radical scavenging potential of PDDP, reduced
glutathione (GSH), enzymatic antioxidants (total superoxide
dismutase and catalase), and total antioxidant capacity (T-
AOC) were estimated in the striatum. Five rats from each
group were sacrificed, and their brains were dissected quickly
on ice pack. Ipsilateral striatum was separated, weighed, and
freshly processed on the same day for the estimation of GSH-
PX (catalog no. A005), total SOD (catalog no. A001-1),
catalase (catalog no. A007-1), and T-AOC (catalog no. A015)
by quantitative analysis using commercial reagent kits (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China) according
to the manufacturer’s instructions.
2.9. Antioxidant and Antiapoptotic Effect of PDDP on
6-OHDA Induced Cytotoxicity toward SH-SY5Y Cells.
2.9.1. Cell Culture and Treatment. SH-SY5Y cells were seeded
in 96-well culture plates (4 × 104 cells/well) for viability assays
or 6-well culture plates (5 × 105 cells/well) for ROS, MMP,
and cell apoptosis tests. On the next day, different treatments
were given to the cells as shown in Table 1. Briefly, cells were
treated with different concentrations of L-DOPA, BPD, and
PDDP for 2 h, after which 100 μM of 6-OHDA was added, and
cells were incubated for another 24 h.
2.9.2. Cell Viability Assay. Cell viability was measured using
MTT reduction assay as described previously.18 At the end of
each treatment, MTT was added to the wells at a final
concentration of 0.5 mg/mL, and cells were further incubated
at 37 °C for 4 h. The insoluble formazan was dissolved in
DMSO, and the determination was measured at 570 nm by a
plate reader (Thermo, Varioskan Flash).
2.9.3. ROS Measurement and Mitochondrial Membrane
Potential (MMP) Determination. Intracellular ROS was
quantified using a commercial kit (catalog no. S0033, Beyotime,
Jiangsu, China) following the manufacturer’s instructions.
Mitochondrial membrane potential was measured using the
fluorescent dye JC-1 (Molecular Probes) according to the
manufacturer’s instructions of a commercial kit (catalog no.
C2006, Beyotime, Jiangsu, China).
2.9.4. Cell Apoptosis Assay. The quantitative analysis of
apoptosis induced by different treatment groups was performed
by Annexin V-FITC/PI double staining using the Cell
Apoptotic analysis kit (Beyotime, Jiangsu, China). Briefly,
SH-SY5Y cells were seeded in 6-well culture plates and treated
with 10 μM L-DOPA, BPD, and PDDP followed by 6-OHDA
as described for the cell viability assays. At the end of the
treatment, cells were harvested, washed with cold PBS,
suspended in 0.5 mL binding buffer and stained by 5 μL
Annexin V-FITC and 5 μL PI. The cells were incubated in the
dark for 15 min and measured by Cytomics FC500 flow
cytometer (Beckman Coulter, U.S.A.).
2.10. Statistical Analysis. Statistical comparisons were
performed by one-way ANOVA for multiple groups, p < 0.05
and p < 0.01 were considered indications of statistical
differences and statistically significantly different, respectively.
3. RESULTS
Table 1. Treatments Given to Different Groups in the
Antioxidant and Antiapoptotic Effect of PDDP on 6-OHDA-
Induced Cytotoxicity Towards SH-SY5Y Cells
3.1. Synthesis of N-3,4-Bis(pivaloyloxy)dopamine-3-
(dimethylamino)propanamide (PDDP). BPD, PDDP and
DDP were synthesized as described in Scheme 1 and Scheme
S1 in the SI. The structures of the conjugates were confirmed
by electrospray ionization mass spectrometry (ESI-MS) and
nuclear magnetic resonance (1H NMR and 13C NMR).
The stability of BPD and PDDP was tested by incubating
with rat plasma and brain homogenate and phosphate buffer
solutions (pH 7.4). After 8 h incubation at 37 °C, 80% of the
original amount of BPD (Figure 1a) and PDDP (Figure 1b)
remained in the buffer, while <50% remained in plasma after 5
min incubation. A sustained release of PDDP was found in the
brain with ∼30% remaining after 1 h incubation, which
indicated a reasonable stability of PDDP. As reported
previously,20 dipivalyloxy derivatives (BPD and PDDP) under-
groups
treatments
control
model
none
treated with 6-OHDA only (100 μM) for 24 h
L-DOPA
pretreated with 10 μM L-DOPA for 2 h before the addition of 6-
OHDA (100 μM)
BPD
pretreated with 10 μM BPD for 2 h before the addition of 6-
OHDA (100 μM)
PDDP2
PDDP5
pretreated with 2.0 μM PDDP for 2 h before the addition of 6-
OHDA (100 μM)
pretreated with 5.0 μM PDDP for 2 h before the addition of 6-
OHDA (100 μM)
PDDD10 pretreated with 10 μM PDDP for 2 h before the addition of 6-
OHDA (100 μM)
Figure 1. Stability of BPD (a) and PDDP (b) in plasma, brain homogenates, and 0.1 M PBS (pH = 7.4) at 37 °C for 8 h. Data represent mean SD
(n = 3).
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Scheme 2. Enzymatic Hydrolysis of BPD and PDDP in Physiological Environment
Figure 2. Cellular uptake of L-DOPA, BPD, and PDDP under various conditions; the intracellular concentration of L-DOPA was below the limit of
detection in all cell uptake assays. Data represent mean SD (n = 3): (a) Cellular uptake of BPD and PDDP (40 μM) in bEnd.3 cells at 0.25, 0.5, 1,
2, and 4 h. PDDP showed significantly higher uptake than BPD at all the time points examined. (b) Cellular uptake of BPD and PDDP in bEnd.3
cells at different doses (10, 20, 40, and 80 μM) at 37 °C for 0.5 h. PDDP showed higher uptake at all the doses examined. (c) Cellular uptake of BPD
and PDDP (40 μM) in bEnd.3 cells at 37 °C, 4 °C, and in the presence of NaN3 for 0.5 h. PDDP displayed a significantly higher uptake at 37 °C
than at 4 °C and in the presence of NaN3. (d) Comparison of the cellular uptake of BPD and PDDP (40 μM) in L929 cells and bEnd.3 cells at 37 °C
for 0.5 h. L929 showed lower uptake of both BPD and PDDP, while in bEnd.3, the uptake of PDDP was about 5 times higher than that of BPD.
went a two-step hydrolysis. Scheme 2 shows the enzymatic
hydrolysis process of BPD and PDDP in physiological
environment.
3.2. Cellular Uptake of PDDP in bEnd.3 Cells. The
uptake of PDDP by bEnd.3 cells was time-, concentration-, and
temperature-dependent (Figure 2), indicating an active trans-
port process. The uptake of PDDP by bEnd.3 cells was higher
than the uptake of BPD during the 4 h incubation period
(Figure 2a). At 0.5 h, the intracellular concentration of PDDP
significantly increased at different given doses compared with
that in the BPD group. The intracellular concentration of
PDDP was 4.6 times higher than that of BPD at the dose of 40
μM.
The uptake amount of PDDP at 37 °C (13.48 0.78 nmol/
mg) was much higher than that at 4 °C (6.19 0.28 nmol/mg)
and in the presence of NaN3 (5.56 0.24 nmol/mg), which
indicated the potential dependence on temperature and energy.
In contrast, the cellular uptake of BPD showed no substantial
differences between 37 and 4 °C or in the presence of NaN3
(Figure 2c). In Figure 2d, the cellular uptake of PDDP in
bEnd.3 cells was much more than that in L929 cells,
was saturated, and reached 17.47
1.42 nmol/mg protein,
which was 3.2-fold that of BPD (5.46 0.38 nmol/mg). Figure
2b shows that cellular uptake in the PDDP group was
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Figure 3. Mechanistic study on the brain-specific uptake of PDDP in vitro. Data represent mean SD (n = 3): (a) Competitive inhibition of cellular
uptake of PDDP (40 μM) by different inhibitors (pyrilamine, propranolol, imipramine, hemicholinium-3, choline, L-carnitine, TEA, L-lysine, L-
arginine, spermine, and spermidine) at 37 °C for 0.5 h. Cationic drugs including pyrilamine, propranolol, and imipramine resulted in significantly
reduced uptake of PDDP. *p < 0.05, **p < 0.01. (b) Effects of sodium ion replacement and membrane potential on PDDP uptake in bEnd.3 cells.
Uptake of PDDP (40 μM) was measured at 37 °C for 0.5 h in sodium-containing (control) or sodium-free (replaced with N-methylglucamine or
potassium) buffer. After pretreatment with valinomycin (10 μM), the uptake was measured in the sodium-containing buffer with valinomycin. (c)
Effect of extracellular pH on PDDP (40 μM) uptake in bEnd.3 cells at 37 °C for 0.5 h.
demonstrating the cell selectivity of PDDP in vitro. However,
no significant differences were observed for BPD between
bEnd.3 and L929 cells. Moreover, the intracellular concen-
tration of L-DOPA was below the limit of detection in all
cellular uptake assays.
Figure 3c. The uptake was decreased by 40% in acidic transport
medium (pH 6.0) and increased by 30% in alkaline medium
(pH 8.4).
3.4. Tissue Distribution and Brain Bioavailability of
PDDP in Rats. To further evaluate the brain targetability of
PDDP, tissue distributions of L-DOPA, BPD, and PDDP were
studied in rats. The brain drug concentration showed that the
concentration of PDDP was significantly higher than those of
L-DOPA and BPD throughout the time course (Figure 4a).
Specifically, the PDDP concentration in brain was about 6.90
times higher than that of the BPD group at 5 min after i.v.
injection in rats. As shown in Figure 4b, at 5 min, a higher
distribution of PDDP was also observed in other tissues, which
is 3.55 times more than that of BPD in heart, 3.69 times more
in liver, 0.44 times more in lung, and 1.5 times more in kidney.
BPD showed a similar distribution property with PDDP in the
systemic circulation except in the brain, which indicated poor
tissue-specific localization. In addition, L-DOPA was found to
accumulate mainly in the kidney due to its short half-life in vivo.
In Figure 4c, the brain drug concentration in the PDDP
group was significantly improved compared with that of L-
DOPA and BPD groups at different time points. Table 2
indicates that the area under the curve (AUC0−t) of PDDP was
116.35 times and 4.63 times higher than that of L-DOPA and
BPD, respectively. The maximal concentration (Cmax) was
3.3. Mechanistic Study on the Brain-Specific Uptake
of PDDP in Vitro. PDDP was proven to improve the cellular
uptake efficacy in bEnd.3 cells by an active transport process.
However, the mechanism behind it remains to be further
addressed. Various transporter inhibitors were thus selected and
incubated with PDDP, and their effects on the uptake were
evaluated by the relative uptake efficiency (Figure 3a and Table
S1 in the SI). The uptake was significantly inhibited by cationic
inhibitors such as pyrilamine, propranolol, and imipramine, but
not by hemicholinium-3 and choline (substrates or inhibitors of
the choline transport system), L-carnitine (an OCTN2-specific
substrate) and tetraethylammonium (classic inhibitor of
OCTs). Alkaline amino acids such as L-lysine, L-arginine,
spermine, and spermidine were shown to increase the cellular
uptake of PDDP to some extent.
Figure 3b shows that the uptake of PDDP was not affected
by the replacement of extracellular Na+ with N-methylgluc-
amine+ or K+. Treatment with valinomycin, a potassium
ionopore, did not change the uptake of PDDP by bEnd.3
cells. pH dependence of the uptake of PDDP is shown in
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Figure 4. Biodistribution in rats after i.v. injection of L-DOPA, BPD, and PDDP. Data represent mean SD (n = 5). (a) Levels of L-dopa, BPD, and
PDDP in brain. (b) Drug levels in other tissues (heart, liver, spleen, lung, kidney, and plasma) at 5 min. PDDP is selectively concentrated in lung,
kidney, and brain, especially in the brain. At 5 min, in brain the concentration of PDDP is ∼7 times that of the BPD concentration. (c) Brain
bioavailability in rats after i.v. injection of L-DOPA, BPD, and PDDP. PDDP displayed significantly higher concentrations than L-DOPA and BPD
during the entire course of the investigation.
269.28 times and 6.41 times higher than that of L-DOPA and
BPD, respectively. The above results demonstrate the
bioavailability improvement of PDDP in the brain.
Table 2. Pharmacokinetic Parameters of L-DOPA, BPD, and
PDDP in Brain after i.v. Injection in Rats
parameters
L-DOPA
BPD
PDDP
3.5. Therapeutic Effect of PDDP on 6-OHDA-Induced
PD Model in Rat. To explore the utility of PDDP in the
treatment of PD, PDDP was injected to the 6-OHDA-lesioned
model rats. The striatal dopamine contents and striatal tyrosine
hydroxylase (TH) immunohistochemistry were used to
evaluate the therapeutic effects of PDDP. On day 21, the
amount of dopamine in the lesioned striatum after different
AUC0‑t,brain (nmol/g*h) 0.24 0.013
6.07 0.73
10.98 1.26
25.11
28.14 1.35
70.39 7.24
116.35
Cmax,brain (nmol/g)
Rebrain
0.26 0.039
−
−
Cebrain
42.00
269.28
Figure 5. Therapeutic effect of L-DOPA, BPD, and PDDP on 6-OHDA rat model of Parkinson’s disease. Data represent mean SD #p < 0.01 with
respect to the sham group, *p < 0.01 with respect to the 6-OHDA lesioned group. (a) Dopamine percentage in the 6-OHDA lesioned striatum of
rats 3 weeks after 6-OHDA lesion (n = 5). (b) TH-immunoreactivity in the striatum of rats of different groups 3 weeks after 6-OHDA lesion. Scale
bar: 100 μm. After PDDP administration, a significant increase was observed in dopamine content and TH-immunopositive neurons (brown
staining), while L-DOPA- and BPD-treated groups showed certain but weaker therapeutic effects.
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Figure 6. Oxidative stress in ipsilateral striatum of different treatment groups in the 6-OHDA rat model of PD. Data represent mean SD (n = 5).
#p < 0.01 vs the sham group, *p < 0.05 and **p < 0.01 vs the 6-OHDA lesioned group. (a) GSH-PX, (b) total SOD, (c) catalase, and (d) total
antioxidant capacity (T-AOC). PDDP-treated group showed a remarkable increase in antioxidant enzymes compared with the model group.
Figure 7. Protective effect of PDDP against cell injury induced by 6-OHDA in SH-SY5Y cells. (a) The cell viability of SH-SY5Y cells treated by L-
DOPA, BPD, and PDDP at concentrations of 0, 5, 10, 20, 50, 100, and 200 μM. After 24 h, the cell viability was determined by MTT assay (n = 3).
L-dOpa and BPD induced severe cytotoxicity in SH-SY5Y cells when the concentration reached 50 μM. *p < 0.05 and **p < 0.01 versus untreated
cells. (b) The effects of L-DOPA, BPD, and PDDP on the cell viability of 6-OHDA-treated (100 μM) SH-SY5Y cells. Cells were pretreated with L-
DOPA (10 μM), BPD (10 μM), and different concentrations of PDDP (2.0, 5.0, and 10 μM) for 2 h before the addition of 6-OHDA. The results
showed PDDP increased the cell viability in a dose-dependent manner. Data represent mean SD (n = 3). #p < 0.01 vs untreated cells, *p < 0.05
and **p < 0.01 vs 6-OHDA-treated group.
treatments is shown in Figure 5a. The amount of DA in 6-
OHDA-lesioned group decreased to 15.22% compared to the
sham group, which demonstrated the successful establishment
of the 6-OHDA model. After treatment with L-DOPA and
conjugates, the amount of DA increased at various degrees
compared with the 6-OHDA group, while PDDP treatment
(44.85%) showed a much higher increase in DA than L-DOPA
(32.32%) and BPD (39.51%) treatment.
Brain sections of rats after different treatments were
performed for the TH-immunohistochemistry on day 21. The
sham group did not show any loss in TH-immunohistochem-
istry in the striatum of the infusion side (Figure 5b). However,
in the 6-OHDA group, the striatum region infused with 6-
OHDA showed extensive lesion as evidenced by significantly
lesser TH-immunoreactive neurons compared to the sham
group. After PDDP administration, there was a remarkable
improvement in the TH-immunopositive neurons in the 6-
OHDA-infused side, while L-DOPA- and BPD-treated groups
showed certain but weaker therapeutic effects.
Long-term usage of L-DOPA could accelerate the neuron
degeneration resulting from oxidative stress. Thus, we studied
the oxidative stress level in the striatum after treatment with
PDDP. The results are summarized in Figure 6. A significant
decrease (p < 0.001) in GSH-PX, T-SOD, CAT, and T-AOC
was observed in striatum of 6-OHDA-lesioned group when
compared to that of the sham group, which was remarkably
restored by 28.92%, 18.80%, 20.65%, and 31.74%, respectively,
in the PDDP treatment group as compared to the model rats.
The BPD treatment group exhibited significant increases in
striatal GSH-PX and CAT by 14.62% and 10.35%, respectively,
compared to the 6-OHDA-lesioned group. However, the L-
DOPA-treated group showed no obvious change in antioxidant
enzyme activities compared with that of the lesioned rats.
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Figure 8. Effect of PDDP on oxidative stress and cell apoptosis induced by 6-OHDA in SH-SY5Y cells. Cells were treated with no 6-OHDA
(control) or with 100 μM 6-OHDA after pretreatment with L-DOPA (10 μM), BPD (10 μM), and different concentrations of PDDP (2.0, 5.0, and
10 μM). Results were normalized to the percentage of control. Data represent mean SD, n = 3. #p < 0.01 with respect to the untreated cells, *p <
0.05 and **p < 0.01 with respect to the 6-OHDA-treated group. (a) Levels of ROS were measured using the DCFH-DA assay; (b) mitochondrial
membrane potential (MMP) was measured using JC-1 staining; (c) proportion of apoptotic cells after different treatments; (d) apoptosis assays
conducted by flow cytometry using annexin V in combination with propidium iodide. The obtained results suggested that PDDP could inhibit
oxidative stress and neuronal apoptosis induced by 6-OHDA.
Scheme 3. Neuroprotection of PDDP in 6-OHDA-Induced SH-SY5Y Cells
3.6. Antioxidant and Antiapoptoptic Effect of PDDP
on 6-OHDA-Induced Cytotoxicity toward SH-SY5Y Cells.
L-DOPA-induced neurotoxicity is thought to be involved in the
side effects of L-DOPA therapy. In this study, we try to
substantiate whether PDDP could induce cytotoxicity to
neurons as L-DOPA did. Catecholaminergic neuroblatoma
SH-SY5Y cells were employed to assess the effects. L-DOPA
could induce cytotoxicity in SH-SY5Y cells when the
concentration reached 50 μM (Figure 7a), whereas PDDP
did not induce any cytotoxicity with the same doses. To further
evaluate the protective effect of PDDP against 6-OHDA-
induced cell injury in SH-SY5Y cells, cells were pretreated with
PDDP at different concentrations for 2 h before addition of 6-
OHDA (100 μM) for another 24 h. As shown in Figure 7b,
treating SH-SY5Y cells with 100 μM 6-OHDA reduced the cell
viability to 65% of the control, while PDDP increased cell
viability in a dose-dependent manner: 2.0 μM, 76.30%; 5.0 μM,
81.09%; and 10 μM, 86.92% of the control, respectively.
Whereas, neither L-DOPA nor BPD at the equal molar
concentration (10 μM) was sufficient to exert the similar
protective effects against 6-OHDA-induced cell injury as PDDP
did.
effect of PDDP on 6-OHDA-induced ROS formation in SH-
SY5Y cells. Figure 8a shows that exposing cells to 100 μM 6-
OHDA for 24 h increased ROS to 120.99% of control value.
Pretreating cells with different concentrations of PDDP for 2 h
ameliorated the 6-OHDA-induced ROS formation in a dose-
dependent manner. In comparison, BPD suppressed the 6-
OHDA-induced ROS formation to a much lesser extent.
Mitochondrial membrane potential (MMP) is essential for cell
survival, particularly cells that are under oxidative stress.
Treating cells with 100 μM 6-OHDA for 24 h decreased
MMP to 31.13% of control value based on JC-1 assay (Figure
8b). Pretreating cells with different concentrations of PDDP for
2 h ameliorated the 6-OHDA-induced MMP reduction in a
dose-dependent manner.
The effects of PDDP pretreatment on 6-OHDA-induced
apoptosis were analyzed by double staining with FITC annexin-
V and propidium iodide. Cells showing FITC-positivity only
were considered to be in an early apoptotic phase. According to
Figure 8c, exposing SH-SY5Y cells to 6-OHDA significantly
increased the level of apoptosis (with apoptotic rate of 60.77
2.83%) than in the absence of 6-OHDA (with apoptotic rate of
19.13
2.80%); however, this increase was attenuated by
6-OHDA is known to selectively kill dopaminergic neurons
by generating reactive oxygen species (ROS) and inducing
oxidative stress in the cells. We thus proceeded to examine the
pretreatment with 10 μM PDDP (with apoptotic rate of 35.37
3.46%). Therefore, PDDP was shown to protect SH-SY5Y
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cells from 6-OHDA-induced cell apoptosis and improve cell
viability (Scheme 3).
Thus, PDDP uptake was not triggered by organic cationic
transporters, choline transporters, or the electrostatic inter-
actions as observed in the transport of cationic peptides.22 This
conclusion was supported by the membrane potential
independence (Figure 3b) and pH dependence (Figure 3c)
of PDDP uptake in bEnd.3 cells. In contrast, the lipophilic
cationic drugs pyrilamine, imipramine, and propranolol
significantly inhibited the uptake of PDDP in bEnd.3 cells.
Yamazaki et al.23,24 demonstrated that pyrilamine and other
basic lipophilic agents25−27 were transferred into the brain by
an organic cation-sensitive transport system, i.e., putative
pyrilamine transporters. Competitive inhibition of these agents
on PDDP uptake indicated that putative pyrilamine cationic
transporters were involved in the brain uptake of PDDP, which
remained to be elucidated at the molecular level.
4. DISCUSSION
PD is an age-related progressive degenerative disease with
motor disorders such as bradykinesia, resting tremor, rigidity,
and postural instability.2 To date, L-DOPA treatment remains
the most effective therapy for PD which compensates for the
dopamine deficiency. However, L-DOPA cannot arrest the
progression of PD, and the long-term application of L-DOPA
induces dyskinesia and accelerates the neuron degeneration.21
Thus, a delivery system with better therapeutic effects and
reduced side effects is highly demanded for the treatment of
PD.
In previous works, our group has shown that N,N-
dimethylethylenediamine-related structures significantly en-
hanced the targeting efficiency of naproxen and dexibuprofen
to the brain.15,16 The ligand offers many advantages such as a
well-defined structure, minimum cytotoxicity, and high
efficiency of brain targeting. Thus, we designed and synthesized
DDP, which is the conjugate of dopamine and N,N-
dimethylamino propanoic acid to improve the brain target-
ability. However, DDP was proven not a qualified candidate for
brain targeting, which did not show brain-specific accumulation
after systemic administration (data not shown). After careful
examination, lipophilicity appeared to be an important impact
factor for achieving brain targeting. Therefore, the dipivaloyloxy
functional group was introduced in the synthesis of N-3,4-
bis(pivaloyloxy)dopamine-3-(dimethylamino)propanamide
(PDDP) to increase the overall lipophilicity of the conjugate,
thus improving brain targetability. To our best knowledge, no
published reports are available regarding N,N-dimethyl amino
group as a brain-targeted ligand. Also, this is the first report to
confirm the essential role of lipophilicity for N,N-dimethyl
amino group-conjugated drug derivatives to achieve brain
targeting. In this regard, the substrates of the novel brain-
targeting ligand would be largely extended. More importantly,
the N,N-dimethyl amino group offers an alternative approach to
the brain-specific delivery of therapeutic agents for central
nervous system diseases.
Previous research confirmed the brain targetability of N,N-
dimethylethylenediamine-related structures by studying the
biodistribution of the conjugate in animals, which did not
explore the mechanism of the improved brain accumulation,
especially the transport process across the BBB. In this study,
the bEnd.3 cell-based BBB model was chosen and established
to study the brain delivery of the conjugate. Enhanced
intracellular delivery of PDDP was observed in bEnd.3 cells,
which was significantly higher than that of L-DOPA and BPD.
Mechanistic investigation further revealed that PDDP entering
bEnd.3 cells required energy which probably occurred through
an active transport process (Figure 2). On the contrary, BPD
did not require energy and showed poor uptake efficiency in
both bEnd.3 cells and L929 cells, which suggested a passive
diffusion process. In addition, DDP could not be uptaken by
either bEnd.3 or L929 cells. Thus, both N,N-dimethyl amino
and dipivaloyloxy groups are essential structures in the PDDP
for transport across the BBB, which confirmed our previous
hypothesis.
In vitro stability is an important prediction of the in vivo fate
for drug conjugates. As previously reported,20 dipivaloyloxy
derivatives were suggested to undergo a two-step enzymatic
hydrolysis process (Scheme 2). PDDP showed a prolonged
release in the brain homogenates, which would likely result in a
sustained therapeutic effect. On the basis of these results, we
proposed the following physiological disposition process for
brain delivery of PDDP: after systemic injection, PDDP may
undergo a rapid distribution to tissues due to its high
lipophilicity, which was attributed to the dipivaloyloxy
structure; meanwhile, with the conjugation of the N,N-
dimethylamino group, PDDP can be specifically recognized
by transporters expressed in the brain microvascular endothelial
cells and then be transported into the brain. Moreover, the
relative lower pH in the brain (intracellular pH ≈ 7.0) than
blood circulation (pH ≈ 7.4)28,29 may induce the ionization of
tertiary amines in the derivative, which may help PDDP to be
retained in the brain, thereby resulting in significantly higher
brain specificity as demonstrated in biodistribution and brain
bioavailability study (Figure 4).
In addition to its selective transport across the BBB and the
rapid accumulation in the brain tissue, PDDP is readily
hydrolyzed to DDP by ubiquitous esterases in brain. The
generation of DDP is a prolonged and continuous process as
discussed above. The primary amine of dopamine is not an
essential requirement for dopaminergic activity,30 and thus,
PDDP may retain the intrinsic activity of dopamine without the
cleavage of the amide bond in PDDP. As a result, effective
therapeutic performance of PDDP was observed in 6-OHDA-
lesioned PD rats. Our data showed that PDDP could effectively
attenuate the striatum lesion induced by 6-OHDA, while L-
DOPA and BPD treatment showed certain but weaker
therapeutic effects (Figure 5). Recent studies revealed that
oxidative stress plays an important role in the neuro-
degenerative pathogenesis of PD.31 Dopamine and L-DOPA
were found to increase oxidative stress and thus aggravate the
condition of PD patients.32 Novel therapeutic strategies have
been developed using neuroprotective agents in the manage-
ment of PD. Here, PDDP was proven effective to increase the
level of antioxidant enzymes including GSH-PX, total SOD,
catalase, and T-AOC (Figure 6). Thus, the suppression of
oxidative injury is likely the primary mechanism for the
protective effect of PDDP in the unilateral 6-OHDA PD rat
model.
Dopamine- and L-DOPA-induced neurotoxicity is thought to
be involved in the side effects of L-DOPA therapy.33 DA and its
metabolites containing two hydroxyl residues were reported to
display dose-dependent cytotoxicity in dopaminergic neuronal
To reveal the driving force behind the specific transport, a
competitive inhibition assay was conducted in bEnd.3 cells.
Cationic compounds, choline and analogs, did not inhibit the
uptake of PDDP, nor did alkaline amino acids (Figure 3a).
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cells mainly due to the generation of highly reactive DA and
DOPA quinones which are dopaminergic neuron-specific
cytotoxic molecules.34 The two hydroxyl groups in PDDP
were protected by dipivaloyloxy, and PDDP displayed a slow
and continuous release in the brain. In consequence, PDDP
was less likely to generate quinone radicals in the brain. In the
cytotoxicity study, L-DOPA showed dose-dependent damages
to catecholaminergic neuroblatoma SH-SY5Y cells in accord
with previous studies,35 while PDDP did not induce
cytotoxicity at the same doses (Figure 7a). Interestingly,
PDDP increased the viability of cells treated by 6-OHDA in a
dose-dependent manner. Furthermore, PDDP showed the
ability to suppress the 6-OHDA-induced ROS formation and
increase 6-OHDA-induced MMP reduction, as well as inhibit
neuronal apoptosis induced by 6-OHDA (Figure 8). Therefore,
we conclude that PDDP could present neuroprotective effect in
6-OHDA-induced SH-SY5Y cells.
PDDP was demonstrated to be selectively delivered to the
brain with improved therapeutic efficacy and reduced side
effects. The study also showed that PDDP displayed good
biocompatibility and safety through in vitro and in vivo
evaluation (Figure S1 in SI). Future studies should focus on
the thorough understanding of the enhanced uptake and
improved efficacy of PDDP.
ABBREVIATIONS:
■
PD, Parkinson’s disease; DA, dopamine; TH, tyrosine
hydroxylase; BBB, blood brain barrier; CNS, central nervous
system; PDDP, N-3,4-bis(pivaloyloxy)dopamine-3-
(dimethylamino)propanamide; BPD, 3,4-bis (pivaloyloxy)-
dopamine; DDP, N-(3,4-dihydroxyphenethyl)-3-
(dimethylamino)propanamide
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We are grateful for the financial support from the National
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Basic Research Program of China (No. 2013CB932504).
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