Large neutral amino acid transporter enables brain drug delivery via prodrugs

Article abstract of DOI:10.1021/jm701175d

The blood-brain barrier efficiently controls the entry of drug molecules into the brain. We describe a feasible means to achieve carrier-mediated drug transport into the rat brain via the specific, large neutral amino acid transporter (LAT1) by conjugatin

Full text of DOI:10.1021/jm701175d

932
J. Med. Chem. 2008, 51, 932–936
Large Neutral Amino Acid Transporter Enables Brain Drug Delivery via Prodrugs
Mikko Gynther,*,† Krista Laine,† Jarmo Ropponen,† Jukka Leppänen,† Anne Mannila,† Tapio Nevalainen,† Jouko Savolainen,‡
Tomi Järvinen,† and Jarkko Rautio†
Department of Pharmaceutical Chemistry, UniVersity of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland, and Fennopharma Ltd.,
Microkatu 1, FI-70210 Kuopio, Finland
ReceiVed September 19, 2007
The blood-brain barrier efficiently controls the entry of drug molecules into the brain. We describe a feasible
means to achieve carrier-mediated drug transport into the rat brain via the specific, large neutral amino acid
transporter (LAT1) by conjugating a model compound to L-tyrosine. A hydrophilic drug, ketoprofen, that is
not a substrate for LAT1 was chosen as a model compound. The mechanism and the kinetics of the brain
uptake of the prodrug were determined with an in situ rat brain perfusion technique. The brain uptake of the
prodrug was found to be concentration-dependent. In addition, a specific LAT1 inhibitor significantly
decreased the brain uptake of the prodrug. Therefore, our results reveal for the first time that a drug-substrate
conjugate is able to transport drugs into the brain via LAT1.
Introduction
be delivered into the brain predominantly via cerebrovascular
LAT1-mediated transport, thus demonstrating the ability of
LAT1 to be utilized in drug delivery. However, these drugs bear
a very close structural resemblance to endogenous LAT1
substrates.
The entry of drug molecules into the central nervous system
is efficiently controlled by the blood-brain barrier (BBB).a1 It
is estimated that more than 98% of small molecular weight drugs
and practically 100% of large molecular weight drugs developed
for central nervous system disorders do not readily cross the
BBB.2 Many of the pharmacologically active drugs fail early
in their development phase because these molecules lack the
structural features essential for crossing the BBB.
The BBB endothelial cells differ from endothelial cells in
the rest of the body by the presence of tight junctions, lack of
fenestrae, and low occurrence of pinocytic vesicles.3 There are
also numerous enzymes4 and efflux proteins present at the
cerebral endothelial cells;5,6 in addition, astrocytes,7 pericytes,8
and neurons take part in the formation of the BBB.9 Because
of these distinctive features of the BBB, it exhibits an efficient
structural and functional barrier for the penetration of drugs into
the central nervous system.4,10 However, as each neuron in the
human brain is perfused by its own blood vessel, a solute that
is able to cross the BBB is distributed rapidly into the whole
brain tissue.11 Thus, vascular drug delivery holds potential in
central nervous system drug delivery.
Several specific endogenous influx transporters have been
identified at the brain capillary endothelium forming the BBB.
These include transporters for nutrients, such as amino acids,
glucose, and vitamins.12 As many drug molecules have similar
structural properties to endogenous substrates, it is clear that
some membrane transporters can take part in drug transport as
well.13 The large neutral amino acid transport system (LAT1)
is expressed on the luminal and abluminal membrane of the
capillary endothelial cells, and it efficiently transports neutral
L-amino acids (e.g., phenylalanine and leucine) into the brain.14,15
Several clinically used amino acid mimeting drugs, such as
L-dopa,16 gabapentin,17 and melphalan,18 have been shown to
Chemical drug modification in a way that the drug can be
recognized by specific transporters but still maintain therapeutic
efficacy has proven to be very challenging. One attractive
approach is to conjugate an endogenous transporter substrate
to the active drug molecule in a bioreversible manner, that is,
to utilize the prodrug approach. The prodrug should be designed
in such a way that it is recognized by a specific transporter
mechanism at the BBB. The endogenous transporter transports
the prodrug across the BBB, and in brain tissue, the active drug
is released from the prodrug to induce pharmacological action.
By prodrug approach, the BBB penetration properties of a drug
molecule can be enhanced without modifying its pharmacologi-
cal properties.19
An amino acid L-tyrosine is a LAT1 substrate20 that has a
phenolic hydroxyl group suitable for the conjugation of various
structurally different drug molecules with a biodegradable
linkage and leaving both carboxyl and R-amino groups
unsubstitutedsa necessary feature for LAT1 recognition. In a
study by Walker et al.,21 a phosphonoformate L-tyrosine
conjugate was able to inhibit the transport of [3H]L-tyrosine in
porcine brain microvessel endothelial cells. In another study,
p-nitro- and p-chlorobenzyl ether conjugates of L-tyrosine
inhibited the transport of [3H]L-tyrosine in rabbit corneal cell
line.22 These results indicate that L-tyrosine conjugates are able
to bind to the LAT1 transporter. However, the ability of these
conjugates to cross the cell membrane has not yet been studied.
We describe a feasible strategy to achieve carrier-mediated
drug transport into the rat brain via LAT1 by conjugating a
model compound to L-tyrosine. A hydrophilic nonsteroidal anti-
inflammatory drug, ketoprofen, was chosen as a model com-
pound, and an amino acid prodrug 2-amino-3-{4-[2-(3-benzoyl-
phenyl)propionyloxy]phenyl}propionic acid (1) (Figure 1) was
synthesized with the aim of utilizing the LAT1 system to bypass
the BBB. By conjugating L-tyrosine, a known LAT1 substrate,
from the phenolic hydroxyl group to the carboxyl group of
* To whom correspondence should be addressed. Tel: +358-17-162825.
Fax: +358-17-162456. E-mail: mikko.gynther@uku.fi.
†
University of Kuopio.
Fennopharma Ltd.
‡
a Abbreviations: BBB, blood-brain barrier; BCH, 2 mM 2-aminobicy-
clo(2, 2, 1)heptane-2-carboxylic acid; DMSO, dimethyl sulfoxide; F, cerebral
perfusion flow rate; LAT1, large neutral amino acid transporter; PA,
permeability-surface area; Vd, volume of distribution; Vv, vascular volume.
10.1021/jm701175d CCC: $40.75
2008 American Chemical Society
Published on Web 01/25/2008

Amino Acid Transporter Enables Brain Drug DeliVery
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 933
addition of 1% (v/v) DMSO does not compromise the integrity
of the tight junctions between the endothelial cells.
A highly diffusible and lipophilic solute [3H]diazepam was
used to determine F under the test conditions.27 The value of F
for 0.2 µCi/mL [3H]diazepam was determined to be 0.02846 (
0.00395 mL/s/g (mean ( SD, n ) 4). The value of F for
[3H]diazepam was also determined using 5 °C perfusion
medium, and it was 0.02561 ( 0.0045 mL/s/g (mean ( SD, n
) 2). Similar F values indicate that the low temperature of the
perfusion medium did not have an effect on the passive diffusion
of [3H]diazepam across the BBB.
Figure 1. Chemical structure of prodrug 1.
ketoprofen via an ester linkage, a zwitterionic prodrug that LAT1
recognizes was formed.23
Results and Discussion
The permeability of rat BBB in the test system was studied
with [14C]urea, a polar molecule with low BBB permeability.27,28
The PA product of 0.2 µCi/mL [14C]urea was determined to be
0.00018 ( 0.000056 mL/s/g (mean ( SD, n ) 3). The low
permeability of urea was in agreement with previous studies.27,28
Chemical and Enzymatic Stability of Prodrug 1. The
degradation of 1 was studied in aqueous buffer solution of pH
7.4 at 37 °C. The degradation followed pseudofirst-order kinetics
with a half-life of 22.5 ( 1.4 h (mean ( SD, n ) 2). Therefore,
1 demonstrated sufficient chemical stability in aqueous solutions
for further evaluation.
The presence of functional LAT1 transporters in rat BBB
was confirmed with L-leucine, which is an endogenous substrate
for the LAT1.14 The PA product of 0.2 µCi/mL [14C]L-leucine
was determined to be 0.02059 ( 0.00323 mL/s/g (mean ( SD,
n ) 5). The PA product was also determined using 5 °C
perfusion medium with PA product of 0.0037 ( 0.00023 mL/
s/g (mean ( SD, n ) 3), suggesting that the uptake of L-leucine
in the in situ rat brain perfusion test method was carrier-
mediated, since the carrier-mediated uptake is reduced when
the temperature is lowered.30 In addition, the determination of
cerebrovascular LAT1 functional expression was carried out
with a competition assay by perfusing [14C]L-leucine (0.2 µCi/
mL) with 2 mM concentration of another known LAT1
substrate, L-phenylalanine.28 This coperfusion resulted in almost
complete inhibition (99%) of [14C]L-leucine brain uptake,
thereby demonstrating functional expression of cerebrovascular
LAT1, which is in agreement with the literature.28
If the target for a prodrug design is the site-selective drug
delivery to the central nervous system, the prodrug must not
only be delivered to its intended site of action, but its
bioconversion should also be selective.4 In the present study, 1
was highly susceptible to enzymatic hydrolysis as it was
quantitatively cleaved to ketoprofen and L-tyrosine in 80% rat
serum, in 20% rat brain homogenate, and in 50% rat liver
homogenate. The enzymatic hydrolysis followed pseudofirst-
order kinetics, the half-lives being 10.2 ( 0.4, 4.4 ( 1.8, and
2.8 ( 0.6 min (mean ( SD, n ) 3) in serum, brain homogenate,
and liver homogenate, respectively. Therefore, 1 undergoes rapid
bioconversion to ketoprofen and L-tyrosine in the brain tissue.
However, 1 is also highly susceptible to enzymatic hydrolysis
in both rat serum and liver, which may compromise its effective
brain drug delivery.
In Situ Rat Brain Perfusion Technique. In the present
study, an in situ rat brain perfusion technique was used for the
determination of the brain uptake mechanism of the prodrug,
since it has advantages over both in vivo and in vitro techniques.
The experimental conditions can be easily manipulated to study
saturable processes such as carrier-mediated uptake, and more-
over, experimental conditions that would be toxic in the in vivo
situation can be used. The most important advantage over in
vivo experiments is the simplicity of the pharmacokinetics that
makes the accurate determination of the uptake mechanism of
the prodrug possible.24–26 In vivo techniques take into account
not only BBB penetration but also binding to plasma proteins,
metabolism, and clearance, and there is significant value in
removing some of this complexity and assessing brain penetra-
tion at the level of the BBB in situ. Furthermore, in situ
techniques provide further insight into the molecular descriptors
that are crucial for BBB penetration.26 The most important
advantage over in vitro techniques is that the BBB is in its
normal physiological state when an in situ technique is used.
An in situ rat brain perfusion technique was used to evaluate
the intravascular volume (Vv) of the rat brain, the cerebral
perfusion flow rate (F), and the brain capillary permeability–
surface area (PA) product of [14C]L-leucine and [14C]urea. The
presence of functional LAT1 transporters was evaluated based
on recent publications.25,27–29 A 0.2 µCi/mL amount of [14C]su-
crose was used as a vascular marker to determine Vv and to
demonstrate the integrity of the BBB during brain perfusion.29
The value of Vv was determined to be 0.0116 ( 0.0013 mL/g
(mean ( SD, n ) 3). The integrity of the brain capillaries was
tested with 0.2 µCi/mL [14C]sucrose after the addition of 1%
(v/v) dimethyl sulfoxide (DMSO). The value of Vv was 0.01032
( 0.00098 mL/g (mean ( SD, n ) 3) indicating that the
Determination of the Brain Uptake Mechanism for
Prodrug 1. The ability of 1 to bind into LAT1 was studied
with the in situ rat brain perfusion technique. In the presence
of 1, the [14C]L-leucine PA product was significantly decreased
from 0.02059 ( 0.00323 mL/s/g (mean ( SD, n ) 5) to
0.000317 ( 0.000136 mL/s/g (mean ( SD, n ) 3) (98.5%
inhibition). This is a clear evidence of significant binding of 1
to the LAT1 (Figure 2). To further study the binding kinetics
of 1 to LAT1, the PA product of [14C]L-leucine was determined
after perfusing rat brain first with 1 at 60 µM for 30 s, followed
by washing the prodrug from the brain capillaries with 30 s of
perfusion of prodrug-free perfusion medium and finally per-
fusing the rat brain with 0.2 µCi/mL [14C]L-leucine for 30 s.
The PA product of [14C]L-leucine (0.01902 ( 0.0058 mL/s/g
mean ( SD, n ) 3) recovered, indicating that the binding of 1
to the LAT1 is reversible (Figure 2).
To confirm the required properties of LAT1 substrate reported
by Uchino et al.,23 we synthesized two ketoprofen prodrugs with
phenylalanine promoiety (2 and 3) and two prodrugs with
leucine promoiety (4 and 5). The synthesis and structural
characterization of these prodrugs are described in the Support-
ing Information. Ketoprofen was conjugated with the carboxyl
group by diethylester linker or the amino group to delete the
zwitterionic properties of the amino acids that are necessary
for LAT1 recognition. None of these four prodrugs were able
to bind to LAT1 (Supporting Information), which is in correla-
tion with the literature.23 Therefore, our results confirm that the
potential substrate should have a positively charged amino
group, a negatively charged carboxyl group, and a hydrophobic
side chain.

934 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Gynther et al.
Figure 2. Mechanism of prodrug 1 rat brain uptake. The PA product of 0.2 µCi/mL [14C]L-leucine in the absence or presence of 1. The control
PA product 0.02059 ( 0.00323 mL/s/g (mean ( SD, n ) 5) is decreased to 0.000317 ( 0.000136 mL/s/g (mean ( SD, n ) 3) in the presence of
70 µM 1 (98.5% inhibition). The uptake of [14C]L-leucine is recovered after washing the prodrug from the brain capillaries, demonstrating the PA
product of 0.01902 mL/s/g. An asterisk denotes a statistically significant difference from the respective control (***P < 0.001, one-way ANOVA,
followed by Dunnett’s t test).
Table 1. Capillary Depletion Analysis after 60 s of Perfusion of 1 with
85 µM Concentration, Followed by Washing the Prodrug from the
Capillaries with 30 s of Perfusiona
right cerebrum
Vd (mL/g)
whole brain
supernatant
pellet
0.0123 ( 0.0016
0.0118 ( 0.0008
b
a Mean ( SD, n ) 3. b Below the lower limit of detection.
perfusion medium.30 The concentration of 1 in brain tissue was
below the lower limit of detection, indicating that there is no
passive diffusion of 1 across the BBB. A lower perfusion
medium temperature did not have any effect on passive diffusion
of [3H]diazepam across the BBB.
Capillary Depletion Analysis. Capillary depletion analysis
of brain samples from perfused brain showed that 1 is present
in the supernatant fraction, which consists of brain parenchyma
(Table 1). The concentration of 1 in the endothelial cell-enriched
pellet fraction was below the lower limit of detection. However,
because the uptake of 1 determined from the whole brain is
higher as compared to brain parenchyma, a fraction of 1 is
captured into the endothelial cells.
Figure 3. Kinetics of prodrug 1 rat brain uptake. (a) Relationship
between 1 concentration of the perfusion medium and 1 brain uptake.
Km and Vmax are 22.49 ( 9.18 µM and 1.41 ( 0.15 pmol/mg/min (mean
( SD, n ) 3), respectively. (b) The uptake of prodrug 1 with 64 µM
concentration is 0.846 ( 0.069 pmol/mg/min (mean ( SD, n ) 3),
whereas in the presence of 2 mM BCH, the uptake of 1 is decreased to
0.362 ( 0.065 pmol/mg/min (mean ( SD, n ) 3). The decrease of the
uptake is statistically significant (***P ) 0.001, t test).
Conclusion
In this study, an L-tyrosine prodrug 1 demonstrated significant
reversible inhibition of brain uptake of the radiotracer [14C]L-
leucine in the in situ rat brain perfusion model, indicating that
1 binds to the LAT1. More importantly, 1 is able to cross the
endothelial cells and penetrate into the brain parenchyma; the
brain uptake of 1 was both concentration-dependent and
saturable. The uptake of 1 was also significantly decreased when
5 °C perfusion medium was used, indicating that the brain
uptake of 1 is carrier-mediated. In addition, the LAT1 inhibitor
BCH significantly decreased the brain uptake of 1. When put
together, these results strongly suggest that the rat brain uptake
of 1 is LAT1-mediated. Importantly, 1 is not only recognized
but also transported across the rat BBB by LAT1; results that
have not been earlier reported for any drug-substrate prodrug.
In the prodrug structure, L-tyrosine acts as a carrier that
possesses the essential structural features needed for LAT1
binding. The systemic pharmacokinetics of 1 is unknown, but
the ester bond between ketoprofen and L-tyrosine is probably
cleaved mostly by esterases present in the peripheral tissues.
Therefore, this prodrug technology demands further development
before being fully applicable to oral drug delivery. In summary,
conjugation of therapeutically active drug with L-tyrosine, with
biodegradable linkage, may represent a strategy to achieve
Brain Uptake Determination of Prodrug 1. The brain
uptake of 1 was determined with the in situ rat brain perfusion
technique using seven concentrations of 1 ranging from 0 to
138 µM at 60 s of perfusion time (Figure 3a). The uptake of 1
was found to be concentration-dependent and exhibited a typical
saturation curve of the Michaelis–Menten type with a Km value
of 22.49 ( 9.18 µM and a Vmax value of 1.41 ( 0.15 pmol/
mg/min (mean ( SD) (Figure 3a). The brain uptake of 64 µM
1 was 0.846 ( 0.069 pmol/mg/min, and addition of 2 mM
2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), a spe-
cific LAT1 substrate,30 to the perfusion medium significantly
decreased the brain uptake to 0.362 ( 0.065 pmol/mg/min
(mean ( SD, n ) 3) (Figure 3b). The brain uptake of 1 was
below the lower limit of detection when 10 and 15 µM
concentrations of 1 were coperfused with BCH. These results
confirm the hypothesis that the brain uptake of 1 is LAT1-
mediated. Finally, a nonsaturable component of 1 brain uptake
was determined using a 60 µM concentration of 1 in a 5 °C

Amino Acid Transporter Enables Brain Drug DeliVery
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 935
The rate of enzymatic hydrolysis of 1 was studied at 37 °C in
rat serum, which was diluted to 80% (v/v) with 0.16 M phosphate
buffer of pH 7.4; in rat brain homogenate, which was diluted to
20% (v/v) with isotonic 0.16 M phosphate buffer of pH 7.4; and in
rat liver homogenate, which was diluted to 50% (v/v) with isotonic
0.16 M phosphate buffer of pH 7.4. The concentration of esterase
enzymes in the homogenates was not determined. The solutions
were kept in a water bath at 37 °C, and 0.2 mL of serum/buffer or
homogenate/buffer mixture was withdrawn and added to 0.2 mL
of acetonitrile to precipitate protein from the sample. After
immediate mixing and centrifugation, the supernatant was analyzed
for remaining prodrug and released ketoprofen by the HPLC. The
pseudofirst-order half-time (t1/2) for the hydrolysis of 1 was
calculated from the slope of the linear portion of the plotted
logarithm of the remaining prodrug against time.
transport of hydrophilic neuropharmaceuticals into the brain by
utilizing carrier-mediated transport and prodrugs.
Experimental Section
General Synthetic Methods. 1H (500.13 MHz) and 13C (125.77
MHz) NMR spectra were recorded on a Bruker Avance 500
spectrometer in CD3OD. Chemical shifts (δ) are reported in ppm
1
relative to CD3OD (3.31 for H and 49.0 for 13C). Electrospray
ionization mass spectra (ESI-MS) were obtained on a LCQ ion trap
mass spectrometer equipped with an electrospray ionization source
(Finnigan MAT, San Jose, CA). Elemental analyses (CHN) were
carried out with a Thermo Quest CE Instruments EA 1110 CHNS-O
elemental analyzer. Flash chromatography was performed on J. T.
Bakers silica gel for chromatography (pore size 60 Å, particle size
50 µm).
In Situ Rat Brain Perfusion Technique. A modified in situ rat
brain perfusion technique 25,29 was used to quantify the brain uptake
and the transport mechanism of 1 at the BBB. Adult male Wistar
rats (200–230 g) were supplied by the National Laboratoty Animal
Centre (Kuopio, Finland) for the rat brain perfusion studies. Rats
were anesthetized with ketamine (90 mg/kg, i.p.) and xylazine (8
mg/mL, i.p.), and their right carotid artery systems were exposed.
The right external carotid artery was ligated, and the right common
carotid artery was cannulated with PE-50 catheters filled with 100
IE/mL heparin. The right occipital and the right pterygopalatine
arteries were left open. The blood flow of the rats was stopped by
severing the cardiac ventricles. The perfusion fluid was infused
through the common carotid artery at the rate of 10 mL/min for
30–60 s using a Harvard PHD 22/2000 syringe pump (Harvard
Apparatus Inc., Holliston, MA). The skull was opened, and brain
sections (10–30 mg) were excised from six different regions of the
right brain hemisphere (frontal cortex, parietal cortex, occipital
cortex, hippocampus, caudate nucleus, and thalamus). The left
parietal cortex was used as a control. All samples were analyzed
for radioactivity by liquid scintillation counting (Wallac 1450
MicroBeta; Wallac Oy, Finland). Brain samples were dissolved in
0.5 mL of Solvable (PerkinElmer, Boston) overnight at 50 °C, and
liquid scintillation cocktail (Ultima Gold, PerkinElmer, Boston) was
added before the samples were analyzed. The perfusion medium
consisted of a pH 7.4 bicarbonate-buffered physiological saline (128
mM NaCl, 24 mM NaHCO3, 4.2 mM KCl, 2.4 mM NaH2PO4, 1.5
mM CaCl2, 0.9 mM MgCl2, and 9 mM D-glucose). The solution
was filtered, heated to 37 °C, and bubbled with 95% O2, 5% CO2
to attain steady-state gas levels within the solution. The in situ rat
brain technique was validated by measuring the Vv as estimated by
[14C]sucrose (PerkinElmer, Boston) and by quantifying the brain
uptake of [3H]diazepam (PerkinElmer, Boston), [14C]urea (Perkin-
Elmer, Boston), and [14C]L-leucine (PerkinElmer, Boston). All
radiolabeled compounds used in the in situ rat brain perfusion
technique were uniformly labeled.
Determination of the Brain Uptake Mechanism for
Prodrug 1. The ability of 1 to bind into LAT1 was studied with
the in situ rat brain perfusion technique. The 100% PA product of
[14C]L-leucine was determined after 30 s of perfusion of 0.2 µCi/
mL [14C]L-leucine solution. In a competition study, [14C]L-leucine
(0.2 µCi/mL) was coperfused with 70 µM concentration of 1 for
30 s. To study whether the binding of 1 to LAT1 was reversible,
the PA product of [14C]L-leucine was determined after perfusing
rat brain first with 1 at 60 µM for 30 s, followed by washing the
prodrug from the brain capillaries with 30 s of perfusion of prodrug-
free perfusion medium, and finally perfusing the brain with 0.2
µCi/mL [14C]L-leucine for 30 s.
Brain Uptake Studies of Prodrug 1. The prodrug 1 brain uptake
studies were performed with the in situ rat brain perfusion technique
described above. Because of relatively low water solubility of 1, it
was first dissolved in DMSO and then added to the perfusion
medium resulting in 1% (v/v) DMSO solution. After the pH was
adjusted to 7.4, the solution was filtered with 0.45 µm Millex-HV
filters. The rat brains were perfused for 60 s with 37 °C perfusion
medium containing prodrug 1. After perfusion, the remaining
prodrug was washed from the brain vasculature with cold prodrug-
Synthesis of 2-Amino-3-{4-[2-(3-benzoyl-phenyl)propionyl-
oxy]phenyl}propionic Acid (1). L-Tyrosine (0.150 g, 0.828 mmol)
was dissolved in trifluoroacetic acid (15 mL) and cooled to 0 °C.
2-(3-Benzoyl-phenyl)propionyl chloride (1.13 g, 4.14 mmol) was
added to the solution, and the mixture was stirred at room
temperature overnight. The solvent was evaporated, and the crude
product was purified by flash chromatography (hexane:ethyl acetate
1:1 gradually increasing to methanol). Combined fractions were
evaporated and dissolved to acetonitrile (20 mL), and HCl gas was
added to the mixture for 5 min. The formed clear solution was
evaporated and dried in vacuo. The yield of white solid was 0.346
g (92%).
1H NMR (CD3OD): δ 1.63 (3H, d, J ) 7.2 Hz, CH3), 3.12–3.35
(2H, m, CH2), 4.16 (1H, q, J ) 7.2 Hz, CH), 4.24 (1H, dd, J ) 7.9
Hz, J ) 5.4, CH), 7.03 (2H, d, J ) 8.6 Hz, ArH), 7.32 (2H, d, J
) 8.5 Hz, ArH), 7.51–7.83 (9H, m, ArH). 13C NMR (CD3OD): δ
18.8 (1H, CH3), 36.6 (1C, CH2),46.5 (1C, CH), 55.1 (1C, CH),
123.2 (2C, ArCH), 129.6 (2C, ArCH), 130.0 (1C, ArCH), 130.1
(1C, ArCH), 130.2 (1C, ArCH), 131.0 (2C, ArCH), 131.7 (2C,
ArCH), 133.0 (1C, ArCH), 133.5 (1C, ArC), 133.9 (1C, ArCH),
138.7 (1C, ArC), 139.4 (1C, ArC), 142.1 (1C, ArC), 151.9 (1C,
ArC), 171.1 (1C, CO), 174.6 (1C, CO), 198.3 (1C, CO). MS: m/z
calcd for C25H23NO5, 417.5; found, 418.1. Anal. calcd for
(C25H23NO5 ·HCl·H2O): C, 63.63; H, 5.55; N, 2.97. Found: C,
63.73; H, 5.29; N, 3.35.
HPLC Assay. The prodrug concentration in rat brain samples
was analyzed by Agilent 1100 HPLC system (Agilent Technologies
Inc., Waldbronn, Karlsruhe, Germany) that consisted of a binary
pump G1312A, a vacuum degasser G1379A, an automated injector
system autosampler Hewlett-Packard 1050, an UV detector Hewlett-
Packard 1050 variable wavelength detector, and an analyst software
Agilent ChemStation for LC Systems Rev. A.10.02. The detector
wavelength was set at 256 nm. A mixture of acetonitrile (50%)
and a 0.02 M phosphate buffer solution of pH 2.5 (50%) at a flow
rate of 1 mL/min was used as a mobile phase. The lower limit of
quantification was 1 µM, and the method was linear over the range
of 1–50 µM. In vitro samples were analyzed by HPLC system
consisting of a Merck Hitachi L-6200A intelligent pump, Merck
Hitachi L-4500 diode array detector, a Merck Hitachi D-6000A
interface module, and a Merck Hitachi AS-2000 autosampler
(Merck LaChrom, Hitachi, Tokyo, Japan). A mixture of 30%
acetonitrile (v/v) and a 0.02 M 70% phosphate buffer solution of
pH 6.0 (v/v) at a flow rate of 1 mL/min was used as a mobile
phase. The lower limit of quantification was 0.4 µM, and the method
was linear over the range of 0.4–211 µM. Reversed-phase HPLC
was conducted with a Zorbax RP-18 column (150 mm × 4.6 mm,
5 µm, Agilent Technologies, Little Falls Wilmington, DE).
Chemical and Enzymatic Stability of the Prodrug 1. The rate
of chemical hydrolysis of 1 was studied in aqueous phosphate buffer
solution of pH 7.4 (0.16 M, ionic strength 0.5) at 37 °C. An
appropriate amount of 1 was dissolved in 10 mL of preheated buffer,
and the solutions were placed in a thermostatically controlled water
bath at 37 °C. At appropriate intervals, samples were taken and
analyzed for remaining 1 by HPLC. Pseudofirst-order half-time (t1/2
)
for the hydrolysis of 1 was calculated from the slope of the linear
portion of the plotted logarithm of remaining 1 vs time.

936 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Gynther et al.
(7) Janzer, R. C.; Raff, M. R. Astrocytes induce blood-brain barrier
properties in endothelial cells. Nature 1987, 325, 253–257.
(8) Ramsauer, M.; Krause, D.; Dermietzel, R. Angiogenesis of the
blood-brain barrier in vitro and the function of cerebral pericytes.
FASEB J. 2002, 10, 1274–1276.
(9) Tontsch, U.; Bauer, H. C. Glial cells and neurons induce blood-brain
barrier related enzymes in cultured cerebral endothelial cells. Brain
Res. 1991, 539, 247–253.
(10) Bodor, N.; Buchwald, P. Recent advances in the brain targeting of
neuropharmaceuticals by chemical delivery systems. AdV. Drug
DeliVery ReV. 1999, 36, 229–254.
(11) Pardridge, W. M. Drug and gene delivery to the brain. Neuron 2002,
36, 555–558.
(12) Pardridge, W. M.; Oldendorf, W. H. Transport of metabolic substrates
through the blood-brain barrier. J. Neurochem. 1977, 28, 5–12.
(13) Tamai, I.; Tsuji, A. Transporter-mediated permeation of drugs across
the blood-brain barrier. J. Pharm. Sci. 2000, 89, 1371–1388.
(14) Duelli, R.; Enerson, B. E.; Gerhart, D. Z.; Drewes, L. R. Expression
of large amino acid transporter LAT1 in rat brain endothelium.
J. Cereb. Blood Flow Metab. 2000, 11, 1557–1562.
(15) Boado, R. J.; Li, J. Y.; Nagaya, M.; Zhang, C.; Pardridge, W. M.
Selective expression of the large neutral amino acid transporter at the
blood-brain barrier. Proc. Natl. Acad. Sci. U.S.A. 1999, 21, 79–84.
(16) Gomes, P.; Soares-da-Silva, P. L-DOPA transport properties in an
immortalised cell line of rat capillary cerebral endothelial cells, RBE
4. Brain. Res. 1999, 829, 143–150.
(17) Cundy, K. C.; Branch, R.; Chernov-Rogan, T.; Dias, T.; Estrada, T.;
Hold, K.; Koller, K.; Liu, X.; Mann, A.; Panuwat, M.; et al. XP13512
[(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-
cyclohexane acetic acid], a novel gabapentin prodrug: I. Design,
synthesis, enzymatic conversion to gabapentin, and transport by
intestinal solute transporters. JPET 2004, 311, 315–323.
(18) Goldenberg, G. J.; Lam, H. Y.; Begleiter, A. Active carrier-mediated
transport of melphalan by two separate amino acid transport systems
in LPC-1 plasmacytoma cells in vitro. J. Biol. Chem. 1979, 254, 1057–
1064.
free perfusion medium (5 °C) for 30 s. The prodrug concentration
of the perfusion medium was analyzed by HPLC after each
perfusion to confirm that the prodrug had stayed intact. To study
the role of passive diffusion (nonsaturable component) in the brain
uptake of 1, the brain capillaries were first washed for 30 s with
cold prodrug-free perfusion medium (5 °C), followed by perfusion
for 60 s with cold 1 solution (5 °C), and washed again with cold
prodrug-free perfusion medium (5 °C).
Capillary Depletion Analysis. Capillary depletion analysis was
carried out as previously described by Triguero et al.31 Brain
samples (right brain hemisphere) were weighed and homogenized
in a glass homogenizer with 1.5 mL of physiological buffer. After
homogenization, 2 mL of 26% dextran solution was added and the
mixture was further homogenized. The homogenate was separated
into two microcentrifuge tubes and centrifuged for 15 min (5400g,
4 °C). The resulting supernatant consisting of the brain parenchyma
and the pellet rich in cerebral capillaries were separated and
prepared for analysis with HPLC. The volume of distribution (Vd)
values for the homogenate, supernatant fraction, and the capillary
pellet were calculated.
Brain Sample Preparation. Because of the short half-life of 1
in the rat brain, the quantification of 1 from the rat brain samples
was performed by analyzing the total concentration of formed
ketoprofen after its enzymatic release from 1. Ketoprofen was
isolated from the rat brain samples by protein precipitation. A
complete brain hemisphere was homogenated with 2.5 mL of water
to produce 3.0 mL of homogenate. The samples were acidified with
300 µL of 2 M hydrochloric acid and vortexed for 5 min.
Ethylacetate (1.0 mL) was added, and aliquots were vortexed for 2
min and centrifuged for 10 min (7500g) after which the supernatants
were collected. This was repeated four times, and the supernatants
were combined. The supernatants were evaporated to dryness under
a nitrogen stream at 40 °C. Prior to analysis, the samples were
reconstituted in 50% (v/v) acetonitrile in water and filtered. External
standards were used for the brain samples. The calibration curve
of the brain method was linear over a range of 0.2–2.0 nmol/brain
hemisphere. The lower limit of quantification for spiked samples
was 0.2 nmol of prodrug/brain hemisphere.
(19) Stella, V. J.; Himmelstein, W. N. A.; Narigrekar, V. H. Prodrugs. Do
they have advantages in clinical practice. Drugs 1985, 29, 455–473.
(20) Smith, Q. R.; Cooper, A. J. L. Mammalian Amino Acid Transport;
Plenum Press: New York, 1992; pp 165–193.
(21) Walker, I.; Nicholls, D.; Irwin, W. J.; Freeman, S. Drug delivery via
active transport at the blood-brain barrier: Affinity of a prodrug of
phosphoformate for the large amino acid transporter. Int. J. Pharm.
1994, 104, 157–167.
Data Analyses. The results from the brain uptake experiments
are presented as means ( SD of at least three independent
experiments. Data analyses for the dose-uptake curves were
calculated as nonlinear regressions using GraphPad Prism 4.0 for
Windows. Statistical differences between groups were tested using
one-way ANOVA, followed by a two-tailed Dunnett’s t test (Figure
2). In Figure 3b, a two-tailed independent samples t test was used.
P < 0.05 was considered as statistically significant. The normality
of the data was tested using a Shapiro-Wilk test. All statistical
analyses were performed using SPSS 14.0 for Windows.
(22) Balakrishnan, A.; Jain-Vakkalagadda, B.; Yang, C.; Pal, D.; Mitra,
A. K. Carrier-mediated uptake of L-tyrosine and its competitive
inhibition by model tyrosine linked compounds in a rabbit corneal
cell line (SIRC)-strategy for design of transporter/receptor targeted
prodrugs. Int. J. Pharm. 2002, 247, 115–125.
(23) Uchino, H.; Kanai, Y.; Kim, D. K.; Wempe, M. F.; Chairoungdua,
A.; Morimoto, E.; Anders, M. W.; Endou, H. Transport of amino acid-
related compounds mediated by L-type amino acid transporter 1
(LAT1): Insights into the mechanisms of substrate recognition. Mol.
Pharmacol. 2002, 61, 729–737.
(24) Foster, K. A.; Roberts, M. S. Experimental methods for studying drug
uptake in the head and brain. Curr. Drug Metab. 2000, 4, 333–56.
(25) Smith, Q. R.; Allen, D. D. Methods in Molecular Medicine: The
Blood-Brain Barrier: Biology and Research Protocols; Humana Press
Inc.: Totowa, NJ, 2003; pp 209–218.
(26) Summerfield, S. G.; Read, K.; Begley, D. J.; Obradovic, T.; Hidalgo,
I. J.; Coggon, S.; Lewis, A. V.; Porter, R. A.; Jeffrey, P. Central
nervous system drug disposition: The relationship between in situ brain
permeability and brain free fraction. JPET 2007, 322, 205–213.
(27) Rousselle, C. H.; Lefauconnier, J. M.; Allen, D. D. Evaluation of
anesthetic effects on parameters for the in situ rat brain perfusion
technique. Neurosci. Lett. 1998, 257, 139–142.
Acknowledgment. This study was supported by the National
Technology Agency of Finland (TEKES). We also thank Helly
Rissanen and Miia Reponen for their technical assistance.
Supporting Information Available: General synthetic proce-
dures and in situ inhibition results for compounds 2–5 and the
combustion analysis data for compounds 1–5. This material is
available free of charge via the Internet at http://pubs.acs.org.
(28) Killian, D. M.; Chikhale, P. J. A bioreversible prodrug approach
designed to shift mechanism of brain uptake for amino-acid-containing
anticancer agents. J. Neurochem. 2001, 76, 966–974.
(29) Killian, D. M.; Hermeling, S.; Chikhale, P. J. Targeting the cere-
brovascular large neutral amino acid transporter (LAT1) isoform using
a novel disulfide-based brain drug delivery system. Drug DeliVery
2007, 14, 25–31.
(30) Kageyama, T.; Nakamura, M.; Matsuo, A.; Yamasaki, Y.; Takakura,
Y.; Hashida, M.; Kanai, Y.; Naito, M.; Tsuruo, T.; Minato, N.;
Shimohama, S. The 4F2hc/LAT1 complex transports L-DOPA across
the blood-brain barrier. Brain. Res. 2000, 879, 115–121.
(31) Triguero, D.; Buciak, J.; Pardridge, W. M. Capillary depletion method
for quantification of blood-brain barrier transport of circulating
peptides and plasma proteins. J. Neurochem. 1990, 54, 1882–1888.
References
(1) Pardridge, W. M. Blood-brain barrier drug targeting: the future of
brain drug development. Mol. InterV. 2003, 3, 90–105.
(2) Pardridge, W. M. The blood-brain barrier: Bottleneck in brain drug
development. NeuroRx 2005, 2, 2–14.
(3) Rubin, L. L.; Staddon, J. M. The cell biology of the blood-brain
barrier. Annu. ReV. Neurosci. 1999, 22, 11–28.
(4) Anderson, B. D. Prodrugs for improved CNS delivery. AdV. Drug
DeliVery ReV. 1996, 19, 171–202.
(5) Cordon-Cardo, C.; O’Brien, J. P.; Casals, D.; Rittman-Grauer, L.;
Biedler, J. L.; Melamed, M. R.; Bertino, J. R. Multidrug-resistance
gene (P-glycoprotein) is expressed by endothelial cells at blood-brain
barrier sites. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 695–698.
(6) Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. The multidrug resistance
protein family. Biochim. Biophys. Acta 1999, 1461, 347–357.
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