Dual system for the central nervous system targeting and blood-brain barrier transport of a selective prolyl oligopeptidase inhibitor

Article abstract of DOI:10.1002/bip.22275

Less than 2% of all potential neurotherapeutics cross the blood-brain barrier (BBB). Here, we sought to build a construct with the capacity to cross this barrier, to behave as a chemical delivery system, and, once inside the central nervous system, to be

Full text of DOI:10.1002/bip.22275

Biopolymers: Peptide Science
Dual System for the Central Nervous System Targeting and Blood-
Brain Barrier Transport of a Selective Prolyl Oligopeptidase Inhibitor
Meritxell Teixidó,*,1 Esther Zurita,1 Laura Mendieta,1 Benjamí OllerꢀSalvia,1 Roger
Prades,1 Teresa Tarragó,1 Ernest Giralt,*,1,2
1
Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, Baldiri Reixac 10,
Barcelona, 08028, Spain
2
Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1ꢀ11, Barcelona, 08028,
Spain
*
Authors to whom correspondence should be addressed: ernest.giralt@irbbarcelona.org,
meritxell.teixido@irbbarcelona.org
Tel.: +34ꢀ93ꢀ4037125; Fax: +34ꢀ93ꢀ4037126
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/bip.22275
© 2013 Wiley Periodicals, Inc.

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Abbreviations: ACH, αꢀcyanoꢀ4ꢀhydroxyꢀcinnamic acid; BAD, bipolar affective
disorder; BBB, bloodꢀbrain barrier; CDS, chemical delivery system; CNS, central
nervous system; Da, dalton; DBU, 1,8ꢀdiazabicyclo[5.4.0]ꢀundecꢀ7ꢀen; DCM,
dichloromethane; DIAD, diisopropylazodicarboxylate; DIEA, diisopropylethylamine;
DIPCDI, N,N'ꢀdiisopropylcarbodiimide; DMAP, 4ꢀdimethylaminopyridine; DMF, N,N’ꢀ
dimethylformamide; DPPꢀIV, dipeptidyl peptidase IV; ESIꢀMS, electrospray ionizationꢀ
mass
spectrometry;
Fmoc,
9ꢀfluorenylmethoxycarbonyl;
HIV,
human
immunodeficiency virus; 1HꢀNMR, nuclear magnetic resonance of proton; HOAt, 7ꢀ
azaꢀ1ꢀhydroxybenzotriazole; HPLC, high performance liquid chromatography; HPLCꢀ
MS, high performance liquid chromatography mass spectrometry; MALDIꢀTOF,
matrixꢀassisted laser desorption ionizationꢀtimeꢀofꢀflight; MeCN, acetonitrile; MeOH,
methanol; NBB, 4ꢀhydroxyꢀ3ꢀnitrobenzoic acid; oꢀNBS, oꢀnitrobenzenesulfonyl
chloride; PAMPA, parallel artificial membrane permeability assay; POP, prolyl
oligopeptidase;
PyBOP,
(benzotriazoleꢀ1ꢀylꢀoxy)tripyrrolidinoꢀphosphonium
hexafluorophosphate; Rf, retention factor; SPPS, solidꢀphase peptide synthesis; SZ,
schizophrenia; TFA, trifluoroacetic acid; THF, tetrahydrofurane; TLC, thin layer
chromatography; tR, retention time.
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Biopolymers: Peptide Science
ABSTRACT:
Less than 2% of all potential neurotherapeutics cross the bloodꢀbrain barrier (BBB).
Here we sought to build a construct with the capacity to cross this barrier, to behave as a
chemical delivery system, and, once inside the central nervous system (CNS), to be
transformed and then act as an enzyme inhibitor. With all this in mind, here we describe
the entire process to obtain such a compound, from the initial candidate selection to
preparation of the compound library and posterior evaluation and final selection of the
most promising candidates in terms of selectivity, serum stability and BBBꢀtransport.
Keywords: bloodꢀbrain barrier; drug delivery; central nervous system;
diketopiperazine; enzyme inhibitor
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INTRODUCTION
Several severe health disorders require treatment of the brain. These include
neurodegenerative diseases, such as Parkinson’s and Alzheimer’s, but also other central
nervous system (CNS) diseases, such as schizophrenia, epilepsy and bipolar disorder.
Cancer, HIV, and even certain aspects of obesity are also considered cerebral
pharmaceutical targets. During recent years we have devoted intensive research effort to
the field of drug delivery to the brain.1ꢀ5
The delivery of pharmacological agents to the brain is not an easy task. In fact, the
scientific community has long dreamt about this challenge. In 1966, a film called “The
Fantastic Voyage” addressed the issue of sending a shuttle to treat the brain. This was a
complex journey as the drugs directed towards targets inside the brain had to overcome
the bloodꢀbrain barrier (BBB). The discovery of the BBB in the late 19th century
stemmed from Paul Ehrlich’s6 observation that intravenously administered dyes stain all
organs except the brain. In 1913, Edwin Goldman7 hypothesized that cerebral capillaries
provide the anatomical basis for this physiological barrier. In the late 1960s, this
hypothesis was confirmed by electron microscopy.8,9 The BBB is present in all
vertebrate brains and acts as a natural defence to protect this organ. In fact, this barrier
is responsible for more than 98% of all potential neurotherapeutics failing to reach the
brain.10 While blood vessels have pores between cells, brain capillaries have tight
junctions, thereby avoiding the paracellular pathway. Thus all transport occurs through
endothelial cells. Various transport mechanisms that allow passage across these cells
ensure that certain nutrients reach the brain. Given the impermeability of the BBB, these
transport mechanisms may prove effective in delivering drugs to the brain.
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Biopolymers: Peptide Science
One of the BBBꢀshuttles that we have been working with in recent years is based on a
diketopiperazine (DKP) scaffold, modified in order to attach cargos and carry them
across the BBB by passive diffusion.2 We focused our attention on the passive diffusion
mechanism as it is a nonꢀsaturable and spontaneous transport process and it can be
evaluated using the PAMPA assay (Parallel Artificial Membrane Permeability Assay).
Introduced by Kansy11 for the prediction of gastrointestinal tract absorption, PAMPA is
a nonꢀcellꢀbased assay with an artificial membrane. During the last decade, this assay,
which presents only nonꢀsaturable transmembrane diffusion, has been modified to be
used as an in vitro model to study permeation of the BBB and skin.12ꢀ14 For a broad
range of CNS drugs, PAMPA correlates with in situ brain diffusion remarkably better
than the Madin Darby canine kidney (MDCK) cellꢀbased model.15 The transport of
compounds across in vitro membranes can be limited by a barrier formed by the
membrane and the two unstirred water layers (UWLs), one on each side of the
membrane. Transport evaluation requires the removal of these layers with a suitable
stirring protocol.16ꢀ19
There are two main purposes of designing a BBBꢀshuttleꢀcargo construct to selectively
reach the brain, namely to maximize therapeutic efficacy and to reduce the sideꢀeffects
of systemic drugs. A compound with the capacity to cross the BBB by passive diffusion
will cross not only brain capillaries but also other capillaries in the body. The chemical
delivery system (CDS) approach is one of the most interesting procedures for
transporting drugs to the CNS in a sustained and specific manner. This approach
involves covalent attachment of a molecular targetor to the drug of interest. The targetor
dihydropyridine (Dhp) is a specific functional group attached to the drug. In addition to
enhancing BBB penetration by virtue of its lipophilicity, this targetor can be converted
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by enzymatic oxidation to a polar quaternary pyridinium salt. This conversion occurs
ubiquitously. The polar oxidized drugꢀconjugate is essentially trapped behind the
lipoidal BBB and remains locked inside the CNS, while in the periphery the ionic
species formed is rapidly removed by the kidney and liver (Figure 1). The conjugate,
which is retained in the CNS, can then undergo biotransformation to give the active
species in a slow and sustained manner.20
This CDS has been successfully applied for brainꢀspecific delivery of several
compounds, such as dopamine,21,22 various estrogens,23 and 4ꢀaminobutanoic acid.24 In
the case of dopamine, the use of the CDS approach reduced the peripheral and central
sideꢀeffects when compared with Lꢀdopa therapy.21,22 Acquired immune deficiency
syndrome (AIDS) is accompanied by AIDS encephalopathy, a condition resulting from
the direct infection of the brain by the human immunodeficiency virus (HIV).
Zidovudine (AZT), the first drug approved for treating AIDS, has contributed to an
amelioration of the neuropsychiatric symptoms associated with AIDS encephalopathy.
AZT shows poor BBB penetration capacity and consequently high doses are required to
elicit this improvement. However, such doses cause severe anaemia, which usually
leads to cessation of this therapy. Administration of an AZTꢀCDS in dogs results in
rapid tissue uptake and conversion to the corresponding quaternary salt with the
subsequent production of AZT in the brain.25 The CDS method has also been used to
deliver several neuropeptides, such as enkephalin, TRH (thyrotropinꢀreleasing hormone)
and kyotorphin analogs (TyrꢀDꢀAlaꢀGlyꢀPheꢀDꢀLeu, GlnꢀLeuꢀProꢀGly and TyrꢀLys,
respectively), to the brain.26ꢀ30
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Biopolymers: Peptide Science
In this study our objective was to build a construct with the capacity to cross the BBB,
to behave as a CDS and, once inside the CNS, to be transformed to the oxidized form
and then act as an enzyme inhibitor. We chose the enzyme prolyl oligopeptidase (POP;
EC 3.4.21.26) as a target. POP is a cytosolic serine protease that hydrolyzes small
prolineꢀcontaining peptides.31 In recent years, POP has gained relevance as a target for
the treatment of cognitive disturbances.32 POP has been reported to be involved in
inositolꢀ1,4,5ꢀP3 (IP3) metabolism, as POP inhibition increases the concentration of
IP3,33,34 a key molecule in the transduction cascade of neuropeptide signalling.
Neuropeptides induce an increase in IP3 that is believed to play a crucial role in
learning and memory. Therefore, an array of strategies is currently being used to
identify POP inhibitors as these compounds show neuroprotective and cognitionꢀ
enhancing effects in experimental animals.35ꢀ38 In spite of promising results, a drug
based on POP inhibition has yet to reach the market.39,40 Here we describe for the first
time a CDS construct that becomes a POP inhibitor once in the CNS.
MATERIALS AND METHODS
Materials and Analyses
Protected amino acids and resins were supplied by Luxembourg Industries, Neosystem,
CalbiochemꢀNovabiochem AG, Bachem AG and Iris Biotech. PyBOP was supplied by
CalbiochemꢀNovabiochem AG. DIEA, ninhydrin and ßꢀmercaptoethanol were obtained
from Fluka Chemika. HOAt was purchased from GL Biochem Shanghai Ltd. Solvents
for peptide synthesis and RPꢀHPLC were obtained from Scharlau or SDS.
Trifluoroacetic acid was purchased from KaliChemie. The other chemicals used were
purchased from Aldrich and were of the highest purity commercially available. PAMPA
plates and PAMPA system solution were from pION. Porcine polar brain lipid extract
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(PBLEP) was from Avantis Polar Lipids. Human serum from male AB plasma was
purchased from Aldrich. Mass spectra were recorded on a MALDI Voyager DE RP
timeꢀofꢀflight (TOF) spectrometer, using an ACH matrix. High resolution mass spectra
were recorded on a LTQꢀFT Ultra (Thermo Scientific). HPLC chromatograms were
recorded on a Waters model Alliance 2695 with Photodiode array detector 996 Waters
using a Sunfire C18 column (100 x 4.6 mm x 3.5 ꢀm, 100 Å, Waters) and Symmetry C18
column (150 x 4.6 mm x 5 ꢀm, 100 Å, Waters), solvents: H2O (0.045% (v/v) TFA) and
MeCN (0.036% (v/v) TFA), flow: 1 mL minꢀ1 and software Millenium version 4.0
software (Waters). HPLCꢀMS [Waters model Alliance 2796, quaternary pump, UV/Vis
Dual absorbance detector Waters 2487, ESIꢀMS model Micromass ZQ and Masslynx
version 4.0 software (Waters)] was done using a Symmetry 300 C18 column (150 × 3.9
mm × 5 ꢀm, 300 Å, Waters), solvents: H2O (0.1% (v/v) formic acid) and MeCN (0.07%
(v/v) formic acid), flow rate: 1 mL minꢀ1. The products were purified in a Waters 600
with Dual absorbance detector (Waters 2487 Waters), and a Symmetry C18 column (100
× 30 mm × 5 ꢀm, 100 Å, Waters), solvents H2O (0.1% (v/v) TFA) and MeCN (0.05%
(v/v) TFA), flow rate: 10 mL minꢀ1. Purity was checked by reverseꢀphase HPLC using a
Sunfire C18 column. Products showed a purity ≥ 98%. ZGPꢀAMC was obtained from
Bachem. POP was obtained by expression in E. coli, and subsequent affinity
purification using a His tail fusion as reported previously.41 Fluorescence was measured
1
using a BioꢀTek FL600 fluorescence plate reader. HꢀNMR spectra were obtained at
25ºC with a Varian Mercury 400 NMR spectrometer using CD3OD as a solvent. UV
spectra were recorded in methanol on a UVꢀ2501 PC Spectrophotometer, Shimadzu.
DPPꢀIV from porcine kidney was obtained from Sigma.
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Biopolymers: Peptide Science
General Protocols for Solid-Phase Synthesis
Syntheses were performed on a 100 ꢀmolꢀscale/each. Solidꢀphase peptide elongation
and other solidꢀphase manipulations were done manually in polypropylene syringes,
each fitted with a polyethylene porous disk. Solvents and soluble reagents were
removed by suction. Washings between synthetic steps were done with DMF (5 × 30 s)
and DCM (5 × 30 s) using 5 mL of solvent gꢀ1 resin each time. During couplings the
mixture was allowed to react with intermittent manual stirring.
Identification Tests
The Kaiser colorimetric assay42 was used for the detection of primary amines bound to
solid phase while the De Clercq test43 was used for secondary amines bound to solid
phase.
Initial Conditioning of Resin
The pꢀMBHA resin was conditioned by washing with DCM (5 x 30 sec) followed by a
40% (v/v) TFA solution in DCM (1 x 30 sec and 2 x 5 min). This acid treatment was
followed by a neutralization step with DIEA 5% (v/v) in DCM (3 x 2 min) and finally
the resin was washed with DCM (5 x 30 sec).
Fmoc Group Removal
The Fmoc group was removed by treating the resin with 20% (v/v) piperidine in DMF
(3ꢀ4 mL gꢀ1 resin, 2 × 1 min and 1 × 10 min). To remove the Fmoc group from FmocꢀLꢀ
Pro(Phe)ꢀOH and FmocꢀDꢀPro(Phe)ꢀOH, an additional treatment with DBU, toluene,
piperidine, and DMF (5%: 5%: 20%: 70%) (1 × 5 min) was performed.
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Synthesis of Nbb Handle
4ꢀbromomethylꢀ3ꢀnitrobenzoic acid (4.5 g, 17.3 mmols) was placed in a roundꢀ
bottoned flask provided with reflux system, and an aqueous saturated solution of
NaHCO3 was added (135 mL). The reaction was stirred and allowed to stand at 90ꢀ
95ºC. The progress of the reaction was monitored by thin layer chromatography (TLC)
(Chloroform/MeOH/AcOEt, 100:50:0.1, v/v) or by HPLC of an acidified aliquot in a
gradient of 0 to 100% of MeCN in 15 min in a Symmetry C18 column (150 x 4.6 mm x
5 ꢀm, 100 Å, Waters). Retention times for the initial and final product were 10.4 min
and 7.1 min respectively. The reaction was completed in 30 min. The solution obtained
was hotꢀfiltered and the reaction was quenched with HCl (12N) to pH 1ꢀ2 and the crude
product was extracted with AcOEt (3x100mL), the organic fractions were combined,
washed with a saturated NaCl aqueous solution and dried over MgSO4 anh. AcOEt was
evaporated in vacuo.
The product was obtained as a pale yellow solid, yield: 80%, 2.73 g. It was
characterized by HPLC (tr) 7.12 min (linear gradient 0ꢀ100% MeCN in 15 min), HPLCꢀ
1
MS 196 Da, TLC Rf = 0.3 (Chloroform/MeOH/AcOEt, 100:50:0.1, v/v) and HꢀNMR
(CD3OD, 400 MHz, δ ppm): 4.98 (s, 2H), 7.96 (d, J = 21, 1H), 8.26 (dd, J = 4, J =20,
1H), 1.56ꢀ1.24 (m, 12H), 8.56 (d, J = 4, 1H).
Coupling Methods
Coupling of Nbb Handle to p-MBHA Resin
Nbb handle (2.95 g, 5 eq.) in DCM (3 mL gꢀ1 resin) and DIPCDI (2.3 mL, 5 eq.) were
sequentially added to the pꢀMBHA resin (3 mmols, 4.9 g). The mixture was left to stir
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Biopolymers: Peptide Science
overnight. The solvent was then removed by suction, the resin was thoroughly washed,
and the reaction was monitored with the Kaiser test.
Method 1, Coupling of the First Amino Acid onto the Nbb Handle
The amino acid derivative (4 eq.), DIPCDI (62 ꢀL, 4 eq.) and DMAP (5 mg, 0.4 eq.) in
DCM (1ꢀ3 mL gꢀ1 resin) were sequentially added to the resin. The mixture was allowed
to react with intermittent manual stirring for 30 min. The solvent was then removed by
suction, the resin was thoroughly washed and the coupling was repeated once.
Method 2, Coupling of Second Amino Acid (Boc-Phe(p-NHFmoc)-OH onto the
Peptidyl-Resin
BocꢁNꢀPhe(pꢀNHFmoc)ꢀOH (4 eq., 200.8 mg), PyBOP (4 eq., 208 mg) and HOAt (12
eq., 163.3 mg) were added sequentially to the resin in DMF (3 mL) followed by DIEA
(12 eq., 205 ꢀL). The mixture was allowed to react with intermittent manual stirring for
1 h. The solvent was removed by filtration; the resin was washed with DMF (5 × 30 s)
and DCM (5 × 30 s). The coupling was repeated two more times and the extent of
coupling was checked by the De Clercq test.
Amino Acid N-Alkylation
Nꢀmethylation was performed using the method described by Miller.44,45 This process
can be divided into three steps: A) protection and activation with oꢀ
nitrobenzenesulfonyl chloride (oꢀNBS); B) Mitsunobu reaction; and C) oꢀNBS removal.
A) Protection and Activation with o-NBS: To perform the protection, oꢀNBS (4 eq.,
89 mg) and collidine (10 eq., 132 ꢀL) in DCM were added to the resin. The reaction
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was left with intermittent manual stirring for 1 h and this step was repeated once and
checked by the Kaiser test.
B) Mitsunobu Reaction: The Mitsunobu reagents, triphenylphosphine (133 mg, 5 eq.)
and MeOH (24 ꢀL, 10 eq.) in dry THF, were added to the resin and left for 1 min.
DIAD (98 ꢀL, 5 eq.) was then added without filtering and the reaction was left for
another 10 min.
C) o-NBS Removal: To remove oꢀNBS, ßꢀmercaptoethanol (10 eq., 70
ꢀL) and DBU
(5 eq., 75 L) in DMF were added to the resin and the mixture was left to react for 5
ꢀ
min under a nitrogen atmosphere. This process was repeated twice.
Coupling of Nicotinic Acid and Methylation
Nicotinoyl chloride hydrochloride (4 eq., 71 mg), PyBOP (4 eq., 208 mg) and HOAt (12
eq., 163.3 mg) in DMF (3 mL) were added sequentially to the resin followed by DIEA
(12 eq., 205 ꢀL). The mixture was allowed to react with intermittent manual stirring for
3 h. The solvent was removed by filtration and the resin was washed with DMF (5 × 30
s) and DCM (5 × 30 s). The extent of coupling was checked by the De Clercq test.
Methylation was achieved by adding CH3I (20 eq., 125 ꢀL) in DMF (3 mL) to the resin.
The mixture was left to stir overnight. The solvent was then removed by suction and the
resin was thoroughly washed.
Boc Group Removal
The Boc group was removed by treating the resin with 40 % (v/v) TFA in DCM (3ꢀ4
mL gꢀ1 resin, 2 x 10 min). In solution, the Boc group was removed using 50% (v/v) TFA
in DCM for 1 h. DCM was then evaporated under reduced pressure.
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Neutralization-Cyclization and Cleavage in a One-Step Process
Only by treating the resin with 10% (v/v) DIEA in DCM (3 x 10 min) was
neutralizationꢀcyclization and cleavage accomplished. The filtrates were collected,
DCM was evaporated under N2, and the residue was solved in H2O:MeCN (1:1) and
lyophilized.
Product Workup and RP-HPLC Purification
After cleavage of the peptides, the solvent was evaporated by N2. The residue was
dissolved in H2O: MeCN (1:1) and then lyophilized. The peptides were purified by
reverseꢀphase HPLC using a symmetry C18 column (100 × 30 mm × 5 ꢀm, 100 Å,
Waters) at a flow rate of 10 mL minꢀ1 with the following solvents: A, H2O with 0.1%
(v/v) TFA; B, MeCN with 0.05% (v/v) TFA.
Product Characterization
The identity of the compounds synthesized was confirmed using MALDIꢀTOF MS, and
HPLCꢀMS. Purity was checked by reverseꢀphase HPLC using a Sunfire C18 column
(100 x 4.6 mm x 3.5 ꢀm, 100 Å, Waters) and a Symmetry C18 column (150 x 4.6 mm x
5 ꢀm, 100 Å, Waters), and the following solvents: H2O (0.045% (v/v) TFA) and MeCN
(0.036% (v/v) TFA), flow rate: 1 mL minꢀ1. Peptides showed purity ≥ 98%. High
resolution mass spectra were recorded on a LTQꢀFT Ultra (Thermo Scientific).
Reduction to Dihydropyridine
Under continuous nitrogen atmosphere and iceꢀcooling, each product was dissolved in
50% (v/v) aqueous methanol, and sodium dithionite (10 eq.) was then added together
with enough sodium bicarbonate to adjust the pH to 6.5ꢀ7.0. This mixture was then
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protected from light. The progress of the reaction was monitored by UV, MS and
HPLC. Once the reduction was complete (usually within 4 h), the reaction mixture was
extracted with cold, degassed DCM. The solvent was then removed with a stream of
nitrogen and each product was subjected to HPLC and MALDIꢀTOF (avoiding the use
of acid in the ACH matrix) and TLC.
Reduction to Dyhidropyridine for PAMPA Evaluation
Under continuous nitrogen atmosphere and iceꢀcooling, each product was dissolved in
PAMPA system solution 10mM in reduced glutathione (1mL), and then sodium
dithionite (10 eq.) was added together with enough sodium bicarbonate to adjust the pH
to 6.5ꢀ7.0. It was then protected from light. The progress of the reaction was monitored
by HPLC. Once the reduction was complete (usually within 4 h) the reaction mixture
was used for PAMPA.
PAMPA Assay
This assay was used to determine the capacity of compounds to cross the BBB by
passive diffusion. The effective permeability of the compounds was measured at an
initial concentration of 200, 100, 25 and 10 ꢀM depending on the experiment. The
buffer solution was prepared from a concentrated one, commercialized by pION, and
following the manufacturer’s instructions. pH was adjusted to 7.4 using a NaOH 0.5 M
solution. The compound of interest was dissolved in buffer solution or buffer with 10
mM reduced glutathione (in the case of Dhp versions) to the desired concentration (200,
100, 25 or 10 ꢀM). The PAMPA sandwich was separated and the donor well was filled
with 195 ꢀL of the compound solution of interest. The acceptor plate was placed into
the donor plate, ensuring that the underside of the membrane was in contact with buffer.
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4 ꢀL of the mixture of phospholipids (20 mg mLꢀ1) in dodecane was added to the filter
of each well and 200 ꢀL of the corresponding buffer solution was added to each
acceptor well. The plate was covered and incubated at room temperature in a saturated
humidity atmosphere for 4 h using a GUTBOX with a UWL of 25 ꢀm. After these 4 h,
150 ꢀL/well of the solution from the donor plate and 150 ꢀL/well from the acceptor
plate were transferred to HPLC vials and 100 ꢀL/per sample was injected into a HPLC
reverseꢀphase Symmetry C18 column (150 × 4.6 mm × 5 ꢀm, 100 Å, Waters). The
phospholipid mixture used was a porcine polar brain lipid extract. Composition:
phosphatidylcholine (PC) 12.6%, phosphatidylethanolamine (PE) 33.1%,
phosphatidylserine (PS) 18.5%, phosphatidylinositol (PI) 4.1%, phosphatidic acid 0.8%
and 30.9% of other compounds. The effective permeability after 4 h was calculated
using equation 1 and the percentage of transport using equation 2.


2⋅CA (t)
CD (t0 )
− 218.3
⋅10−6 cm/ s
[Equation 1]
[Equation 2]
P =
⋅ Log 1−
e



t

where t is time (h); CA(t) is the compound concentration in the acceptor well at time t
and CD(t0) is the compound concentration in the donor well at 0 h. Membrane retention
(%R) was calculated from the difference between the total starting amount and the
amounts in donor and acceptor compartments at the end of the experiment (t = 4 h).
Blood-Brain Barrier Cell-Based Assay
This experiment was performed using the BBBꢀRTU 24 well kit by Cellial
Technologies S.A. This kit contains the plates with and without cells as well as the
mediums and buffers required for the assay. The protocol for cell culture and transport
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assay followed is briefly summarized in the following lines.
The complete culture medium was defrosted the day of manipulation and preꢀheated
at 37ºC. As soon as the RTU plate was removed from ꢀ80ºC, BBB complete culture
medium (CCM) was poured in the lower compartment (24 mL) without separating the
plates and in each donor well (0.5 mL). The cells were resuspended in each insert
(donor well) and the plates were placed in a cell culture incubator (Binder BD 53) at 37
ºC, 5 % CO2 and 95 % humidity during 4h. After that time, the medium from all
compartments was removed gently using an aspiration system. The plate was washed
one time with BBB CCM and refilled with new BBB CCM (24 mL in the lower
compartment and 0.5 in each donor well). The plates were put into the cell culture
incubator and the cells were allowed to grow for three days. BBB properties induction
was achieved by incorporating BBB inducing medium (IM) on the third day. BBB IM
was thawed and heated at 37ºC. The BBB CCM was gently removed by aspiration and
all compartments were refilled with the volume they had. The plate was placed in a cell
culture incubator for 48h.
The day of the transport experiment, Ringer Hepes (RH) buffer was defrosted and
warmed up to 37ºC to avoid temperature stress when performing the experiment. The
stock solutions of test compounds were prepared in RH buffer in a 200
concentration containing Lucifer Yellow (LY) 20 M. Transport plates (24ꢀwell
Receiver Tray, Millipore ref. PSMW010R5) were filled by adding 800 L of warm RH
ꢀM
ꢀ
ꢀ
buffer and put into a cell culture incubator. The insert plate was removed from the
incubator and the filters were separated from the lower compartment of the plate. The
medium in both the insert and the lower plates was discarded and put upside down on
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paper towels for less than 30 seconds. The inserts and lower compartment were washed
with RH buffer, which was removed after less than 5 minutes. At time zero, the insert
plate was gently transferred in the 24ꢀwell plate containing RH buffer and 400 ꢀL of the
compound stock solution were applied to each insert. The plate was covered with its lid
and placed in the incubator. After 2 h the samples from both donor and acceptor wells
were recovered and frozen until HPLC analysis.
LY fluorescence was measured using a 96ꢀwell plate in a Fluoroskan Ascent
Microplate Fluoromoeter (Thermo Fisher Scientific). The samples were injected in an
HPLC, and the areas under the peaks were integrated and they were used to calculate
transport and to obtain Pe.
Pe= (dQ/dt)*(1/A)*(1/C0) (cm/s)
[Equation 3]
In addition, mass balances were also calculated to estimate the amount of peptide
that is degraded or trapped in the cells or membranes. Samples were evaluated in
triplicates.
POP Activity Assay
POP activity was determined as follows. For each reaction, activity buffer (131 ꢀl, 100
mM of Na/K phosphate buffer, pH 8.0) was preꢀincubated for 15 min at 37 °C, agitation
90 rpm with POP (7 nM) and the corresponding inhibitor stock solution (3 ꢀl). A 5 mM
stock solution of inhibitor was prepared in MeOH. After preꢀincubation, ZGPꢀAMC (10
ꢀl, 3 mM in 40 % of 1,4ꢀdioxane) was added, and the reaction was incubated for 1 h at
37 °C under agitation at 90 rpm. The reaction was stopped with sodium acetate (150 ꢀl,
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1M, pH 4) and the formation of AMC was measured fluorimetrically. The excitation
and emission wavelengths were 360/40 and 485/20 nm, respectively. A control with the
same amount of MeCN was performed.
DPP-IV Activity Assay
DPPꢀIV activity was determined as follows. For each reaction, activity buffer (131 ꢀl,
100 mM of Na/K phosphate buffer, pH 8.0) was preꢀincubated for 15 min at 37 °C,
agitation 90 rpm with DPPꢀIV (7 nM) and the corresponding inhibitor stock solution (3
ꢀl). A 5 mM stock solution of inhibitor was prepared in MeOH. After preꢀincubation,
HGPꢀAMC (10 ꢀl, 3 mM in 40 % of 1,4ꢀdioxane) was added, and the reaction was
incubated for 1 h at 37 °C with agitation at 90 rpm. The reaction was stopped with
sodium acetate (150 ꢀl, 1M, pH 4), at which point the formation of AMC was measured
fluorimetrically. The excitation and emission wavelengths were 360/40 and 485/20 nm,
respectively. A control with the same amount of MeCN was performed.
Human Serum Stability
The compound (200 ꢁM) was dissolved in 300 ꢁL of human serum from male AB
plasma and incubated at 37 °C on an orbital shaker (100 rpm). After the desired time,
300 ꢁL of MeCN was added to the solution to precipitate the serum. The sample was
then centrifuged (15 min, 10000 rpm) at 4 °C, and then 150 ꢁL of supernatant was taken
and analyzed by HPLC. The presence of the compound was demonstrated by MALDI.
The percentage of intact compound was calculated by dividing the peak area of
compound at a given time to that of the compound at time zero.
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Biopolymers: Peptide Science
RESULTS AND DISCUSSION
Designing a Selective POP Inhibitor with Effective CNS-Targeting and BBB
Transport, Scaffold Selection
From our family of DKPꢀbased BBBꢀshuttles,2 we chose DKP PheꢀNꢀMePhe as a
starting scaffold for the design of a CDS construct that is also a selective POP inhibitor.
To achieve brain targeting, we selected the dihydropyridine/trigonellyl targetor
moieties. Trigonellyl is formed in the brain after enzymatic oxidation of
dihydropyridine analogously to that of NAD(P)H to NAD(P)+. We addressed two CDS
scaffold options, namely to change one of the amino acids of the DKP ring (Scaffold A)
or to attach the targeting dihydropyridine moiety to a side chain (Scaffold B). In our
case, the dihydropyridine moiety does not need to be removed from the construct once
inside the CNS. In fact, it masks the active trigonellyl form, which is the one that shows
POP inhibitory activity. This Nꢀalkylpyridinium structure is also present in berberine, a
POP inhibitor isolated from Rhizoma Coptidis extract, used in traditional Chinese
medicine (Figure 2). 39
Solid-Phase Peptide Synthesis and POP Inhibition Evaluation of the Scaffold
The two CDS scaffolds (Scaffold A & B) were prepared (Figure 3) and evaluated
(Figure 4). Only the one maintaining the intact DKP ring and having the trigonellyl
moiety attached to the Phe side chain (Scaffold B) showed POP inhibitory activity. We
then proceeded with Scaffold B and the active compound was reduced to its
dyhidropyridine version and oxidized (Box) and reduced (Bred) forms were evaluated.
Only the former showed inhibited the enzyme (IC50: 101 ꢀM). On the basis of the UV
bands detected (263 and 363 nm), we determined that Bred is as a mixture of 1,4ꢀ and
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1,6ꢀdihydropyridines. These two structures are converted to the Nꢀalkylpyridinium form
by oxidation and it is precisely this conversion that triggers POP inhibitory activity.
The observation that the reduced form (Bred) is not a POP inhibitor is relevant because
it decreases the risks of side effects. Also it is important to highlight that Box presents a
better POP inhibition activity than berberine that has an IC50 of 145 ꢀM.
PAMPA BBB Transport Evaluation of the Scaffold
We evaluated the reduced (Bred) and oxidized (Box) CDS versions by PAMPA assay.
This assay consists of two compartments separated by a filter that can be coated with a
mixture of phospholipids thus mimicking the membrane of study. We chose a porcine
polar brain lipid extract to mimic the BBB. Also, the pH of the two compartments was
selected to mimic the pH of blood and brain. The agitation produced in the capillaries
caused by the passage of red blood cells is also mimicked by magnetic stirring of the
donor compartments. In all the experiments, propranolol and carbamazepine, two CNS
active drugs, were used as positive controls. Only the reduced version (Bred), with the
dihydropyridine targetor, crossed the BBB model, showing an effective permeability
(Pe) of 0.24ꢂ10ꢀ6 cm sꢀ1. This value can be classified as medium permeability.46 On the
basis of these results, we propose that the compound Bred will reach the brain and after
oxidation Box will be locked inside the CNS. In summary, up to this point, we have a
construct that crosses the BBB but that is not yet active as POP inhibitor. However,
once oxidized, this construct becomes active against POP and remains locked in the
brain (Figure 5).
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Biopolymers: Peptide Science
Preparation and Evaluation of Inhibitor Library
On the basis of these initial results, we prepared a library of 14 compounds (Scheme 1),
modifying the size, flexibility, as well as the amino acid configuration in the DKP ring
of the Scaffold B (See supporting information). The selection of amino acids was based
on those that show the best performance as BBBꢀshuttles in our conditions.2
Our workingꢀpipeline is shown in Scheme 2. After the syntheses, the POP inhibitory
activity of the compounds was first evaluated (Figure 6). Four were selected as they
showed high POP inhibition at a concentration of 100 ꢁM, having better activity than
berberine (IC50: 145 ꢀM). In some cases, the reduced forms (Dhp) of the selected
products showed improved PAMPA transport (Figure 7). The dyhidropyridine
constructs showed excellent transport across the BBB (high permeability region),
comparable with compounds known to cross the BBB in vivo such as propranolol and
carbamazepine, while their trigonellyl versions continued to show poor transport and
berberine show no transport in the PAMPA assay (Pe <10ꢀ9 cm/s). These findings
support the feasibility of achieving a POP inhibitor that is locked inside the CNS. Of the
entire library, LꢀNꢀMeCha and LꢀNꢀMe2Nal were the most promising candidates.
To confirm the passive transport and the effect of initial concentrations in the transport
rates, the best two candidates LꢀNꢀMeCha and LꢀNꢀMe2Nal were evaluated by PAMPA
at different initial concentrations (200, 100, 25, 10 ꢀM). The results can be found in
figure 8 and confirms the passive transport of the candidate compounds, as well as,
confirms the absence of transport in the case of berberine and the membrane retention in
the case of Dhp of LꢀNꢀMeCha.
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Based on the results shown in figures 7 and 8, the best pairs of oxidized and reduced
versions were selected as the best candidates to be further developed. We select the
promising pairs where the reduced version (Dhp) has a Pe classified as high
permeability while their corresponding oxidized forms (Trg) are in the medium or low
permeability region. With this criterion in our hands, LꢀNꢀMeCha and LꢀNꢀMe2Nal
were selected to move on and be further developed.
To study their selectivity, dipeptidyl peptidaseꢀIV (DPPꢀIV), which belongs to the same
family as POP (DPPꢀIV; EC 3.4.14.5), was selected. DPPꢀIV, an integral membrane
serine protease, hydrolyzes peptides up to about 80 residues long that contain proline at
the second position.47 The active form of DPPꢀIV is a homodimer, the Nꢀterminus of
each subunit is located in the cytosol and after a transmembrane segment the rest of the
dimer is extracellular.48,49 Therapeutically, DPPꢀIV is a key target for the treatment of
type 2 diabetes mellitus (T2DM) since it inactivates glucagonꢀlike peptideꢀ1 (GLPꢀ
1).50,51
Our evaluation confirmed that the oxidized versions of the most promising candidates
(Trg) are selective POP inhibitors that do not inhibit DPPꢀIV (Figure 9). This is an
interesting result as berberine, the POP inhibitor that presented a trigonellyl moiety in
its structure, has been described to have DPPꢀIV inhibitory properties.52 Our best
candidates show a 12 and <5 % of DPPꢀIV inhibition whether berberine at the same 500
ꢀM concentration shows 80% of inhibition activity. Finally, the stability of compounds
was checked in human serum and they were found to be stable for periods longer than
24h.
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Biopolymers: Peptide Science
In addition to PAMPA assay, the capacity of LꢀNꢀMe2Nal and LꢀNꢀMeCha in the
reduced and oxidized forms to cross the bloodꢀbrain barrier was also checked by BBB
permeability experiments using an in vitro cellular model of the bloodꢀbrain barrier.53
This model is one of the most suitable approaches to mimic the bloodꢀbrain barrier and
it has been used previously, 53,54 showing a good correlation with in vivo brain perfusion
(Figure 10). The idea is to achieve conditions that most closely reproduce the cellular
organization of the bloodꢀbrain barrier. Endothelial cells are seeded in the upper
compartment (apical) of the system, thereby mimicking the luminal side (blood stream)
of a brain capillary while astrocytes are seeded on the other side of the filter, thus
helping endothelial cells to become specialized, polarized and functional. Alternatively
astrocytes conditioned medium could be used in the basal compartment instead of
astrocytes. This lower compartment (basal) mimics the abluminal side of a brain
capillary (brain tissue). Experiments are done by loading a solution of the compound to
be tested in the upper compartment while the lower compartment is loaded with buffer
only. The model is incubated for 2 h with the sample and afterwards the content of each
compartment is analyzed and quantified by HPLC.
During the assay the samples were coꢀincubated with Lucifer Yellow (LY), which
was used as an integrity marker of the barrier. When effective permeability (Pe) for LY
is equal to or higher than 1.7ꢂ10ꢀ5 cm/s, it is considered that there is breakage of the cell
barrier,55 and in this situation results cannot be considered valid.
Candidates LꢀNꢀMeCha and LꢀNꢀMe2Nal were evaluated at a concentration of 200
ꢀM. Dhp forms crossed the cellular layer of the model with Pe values of 2.25 and
2.6ꢂ10ꢀ5 cm/s respectively for LꢀNꢀMeCha and LꢀNꢀMe2Nal. It has been observed that
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compounds with a Pe higher than 1ꢂ10ꢀ6 cm/s show satisfactory permeability across the
bloodꢀbrain barrier in vivo.56
CONCLUSIONS
We have opened the way to a novel family of enzyme inhibitors which, when combined
with the CDS, are inactive until they are oxidized to the active form. We thus designed
two CDS constructs (DKP Phe(pꢀNHꢀDhp)ꢀLꢀNꢀMeCha and DKP Phe(pꢀNHꢀDhp)ꢀLꢀ
NꢀMe2Nal) with the capacity to cross the BBB model (effective permeability 1.75ꢂ10ꢀ6
and 2.31ꢂ10ꢀ6 cm sꢀ1 respectively) and once inside the CNS to transform and thus
become locked inside this system and then act as selective POP inhibitors (IC50: 30.7
and 80.3 ꢀM respectively). We have achieved an effective CNSꢀtargeting and BBB
transport of a selective POP inhibitor.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
Acknowledgements
This work was supported by MCIꢀFEDER (Bio2008ꢀ00799), the MARATÓ TV3, and the
Generalitat de Catalunya (XRB and 2009SGRꢀ1005).
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Biopolymers: Peptide Science
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Biopolymers: Peptide Science
Figure and schemes:
FIGURE 1 Dihydropyridine/trigonellylꢀbased chemical delivery strategy.
FIGURE 2 Structure of berberine, a POP inhibitor found in Rhizoma Coptidis
extracts, IC50: 145 ± 19 ꢀM.
FIGURE 3 Two chemical delivery system options: TrgꢀNꢀMePhe (a) and Phe(pꢀNHꢀ
CoꢀTrg)ꢀNꢀMePhe (b). HPLC chromatograms 0ꢀ100% MeCN in 15 min RPꢀHPLC
Symmetry C18 column (150 x 4.6 mm x 5 ꢀm, 100 Å, Waters), solvents: H2O (0.045%
TFA) and MeCN (0.036% TFA), flow: 1 mL/min. Confirmed by HPLCꢀMS and
MALDI.
FIGURE 4 Doseꢀresponse plot of POP inhibition for compound DKP Phe(pꢀNHꢀTrg)ꢀ
LꢀNMePhe (Option B) IC50: 101.2 ꢀM.
FIGURE 5 Properties of the chemical delivery systems in the oxidized and reduced
forms.
FIGURE 6 Comparison of POP inhibitory potency for the compound library. All
compounds were tested at 100 ꢀM.
FIGURE 7 PAMPA results for the selected candidates in the trigonellyl and
dihydropyridine forms at 200 ꢀM initial concentration. n.d. non detectable.
FIGURE 8 PAMPA results at different concentrations for the best two candidates ( Lꢀ
NꢀMeCha and LꢀNꢀMe2Nal) in the trigonellyl and dihydropyridine forms and berberine.
n.d. non detectable
FIGURE 9 Doseꢀresponse plots for compound DKP Phe(pꢀNHꢀTrg)ꢀLꢀNMeCha (a)
and compound DKP Phe(pꢀNHꢀTrg)ꢀLꢀNMe2Nal (b).
FIGURE 10 Correlation between bloodꢀbrain barrier cellꢀbased assay and in vivo brain
perfusion
SCHEME 1 Solidꢀphase synthesis of the POP inhibitor library.
SCHEME 2 Workingꢀpipeline approach followed to identify an effective selective POP
inhibitor with central nervous systemꢀtargeting and bloodꢀbrain barrier transport.
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TOC:
Text for the TOC:
Less than 2% of all potential neurotherapeutics cross the bloodꢀbrain barrier (BBB).
Here we sought to build a construct with the capacity to cross this barrier, to behave as a
chemical delivery system, and, once inside the central nervous system (CNS), to be
transformed and then act as an enzyme inhibitor.
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FIGURE 1 Dihydropyridine/trigonellyl-based chemical delivery strategy.
231x233mm (300 x 300 DPI)
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