Oxazepam-Dopamine Conjugates Increase Dopamine Delivery into Striatum of Intact Rats

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00405

The neurotransmitter dopamine (DA) was covalently linked to oxazepam (OXA), a well-known positive allosteric modulator of γ-aminobutyric acid type-A (GABA_A) receptor, through a carbamate linkage (4) or a succinic spacer (6). These conjugates were synthesized with the aim of improving the delivery of DA into the brain and enhancing GABAergic transmission, which may be useful for the long-term treatment of Parkinson disease (PD). Structure-based permeability properties, in vitro stability, and blood-brain barrier (BBB) permeability studies led to identify the OXA-DA carbamate conjugate 4a as the compound better combining sufficient stability and ability to cross BBB. Finally, in vivo microdialysis experiments in freely moving rats demonstrated that 4a (20 mg/kg, i.p.) significantly increases extracellular DA levels into striatum, with a peak (more than 15-fold increase over the baseline) at about 80 min after a single administration. The stability and delivery data proved that 4a may be a promising candidate for further pharmacological studies in animal models of PD.

Full text of DOI:10.1021/acs.molpharmaceut.7b00405

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Article
Oxazepam-dopamine conjugates increase
dopamine delivery into striatum of intact rats
Tommaso Cassano, Antonio Lopalco, Modesto de Candia, Valentino Laquintana,
Angela Lopedota, Annalisa Cutrignelli, Mara Perrone, Rosa M. Iacobazzi,
Gaurav Bedse, Massimo Franco, Nunzio Denora, and Cosimo D. Altomare
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00405 • Publication Date (Web): 05 Aug 2017
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Oxazepam-dopamine
conjugates
increase
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dopamine delivery into striatum of intact rats
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Tommaso Cassano,a,# Antonio Lopalco,b,#,* Modesto de Candia,b,# Valentino
Laquintana,b Angela Lopedota,b Annalisa Cutrignelli,b Mara Perrone,b Rosa M.
Iacobazzi,c Gaurav Bedse,d,e Massimo Franco,b Nunzio Denora,b,* Cosimo D. Altomare.b
a
Department of Clinical and Experimental Medicine, University of Foggia, Foggia
71100, Italy
b Department of Pharmacy ꢀ Drug Sciences, University of Bari Aldo Moro, Bari 70125,
Italy
c Istituto tumori IRCCS "Giovanni Paolo II", Flacco, St. 65, 70124 Bari, Italy.
d Department of Physiology and Pharmacology "V. Erspamer," Sapienza University of
Rome, 00185 Rome, Italy
e Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN 37232,
USA
*Corresponding authors. Phone: +39 080ꢀ544ꢀ2767. Eꢀmail: nunzio.denora@uniba.it
(N.D.). Phone: +39 080ꢀ544ꢀ2767. Eꢀmail: antonio.lopalco@uniba.it (A.L.).
#These authors have equally contributed to this work.
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ABSTRACT
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The neurotransmitter dopamine (DA) was covalently linked to oxazepam (OXA), a wellꢀ
known positive allosteric modulator of γꢀaminobutyric acid typeꢀA (GABAA) receptor,
through a carbamate linkage (4) or a succinic spacer (6). These conjugates were
synthesized with the aim of improving the delivery of DA into the brain and enhancing
GABAergic transmission, which may be useful for the longꢀterm treatment of Parkinson
disease (PD). Structureꢀbased permeability properties, in vitro stability and bloodꢀbrain
barrier (BBB) permeability studies led to identify the OXAꢀDA carbamate conjugate 4a
as the compound better combining sufficient stability and ability to cross BBB. Finally,
in vivo microdialysis experiments in freelyꢀmoving rats demonstrated that 4a (20 mg/kg,
i.p.) significantly increases extracellular DA levels into striatum, with a peak (more than
15ꢀfold increase over the baseline) at about 80 min after a single administration. The
stability and delivery data proved that 4a may be a promising candidate for further
pharmacological studies in animal models of PD.
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Keywords:
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Blood Brain Barrier
Prodrugs
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Codrugs
Dopamine
GABA transmission
Parkinson disease.
Abbreviations: ANOVA, analysis of variance; BBB, bloodꢀbrain barrier; CDI, 1,1ꢀ
carbonyldiimidazole; CNS, central nervous system; DA, dopamine; DMEM, Dulbecco’s
modified eagle’s medium – high glucose; DMSO, dimethylsulfoxide; EDC, Nꢀ(3ꢀ
Dimethylaminopropyl)ꢀN’ꢀethylcarbodiimide
hydrochloride;
EDTA,
Ethylenediaminetetraacetic acid; ESI, electrospray ionization mass spectrometry; FD4,
Fluorescein isothiocyanateꢀdextran; FTꢀIR, Fourier transform infrared spectroscopy;
GABA, γꢀaminobutyric acid; 1HꢀNMR, Proton nuclear magnetic resonance; HPLC, highꢀ
performance liquid chromatography; i.p., intraperitoneal; LAT1, neutral aminoꢀacid
transporter 1; LC–MS, LC–mass spectrometry; LD, LꢀDopa; MDCKIIꢀMDR1, Madin–
Darby canine kidney cells retrovirally transfected with the human MDR1 cDNA; PD,
Parkinson’s disease; S.E.M., Standard Error of Mean; tPSA, topological Polar surface
area; TEA, triethylamine; TEER, transꢀepithelial electrical resistance; TFA,
Trifluoroacetic acid; THF, tetrahydrofurane; TLC, thin layer chromatography.
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1. Introduction
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The neurotransmitter dopamine (DA), like many other CNS active molecules, does not
cross the bloodꢀbrain barrier (BBB) to enter the central nervous system (CNS), because
of its hydrophilicity and the absence of endogenous transport systems through the brain
capillary endothelium. Only small molecules with moderate lipophilicity and low
molecular mass are able to cross the BBB via passive transport, if not substrates for the
ATPꢀdependent drug efflux pumps.1ꢀ3 Various approaches have been pursued to increase
the brain delivery of therapeutic small molecules4ꢀ6, including the development of
prodrugs.7,8 It has been well established that a CNSꢀtargeted prodrug or codrug should
possess either optimal physicochemical properties (e.g., lipophilicity), which allow
passive diffusion through the BBB via the transcellular route, or structural features to
serve as a substrate for one of the endogenous influx transport systems of the BBB.9
In this context, LꢀDopa (LD), the most effective therapy for the treatment of Parkinson
disease (PD)10, is considered a prodrug of DA. LD crosses the BBB through the type 1
large neutral aminoꢀacid transporter (LAT1) and, once in the brain, is rapidly converted
to DA by aromatic aminoꢀacid decarboxylase.11 Although LD improves PD symptoms in
the initial stages of the disease, clinical and preclinical studies have demonstrated that its
longꢀterm use is limited by the development of severe side effects, including abnormal
involuntary movements (dyskinesia) and psychiatric complications.12,13
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In PD an altered activity of the GABAergic neurotransmission and a dominance
of the excitatory transmission in the basal ganglia and substantia nigra, may determine
the loss of dopaminergic neurons in the pars compacta of the substantia nigra.14 To
overcome this drawback, it has been suggested that drugs enhancing γꢀaminobutyric acid
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(GABA) transmission could be a useful therapy for PD.15 Indeed, it has been shown that
GABAA receptors contribute to the modulation of the activity of mesocortical and
mesolimbic dopaminergic neurons and to the neuroprotection of dopaminergic cells.16 To
this regard, selective GABAergic agonists, such as zolpidem, that acts within the basal
ganglia, and muscimol, that decreases the output of the basal ganglia to thalamus, could
be beneficial in patients who experience complications associated with longꢀterm LD or
dopaminomimetic treatments15,16, and may also potentially be a useful treatment to
modulate GABAergic tone in the basal ganglia.17
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Recently, we have studied novel codrugs with GABAergic activity for DA delivery in the
brain.18 In this study, we investigated new conjugates obtained by linking DA and
oxazepam (OXA), a wellꢀknown benzodiazepine GABAꢀagonist.19 OXA (1, Fig. 1) and
DA (2a), and the respective 3,4ꢀOꢀdiacetyl ester derivative (2b), were covalently linked
through a carbamoyl group (4a,b; Scheme 1). In addition, one LD carbamate ester (4c)
and two amide derivatives (6a,b; Scheme 2) of the OXA hemisuccinate 5 were
synthesized and tested for their hydrolytic stability. These molecules were synthesized
taking advantage of two features concerning benzodiazepine compounds. The first one
relates to the lipophilic nature of the benzodiazepine moiety that could serve as carrier for
DA (or LD derivatives) leading to increased brain levels. In this case, the benzodiazepine
itself is the lipophilic proꢀmoiety of the hydrophilic neurotransmitter DA or LD ethyl
ester. The second one is the high affinity for the GABAꢀbenzodiazepine receptor
complex shown by OXA 1 and its hemisuccinate 5. Synthesis, hydrolytic stability,
assessment of permeabilityꢀrelated properties, including in vitro transport assays for
estimating BBB penetration, and in vivo rat brain microdialysis are herein reported. The
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striatum, which is rich in dopaminergic nerve terminals, is the main target region for
symptomatic treatment of PD. Therefore, we have evaluated the pharmacological effects
of oxazepamꢀDA conjugates in the striatum of intact rats.
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2. Materials and methods
2.1 Synthesis
Melting points were determined in open capillary tubes with a Buchi apparatus and are
uncorrected. Final compound purities were assessed by elemental analyses (C, H, N),
performed on Euro EA3000 analyzer (Eurovector, Milan, Italy) by the Analytical
Laboratory Service of the Department of Pharmacy−Drug Sciences of the University of
Bari (Italy), and the results agreed to within ± 0.40% of theoretical values. IR spectra
were recorded on a PerkinꢀElmer Spectrum One Fourier transform infrared
spectrophotometer (PerkinꢀElmer Ltd., Buckinghamshire, U.K.), and the most significant
1
absorption bands are listed. H NMR spectra were recorded at 300 MHz on a Varian
Mercury 300 instrument. Chemical shifts are expressed in δ. The following abbreviations
are used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad signal. Signals due to NH
and OH protons were located by deuterium exchange with D2O.20 Mass spectra were
obtained by Agilent 1100 Series LCꢀMSD Trap System VL, equipped with ESI
(electrospray ionization) source (Agilent Technologies Italia S.p.A., Cernusco sul
Naviglio, Milan, Italy). Chromatographic separations were performed on silica gel 60 for
column chromatography (Merck 70−230 mesh). All the reactions were carried out under
a nitrogen atmosphere and the progress of the reaction was monitored by thinꢀlayer
chromatography (TLC) by using Kieselgel 60 F254 (Merck) plates. Unless otherwise
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stated, starting materials and all chemicals and solvents were purchased from Sigmaꢀ
Aldrich (Milan, Italy).
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Dopamine 3,4ꢀOꢀdiacetyl ester derivatives (2b), LꢀDopa ethyl ester hydrochloride (2c)
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and (± ;
) oxazepam hemisuccinate (5) were prepared according to known procedures21,22
their melting points and spectral data were in full agreement with those reported in
literature. No effort was made at this stage to optimize the synthesis yields.
2.1.1. Synthesis of compounds 4a-c
To a stirred solution of racꢀoxazepam 1 (0.35 mmol) in anhydrous THF (10 ml), TEA
(0.42 mmol) and bisꢀ(4ꢀnitrophenyl) carbonate (0.7 mmol) were added. The reaction
mixture was stirred at room temperature under N2 atmosphere for 3 h. The progress of the
reaction was monitored by thin layer chromatography (TLC) using petroleum ether/ethyl
acetate 1:1 (v/v) as the eluent. Subsequently, a solution of 2a-c in dry DMF was added,
and stirring was prolonged for 12ꢀ24 h. The progress of the reaction was monitored by
TLC using petroleum ether/ethyl acetate 2:8 (v/v) as the eluent. The solvent was then
removed under reduced pressure and the residue was purified by flash silica gel column
chromatography using petroleum ether/ethyl acetate 1:1 (v/v) as the eluent to yield the
desired conjugates 4a-c.
4a: 45% yield; mp 83ꢀ85 °C; IR (KBr): 3367, 1699 cmꢀ1; 1H NMR (DMSOꢀd6) δ: 2.5 (m,
2 H, CH2Ar), 3.1 (m, 2 H, NHCH2), 5.67 (s, 1 H, CH), 6.4ꢀ6.7 (m, 3 H, Ar), 7.2ꢀ7.35 (m,
2 H, Ar), 7.4ꢀ7.55 (m, 4 H, Ar), 7.69 (m, 1 H, Ar), 7.8 (br, 1 H, NH), 7.93 (s, 1 H, Ar);
MS ESI+ m/z: 488.0 [M+Na]+; MS ESIꢀ m/z: 463.8 [MꢀH]ꢀ.
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4b: 10% yield; mp 133ꢀ134 °C; IR (KBr): 1770, 1720 cmꢀ1; 1H NMR (CDCl3) δ: 2.2 (s, 6
H, CH3), 2.85 (t, 2 H, CH2Ar), 3.46 (m, 2 H, CH2), 5.95 (s, 1 H, CH), 6.4ꢀ6.7 (m, 3 H,
Ar), 7.4ꢀ7.8 (m, 8 H, Ar), 9.3 (br, 1 H, NH); MS ESI+ m/z: 571.9 [M+Na]+; MS ESIꢀ m/z:
547.7 [MꢀH]ꢀ.
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4c: 14% yield; 146ꢀ149 °C; IR (KBr): 3300, 1700 cmꢀ1; 1H NMR (CDCl3) δ: 1.05 (t, 3 H,
CH3), 3.2 (m, 2 H, CH2), 4.1 (m, 2 H, CH2), 4.2 (m, 1 H, CH), 6.0 (s, 1 H, COCHN), 6.6ꢀ
7.2 (m, 3 H, Ar), 7.18ꢀ7.75 (m, 6 H, CHAr), 8.63 (m, 2 H, CH Ar), 9.8 (br, 1 H, NH). MS
ESI+ m/z: 560.0 [M+Na]+; MS ESIꢀ m/z: 535.8 [MꢀH]ꢀ.
2.1.2. Synthesis of compound 6a
A solution of oxazepam hemisuccinate 5 (0.26 mmol) and CDI (0.39 mmol) in anhydrous
DMF (10 ml) was stirred at room temperature under N2 atmosphere for 15 min. Then 2a
(0.39 mmol) was added, and stirring was prolonged for 12 h. The reaction was monitored
by TLC using petroleum ether/ethyl acetate 2:8 (v/v) as the eluent. The solvent was
removed under reduced pressure and the residue purified by flash silica gel column
chromatography using petroleum ether/ethyl acetate 2:8 (v/v) as the eluent to yield 6a.
6a: 12% yield; mp 99ꢀ102 °C; IR (KBr): 3400 cmꢀ1, 1700 cmꢀ1;1H NMR (DMSO d6) δ:
2.9 (m, 4 H, OCOCH2), 3.1 (m, 2 H, CH2), 4.1 (m, 2 H, CH2OCO), 5.95 (s, 1 H, NꢀCHꢀ
CO), 6.6ꢀ7.2 (m, 3 H, CH Ar.), 7.1ꢀ7.75 (m, 8 H, CH Ar.); MS ESI+ m/z: 544.0 [M+Na]+;
MS ESIꢀ m/z: 520.0 [MꢀH]ꢀ.
2.1.3. Synthesis of compound 6b
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EDC (0.62 mmol) was added to a stirred and cooled (on ice bath) solution of 5 (0.52
mmol) in 20 ml of CH2Cl2. After 30 min, a solution of 2b in 5 ml of CH2Cl2 and then
TEA (0.78 mmol) were added dropwise. Stirring was continued for 4 h at room
temperature and the progress of the reaction was monitored by TLC using petroleum
ether/ethyl acetate 2:8 (v/v) as the eluent. The solvent was then removed under reduced
pressure to yield compound 6b.
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6b: 32% yield; mp 120ꢀ123 °C; IR (KBr): 3380 cmꢀ1, 1760 cmꢀ1, 1740 cmꢀ1, 1700 cmꢀ1,
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1650 cmꢀ1; H NMR (CDCl3) δ: 2.0 (s, 6 H, OCOꢀCH3), 2.5ꢀ3.0 (m, 6 H, CH2), 5.95 (s,
1H, CH), 6.6 (t, 1 H, NH), 7.0ꢀ7.6 (m, 11 H, CH Ar); MS ESIꢀ m/z: 603.8 [MꢀH]ꢀ.
2.2. Stability studies
2.2.1. Chemical hydrolysis
The chemical hydrolysis of each test compound was monitored at pH 7.4 in 0.05 M
phosphate buffer (PBS) at 37 ± 0.5 °C using a shaking water bath. Reactions were
initiated by adding 50 µl of a DMSO stock solution to 5 ml of the preheated buffer
solution. The resulting final concentration was 50 µM. The test solutions were vortexed
and kept in a shaking water bath at constant temperature (37 ± 0.5 °C). Pseudoꢀfirstꢀorder
rate constants (kobs) for the hydrolysis of the compounds were then calculated from the
slopes of the linear plots of log (% residual compound) against time.23,24 The % residual
compound was measured by HPLC at several time intervals. The experiments were run in
triplicate.
2.2.2. Serum hydrolaseꢀcatalyzed hydrolysis
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Fetal bovine serum (FBS) was diluted with 0.05 M PBS (pH 7.4, 0.14 M NaCl) to 50%
v/v, and incubation of each test compound was performed at 37 ± 0.5 °C using a shaking
water bath. The reaction was initiated by adding 10 µl of a stock solution of the test
compound in DMSO (10 mM) to 1.6 ml of the preheated FBS solution. Aliquots (100 µl)
were withdrawn at various times and deproteinized by mixing with 500 µl of cold
acetonitrile (MeCN). After centrifugation for 10 min at 3500 rcf, 10 µl of the supernatant
was analyzed by HPLC. Pseudoꢀfirstꢀorder rate constants for the disappearance of the test
compounds were determined from the slope of the linear plots of the log (% residual
compound) against time. Degradation products of the test compounds in FBS were
analyzed by direct injection of an aliquot of deproteinized supernatant (further diluted
1:10 with blank) into an ESI mass spectrometer (both positive and negative mode).
The stability of 4a and 4c was also assessed in 100% rat and human serum (lyophilized
and reconstituted with 4 ml of deionized water). The reaction was initiated by adding 10
µl of a DMSO stock solution of the test compound (10 mM) to 0.990 ml of preheated
serum, and incubation was performed at 37 ± 0.5 °C using a shaking water bath. At
appropriate time intervals, aliquots (200 µl) were withdrawn and deproteinized by mixing
with 0.800 ml of iceꢀcold MeCN. After centrifugation for 5 min at 3500 rcf, the
supernatant was filtered (0.2 ꢀm PTFE membrane) and analyzed by HPLC. The
percentage of the residual compound was measured by monitoring the peak area of the
chromatogram (external standard).
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2.2.3. HPLC analyses
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The HPLC analyses for monitoring the disappearance of the starting test compounds were
carried out at temperature of 25 ± 0.2 °C and UV detection at 254 nm wavelength, using
an Agilent 1260 infinity HPLC system equipped with a diode array detector (Agilent
Technologies Italia). The analyses were performed on a Phenomenex Kinetex C18
column (150 × 4.6 mm i.d.; 5 µm particles), using MeOH and 10 mM ammonium
formate solution (pH 5.0) mixed in different fraction composition (typically, 60:40 v/v,
respectively) depending upon the retention of the analyte (injection volume: 10 µl; flow
rate: 1 ml/min). Calibration curves were obtained by measuring peak areas for each test
compound at known concentrations.
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The stability in rat serum of the enantiomers of racemic 4a has been monitored using as
the chiral stationary phase a Chirobiotic TAG steel column (250×4.6 mm i.d., 5 µm
particles), equipped with the teicoplanin aglycone bonded to silica gel (Astec, Whippany,
NY, USA). The 4a enantiomers were eluted in polar mode using MeOH as the mobile
phase (injection volume: 2 ꢁl; flow rate 0.3 ml/min).
2.3. Lipophilicity and bloodꢀbrain barrier permeability descriptors
Lipophilicity was assessed by calculated log P (octanol/water partition coefficient) using
ClogP software (v. 4, BioByte Corp.). Polar surface area, that is the surface area in Å2
occupied by nitrogen and oxygen atoms, and the connected polar hydrogens, was
estimated using a simple protocol proposed by Ertl et al. for calculating the soꢀcalled
topological PSA (tPSA).25 The likelihood that a compound penetrates the bloodꢀbrain
barrier (BBB) was estimated by calculating log(Cbrain/Cblood), expressed as logBB value,
according to Clark’s equation26:
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LogBB = ꢀ0.0148 (±0.001) PSA + 0.152 (±0.036) ClogP + 0.139 (±0.073)
(1)
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Cell cultures were grown under controlled conditions as previously described by Denora
et al.21 The bloodꢀbrain transport of compounds was investigated using MDCKIIꢀMDR1
cell monolayers (passage 8ꢀ11). Cells were seeded at a density of 100,000 cell/cm2 onto
polyester Transwell inserts (pore size 0.4 µm, diameter 6.5 mm, apical volume 0.5 ml,
basolateral volume 1.5 ml). MDCKIIꢀMDR1 cell barrier function was verified prior to
the transport experiments by means of transꢀepithelial electrical resistance (TEER) using
an EVOM apparatus and the measurement of the flux of FD4 and diazepam as
paracellular and transcellular markers of cell monolayers integrity. The TEER was
measured in growth media (DMEM) at room temperature and calculated as the measured
resistance minus the resistance of an empty Transwell (blank without cells). Cell
monolayers with TEER values above 200 Ohm cm2 were used. Following the TEER
measurements, the cells were equilibrated in transport medium in both the apical and
basolateral chambers for 30 minutes at 37 °C. At time 0, a solution of compounds diluted
in the transport medium was added to the apical chamber. The transport experiments
were carried out under cell culture conditions (37 °C, 5% CO2, 95% humidity) with
constant stirring (70 rpm). At 15 min, 30 min, 45 min, 1 h, 1 h and 30 min, 2 h, 3 h, 200
µl samples were removed from the basolateral chamber and replaced with 200 µl of fresh
transport medium. At the final time point, a 200 µl sample was also removed from the
apical chamber. The transport of compounds was investigated at a concentration of 50
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µM. The apparent permeability coefficient (Papp) was determined according to the
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following equation:
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V ∙ dC
P
ꢀꢁꢁ
=
(2)
A ∙ Cꢂ ∙ dt
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where A is the filter/cell surface area, C0 is the initial concentration of the test compound
4a, and V(dC/dt) is the linear appearance rate of mass in the receiver solution.
Concentrations of 4a, FD4, diazepam and dopamine were assessed by HPLC.
The experiments were performed in triplicate and results are expressed as means ± SEM.
Statistical analysis was carried out using Student’s tꢀtest between two mean values.
2.5. Brain microdialysis
2.5.1. Animals
Animal care and all experiments were conducted in accordance with the guidelines of the
European Communities Council Directive of 24 November 1986 (86/609/EEC) and the
Italian Department of Health (D.L. 116/92), and approved by the Institutional Animal
Care and Use Committees of the University of Foggia, Italy. All efforts were made to
minimize animal suffering and to reduce the number of animals used in the study. Male
Wistar rats (225ꢀ250 g; Harlan, San Pietro al Natisone, Udine, Italy) were housed on 12ꢀh
darkꢀlight cycle, at 22 ± 1° C with food and water available ad libitum and habituated to
housing conditions for 1 week before the experiments.
2.5.2. Microdialysis and treatment schedule
In vivo microdialysis experiments were carried out as previously described.18,27 Briefly,
animals were anesthetized with equithesin (3 ml/kg, i.p.) and a customꢀmade
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microdialysis probe of concentric design (AN69 Hospal S.p.A; 20 kDa cutꢀoff;
membrane length, 3ꢀmm) was implanted stereotaxically into the striatum according to the
following stereotaxic coordinates: AP +1.0, ML ±2.8 from bregma and DV ꢀ6.8 from
dura.28 The membranes were tested for in vitro recovery of DA on the day before surgery
to verify that recoveries were within a desired range (∼ 30 % of DA). Twentyꢀfour hours
after surgery, the microdialysis probe was perfused with Krebs Ringer solution (NaCl
145 mmol/l, KCl 2.7 mmol/l, CaCl2 2H2O 1.2 mmol/l, MgCl2 6H2O 1 mmol/l, Na2HPO4
2 mmol/l, pH 7.4) at a constant flow rate of 2 ꢁl/min. Perfusates were collected every 20
min into miniꢀvials. After a washꢀout period of 2 hours, 4 samples were collected to
determine the baseline levels of the DA (no more than 10% difference among 4
consecutive samples).
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Compound 4a was freshly dissolved in saline/Tween 80/PEG (90/5/5) and administered
i.p. in a volume of 3 ml/kg. Rats were acutely treated with 4a (10 and 20 mg/kg, i.p.) or
vehicle and consecutive microdialysate samples were collected every 20 min over a 4 h
period.
A second set of animals was treated with a single injection of LD + the inhibitor of
aromatic aminoacid decarboxylase, benserazide (8.5 mg/kg and 12.5 mg/kg i.p.,
respectively) or vehicle (saline), and dialysates were collected over a 2ꢀh period. DA
concentration, obtained from the same samples, were detected and quantified by HPLC.
At the end of each experiment, the correct placement of dialysis probes was verified
histologically.
2.5.3. HPLC Analyses
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The extracellular level of DA was determined by HPLC using a ODS Hypersil column
(150×3 mm i.d.; 5 µm particles) (Thermo Fisher Scientific, Milan, Italy) with an Unijet
cell (BAS, Bioanalytical Systems, Kenilworth Warwickshire, United Kingdom) equipped
with a 6ꢀmmꢀdiameter glassy carbon electrode (set at + 650 mV) and connected to an
electrochemical amperometric detector (INTRO, Antec Leyden, The Netherlands), as
previously described.27,29 The flow rate of the mobile phase (85 mM sodium acetate, 0.34
mM EDTA, 15 mM sodium chloride, 0.81 mM octanesulphonic acid sodium salt, 5%
methanol (v/v), pH 4.85) was 0.7 ml/min and the total runtime 15 min.
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2.5.4. Statistical analysis
Neurochemical data were expressed as percentages of baseline (± S.E.M.), which was
defined as the average of four consecutive samples with stable level of neurotransmitters
before drug administration. Data were analyzed by twoꢀway analysis of variance
(ANOVA) for repeated measures with treatment (tr) as the between variable and time (t)
as the within variable. Violations of the sphericity assumption were corrected using the
GreenhouseꢀGeisser epsilon correction to adjust the degrees of freedom for each test.
Postꢀhoc test was made by Bonferroni’s multiple comparison test. The overall effect of
drug treatments on the striatal DA outputs was estimated by oneꢀway ANOVA and by
comparing the areas under the curve (AUC) by the unꢀpaired Student’s tꢀtest. The AUC
was calculated using the standard trapezoid method30 using neurotransmitter levels over a
time window of 0–240 min for each experimental group. The threshold for statistical
significance was set at P < 0.05.
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3. Results and discussion
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Based on previous findings18,21, in this study the neurotransmitter DA and its 3,4ꢀOꢀ
diacetyl ester derivative were coupled with OXA 1 (Fig. 1) through carbamate linkage
(4a and 4b) and succinic spacer (6a and 6b).
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Fig. 1. Structures of racꢀoxazepam (1), HCl salts of dopamine (2a) and its 3,4ꢀOꢀdiacetyl
ester (2b), and LꢀDopa ethyl ester (2c).
As outlined in Scheme 1, compounds 4a and 4b were synthesized through the formation
of the carbonate intermediate 3 and subsequent reaction with 2a,b in dry DMF.
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Scheme 1. Synthesis of oxazepamꢀdopamine (LꢀDOPA) carbamoyl derivatives. Reagents
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and conditions: (a) Dry THF, TEA, N2 atm., r.t., 3 h; (b) dry DMF, r.t., 12ꢀ24 h.
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The starting 3,4ꢀOꢀdiacetyl ester of DA (2b) was prepared in almost quantitative yield by
treating DA, dissolved in anhydrous trifluoroacetic acid, with acetyl chloride. This
method, that is a modification of the classic acetylation of catechol OH groups, provided
satisfactory yields, due to the dual advantage of the simplicity of direct acetylation of
OHs (i.e., without the preparation of Nꢀcarbobenzyloxy derivative of DA) and higher
yield. The conjugate 4c was similarly prepared by reacting LD ethyl ester 2c with 3.
Unfortunately, because of their low stability, chromatographic purification of the end
products 4b and 4c on a normal phase silica column decreased the total yields compared
to compound 4a.
The DAꢀamide derivatives 6a and 6b of OXA hemisuccinate 5 were synthesized using
CDI and EDC as suitable coupling agents, respectively (Scheme 2).
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Scheme 2. Synthesis of oxazepam hemisuccinateꢀdopamine amide derivatives. Reagents
and conditions: (a) CDI, dry DMF, N2 atm., r.t., 15 min; then, 2a, r.t., 12 h; (b) EDC,
CH2Cl2, 0 °C, 30 min; then, 2b, CH2Cl2, TEA, r.t., 4 h.
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The BBB penetration of the five synthesized conjugates was estimated using the Clark’s
model26, a structureꢀbased quantitative property relationship relating logBB (i.e., log
bloodꢀbrain partitioning) to calculated log P and PSA (Table 1). As a general trend,
compounds with logBB < ꢀ1.0 may be unable to enter the CNS, whereas compounds with
values 0.3ꢀ0.5 should cross to some extent the BBB and compounds with values > 1.0
should have a desirable BBB permeability. The logBB values of the newly synthesized
conjugates were all lower than ꢀ1, suggesting a low probability for crossing the BBB.
However, this can not lead to rule out that at least 4a, the conjugate showing the highest
predicted logBB, may penetrate the BBB.
An ideal brainꢀtargeted prodrug or codrug should have a sufficient linker stability in
plasma to enable the conjugate to circulate intact in the bloodstream before penetrating
and distributing within the CNS. Once the intact conjugate molecule permeated the BBB,
the linker should be labile enough within the brain to efficiently release the active
compound/s, such as DA and OXA in this case.
Table 1
Lipophilicity, bloodꢀbrain permeability and stability of the OXAꢀDA conjugates.
Half-life (37°C)d
ClogPa
tPSAb
Log BBc
Cmpd
4a
PBS (pH 7.4), h
FBS, min
3.51
120.25
ꢀ1.11
24.3 ± 0.2
145 ± 23
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3.30
3.67
3.01
2.79
132.39
146.55
137.32
149.46
ꢀ1.32
ꢀ1.47
ꢀ1.44
ꢀ1.65
7.52 ± 0.50
15.4 ± 1.2
26.9 ± 0.1
68.2 ± 16.2
9.40 ± 0.10
4b
4c
6a
6b
9.13 ± 0.30
1.70 ± 0.14
1.19 ± 0.01
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a Calculated logP using ChemDraw Ultra 10.0 software.
b Topological polar surface area calculated according to Ertl’s equation.25
c Log (Cbrain/Cblood) calculated according to Clark’s equation.26
d Stability evaluated in 0.05 M phosphate buffer solution (PBS, pH 7.4, 0.15 M KCl) and
diluted fetal bovine serum (FBS) solution (50%, v/v) at 37 °C; halfꢀlife, t1/2 (mean ±
SEM, n = 3), refers to disappearance of the starting compound and is expressed in hours
(h) and minutes (min), respectively.
The study of chemical and enzymatic stability was performed at 37 °C in 0.05 M PBS
and in FBS solution (the most widely used serumꢀsupplement for the in vitro cell culture
of eukaryotic cells), respectively, at pH 7.4. In both media, all the test compounds
disappeared following pseudoꢀfirstꢀorder hydrolysis kinetics, and the observed halfꢀlives
(t ) are reported in Table 1. Although the degradation products of the carbamateꢀ
½
containing compounds 4a-c and hemisuccinate amide derivatives 6a-b were not fully
characterized, HPLC analyses, carried out at regular intervals along the monitoring time
between t0 and 2×t , were combined with ESI MS. In the chromatographic conditions
½
used, the HPLC chromatograms of the test compounds incubated both in PBS and diluted
FBS at pH 7.4 revealed several degradation products. In particular, the main fractions
collected by HPLC and analyzed by ESI MS in negative mode revealed the appearance of
OXA 1 (m/z 285) as a major hydrolytic decomposition product from 4a and 4c,
compound 4a (the deacetylation product) and 1 from 4b. OXA 1, and to a lesser extent 5
(m/z 385), were revealed as the hydrolysis products of 6a and 6b. The retention times of
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the observed degradation products were also confirmed in HPLC by comparison to the
standards, suggesting that the hydrolysis at the carbamate bond in compounds 4 as well
as at the ester and amide bonds in compounds 6 occurred with different rates in PBS and
FBS at physiological pH and temperature.
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The study of the stereoselective (enantioꢀ and diastereoselective) enzymeꢀcatalyzed
hydrolysis is out of the purposes of this work, given that the examined conjugates could
release by hydrolysis as major active products only the achiral DA (2a), or LꢀDOPA from
4c, and OXA 1 in racemic form (racꢀOXA). On the other hand, it is known that the R and
S enantiomers of OXA can undergo racemization (bidirectional chiral inversion at C3) in
aqueous solution in vitro.31,32 The halfꢀlives in Table 1 refer to disappearance of racemic
4a, 4b, 6a and 6b, and diastereomeric mixture of 4c (i.e., sum of the peak areas of the
two diastereoisomers).
Compounds bearing the succinic spacer (6a and 6b) proved to be in PBS much less stable
than the respective carbamate derivatives (4a and 4b), and the hydrolytic disappearance
of the 3,4ꢀOꢀdiacetyl ester derivatives 4b and 6b occurred faster than the parent
compounds 4a and 6a. The disappearance in PBS of the LD ethyl ester conjugate 4c (t1/2
= 9.13 h), due to hydrolysis of the COOCH2CH3 group, was about threeꢀfold faster than
the respective DA conjugate 4a (t1/2 = 24.32 h).
The enzymeꢀcatalyzed hydrolysis of the carbamateꢀbased conjugates 4 in FBS proved to
be tenꢀtoꢀthirty faster than in pH 7.4 aqueous solution and comparatively much less
effective for the OXA hemisuccinate derivatives 6. Compound 4a, with halfꢀlife slightly
more than two hours, proved the most stable one in FBS. The stability of 4a, and 4c for
comparison, was also studied in rat and human sera at 37 °C (Table 2).
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Table 2
Average halfꢀlives of 4a and 4c in 100% rat and human serum at 37 °C.
Rat (min)
Human (h)
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109
6.10
4a
4c
< 1
11.3 (12.4) a
a Data in parentheses refer to the second (more retained) diastereoisomer of 4c.
The rate of hydrolysis of 4a to produce DA and racꢀOXA was just 1.3ꢀfold faster in
100% rat serum than in diluted FBS. In contrast, its halfꢀlife in 100% human serum
exceeded 6 h. This so marked difference in serum stabilities could depend upon the
different ester hydrolase activities in human compared to rodent serum.33,34 Indeed, rats
and humans differs in serum esterase composition, mainly for carboxylesterases which
are present in rat serum and not in human serum. That carboxylesterases can effectively
contribute to catalyze the hydrolysis of the compounds under examination is also shown
by the decomposition rate of the double ester 4c, which is hydrolyzed in rat serum with a
t1/2 of less than 1 min and highly stable in human serum (t1/2 > 11 h).
A preliminary enantioselective HPLC determination of racꢀ4a at several times in rat
serum showed that the two enantiomers should be hydrolyzed with very similar rates,
their halfꢀlives resulting 135 and 126 min (Fig. 2).
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Fig. 2. Overlay of chromatograms for the chiral separation of racꢀ4a in rat serum at
different incubation times: t = 0 min (blue), t = 60 min (magenta), t = 120 min (green).
The chiral stationary phase resolved the enantiomers (*) with peaks at 17.50 and 18.90
min. Column: Astec CHIROBIOTIC TAG, 250×4.6 mm i.d., 5 µm particles (Whippany,
NY, USA). Mobile phase: Methanol. Column temperature: 25 °C. Flow rate: 0.3 ml/min.
Detection: UV, 254 nm. Injection: 2 µl.
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Stability data in Tables 1 and 2 suggest that the carbamateꢀbased conjugate 4a could be
stable enough in the circulatory system to cross intact the BBB (the average halfꢀlive of
4a in 100% human serum at 37 °C is 6.1 h), possibly releasing DA and OXA in the brain
parenchyma after hydrolysis. Compound 4a, showing the highest plasma stability and
logBB value was screened for its permeability across a BBB model.
The ability of 4a to cross the BBB was furnished by in vitro transport assays performed
with MDCKIIꢀMDR1 monolayers, a cell model that has been found to be predictive of
passive BBB permeability. Data in Table 3 supported the ability of 4a to cross the BBB;
4a has a Papp value 20ꢀfold higher than that of DA (2a), only 3ꢀfold lower than Papp of
diazepam, and about 7ꢀfold greater than that of FD4 (a marker for the paracellular
pathway).
Table 3
MDCKIIꢀMDR1 permeability value of compound 4a compared to those of dopamine
(2a), diazepam and FD4.
Compound
Papp×10-6(cm/sec) a
6.73 ± 0.82
4a
2a
0.39 ± 0.28
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Diazepam
FD4
18.9 ± 1.2
0.977 ± 0.292
a Mean ± SEM, n = 3.
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To assess whether the peripheral (i.p.) administration of 4a affects brain dopaminergic
transmission, in vivo microdialysis experiments were performed to determine the
extracellular output of DA in the striatum of freelyꢀmoving rats. To this regard, since
long time in vivo microdialysis has been increasingly applied to monitor the penetration
of various agents across the BBB.35 Continuous perfusion of the probe allowed the
sequential collection of samples of dialysate containing, among other solutes in the
extracellular fluid, the neurotransmitters released by neurons so that changes in their
concentrations provides information on the effects of centrally acting drug treatment.35
According to our experimental protocol, a series of dialysis samples was first obtained for
determination of basal neurotransmitter levels and then vehicle and drug solutions (4a or
LD) were administered. In particular, the effects of single injections (10 and 20 mg/kg,
i.p.) of 4a on the basal extracellular concentrations of DA (524 ± 80 pg/ml, value not
corrected for in vitro probe recovery) were monitored for 4 h after administration (Fig. 3
A). Twoꢀway ANOVA statistics revealed a significant main effect of treatment (F(2,154)
=
4.782, P < 0.05), a significant main effect of time (F(14,154) =3.232, P < 0.001), and any
significant interactions between factors (F(28,154) =1.499, n.s.). The postꢀhoc test showed
that 4a evoked a doseꢀdependent increase of DA extracellular outflow in the striatum.
Following the administration of the highest dose of 4a (20 mg/kg, i.p.), a significant
increase of DA level was observed at 60 min after administration that peaked (1663 %
increase above the baseline) at 80 min and disappeared at 120 min after treatment. On the
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significant effect on striatal DA release (Fig. 3 A, B).
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Fig. 3. A) Effect of single injections of 4a (10 and 20 mg/kg, i.p.) on the DA outflow in
the striatum of awake rats over 4 h. Data are expressed as mean percentages of baseline
(± SEM), n = 4ꢀ5 animals per group; twoꢀway ANOVA followed by Bonferroni’s
multiple comparison test: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. respective vehicle; ##
P < 0.01 vs. respective 4a lower dose (10 mg/kg). Arrow indicates the time of 4a or
vehicle administration. B) Effect of single injections of LD (8.5 mg/kg, i.p.) and vehicle
(saline) on the DA outflow in the striatum of awake rats over 4 h. Data are expressed as
mean percentages of baseline (± SEM), n = 5 animals per group. Statistical analysis was
performed by twoꢀway ANOVA. Arrow indicates the time of LD or vehicle
administration.
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The brain microdialysis study has been carried out also with the golden standard
treatment of PD. A second set of animals was treated with LD (8.5 mg/kg, i.p.), coꢀ
administered with the inhibitor of aromatic aminoacid decarboxylase benserazide (12.5
mg/kg, i.p.), and the extracellular level of DA in the striatum was monitored. The dose of
LD was equimolar to the higher dose of 4a (20 mg/kg) that significantly increases the
level of DA. Statistical analysis showed that LD did not modify the extracellular level of
DA in the striatum compared to vehicleꢀtreated rats (Ftime(14,112) = 0.8605, n.s.,
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Ftreatment(1,112) = 0.2847, n.s., Ftime x treatment(14,112) = 0.4143, n.s.) (Fig. 3 B, n=5).
Fig. 4. AUC values of striatal dopamine outflow after single injection of vehicle, 4a (10
and 20 mg/kg. i.p.) or LD (8.5 mg/kg, i.p.) in freely moving rats. Statistical analysis was
performed by oneꢀway ANOVA followed by Bonferroni’s multiple comparison test: * P
< 0.05 vs. respective vehicle.
The overall effect of treatments was assessed by comparing their respective AUCs (Fig.
4). Statistical analysis confirmed that only 4a at the highest dose significantly increases
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the striatal levels of DA (+ 90% compared to vehicleꢀtreated rats). The lowest dose of 4a
increases the DA output in the striatum up to 75%, although it did not attain statistical
significance. Conversely, the dose of LD, equimolar to the highest dose of 4a, did not
increase DA release in the striatum. The latter results are in line with our previous
microdialysis experiments, where we found no increase in striatal DA efflux immediately
after a single LD/benserazide injection (6 mg/kg and 12.5 mg/kg, s.c., respectively) in
rats treated acutely or in rats that had been treated chronically with LD/benserazide (6
mg/kg and 12.5 mg/kg, s.c., respectively) for 11 days.27
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In the literature, a wide range of LD dosages has been reported in microdialysis
experiments (from 6 up to 120 mg/kg), and an increase of the extracellular DA levels in
the striatum has been observed only with very high concentrations of LD (99 and 120
mg/kg, i.p.) compared to the concentration of 4a used in the present study.36
Moreover, other authors have demonstrated that chronic treatment (21 days) with
LD/benserazide (6 or 24 mg/kg and 15 mg/kg p.o., respectively) is able to produce a
doseꢀrelated increase of the extracellular levels of DA only in the 6ꢀhydroxyDAꢀ (6ꢀ
OHDA)ꢀlesioned hemisphere of the hemiꢀparkinsonian rats.37,38 These findings are in line
with the results of many other groups.36,39ꢀ41
Keeping in mind that these doses are typically associated with a marked increment
of brain LD levels39,42ꢀ44, the low response of DA on the intact brain (or intact hemisphere
in hemiparkinsonian rats) after LD systemic injection indicates that under physiological
conditions dopaminergic neurons are able to handle extracellular DA very efficiently,
thus keeping physiological homeostasis. Differently, DA availability markedly increased
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(+ 600%) after DAergic denervation of the striatum, indicating a disruption of the normal
regulation of extrasynaptic DA.36
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From the present investigation and several in vivo microdialysis studies, it is clear
that the peripherally administered LD, irrespective of the administration route (i.p., s.c. or
p.o.), does not increase the extracellular DA release in the striatum of intact rats, at least
at doses comparable with those tested for 4a. It will be interesting in the next future to
compare the effects of the new conjugate 4a with those of LD in models of 6ꢀOHDA
lesioned rats.
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As a matter of facts, following i.p. administration of 4a, DA levels significantly increased
in striatum at the higher concentration (20 mg/kg), ultimately supporting the hypothesis
that the OXAꢀDA carbamate derivative 4a, sufficiently stable in rat serum, is able to
cross the BBB and undergo hydrolytic decomposition in the brain parenchyma, achieving
sustained DA levels within 3 h. This hypothesis is consistent with the behavior of
previously reported imidazopyridine derivatives of DA.18
Although the overall results of 4a seem promising, other preclinical studies should be
conducted to further warrant the usefulness of this compound in PD therapy. As
mentioned before, drugs acting as dopaminergic agonists may elicit different effects on
DA extracellular release depending upon the animal model used. Therefore, in future
experiments the efficacy of 4a in 6ꢀOHDAꢀlesioned rats after acute and subꢀchronic
treatment will be tested. The latter may be conducted with the lower dose (10 mg/kg) of
4a, since the magnitude of DA release by the higher dose may cause side effects after
subꢀchronic treatment. Recent in vitro and in vivo studies have demonstrated that long
term LD therapy can cause DAꢀ or LDꢀinduced neurotoxicity,45,46 due to the reactive
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oxygen species (ROS) or reactive nitrogen species (RNS) and quinones generated in the
enzymatic oxidation or autoꢀoxidation of an exceeding amount of DA. Specifically, the
primary consequences of DAꢀ or LDꢀinduced oxidative stress are depletion of
endogenous antioxidants, mitochondrial dysfunction, neuronal damage, apoptotic or nonꢀ
apoptotic cell death. To this regard, the use of exogenous antioxidants could scavenge
ROS and RNS in the brain, preventing the DAꢀinduced damage and/or maintaining
optimal DA levels.20,23,24,47ꢀ49
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Of note, Table 2 shows that halfꢀlives of compounds 4a and 4c are higher in human
serum than in rat serum. This must not be surprising, taking into account the different
plasma esterases’ composition in humans and rats.33,34 A possible consequence of these
findings could be that 4a, which is more stable in human than in rat plasma, may
penetrate the BBB more efficiently in humans, and be active at lower concentrations with
less side effects. This is just a hypothesis that, however, points out the need for a critical
examination of qualitative and quantitative differences among animal species, among
humans, and between healthy people and people affected by pathological conditions.
3. Conclusions
In this study, we synthesized some novel compounds in which DA was covalently joined
to OXA, a wellꢀknown positive allosteric modulator of GABAA receptor, through a
carbamate linkage (4) or a succinic spacer (6). These potential antiparkinson codrugs
have been designed with the aim of improving the delivery of the neurotransmitter DA
into the brain, while enhancing GABAergic transmission. Based on the in vitro stability
data in animal serum, and using as ‘filters’ for estimating the potential CNS penetration
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computational descriptors and data from a cell model of transport through BBB
(MDCKIIꢀMDR1 monolayers), the OXAꢀDA carbamate derivative 4a was identified as
the conjugate achieving sufficient stability in rat serum and ability to cross BBB to some
extent. With regard to serum stability, the kinetic data showed that no advantage could
come from acetylation of the catechol OH groups in DA, given that the 3,4ꢀOꢀdiacetyl
ester derivative 4b (and 6b as well) disappeared rapidly in serum due to the lability of the
OCOCH3 groups. Interestingly, brain microdialysis experiments in rats provided an in
vivo proof that 4a (20 mg/kg, i.p.) should diffuse passively across the BBB and release
DA into the brain after enzymatic hydrolysis, with a peak (more than 15ꢀfold increase
over the baseline) at 80 min after administration. In vivo toxicity studies will be
performed in the next future, but the stability and DAꢀrelated delivery data of this study
highlight compound 4a as a noteworthy codrug candidate for further pharmacological
studies in animal models of PD, such as the hemiparkinsonian rats (i.e., rats with 6ꢀ
OHDAꢀlesioned hemisphere), in comparison with the golden therapy with LD.
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Disclosure of interest
The authors report no conflicts of interest.
ACKNOWLEDGMENTS
We thank Mr. Giovanni Dipinto and Mr. Antonio Palermo for skillful technical assistance
in recording mass spectra and NMR spectra, respectively. The University of Bari (Italy)
and the InterꢀUniversity Consortium for Research on the Chemistry of Metal Ions in
Biological Systems (C.I.R.C.M.S.B.) are also gratefully acknowledge for their financial
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