CNS delivery of L-dopa by a new hybrid glutathione-methionine peptidomimetic prodrug

Article abstract of DOI:10.1007/s00726-010-0804-z

Parkinson's disease (PD) is a neurodegenerative disorder associated primarily with loss of dopamine (DA) neurons in the nigrostriatal system. With the aim of increasing the bioavailability of L-dopa (LD) after oral administration and of overcoming the pro-oxidant effect associated with LD therapy, we designed a peptidomimetic LD prodrug (1) able to release the active agent by enzyme catalyzed hydrolysis. The physicochemical properties, as well as the chemical and enzymatic stabilities of the new compound, were evaluated in order to check both its stability in aqueous medium and its sensitivity towards enzymatic cleavage, providing the parent LD drug, in rat and human plasma. The radical scavenging activities of prodrug 1 was tested by using both the DPPH-HPLC and the DMSO competition methods. The results indicate that the replacement of cysteine GSH portion by methionine confers resistance to oxidative degradation in gastric fluid. Prodrug 1 demonstrated to induce sustained delivery of DA in rat striatal tissue with respect to equimolar LD dosages. These results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain. Springer-Verlag 2010.

Full text of DOI:10.1007/s00726-010-0804-z

Amino Acids (2012) 42:261–269
DOI 10.1007/s00726-010-0804-z
ORIGINAL ARTICLE
CNS delivery of L-dopa by a new hybrid glutathione–methionine
peptidomimetic prodrug
•
Francesco Pinnen Ivana Cacciatore Catia Cornacchia Adriano Mollica
•
•
•
•
•
•
Piera Sozio Laura S. Cerasa Antonio Iannitelli Antonella Fontana
•
•
Cinzia Nasuti Antonio Di Stefano
Received: 20 August 2010 / Accepted: 29 October 2010 / Published online: 17 November 2010
Ó Springer-Verlag 2010
Abstract Parkinson’s disease (PD) is a neurodegenera-
tive disorder associated primarily with loss of dopamine
(DA) neurons in the nigrostriatal system. With the aim of
increasing the bioavailability of L-dopa (LD) after oral
administration and of overcoming the pro-oxidant effect
associated with LD therapy, we designed a peptidomimetic
LD prodrug (1) able to release the active agent by enzyme
catalyzed hydrolysis. The physicochemical properties, as
well as the chemical and enzymatic stabilities of the new
compound, were evaluated in order to check both its sta-
bility in aqueous medium and its sensitivity towards
enzymatic cleavage, providing the parent LD drug, in rat
and human plasma. The radical scavenging activities of
prodrug 1 was tested by using both the DPPH–HPLC and
the DMSO competition methods. The results indicate that
the replacement of cysteine GSH portion by methionine
confers resistance to oxidative degradation in gastric fluid.
Prodrug 1 demonstrated to induce sustained delivery of DA
in rat striatal tissue with respect to equimolar LD dosages.
These results are of significance for prospective therapeutic
application of prodrug 1 in pathological events associated
with free radical damage and decreasing DA concentration
in the brain.
Keywords L-Dopa ꢀ Glutathione ꢀ Methionine ꢀ
Parkinson’s disease
Introduction
Parkinson’s disease (PD) and Alzheimer’s disease (AD) are
progressive neurodegenerative disorders that affect an
increasing number of the elderly population. Although
current drug therapy of PD turned out to be more suc-
cessful compared to that of AD, it does not stop the
degenerative process, and pharmacological treatments lose
effectiveness with progression of the disease (Everts et al.
2001). Dopamine (DA) shortage is mainly responsible for
the motor deficits of the disorder and several drugs, that
boost the levels of DA or mimic its effects, are available
for treating PD; nevertheless, none of them exceeded the
clinical efficacy of their biological precursor L-dopa (LD)
(LeWitt 2008). Since its introduction in the late 1960s, LD,
which counteracts parkinsonian motor symptoms by
restoring the nigrostriatal DA deficiency, still remains the
key compound of pharmacotherapy for PD to which all
other therapies are compared. Up to now, several com-
pounds have been studied with the aim of enhancing LD
chemical stability and water or lipid solubility, and
diminishing its susceptibility to enzymatic degradation.
During the last two decades the prodrug approach was
demonstrated to be quite promising in addressing the
problem of the rapid in vivo degradation of LD, and con-
sequently, in improving its bioavailability (Wang et al.
1995; Marrel et al. 1985; Cooper et al. 1984; Garzon-
Aburbeh et al. 1986; Cooper et al. 1987; Ihara et al. 1989;
Cingolani et al. 2000; Giorgioni et al. 2010). In recent
years an ameliorative version of the prodrug approach,
developed for favoring the delivery of LD to the central
F. Pinnen ꢀ I. Cacciatore ꢀ C. Cornacchia ꢀ A. Mollica ꢀ
P. Sozio ꢀ L. S. Cerasa ꢀ A. Iannitelli ꢀ A. Fontana ꢀ
A. Di Stefano (&)
Dipartimento di Scienze del Farmaco,
`
Universita ‘‘G. D’Annunzio’’, Via dei Vestini 31,
66100 Chieti, Italy
e-mail: adistefano@unich.it
C. Nasuti
Dipartimento di Medicina Sperimentale e Sanita Pubblica,
`
Universita di Camerino, Via Scalzino, 62032 Camerino,
MC, Italy
`
123

262
F. Pinnen et al.
nervous system (CNS), has involved the development of
dual-acting prodrugs. A few papers reported on the efficacy
of LD, covalently linked to different antioxidant molecules,
to induce sustained release of drug in rat striatum and, at
the same time, to protect against the oxidative stress
deriving from autoxidation of DA (More and Vince 2008;
Sozio et al. 2008; Di Stefano et al. 2006; Pinnen et al.
2007; Pinnen et al. 2009). In order to efficiently deliver
small, hydrophilic molecules across the BBB two different
strategies are currently used: (1) the chemical modification
of hydrophilic molecules to more lipophilic derivatives,
named lipidization; and (2) the transporter-mediated
delivery, in which the substrate of one of the numerous
transporters located within the BBB is exploited as the
carrier moiety of the drug (Pardridge 2009).
transporters located on the luminal side of the BBB. The
latter demonstrated the capacity of different peptides to
transfer levodopa through an artificial membrane by
means of passive diffusion via peptide BBB-shuttles. The
replacement of cysteine by methionine in GSH has been
shown to favor its antioxidant activity at pH 4 rather than
at pH 1, as is the case of native GSH, thus conferring
resistance to oxidative degradation in the gastric fluid
(Bobrowski et al. 2007). The release of radical scavenging
molecules such as the here investigated glutathione pep-
tidomimetic residue inside the CNS would most likely
contribute in attenuating the damage caused by the oxi-
dative stress commonly associated with PD and by the
pro-oxidant effects of traditional LD therapies. Further-
more, the incorporation of methionine in the GSH residue
would add to the prodrug the benefit of conferring stability
against c-glutamyl transpeptidase (c-GT) cleavage. In
order to propose the new compound 1 as a prospective
prodrug for PD, we have investigated in the present study
its biological and physicochemical properties as well as
the rates of chemical and enzymatic hydrolysis.
Several studies showed that the transport of many
peptides can be accounted for on the basis of their size
and lipophilicity and a number of peptide transport sys-
tems have been described in the BBB. In this respect,
several di- and tri-peptide mimetic prodrugs of DA and
LD have been shown to increase the oral bioavailability of
LD together with improved BBB permeability (Bai 1995;
Malakoutikhah et al. 2008; Wang et al. 1995; More and
Vince 2008). On the basis of these findings and in an
attempt to increase the BBB penetration of LD, we
developed a novel hybrid glutathione–methionine pepti-
domimetic operating as a dual-acting LD prodrug. This
hybrid peptidomimetic is constituted by the following
components: the carrier, a modified GSH in which cys-
teine is replaced with methionine, the drug, an acetylated
LD methylester, and the linker, an amide bond which
connect LD with the glycine residue of the modified GSH
(Fig. 1). The rationale behind the design of this new
compound is based on previous studies on glutathione
peptidomimetics as components of antiParkinson prodrugs
performed by More and Vince (2008) and Pinnen et al.
(2007) and on the observation of Malakoutikhah et al.
(2008). The former demonstrated an interaction between
the GSH residues of the investigated prodrug and the GSH
Chemistry
Fmoc-Met-Gly-OBut (2) and H-LD(Ac)2-OMe.HCl were
¨
synthesized as previously reported (Stober et al. 1997;
Bodor et al. 1977).
Prodrug 1 was synthesized as outlined in Scheme 1 by
employing solution phase procedures through elongation of
the protected peptide chain 5 in the C-direction with the
LD derivative.
Experimental section
Microanalyses were performed on a 1106 Carlo Erba CHN
1
analyzer. H- and 13C-NMR spectra were recorded on a
Varian VXR 300-MHz spectrometer. Chemical shifts are
reported in parts per million (d) downfield from the internal
standard tetramethylsilane (Me4Si). The LC–MS/MS sys-
tem consisted of an LCQ (Thermo Finnigan) ion trap mass
spectrometer (San Jose, CA, USA) equipped with an
electrospray ionization (ESI) source. The capillary tem-
perature was set at 300°C and the spray voltage at 4.25 kV.
The fluid was nebulized using nitrogen (N2) as both the
sheath gas and the auxiliary gas. The identity of the new
prodrug was confirmed by elemental analysis, NMR data
and LC–MS/MS measurements; homogeneity was con-
firmed by TLC on silica gel Merck 60 F254. Solutions were
routinely dried over anhydrous sodium sulphate prior to
evaporation. Chromatographic purifications were per-
formed by Merck 60 70–230 mesh ASTM silica gel
column.
methionine
(stability against γ -GT cleavage)
OAc
S
linker
O
O
OAc
H
N
AcHN
N
H
N
H
COOMe
COOMe
O
carrier 2
carrier 1
(target of BBB-GSH transporter)
(target of peptide-BBB shuttle)
Fig. 1 Chemical structure of multifunctional prodrug 1
123

CNS delivery of L-dopa
263
Scheme 1 Reagents and
conditions. a DBU, DCM, DCC,
20 min, rt; b Ac-Glu-OMe,
HOBt, DCC, DMF, 3 h, 0°C
then 15 h, 4°C; c TFA, 2 h, rt;
d H-LD(Ac)2-OMe.HCl, IBCF,
TEA, DMF, 3 h, 0°C then 15 h,
4°C
S
S
b
O
H
H
N
COOBut
AcHN
COOR
N
RHN
N
H
O
COOMe
O
2 R = Fmoc
3 R = H
4 R = But
5 R = H
a
c
OAc
OAc
S
d
O
O
H
N
AcHN
N
N
COOMe
H
H
COOMe
O
1
Chemicals
The residue obtained after drying and evaporation was
chromatographed on silica gel by using CHCl3:MeOH
(95:5) as the eluant to give the corresponding pure tri-
Ac-Glu-OMe was obtained from Bachem. All other
chemicals were of the highest commercially available
purity.
peptide t-butyl ester
4 (1.97 g, 77%). Rf = 0.30,
1
CHCl3:MeOH (95:5); H-NMR (CDCl3) d: 1.44 (9H, s,
OBut), 1.96–2.01 (2H, m, Met b-CH2), 2.02 (3H, s, Met
CH3), 2.11 (3H, s, Glu Ac), 2.16–2.20 (2H, m, Glu b-CH2),
2.33-2.38 (2H, m, Glu c-CH2), 2.55–2.63 (2H, m, Met
c-CH2), 3.74 (3H, s, OMe), 3.91–3.93 (2H, m, Gly a-CH),
4.56 (1H, m, Glu a-CH), 4.65 (1H, m, Met a-CH), 6.65
(1H, d, J = 7.2 Hz, Glu NH), 6.89 (2H, m br, Mat and Gly
NH); 13C-NMR (CDCl3) d: 15.41 (Met CH3), 23.25 (Glu
Ac), 28.25 (OBut), 29.89 (Glu b-CH2), 30.36 (Met b-CH2),
31.38 (Glu c-CH2), 32.45 (Met c-CH2), 42.21 (Gly a-CH),
52.02 (OCH3), 52.33 (Glu a-CH), 52.77 (Met a-CH), 82.57
(OBut), 168.92, 170.81, 171.47, 172.53 and 172.75
(5 9 CO).
H-Met-Gly-OBut (3) To a solution of the N-protected
dipeptide t-butyl ester 2 (5 g, 10.3 mmol) in CH2Cl2
(70 mL) DBU (1.57 g, 10.3 mmol) was added at room
temperature. After 20 min the solution was evaporated to
dryness and the residue chromatographed on silica gel
using CHCl3:MeOH (95:5) as eluant to yield pure
N-deprotected dipeptide t-butyl ester 3 (1.68 g, 62%).
Rf = 0.43, CHCl3:MeOH (95:5); 1H-NMR (CDCl3) d: 1.44
(9H, s, OBut), 2.45 (2H, s br, NH2), 1.65–1.80 (1H, m, Met
b-CHA), 2.15 (3H, s, Met CH3), 2.16–2.20 (1H, m, Met
b-CB), 2.45–2.55 (2H, m, Met c-CH2), 3.50 (1H, m,
Met a-CH), 3.85–3.90 (2H, m, Gly a-CH), 7.84 (1H, s br,
Gly NH). 13C-NMR (CDCl3) d: 15.48 (Met CH3), 28.24
(OBut), 30.77 (Met b-CH2), 34.19 (Met c-CH2), 41.88
(Gly a-CH), 54.31 (Met a-CH), 82.33 (OBut), 169.28 and
175.02 (2 9 CO).
Ac-Glu-[Met-Gly-LD(Ac)2-OMe]-OMe (1) The above
reported tripeptide t-butyl ester 4 (1.80 g, 4.02 mmol) was
dissolved in TFA (5.1 mL). After 2 h at room temperature
the solution was evaporated to dryness and the residue
repeatedly evaporated with ether to give 5 in quantitative
yield. This product was used without further purification
(1.54 g, 98%).
Ac-Glu[-Met-Gly-OBut]-OMe (4) To a stirred solution of
N-deprotected dipeptide t-butyl ester 3 (1.5 g, 5.7 mmol)
in dry DMF (7 mL) Ac-Glu-OMe (1.16 g, 5.7 mmol) in
dry DMF (7 mL) was added at 0°C followed by portion-
wise addition of HOBt (0.77 g, 5.7 mmol) and DCC
(1,10 g, 5.7 mmol) in dry DMF (8 mL). After 3 h at 0°C
and 16 h at 5°C, the reaction mixture was filtered and the
resulting solution was evaporated under vacuum. The res-
idue was taken up in EtOAc and the organic layer washed
with 1 N KHSO4, saturated aqueous NaHCO3 and brine.
To an ice-cold solution of deprotected tripeptide 5
(1.40 g, 3.58 mmol) in dry DMF (5 mL) TEA (0.50 mL,
3.58 mmol) and isobutyl chloroformate (IBCF) (0.47 mL,
3.58 mmol) were added under stirring. After 15 min at
-15°C, H-LD(Ac)2-OMe.HCl (1.19 g, 3.58 mmol) in TEA
(0.50 mL, 3.58 mmol) and dry DMF (5 mL) were added to
the mixture at -15°C with stirring. After 3 h at 0°C and
16 h at 5°C, the reaction mixture was filtered and the
123

264
F. Pinnen et al.
resulting solution was evaporated under vacuum. The res-
idue was taken up in AcOEt and the organic layer washed
with 1 N KHSO4, saturated aqueous NaHCO3 and brine.
The residue obtained after drying and evaporation was
crystallized from AcOEt (0.96 g, 40%). Rf = 0.41,
solutions were freshly prepared every week and stored at
4°C for use right away. The monoamine and their metab-
olites were identified on the basis of retention time. Mea-
surements were performed in triplicate for each original
sample.
1
CHCl3:MeOH (95:5); H-NMR (d6-DMSO) d: 1.77–1.94
(4H, m, Glu and Met b-CH2), 1.90 (3H, s, Met CH3), 2.01
(3H, s, Glu Ac), 2.09–2.55 (4H, m, Glu and Met c-CH2),
2.23 and 2.25 (6H, 2 9 s, LD Ac), 2.95–3.10 (2H, m, LD
b-CH2), 3.65 (3H, s, OMe), 3.70 (3H, s, OMe), 3.72–3.80
(2H, m, Gly a-CH2), 4.10–4.25 (2H, m, Glu and Met
a-CH), 4.45 (1H, m, LD a-CH), 7.10–7.20 (3H, m, Ar),
8.05 (1H, d, J = 7.2 Hz, Glu NH), 8.10 (1H, t, J = 5.8 Hz,
Gly NH), 8.15 (1H, d, J = 6.89 Hz, Met NH), 8.25 (1H, d,
J = 7.1 Hz, LD NH); 13C-NMR ((d6-DMSO) d: 15.21
(Met CH3), 21.03 and 21.05 (LD 2 9 Ac), 22.94 (Glu Ac),
27.36 (Glu b-CH2), 30.21 (Met b-CH2), 31.90 (Glu c-CH2),
32.17 (Met c-CH2), 36.53 (LD b-CH2), 42.19 (Gly a-CH),
52.14 (OCH3), 52.47 (Glu a-CH), 52.57 (OCH3), 52.76
(Met a-CH), 54.09 (LD a-CH), 123.97, 124.83, 127.89,
136.50, 141.34 and 142.31 (Ar), 168.87, 168.92, 169.57,
170.13, 172.23, 172.32 and 173.22 (6 9 CO). Anal. cal-
culated for (C29H40N4O12S): C, 52.09; H, 6.03; N, 8.38; S,
4.80; found: C, 52.29; H, 6.02; N, 8.40; S, 4.65. MS (ESI)
m/z 669 (M–H)-.
Aqueous solubility
Compound 1 (50 mg) was placed in a microtube containing
1 mL of deionized water or buffer solutions (at pH 7.4, 5.0
and 1.3 by using 0.02 M phosphate buffer, 0.02 M acetate
buffer or 0.02 M hydrochloridic acid buffer, respectively)
and shaken at 25°C for 1 h to ensure the solubility equi-
librium. After centrifugation, a 20-lL portion of the
supernatant was analyzed by HPLC (Pinnen et al. 2009).
Lipophilicity
clog P
The calculated clog P was determined by using ACD Log
P software package, version 4.55 (Advanced Chemistry
Development Inc., Toronto, Canada).
Octanol/water partition coefficient
Chromatographic conditions
Octanol/water partition coefficient (log P) was determined
by placing approximately 5 mg of compound 1 in 1 mL of
aqueous saturated n-octanol, shaking vigorously for about
2 min and filtering. An equal volume of phosphate buffer
pH 7.4 was added and the mixture was equilibrated by
repeated inversions of up to 200 times for 5 min and then
allowed to stand for 30 min for the phases to fully separate.
Thereafter the respective phases were analyzed by HPLC.
Analytical HPLC measurements were run on a Waters
1525 Binary HPLC pump, equipped with a Waters 2996
photodiode array detector, a 20-lL Rheodyne injector and
a computer integrating apparatus. The column was a
Waters Symmetry RP-C18 column (4.6 9 150 mm, 5 lm),
the mobile phase was a mixture of water/methanol (10:90)
at a flow rate of 1 mL/min, the UV-detector was set at a
length of 264 nm.
Log k0
Pharmacokinetic analysis were run on the HPLC system
consisted of a Waters 600 controller pump, a Rheodyne
7295 injector with a 10-lL loop and an Antec Leyden
Decade II detector; the operating potential was 0.75 V.
Separation was achieved on a Waters Symmetry RP-C18
column (4.6 9 150 mm, 5 lm). The mobile phase con-
sisted of 0.045 M monobasic sodium phosphate, 0.001 M
1-octanesulphonic acid sodium salt, 0.006% triethylamine,
0.015% 100 lM sodium EDTA and 6% acetonitrile. The
pH of the mobile phase was adjusted to 3.0 by o-phosphoric
acid. The mobile phase was filtered and degassed by vac-
uum. A flow rate of 1 mL/min was used in all experiments.
Monoamine stock solutions were prepared at a concentra-
tion of 1 mg/mL (as a free base) in 0.05 N perchloric acid
containing 0.064% 1-octanesulphonic acid sodium salt,
0.060% heptanesulphonic acid sodium salt, 0.004% sodium
EDTA, and 0.010% sodium metabisulphite. These standard
Solute hydrophobicity of different compounds can be
estimated from the corresponding retention times (RTs)
due to the good relationship between log octanol/water
partition coefficient and logarithmic retention factors in
reverse-phase chromatography (log k value) determined by
´
using octadecyl silica columns. (Vailaya and Horvath
1998; Angelini et al. 2005). Retention in reverse-phase
chromatography increases with solute lipophilic character
and, for a given combination of solute and stationary phase,
with the water content of the mobile phase.
Compound 1 was dissolved in methanol (concentration
1 mg/mL). Aliquots of this solution were filtered and
analyzed by HPLC. The mobile phase consisted of aceto-
nitrile and water with acetonitrile content varying in the
range 90–55% (v/v) with 5% increments (Bajda et al.
2007).
123

CNS delivery of L-dopa
265
The dead time was measured by injection of methanol as
a non-retained compound. The isocratic capacity factor, log
k, was calculated from the dead time (t0) and the retention
time (tr) values by using the following equation (1):
2010). The DPPH peak was monitored at 517 nm and the
radical scavenging activity was calculated from the peak
area (PA) as reported above:
ꢀ
ꢁ
PAblank ꢁ PAsample
ꢀ
ꢁ
Radical scavenging ð%Þ ¼
ꢂ 100
tr ꢁ t0
PAblank
log k ¼ log
ð1Þ
t0
DMSO competition method
By plotting log k values against the acetonitrile content
it is possible to calculate, by extrapolation to zero
acetonitrile percentage, the log k0 value, that is the solute
retention with pure water as the eluent.
The hydroxyl radical production was determined accord-
ing to Nash (1953). When the hydroxyl radicals, gener-
ated by mixing the Fe3?/ascorbic acid system, react with
DMSO, the methyl radical forms and then partly trans-
forms into formaldehyde. The reaction mixture (300 lL)
was prepared as previously reported (Melagraki et al.
2009): EDTA (0.1 mM), Fe3? (167 mM), DMSO
(33 mM) in phosphate buffer (50 mM, pH 7.4), the tested
compounds (concentration 0.1 mM) and ascorbic acid
(10 mM). After 30 min of incubation (37°C) the reaction
was stopped with 100 lL of CCl3COOH (17% w/v) and
then 400 lL of ethyl 3-oxobutanoate were added. After
20 min at 60°C, the mixture was filtered and the form-
aldehyde derivative peak was detected by HPLC–UV at
365 nm (Burini and Coli 2004). The competition with
DMSO was expressed as percent of inhibition of form-
aldehyde production and was calculated from peak area
(PA) as reported above:
Kinetics of chemical hydrolysis
A 0.02 M hydrochloridic acid buffer of pH 1.3 as non-
enzymatic simulated gastric fluid (SGF) and a 0.02 M
phosphate buffer of pH 7.4 were used in this study.
Reactions were initiated by adding 1 mL of 10-4
M
stock solution (in acetonitrile) of prodrug 1 to 10 mL of
the appropriate thermostatted (37 0.5°C) aqueous buf-
fer solution, containing 20% acetonitrile. At appropriate
time intervals, samples of 20 lL were withdrawn and
analyzed by HPLC. Pseudo-first-order rate constants
(kobs) for the hydrolysis of the prodrug were then cal-
culated from the slopes of the linear plots of log (%
residual prodrug) against time. The experiments were run
in triplicate and the mean values of the rate constants
were calculated.
ꢀ
ꢁ
PAblank ꢁ PAsample
inhibition ð%Þ ¼
ꢂ 100
ð2Þ
PAblank
Kinetics of enzymatic hydrolysis
Animals
Plasma from rats and human was obtained by centrifuga-
tion of blood samples containing 0.3% citric acid at
3,0009g for 15–20 min. Plasma fractions (4 mL) were
diluted with 0.02 M phosphate buffer (pH 7.4) to give a
final volume of 5 mL (80% plasma). Incubations were
performed at 37 0.5°C using a shaking water bath. The
reactions were initiated by adding 100 lL of a stock
solution of drug (1 mg/mL in acetonitrile) to 5 mL of
preheated plasma. Aliquots (100 lL) were taken at various
times and deproteinized by mixing with 200 lL of 0.01 M
HCl in methanol. After centrifugation for 5 min at
5,0009g, 10 lL of the layer supernatant was chromato-
graphed as described above. The logarithms of the
remaining original prodrug were plotted as a function of
Male Wistar rats (n = 75) (Harlan, UD, Italy) weighing
250–300 g were employed. Five rats were assigned to
each treatment group. The animals were housed in plastic
(Makrolon) cages in
a temperature-controlled room
(21 5°C) and maintained on a laboratory diet and
water ad libitum. The light/dark cycle was from 7 a.m. to
7 p.m.
Drug administration
Benserazide hydrochloride, a peripheral dopa-decarbox-
ylase inhibitor, was dissolved in water whereas the pro-
drug 1, LD and tripeptide were dissolved in dimethyl
sulfoxide. All animals received a dose of benserazide
(0.083 mmol/kg) combined with 1, LD or tripeptide in
equimolar doses (0.332 mmol/kg). The drugs were given
at a volume of 5 ml/kg in a single oral administration by
intragastric tube. This study was carried out in accordance
with the Italian government’s guidelines for the care and
use of laboratory animals (D.L. n. 116 of January 27,
1992).
incubation time in order to give the corresponding kobs
.
Radical scavenging activity
DPPH–HPLC method
The DPPH radical scavenging activity was estimated
according to the method previously reported (Sozio et al.
123

266
F. Pinnen et al.
Pharmacokinetic analysis
Results and discussion
After slight anesthesia with carbon monoxide, the blood of
rats, was collected, by cardiac puncture, in vials containing
heparin (250 IU), centrifuged at 2,0009g for 10 min and
kept at -80°C until analysis. Aliquots (400 lL) were taken
at various times and deproteinized by mixing with 40 lL of
4 M perchloric acid. After centrifugation for 5 min at
5,0009g and filtration (Millipore 0.45 lm), 10 lL of the
layer supernatant was chromatographed as described
below. The amounts of LD were plotted as a function of
incubation time.
The physicochemical properties, water solubility and n-
octanol/water partition coefficient for prodrug 1 were
determined in order to predict its absorption, distribution
and brain bioavailability. It is generally accepted that good
absorption of an orally administered drug could be
obtained when the aqueous solubility is more than 10 lg/
mL (Yalkowsky and Morzowich 1980) and therefore, to
assess this potential, solubility of the prodrug 1 was
determined in water and buffer solutions (at pH 1.3, 5.0 and
7.4). From the data reported in Table 1 it appears that the
solubility is relatively high in water (6.69 mg/mL) and it
decreases in buffers. As expected, the solubility slightly
decreases upon increasing the pH of the buffer, although it
remains in all cases much higher than the critical value
reported by Madhukar (Madhukar et al. 2010).
The striatum tissue of the rats were dissected out, frozen
as well in liquid nitrogen and stored at -80°C until use.
The sampling schedules were 1, 2, 4 and 6 h after treat-
ment with drugs. All striatal tissues were individually
homogenized for 2 min with a Dyna-Mix homogenizer
(Fisher Scientific) in 500 lL of 0.05 N perchloric acid
solution containing (by weight/volume) 0.064% 1-octane-
sulphonic acid sodium salt, 0.060% heptanesulphonic acid
sodium salt, 0.004% sodium EDTA, 0.010% sodium
metabisulfite and 25 ng/mL dihydroxybenzylamine
(DHBA) as an internal standard. The whole procedure was
carried out on ice. The resulting homogenate was then
centrifuged at 4,500 g for 10 min and the supernatant was
filtered by using 0.45 lm Millipore filters. For each mea-
surement, 10 lL of the obtained filtrate were injected into
the liquid chromatography equipment (Giorgioni et al.
2010).
In order to evaluate the penetration of a molecule
through the BBB, the lipophilicity was estimated by using
the apparent partition coefficient (log Papp) measured in n-
octanol/phosphate buffer at pH 7.4 and the log capacity
factor (log k0) calculated from reverse-phase chromato-
graphic retention times with acetonitrile and water as the
eluents (Bonina et al. 1996). For the sake of comparison,
the lipophilicity of prodrug 1 was also calculated using the
ACD Log P software package, version 4.55 (Advanced
Chemistry Development Inc., Toronto, Canada). Data
reported in Table 1 point out that compound 1 is quite
hydrophilic as evidenced by negative log P and clog
P values. The value of log k0 = 1.21 is in agreement with a
low retention in reverse-phase chromatography as expected
for not very lipophilic molecules (Di Stefano et al. 2008).
Stability studies of the new compound were performed
at 37°C in isotonic sodium phosphate buffer (pH 7.4), in
simulated gastric fluid (SGF, pH 1.3) and in rat and human
plasma diluted to 80% with isotonic sodium phosphate
buffer (pH 7.4) as previously described (Di Stefano et al.
2006). The prodrug 1 showed good stability toward gas-
trointestinal hydrolysis, being the half time for chemical
hydrolysis in SGF of ca. 20 h and twice as much as that in
isotonic sodium phosphate buffer, thus meeting the
requirements for gastrointestinal absorption after oral
administration (Table 2). On the contrary, in rat and human
Statistical analysis
DA concentrations in rat striatal tissue are given as per-
centages of mean values with respect to basal levels. Basal
concentrations were determined as the mean of at least
three measurements with B5% variation obtained at the
beginning of the experiment. Data are expressed as means
with error bars for standard deviations of five rats. One-
way ANOVA was used to evaluate the effect of procedures
on each group of animals. If a general effect was deter-
mined by ANOVA, post hoc analysis was performed with
the Scheffe or Student’s test with P B 0.05 used as the
level of significance.
Table 1 Physicochemical properties of prodrug 1
Solubilitya (mg/mL)
Lipophilicitya
Water
Buffer solutions
pH 1.3
log Papp
clog P
log k0
pH 5.0
pH 7.4
6.69 ( 0.21)
5.38 ( 0.27)
5.00 ( 0.22)
4.54 ( 0.16)
-0.25 ( 0.02)
-0.90 ( 0.88)
1.21 ( 0.04)
a
Values are means of three experiments, standard deviation is given in parentheses
123

CNS delivery of L-dopa
267
Table 2 Rate constants for the chemical and enzymatic hydrolysis of prodrug 1 at 37°C
Chemical hydrolysisa
Enzymatic hydrolysisa
Rat plasma
pH 1.3
pH 7.4
Human plasma
t‘ (min)
kobs (min-1
a
1240 ( 50)
5.67 ( 0.17) 10-4
648 ( 24)
1.07 ( 0.05) 10-3
4.74 ( 0.12)
0.15 ( 0.01)
7.32 ( 0.30)
0.10 ( 0.01)
)
Values are means of three experiments, standard deviation is given in parentheses
plasma, liquid chromatography/mass spectrometry (LC/
MS) measurements on extracts, performed by using the
previously proposed LC/MS/MS method and electrospray
ionization (ESI) mode for MS experiments (Sozio et al.
2010), pointed out a rapid bioconversion of prodrug 1 into
LD.
Pharmacological studies were performed to test the
ability of prodrug 1 to increase basal levels of striatal DA
and LD and therefore affecting brain neurochemistry
associated with dopaminergic activity. Figures 2 and 3
show plasma concentrations–time profiles of LD and DA
striatal levels, respectively, in rats following oral admin-
istration by intragastric tube of prodrug 1, LD itself or
tripeptide 5. With regard to basal levels of DA (1.5 ng/mg
Fig. 2 Effect of oral administration of compound 5, LD or prodrug 1
(0.332 mmol/kg) on DA striatal levels
tissue), prodrug 1 elicited a sustained release compared to
that induced by LD (P \ 0.05). The effect reached its
maximum 4 h after administration, and maintained higher
levels of striatal DA, compared to those induced by LD,
from the third to the twelfth hour following the treatment.
The values of LD plasma concentration 4 h post dose were
ca. 110 lg/ml for prodrug 1 and 75 lg/ml for LD, with
rapid decrease of plasma levels 2 h after administration in
the latter case. The prolonged plasma LD levels induced by
prodrug 1 with respect to equimolar doses of LD could be
beneficial in the treatment of motor fluctuation in advanced
state of the disease because they prevent the well known
wearing-off phenomenon (or end-of-dose deterioration)
directly related to LD plasma level fluctuation after long-
term treatment of PD with chronic LD therapy (Koller et al.
1999). This feature of prodrug 1 administration is even
more intriguing if one considers the relatively short half-
life of prodrug 1 in plasma (from 4 to 5 min in rat plasma
and from 7 to 8 min in human plasma, see Table 2). Fur-
thermore, the plasma concentration profiles of Figs. 2 and
3 are consistent with a slow release of LD in the brain
afterwards 1 administration and may represent a desirable
therapeutic feature in order to reduce the frequency of drug
administration.
Fig. 3 Effect of oral administration of compound 5, LD or prodrug 1
(0.332 mmol/kg) on LD plasma levels
absorption at 517 nm whose peak area decreases if the
tested compound is a free radical scavenger and, by
reacting with DPPH, converts it to the stable diamagnetic
molecule 1,1-diphenyl-picrylhydrazine (Panteleon et al.
2008). Since the decrease in the peak depends on the
concentration of the tested compound, four different con-
centrations were investigated (50, 100, 300 and 600 lM).
Our results pointed out that the radical scavenging activity
of the investigated compounds increased with concentra-
tion (Fig. 4) and with incubation time (data not shown).
Moreover, tripeptide 5 showed a stronger activity than
In the present study, we assessed also the antioxidant
efficacy of prodrug 1 and tripeptide 5 by using two dif-
ferent assays, the DPPH–HPLC and the DMSO competi-
tion methods. This activity could be efficaciously exploited
in therapy as potential neuroprotective agent. The 1,1-
diphenyl-2-picrylhydrazyl (DPPH) has a characteristic
123

268
F. Pinnen et al.
In conclusion, we have synthesized a new peptidomi-
metic prodrug constituted by a modified GSH, as the carrier
towards CNS, and LD methylester, as the active antipar-
kinson agent. This compound demonstrated to cross unal-
tered the acidic environment of the stomach, to be stable
enough to be absorbed from the intestine, to have radical
scavenging activity and to release LD in human plasma
after enzymatic hydrolysis. Compared to other drugs
intended for PD therapy, prodrug 1 appears to be particu-
larly promising because it showed to induce sustained
delivery of DA in rat striatal tissue with respect to equi-
molar administration of LD itself. Taken together, these
results are of significance for prospective therapeutic
application of prodrug 1 in pathological events associated
with free radical damage and decreasing DA concentration
in the brain.
Fig. 4 % DPPH radical scavenging activity after 20 min incubation
Acknowledgments Financial support from Ministero dell’
`
Istruzione, dell’Universita e della Ricerca (MIUR) is gratefully
acknowledged.
Conflict of interest The authors declare that they have no conflict
of interest.
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