A kinetic analysis of the effects of β-phenylethylamine on the concentrations of dopamine and its metabolites in the rat striatum

Article abstract of DOI:10.1021/js960192p

The purpose of this investigation was to determine whether the increase in the dopamine (DA) concentration in the rat striatum after a rapid iv injection of β-phenylethylamine (PEA) can be quantitatively explained by the alteration of the striatum PEA con

Full text of DOI:10.1021/js960192p

A Kinetic Analysis of the Effects of
â-Phenylethylamine on the Concentrations
of Dopamine and Its Metabolites in the Rat Striatum
S
HINJI
S
ATOX, ASTUSHI
T
AMURA, SHUJI
KITAGAWA
,
AND
A
KIRA
K
OSHIRO
Received April 29, 1996, from the Department of Pharmaceutics, Niigata College of Pharmacy, 5-13-2 Kamishinei-cho,
Niigata, 950-21, Japan.
Accepted for publication January 6, 1997X.
extremely rapid.5,6 With respect to the pharmacological
responses of PEA, it has been reported that (1) PEA can
inhibit the reuptake of dopamine (DA) into DA neuronal
terminals and stimulate the release of radiolabeled DA from
slices of rat striatum.7,8 (2) PEA is a typical substrate for
monoamine oxidase-B (MAO-B),9 (3) the effect of PEA on the
release of DA and the reduction of its metabolite 3,4-dihy-
droxyphenylacetic acid (DOPAC) in rat striatum slices is
attributed to the competitive inhibition of PEA on the re-
uptake of DA into DA neuronal terminals and the inhibition
of MAO-B by PEA,10 (4) the specific PEA receptors or recogni-
tion sites are present in the central nervous system,11,12 and
(5) the endogenous PEA concentration in guinea pig striatum
was significantly increased by MAO-B inhibitors.13 As for the
clinical responses of PEA, it has been reported that (1) the
significant alterations of PEA metabolism are linked to the
etiology of schizophrenia14 and depression,15 (2) phenylacetic
acid (PAA), the main metabolite of PEA, is decreased in the
biological fluids of depressed subjects,15 and (3) the adminis-
tration of PEA or of its precursor L-phenylalanine improves
mood in depressed patients treated with a selective MAO-B
inhibitor and may be therapeutic in selected depressed
patients.16 Thus, it is thought that the establishment of the
quantitative relationship between the PEA concentration and
its pharmacological responses, especially the effect of PEA on
the disposition of catecholamines in the central nervous
system, is important for the elucidation of the pharmacological
mechanism of PEA. However, comprehensive pharmacoki-
netic studies of PEA and the quantitative studies of the effects
of PEA on the disposition of catecholamines have not been
performed. A previous study showed that the time courses
of the concentrations of DA and its metabolites DOPAC and
homovanillic acid (HVA) in the striatum after L-dopa injection
can be described quantitatively by a constructed DA metabo-
lism model,17 which was also able to describe quantitatively
the relationship between the striatum chlorpromazine con-
centration and the alteration of the striatum DA, DOPAC,
and HVA concentration in rat after the iv administration of
chlorpromazine.18 The goals of the present investigation were
(1) to develop a pharmacokinetic model for PEA in plasma
and the striatum, (2) to determine whether the quantitative
relationship between PEA concentration and the enlargement
of DA concentration in rat striatum is present after PEA
injection, and (3) to examine whether the time courses of
DOPAC and HVA concentrations in the striatum after PEA
injection can be explained by the constructed DA metabolism
model.
Abstract
the increase in the dopamine (DA) concentration in the rat striatum after
a rapid iv injection of -phenylethylamine (PEA) can be quantitatively
explained by the alteration of the striatum PEA concentration using a
constructed DA metabolism model and to examine whether the time
courses of the striatum DA metabolites 3,4-dihydroxyphenylacetic acid
(DOPAC) and homovanillic acid (HVA) concentration can be described
by this DA metabolism model. The time courses of PEA concentration
0 The purpose of this investigation was to determine whether
â
in plasma and the striatum were determined by gas chromatography
−
mass spectrometry. The plasma PEA concentration was described by a
two-compartment model with nonlinear elimination kinetics. The striatum
PEA concentration was about 10 times higher than the plasma PEA
concentration. The time course of the striatum PEA concentration was
described by a diffusion-limited model including a Michaelis−Menten type
transport system from plasma to the striatum and nonlinear elimination
from the striatum. The DA concentration in the striatum increased
immediately after PEA injection. In contrast, the DOPAC concentration
in the striatum decreased immediately. HVA concentration in the striatum
increased gradually. Assuming that the enhancement of DA concentration
in the striatum after PEA injection is caused by the competitive inhibition
of PEA on the reuptake of DA into DA neuronal terminals (and the
metabolism from DA to DOPAC is then competitively inhibited by PEA in
the DA neuronal terminals), the relationship between the enhancement
of DA concentration and PEA concentration in the striatum was analyzed
using a constructed DA metabolism model. The enhancement of the
DA concentration in the striatum was described quantitatively by this
model. Thus, it was clarified that a quantitative relationship between
PEA concentration and the enhancement of DA concentration in the
striatum is present after PEA injection. However, the time courses of
the striatum DOPAC (lower dose) and HVA (time delay) concentrations
could not be described by this model. These results indicated that other
factors might be necessary to explain the time courses of the DOPAC
and HVA concentrations in the striatum after PEA injection, such as the
separate evaluation of the effect of PEA on the reuptake of DA into DA
neuronal terminals and on the monoamine oxidase-B (MAO-B) activity in
the DA neuronal terminals, and the metabolic pathway from DOPAC to
HVA.
Introduction
â-Phenylethylamine (PEA) is an endogenous compound and
can act as a neuromodulator of dopaminergic responses.1 PEA
has a very complex mode of action in pharmacokinetics and
pharmacological and clinical responses. Concerning the phar-
macokinetics of PEA, it has been reported that (1) PEA is a
lipophilic compound that passes readily through the blood-
brain barrier2 and has a heterogeneous distribution in the
mammalian brain in spite of the low endogenous PEA
concentration in the brain3 and (2) the synthesis rate of PEA
is quite low,4 and the turnover rate of PEA in the brain is
Experimental Section
Ch em ica lssâ-Phenylethylamine hydrochloride (Sigma Chemicals,
St. Louis, MO) was purchased commercially and used without further
purification. This drug was dissolved in isotonic sodium chloride
solution (Otsuka Pharmaceuticals, Tokyo, J apan) and was adminis-
tered intravenously. All other chemicals were of reagent grade and
were obtained commercially (Wako Pure Chemical Industries, Osaka,
J apan).
An im a lssMale wistar rats (Sankyo Lab Service Corporation, Inc.,
Shizuoka, J apan) were used. The rats were separated into two
X Abstract published in Advance ACS Abstracts, March 15, 1997.
© 1997, American Chemical Society and
American Pharmaceutical Association
S0022-3549(96)00192-X CCC: $14.00
Journal of Pharmaceutical Sciences / 487
Vol. 86, No. 4, April 1997

groups, one group for measurement of the plasma PEA concentration
after PEA injection and the other group for measurement of the PEA,
DA, DOPAC, and HVA concentrations in the striatum after PEA
injection. The rats were housed individually in metal cages under
controlled temperature (22-24 °C) and alternating 12 h light (7
a.m.-7 p.m.) and dark cycles. Food and water were withdrawn in
the morning on the day of the experiment, and the rats were then
placed in individual plastic metabolic cages. The animals had an
indwelling cannula implanted in the right jugular vein 1 day before
the experiments.
An im a l Exp er im en ts for P la sm a P EA Con cen tr a tion sTo
characterize the PEA disposition in plasma, PEA (10, 25, 50, and 75
mg/kg) was injected rapidly into the right jugular vein. Blood samples
(0.2 mL) were collected from the same right jugular vein cannula at
2, 5, 10, 15, 20, 30, 40, 50, and 60 min after PEA injection. The blood
was then replaced by injection of an equal volume of citrated blood
from a donor rat. The obtained plasma samples were stored at -80
°C until the analysis for plasma PEA concentration.
Figure 1sSchematic representation of the pharmacokinetic model for PEA in rat
An im a l Exp er im en ts for P EA, DA, DOP AC, a n d HVA Con -
cen tr a tion in th e Str ia tu m sTo characterize the alteration of PEA,
DA, DOPAC, and HVA concentrations in the striatum, PEA (25 and
50 mg/kg) was injected rapidly into the right jugular vein. At 2, 5,
10, 15, 20, 30, 45, and 60 min after the dosing, rats were respectively
sacrificed by 2 nmol/mL KCl (1 mL) injection and the entire brain
was quickly excised, rinsed with cold physiological saline, and
dissected into the corpus striatum by a modified method of Glowinski
and Inversen.19 The striatum samples were stored at -80 °C until
the analysis for the striatum DA, DOPAC, and HVA concentrations.
The remaining striatum filtrate samples (which were used for the
striatum DA, DOPAC, and HVA concentrations) were used for the
analysis of the striatum PEA concentration using gas chromatogra-
phy-mass spectrometry (GC-MS) (described below).
plasma and the striatum.
i.d. The injector port temperature was 220 °C and the helium carrier
gas flow rate was 0.8 mL/min. The initial column oven temperature
was 200 °C, and the increasing temperature was at 30 °C/min up to
280 °C. The ionization potential was 70 eV. Ion currents at m/z 261
for PEA (N-benzenesulfonamide derivation) and m/z 275 for 3-phenyl-
1-propylamine (N-benzenesulfonamide derivation, the internal stan-
dard) were recorded by computer (HP Vectra QS/16S) with an MS
chemistation program. Retention times were 5.2 and 6.1 min for the
N-benzenesulfonamide derivative of PEA and 3-phenyl-1-propyl-
amine, respectively. To test the linearity of the calibration graph,
various amounts of PEA ranging from 0.25 to 1000 ng for the plasma
PEA concentration and from 2.5 to 10000 ng for the striatum PEA
concentration were derivatized, respectively. Linear relationships
were obtained from both logarithmic plots, and the regression lines
for the plasma PEA concentration and the striatum PEA concentra-
tion were ln y ) 0.886 ln x - 4.419 (γ ) 0.9957) and ln y ) 0.895 ln
x - 4.065 (γ ) 0.9938), respectively, where y is the peak-area ratio
and x is the amount of PEA. The coefficient of variation was 5% or
less in these concentration ranges for calibration graphs. Detection
limits of sensitivity to PEA based on the signal to noise ratio of 3
were determined by injection of diluted standard solutions. The
detection limits of the assay for the plasma PEA and the striatum
PEA concentrations were about 0.1 ng (1 ng/mL) and 1 ng (10 ng/g),
respectively.
Assa y Meth od s for DA, DOP AC, a n d HVA Con cen tr a tion s
in th e Str ia tu m sThe striatum concentrations of DA, DOPAC, and
HVA were determined by a modification of the high-performance
liquid chromatographic (HPLC) assay of Murai et al.21 with isopro-
terenol (Wako) as the internal standard. The striatum samples (0.05
g) were put into glass test tubes and homogenized with a Polytron
homogenizer (PT 10-35, Kinematica, Switzerland) at 15 000 rpm for
10 s in 500 µL of 0.1 M perchloric acid containing 10 µM 2Na EDTA
and isoproterenol (1000 ng) and 3-phenyl-1-propylamine (500 ng,
which is the internal standard for the striatum PEA concentration)
for the precipitation of protein. After centrifugation at 4000 rpm for
10 min at 4 °C, the clear supernatants were filtered through a 0.45
µm filter (disposable syringe filter unit, dismic-3cp cellulose acetate,
ADVANTEC, Tokyo, J apan), and 10 µL of the filtrates was injected
onto the HPLC system. The resultant filtrate was loaded onto a
reversed-phase HPLC column (Supelcosil LC-18-DB, SUPELCO). The
solvent delivery system (L-5000 LC controller and 655A-11 pump,
Hitachi, Tokyo, J apan) was equipped with an electrochemical detector
(ECD-100, EICOM, Kyoto, J apan) at +0.7 V vs an Ag-AgCl reference
electrode with an auto sampler (AS-8010, Tosoh, Tokyo, J apan) and
with a chromatointegrator (D-2500, Hitachi). A guard column (Su-
pelcosil LC-18-DB, SUPELCO) was placed between the autosampler
and the analytical column. The mobile phase was 0.01 M citrate
buffer (pH 4.4)-MeOH (90:10 v/v) containing 10 µM 2Na EDTA and
0.5 mM sodium 1-octanesulfonate, and the flow rate was 1.0 mL/min.
Retention times were 9, 24, 27, and 45 min for DOPAC, HVA, DA,
and internal standard, respectively. To test the linearity of the
calibration graph, various amounts of DA, DOPAC, and HVA ranging
from 25 to 1000 ng for the striatum DA, DOPAC, and HVA concentra-
tions were prepared. Linear relationships were obtained and the
regression lines for the striatum DA, DOPAC, and HVA concentra-
tions were y ) 0.001577x + 0.002426 (γ ) 0.9989), y ) 0.001326x +
Assa y Meth od s for P EA Con cen tr a tion in P la sm a a n d th e
Str ia tu m sThe PEA concentration in plasma and the striatum was
determined by a modification of the GC-MS assay of Kataoka et al.20
with 3-phenyl-1-propylamine (Aldrich Chemicals, Milwaukee, WI) as
the internal standard. For the plasma samples, 0.1 mL of the plasma
sample was pipetted into a glass tube. To the glass tube was added
0.1 mL of the internal standard solution (3-phenyl-1-propylamine, 500
ng) and 0.05 mL of 50% potassium hydroxide; all were mixed well.
The mixture was extracted with 3 mL of n-hexane and centrifuged
at room temperature at 3000 rpm for 3 min. The aqueous layer was
frozen in a chilled methanol bath using a cooler (Eyela Cool ECS-50,
Tokyo Rikakikai, Tokyo, J apan). The upper organic layer was then
decanted into another glass tube. The organic solvent was evaporated
to dryness. To the glass tube containing this residue were added 0.3
mL of water, 0.05 mL of 50% potassium hydroxide, and 0.02 mL of
benzenesulfonyl chloride (BSC, Wako) to convert PEA to its N-
benzenesulfonamide derivative. The mixture was then shaken at 300
rpm (up and down) for 15 min at room temperature. The reaction
mixture was extracted with 3 mL of n-hexane to remove excess regent
and the N-benzenesulfonamide derivative of secondary amines. After
the hexane extract was discarded, 0.05 mL of 65% potassium
hydroxide solution containing 30% methanol was added to the
aqueous layer. The aqueous layer was also extracted with 3 mL of
n-hexane to remove excess regent and N-benzenesulfonamide deriva-
tive of secondary amines. After the hexane extract was discarded,
0.5 mL of 15% hydrochloric acid was added to the aqueous layer, and
the mixture was extracted with 3 mL of n-hexane. The aqueous layer
was frozen in a chilled methanol bath using the cooler. The upper
organic layer was then decanted into another glass tube. The organic
solvent was evaporated to dryness, and the residue was dissolved in
0.02 mL of ethyl acetate. One microliter of the ethyl acetate solution
was injected into the GC-MS system. For the striatum samples, the
remaining striatum filtrate samples which included the internal
standard (3-phenyl-1-propylamine, 500 ng) and were used for DA,
DOPAC and HVA concentrations (the preparation methods for these
filtrate samples are described next) were employed. The extraction
and reaction of these striatum filtrate samples were performed by
the same procedure as described above.
Mass fragmentography was performed on a Hewlett-Packard 5890
series II gas chromatograph, a 5971 A mass selective detector, and a
7673 autoinjector. The instrument was operated in the electron
impact (EI) mode with the interface and ion source at 300 and 280
°C, respectively. The chromatographic column was
a capillary
column, DB-1301 (cross-linked, J & W Scientific) 15 m × 0.25 mm
488 / Journal of Pharmaceutical Sciences
Vol. 86, No. 4, April 1997

Table 1sList of Symbols and Definitions
DA
Dopamine
3,4-Dihydroxyphenylacetic acid
Homovanillic acid
Monoamine oxidase-B
Phenylacetic acid
DOPAC
HVA
MAO-B
PAA
PEA
CDO
CDO0
CHV
CHV0
CPA
CPA0
CPE
CPL
â
(
(
(
(
(
(
(
µ
µ
µ
µ
µ
µ
(mg/L) Plasma PEA concentration
CLDE
CLDH
CLm
(mL/h) Elimination (conversion) clearance from DA to DA metabolites except DOPAC and HVA in striatum, CLDE ) kDEVst
(mL/h) Metabolism clearance from DA to HVA in striatum, CLDH
) kDHVst
(mL/h) Apparent metabolism clearance from PEA to PAA in striatum, CLm
)
koutVst
−
PA
EKm(PE)
EVmax(PE)
EVmax(PE)
INFDO
KI(PE)
Km(DO)
Km(HV)
Km(PA)
Km(PL)
kDE
(
[(
µ
g/g) Striatum PEA concentration at half-maximum elimination rate of PEA in striatum
g/g)/h] Maximum elimination rate of PEA in striatum, EVmax(PE) EVmax(PE)/Vst
EVmax(PE)′Vst
g/g)/h] Zero-order production rate of endogenous DA in striatum, INFDO
µ
)
′
′
(
[(
µ
g/h) Maximum elimination rate of PEA in striatum, EVmax(PE)
µ
)
)
k0DO/Vst
(
(
(
(
µg/g) Inhibitor constant of PEA for metabolism from DA to DOPAC in striatum
µ
µ
µ
g/g) Striatum DA concentration at half-maximum metabolism rate from DA to DOPAC in striatum
g/g) Striatum HVA concentrations at half-maximum elimination rate of HVA in striatum
g/g) Striatum DOPAC concentration at half-maximum elimination rate of DOPAC in striatum
(mg/kg) Amount of PEA in plasma compartment at half maximum elimination rate of PEA
(h-1) Elimination (conversion) constant from DA to DA metabolites except DOPAC and HVA in striatum, kDE
) CLDE/Vst
kDH
kin
kout
(h-1) Metabolism constant from DA to HVA in striatum, kDH
)
CLDH/Vst
(h-1) Apparent first-order constant of PEA from plasma to striatum, kin
(h-1) Apparent first-order constant of PEA from striatum to outside striatum including the apparent metabolism constant from PEA to PAA,
kout (PA CLm)/Vst
g/h) Zero-order production rate of endogenous DA in striatum, k0DO
(h-1) Intercompartmental transfer rate constant
(h-1) Apparent diffusion constant of PEA between plasma and striatum, PA′ ) PA/Vst
(mL/h) Apparent diffusion clearance of PEA between plasma and striatum, PA PA Vst
g/g) Plasma PEA concentration at half-maximum transport rate of PEA from plasma to striatum
g/g)/h] Maximum transport rate of PEA from plasma to striatum, PVmax(PE) PVmax(PE)/Vst
PVmax(PE)′Vst
) PA/Vst
)
+
k0DO
k12, k21
(µ
) INFDOVst
PA
PA
′
)
′
PKm(PE)
PVmax(PE)
PVmax(PE)
t
Vd
Vst
(µ
[(
µ
)
′
′
(µg/h) Maximum transport rate of PEA from plasma to striatum, PVmax(PE) )
(h) Time after PEA administration
(L/kg) Apparent volume distribution of PEA in plasma
(g) Striatum weight
Vmax(DO)
Vmax(DO)
µ
g/g)/h] Maximum metabolism rate from DA to DOPAC in striatum, Vmax(DO)
)
Vmax(DO)/Vst
Vmax(DO)′Vst
Vmax(HV)/Vst
Vmax(HV)′Vst
g/g)/h] Maximum elimination rate of DOPAC in striatum, Vmax(PA) Vmax(PA)/Vst
Vmax(PA)′Vst
′
′
g/h) Maximum metabolism rate from DA to DOPAC in striatum, Vmax(DO)
)
Vmax(HV)
g/g)/h] Maximum elimination rate of HVA in striatum, Vmax(HV)
)
′
′
Vmax(HV)
g/h) Maximum elimination rate of HVA in striatum, Vmax(HV)
)
Vmax(PA)
)
′
′
Vmax(PA)
Vmax(PL)
X1
g/h) Maximum elimination rate of DOPAC in striatum, Vmax(PA)
)
X2
dX2
dt
0.009285 (γ ) 0.9983) and y ) 0.001383x + 0.014338 (γ ) 0.9956),
respectively, where y is the peak-area ratio and x is the amount of
DA, DOPAC, or HVA. The coefficient of variation was 3% or less in
these concentration ranges for calibration graphs. Detection limits
of sensitivity to these substrates (DA, DOPAC, and HVA) based on
the signal to noise ratio of 3 were determined by injection of diluted
standard solutions. The detection limits of the assay for DA, DOPAC,
and HVA amounts were about 2.5, 2.5, and 2.5 ng, respectively.
P h a r m a cok in etic An a lysis for P EA Con cen tr a tion in P la sm a
a n d th e Str ia tu m sIn order to quantitatively describe the time
course of PEA concentration in plasma and the striatum after PEA
injection, a pharmacokinetic model for PEA was constructed and is
shown in Figure 1. It has been well-recognized that PEA is rapidly
and almost quantitatively metabolized to PAA by MAO-B.6 The time
course of PEA concentration in plasma (CPL) after iv administration
of PEA was fitted to a two-compartment model with Michaelis-Menten
type elimination kinetics as follows.
) k12X1 - k21X2
CPL ) X1/Vd
(2)
(3)
Definitions of the symbols used in all the equations are listed in Table
1. It has been reported that (1) PEA is a lipophilic compound that
passes readily through the blood-brain barrier2 and has a hetero-
geneous distribution in the mammalian brain in spite of the low
endogenous PEA concentration in the brain,3 (2) PEA is synthesized
by the decarboxylation of phenylalanine and this synthesis rate is
quite low,4 and (3) PEA is metabolized via oxidative deamination to
PAA by MAO-B very quickly and the turnover rate of PEA in the
brain is extremely rapid.5,6 Therefore, in this analysis, the time course
of the striatum PEA concentration was analyzed using a diffusion-
limited model including a Michaelis-Menten type transport system
from plasma to the striatum and nonlinear elimination from the
striatum (Figure 1). The time course of the PEA concentration in
the striatum (CPE) can thus be expressed as follows.
Vmax(PL)
dX1
dt
) k21X2 - k12
+
X1
(1)
(
)
Km(PL) + X1
Journal of Pharmaceutical Sciences / 489
Vol. 86, No. 4, April 1997

assumed to be complete when the iteration for the relative change in
the sum of weighted squares was less than 10-6 26
PVmax(PE)CPL
EVmax(PE)CPE
dCPE
dt
Vst
) PA(CPL - CPE) +
-
(4)
.
PKm(PE) + CPL EKm(PE) + CPE
P h a r m a cod yn a m ic An a lysis of th e Effect of P EA on th e
Str ia tu m DA, DOP AC, a n d HVA Con cen tr a tion ssIn order to
determine whether the quantitative relationship between PEA con-
centration and the increase in DA concentration in rat striatum is
present after PEA injection and to examine whether the time courses
of the DOPAC and HVA concentrations in the striatum after PEA
injection can be explained by the above model’s assumptions on the
effect of PEA on the disposition of catecholamines in the striatum,
the DA metabolism model17 was applied, as shown in Figure 2. It
has been reported that (1) MAO exists on the outer surface of
mitochondria within the DA neuronal terminals22 and the reuptake
of DA into DA neuronal terminals is performed by the carrier-
mediated transport system,23,24 (2) PEA can inhibit the reuptake of
DA into DA neuronal terminals and stimulate the release of radio-
labeled DA from slices of rat striatum,7,8 (3) PEA is a typical substrate
for MAO-B,9 and (4) the effect of PEA on the release of DA and the
reduction of DOPAC in rat striatum slices is attributed to the
competitive inhibition of PEA on the reuptake of DA into DA neuronal
terminals and the inhibition of MAO-B by PEA.10 Therefore, in the
present analysis using the DA metabolism model, it was assumed
that (1) the metabolism from DA to DOPAC can be explained by
Michaelis-Menten type kinetics, (2) the enhancement of DA concen-
tration in the striatum after PEA injection is caused by the competi-
tive inhibition of PEA on the reuptake of DA into DA neuronal
terminals, and (3) the relationship between the alteration of the
striatum DA concentration and the striatum PEA concentration can
be explained by the competitive inhibition equation. The changes of
the striatum DA, DOPAC, and HVA concentrations (CDO, CPA, and
Results and Discussion
P EA Con cen tr a tion in P la sm a a n d th e Str ia tu m sPEA
is an endogenous compound. It has been reported that the
basal endogenous plasma PEA concentration in normal
volunteers and the basal rat striatal PEA concentration were
335 ( 255 pg/mL and 2.89 ( 1.03 ng/g, respectively.27
However, in the present study, the control concentration of
PEA in plasma and the striatum before PEA injection could
not be determined by GC-MS. The quantitative limit of PEA
concentration in plasma and the striatum by GC-MS were
about 1 ng/mL and 10 ng/g, respectively. In the analysis of
the alterations of the striatum DA concentrations after PEA
injection, the competitive inhibition equation was applied (eqs
5 and 6). The striatum PEA concentration (CPE) in eqs 5 and
6 is the difference of the striatum PEA concentration between
the control value and the enhancement value. The maximum
PEA concentrations in plasma and the striatum after PEA
injection were about 20 µg/mL and 100 µg/g, respectively, and
these concentration values were extremely high compared
with the control values. Therefore, in this study, the control
values of plasma and the striatum PEA concentrations before
PEA injection were ignored.
P h a r m a cok in et ic An a lysis of P la sm a P E A Con cen -
tr a tion sThe time courses of plasma PEA concentration after
the iv administration of PEA 10, 25, 50, and 75 mg/kg in the
rats are shown in Figure 3a. The data are plotted semiloga-
rithmically as a function of time. The disappearance of the
plasma PEA concentration seemed to follow the nonlinear
elimination kinetics. In order to confirm whether the disap-
pearance of the plasma PEA concentration can be explained
by the nonlinear elimination kinetics, the values of the dose-
normalized area under the curve (AUC) of the plasma PEA
concentration (AUC/DOSE) were calculated. The values of
C
HV) after PEA injection can be expressed by the following equations.
V
max(DO)CDO
Km(DO)(1 + CPE/KI(PE)) + CDO
CLDHCDO - CLDECDO (5)
dCDO
dt
Vst
) k0DO
-
-
V
max(DO)CDO
V
max(PA)CPA
dCPA
Vst
)
-
(6)
(7)
dt
Km(PA) + CPA
Km(DO)(1 + CPE/KI(PE)) + CDO
AUC/DOSE at 10, 25, 50, and 75 mg/kg iv were 8.43 × 10-3
,
29.30 × 10-3, 65.71 × 10-3 and 77.68 × 10-3 ((µg/mL)h)/(mg/
kg), respectively. The values of AUC/DOSE were increased
with the increase in the dose of PEA. These results indicated
that the assumption of the nonlinear elimination kinetics is
necessary to explain the pharmacokinetic behavior of the
plasma PEA concentration after PEA injection. In the case
of the lowest dose (10 mg/kg), the disappearance of plasma
PEA concentration followed a two-exponential curve. There-
fore, in this analysis, the two-compartment model with the
nonlinear elimination kinetics was used to explain the plasma
PEA concentration after PEA injection (Figure 1). The solid
lines in Figure 3a represent the calculated values. The
pharmacokinetics of PEA in plasma could be described
quantitatively using this model. The pharmacokinetic pa-
rameters (k12, k21, Vmax(PL), Km(PL), and Vd) were computed by
the nonlinear least squares method and are listed in Table 2.
V
max(HV)CHV
dC
Vst HV ) CLDHCDO
-
dt
Km(HV) + CHV
At the steady state before PEA administration, the left sides of eqs
5-7 equal zero. Rearrangement of eqs 5, 6, and 7 yield the following
eqs 8, 9, and 10, respectively.
V
max(DO)′CDO0
INFDO
)
+ kDHCDO0 + kDECDO0
(8)
(9)
Km(DO) + CDO0
Vmax(DO)′CDO0(Km(PA) + CPA0)
Vmax(PA)′
)
(Km(DO) + CDO0)CPA0
k
DHCDO0(Km(HV) + CHV0)
Vmax(HV)′
)
(10)
CHV0
P h a r m a cok in etic An a lysis of th e Str ia tu m P EA Con -
cen tr a tion sThe time course of PEA concentration in the
striatum after iv administration of PEA (25 and 50 mg/kg) is
shown in Figure 3b. The striatum PEA concentration was
about 10 times higher than the plasma PEA concentration.
First, the time course of the striatum PEA concentration was
analyzed using the hybrid model28 in which the striatum
compartment is independently connected with the plasma
compartment by the apparent diffusion clearance. The changes
in the striatum PEA concentration can be expressed as follows.
The value of the production rate constant of endogenous DA concen-
tration in the striatum (INFDO) and the value of the maximum
elimination rate of DOPAC [Vmax(PA)′] and HVA [Vmax(HV)′] for nonlinear
elimination kinetics can be calculated by these equations (eqs 8, 9,
and 10, respectively).
Lea st Squ a r es Mod el Ad a p ta tion sIn order to estimate the
pharmacokinetic and pharmacodynamic parameters of PEA, the data
on the concentrations of PEA (in plasma and the striatum), DA,
DOPAC, and HVA (in the striatum) after the iv administration of
PEA were fitted to eqs 1-10 by a nonlinear least squares regression
program, FKDM,25 using a digital computer (PC-9821 Ae, NEC,
Tokyo, J apan). The inverse value of each datum was used as the
weighting value of the least squares method. Convergency was
dCPE
Vst
) PACPL - PACPE - CLmCPE
(11)
dt
490 / Journal of Pharmaceutical Sciences
Vol. 86, No. 4, April 1997

Figure 2
s
Schematic representation of pharmacodynamic models for PEA in DA, DOPAC, and HVA concentrations in rat striatum.
Figure 3
)
s
b
Time course of PEA concentrations in rat plasma and the striatum after the intravenous administration of PEA: (a) plasma PEA concentration:
0
, 75 mg/kg
(n 3);
, 50 mg/kg (n
)
3);
O, 25 mg/kg (n
)
3); and
9, 10 mg/kg (n
)
3). (b) the striatum PEA concentration:
b, 50 mg/kg (n
)
3) and
O
, 25 mg/kg (n )
3). The plotted points represent the observed data. The plasma PEA concentrations at 50 and 60 min (25 mg/kg) and at 40, 50, and 60 min (10 mg/kg) could not
be detected by GC MS. The striatum PEA concentration at 60 min (50 mg/kg) could not be detected by GC MS. The solid lines represent the calculated values
using eqs 1 4 in the text. The dotted lines represent the calculated values using eq 12 in the text. Each experimental point is shown as the mean SD.
−
−
−
±
Equation 11 was simplified and the following equation was
given:
Karoum et al. reported that the administration of deuter-
ated PEA leads to an unexpected elevation in brain endog-
enous PEA concentration, and they speculated that the
enhancement of brain endogenous PEA concentration may be
the result of competitive inhibition of endogenous PEA
metabolism by the deuterated PEA.31 These results suggested
that the separated pharmacokinetic analyses for exogenous
and endogenous PEA concentration in the striatum are
necessary to explain the time course of the striatum PEA
concentration after PEA injection. However, the exogenous
and endogenous PEA concentration in the striatum were not
measured separately in the present study. Therefore, the
Michaelis-Menten type transport system from plasma to the
striatum was assumed to explain the time course of the
striatum PEA concentration after PEA injection (eq 4).
Moreover, it has been demonstrated that the oxidative deami-
nation of PEA by MAO-B is the main route of catabolism of
PEA in rat brain.5,6 Therefore, in the present analysis, it was
dCPE
) kinCPL - koutCPE
(12)
dt
The dotted lines in Figure 3b represent the calculated values
using eq 12. The time course of the striatum PEA concentra-
tion could not be described by this equation. The estimated
values of kin and kout were 584.62 ( 392.98 h-1 and 71.13 (
48.26 h-1, respectively. The value of kin (PA/Vst) was greater
than the value of kout((PA + CLm)/Vst). Moreover, the value
of kin was about 16 times greater than the value of the serum
flow rate constant to the brain29 (QBR′ ) 37.2 h-1: QBR′ ) QBR
BR; QBR ) 64.728 mL/h, VBR ) 1.74 mL30). These results
/
V
indicated that the time course of the striatum PEA concentra-
tion after PEA injection cannot be described by the general
diffusion-limited model.
Journal of Pharmaceutical Sciences / 491
Vol. 86, No. 4, April 1997

Table 2sValues of Pharmacokinetic Parameters of PEA in Rats, Obtained
from Computer Fitting of Plasma and the Striatum Concentration Data
after Intravenous Administration of PEA (10, 25, 50, and 75 mg/kg)
calculated by eq 8 and the values of these parameters are
listed in Table 3.
The endogenous DA, DOPAC, and HVA concentrations in
the striatum before PEA injection were 6.18 ( 0.13, 2.23 (
0.25, and 0.59 ( 0.04 µg/g (mean ( SD), respectively, and
these values are consistent with the report of the previous
L-dopa study17 (DA, 5.9 ( 0.7 µg/g; DOPAC, 3.6 ( 0.4 µg/g;
and HVA, 1.0 ( 0.2 µg/g). The values of the parameters for
the production rate constant of endogenous DA concentration
in the striatum (INFDO), and the maximum elimination rate
of DOPAC [Vmax(PA)′] and HVA [Vmax(HV)′] can be calculated by
eqs 8, 9, and 10, respectively. Since the values of these
parameters [INFDO, Vmax(PA)′, and Vmax(HV)′] contain the values
of the endogenous DA, DOPAC, and HVA concentrations
(CDO0, CPA0, and CHV0), the values of these parameters are
1
k21 (h-
)
)
5.91
8.62
97.39
1.39
2.97
45.94
10213
10.97
±
±
±
±
±
3.93a
4.53a
1
k12 (h-
Vmax(PL) [(mg/kg)/h]
Km(PL) (mg/kg)
10.59a
0.87a
Vd (L/kg)
0.47a
1
PA
′
(h-
)
′
±
17.81a
PVmax(PE)
PKm(PE)
EVmax(PE)
EKm(PE)
Vst (g)
PA (mL/h)
PVmax(PE)
EVmax(PE)
(
µ
g/g)/h
±
4075a (84.3
×
×
10-3 M/h)b
10-6 M)b
(
µ
′
g/g)
±
±
13.12a (90.5
(
µ
g/g)/h
1037
1.45
1396a (8.6
×
×
10-3 M/h)b
10-6 M)b
(
µ
g/g)
±
3.47a (12.0
0.082c
3.77d
(
µ
g/h)
g/h)
837.5d
85.0d
changed by the alterations of the alues of CDO0, CPA0, and CHV0
.
(µ
Therefore, in the present study, the values of CDO0, CPA0, and
CHV0 were used with the obtained values of the L-dopa study.
The values of DA, DOPAC, and HVA concentrations in
Figures 4 and 5 were the recalculated values using the control
values from the L-dopa study and the enhanced values of the
a The pharmacokinetic parameters of PEA in plasma and the striatum were
estimated by fitting the data to eqs 1 4 using the computer program FKDM. All
values are expressed as the mean
SD of the estimated parameter. b The values
of these parameters were expressed using molar concentrations. c The value of
this parameter was fixed to the value obtained from the
-dopa study.17 d The
values of these parameters were calculated by the following equations: PA
PA Vst, PVmax(PE) PVmax(PE)′Vst, and EVmax(PE) EVmax(PE)′Vst.
−
±
L
present study. In Table 3, although the values of kDH, Km(PA)
,
)
K
m(HV), Vmax(HV)′, CLDH, and Vmax(HV) are the same as the values
′
)
)
of the L-dopa study, the values of INFDO, Vmax(PA)′, k0DO
,
V
max(DO), and Vmax(PA) are different from those of the L-dopa
assumed that the disappearance of the striatum PEA concen-
tration can be explained by the nonlinear elimination kinetics
(eq 4). The analysis of the striatum PEA concentration was
performed using eq 4. The solid lines in Figure 3b represent
the calculated values. The time course of the striatum PEA
concentration after PEA injection can be described by the
constructed pharmacokinetic model for PEA (Figure 1). The
study. These differences were dependent on the differences
(modifications) of the DA metabolism model in the L-dopa
study and the PEA study.
In the L-dopa study,17 in order to construct a simple model
for DA metabolism and to obtain the stable values of the
parameters for DA and DOPAC, it was assumed that the
metabolism from DA to DOPAC can be explained by the first-
order clearance term. In contrast, in the PEA study, it was
assumed that the metabolism from DA to DOPAC can be
explained by Michaelis-Menten type kinetics and that the
inhibition of PEA on the reuptake of DA into DA neuronal
terminals can be explained by the competitive inhibition
equation (eqs 5 and 6). These assumptions were based on the
pharmacological mechanism of the effect of PEA on the DA
disposition in the central nervous system. Moreover, in the
L-dopa study,17 the elimination (conversion) parameter from
DA to DA metabolites except DOPAC and HVA (CLDE) was
not assumed. In order to confirm whether the time course of
the striatum DA concentration after PEA injection can be
explained without this conversion parameter (CLDE), the
analysis was performed using the following equation (eq 13).
pharmacokinetic parameters [PA, PVmax(PE), PKm(PE), EVmax(PE)
,
and EKm(PE)] were computed by the nonlinear least squares
method and are listed in Table 2. Although the constructed
pharmacokinetic model for the striatum PEA concentration
in the present study is not a unique model for explaining the
kinetic behavior of PEA in the striatum, this pharmacokinetic
model was a useful compartment model for explaining the
disposition of PEA in the striatum after PEA injection. The
estimated value of the apparent diffusion constant between
plasma and the striatum (PA′) was 45.94 h-1, as shown in
Table 2. The value of PA′ was almost same as the value of
the serum flow rate constant to the brain (QBR′ ) 37.2 h-1).
This result indicated that the time course of the striatum PEA
concentration after PEA injection can be described using the
serum flow rate to the brain.
P h a r m a cod yn a m ic An a lysis of th e Effect of P EA on
th e Str ia tu m DA Con cen tr a tion sThe time course of the
striatum DA concentration before and after the iv administra-
tion of PEA 25 and 50 mg/kg is shown in Figure 4. The DA
concentration in the striatum increased immediately after
PEA injection, with the peak concentration (8.0 ( 0.3 µg/g)
occurring at 2 min; then it returned to the premedication level
until 45 min at 50 mg/kg dosing. In order to determine
whether the quantitative relationship between the striatum
PEA concentration and the increase in the striatum DA
concentration is present after PEA injection, the DA metabo-
lism model17 was applied (Figure 2) and the analyses were
performed using eqs 5-10. The solid lines in Figure 4
represent the calculated values. The time course of the
striatum DA concentration after PEA injection was reasonably
well-described using this constructed DA metabolism model.
Thus, it was clarified that the quantitative relationship
between the striatum PEA concentration and the enhance-
ment of the striatum DA concentration is present after PEA
Vmax(DO)CDO
dCDO
dt
Vst
) k0DO
-
-
Km(DO)(1 + CPE/KI(PE)) + CDO
CLDHCDO (13)
However, the calculation was unsuccessful; the parameters
for DA concentration could not be estimated by this equation.
This result indicated that the elimination parameter (CLDE
is necessary to describe the time course of the striatum DA
concentration after PEA injection.
Horn examined the effect of DA and PEA on the inhibition
of [3H]DA uptake into the rat corpus striatum homogenate.32
The values of IC50 (which represent the concentration of
inhibitor required to produce a 50% inhibition of the uptake
of [3H]DA) of DA and PEA were 3.5 × 10-7 and 1.4 × 10-6 M,
respectively. In order to compare these values with the data
obtained in the present study, the values of IC50 of DA and
PEA were calculated using the obtained values of Km(DO) and
)
injection. The parameters [KI(PE), Vmax(DO)′, Km(DO), and kDE
]
KI(PE) (which represent the striatum DA concentration at half
for PEA and DA were computed by the nonlinear least squares
method. The parameter kDH was fixed to the obtained values
of the L-dopa study,17 and the other parameter (INFDO) was
the maximum metabolism rate from DA to DOPAC and the
inhibitor constant (concentration) of PEA for the metabolism
from DA to DOPAC) in this analysis. The calculated values
492 / Journal of Pharmaceutical Sciences
Vol. 86, No. 4, April 1997

Figure 4
3); , DA concentration;
using the values of the parameters which were obtained in the
values of the parameters which were estimated by the nonlinear least squares method. Each experimental point is shown as the mean
s
Time courses of DA and DOPAC concentration in the striatum before and after intravenous administration of PEA: (a) 50 mg/kg (n
, DOPAC concentration. The plotted points represent the observed data. The solid lines represent the calculated values by eqs 5
-dopa study17 in the text. The dotted lines represent the calculated values by eqs 5
10 using the
SD.
)
3); (b) 25 mg/kg (n
)
b
O
−
10
L
−
±
Figure 5
plotted points represent the observed data. The solid lines represent the calculated values by eqs 5
the 10 using the values of the parameters which were estimated by the nonlinear
-dopa study17 in the text. The dotted lines represent the calculated values by eqs 5
least squares method. Each experimental point is shown as the mean SD.
s
Time course of HVA concentration in the striatum before and after intravenous administration of PEA: (a) 50 mg/kg (n
) 3), (b) 25 mg/kg (n ) 3). The
−
10 using the values of the parameters which were obtained in
L
−
±
of IC50 of DA and PEA were 0.14 × 10-9 and 1.80 × 10-9 M,
and HVA concentration before and after PEA injection are
shown in Figures 4 and 5, respectively. The striatum DOPAC
concentration after PEA injection (50 mg/kg) decreased im-
mediately and recovered to the premedication level 45 min
later. The HVA concentration in the striatum increased
gradually after the PEA injection. In order to determine
whether the time courses of DOPAC and HVA concentrations
after PEA injection can be described by this DA metabolism
model, the analyses were performed using eqs 5-10. The solid
respectively. These calculated values of IC50 [Km(DO) and KI(PE)
]
of DA and PEA were consistent with the values of IC50 of DA
and PEA in Horn’s study. These results indicated that the
calculation of the IC50 values using this DA metabolism model
might be a useful method for the evaluation of the inhibitor’s
ability on the DA uptake into the DA neuronal terminals.
Effect of P EA on th e DOP AC a n d HVA Con cen tr a tion
in th e Str ia tu m sThe time courses of the striatum DOPAC
Journal of Pharmaceutical Sciences / 493
Vol. 86, No. 4, April 1997

Table 3
sValues of Parameters for PEA, DA, DOPAC, and HVA in Rats, Obtained from Computer Fitting and Calculating of Striatum Concentration Data
after Intravenous Administration of PEA (25 and 50 mg/kg)
Characteristics
Fixeda
10-6 M)e
Estimatedb
L
-Dopa Studyc
CDO0
CPA0
CHV0
(µg/g)
5.9d (38.5
3.6d (21.4
×
×
5.9d
3.6d
1.0d
1506
5.9
3.6
1.0
(kDP
s
s
−
2.128
3.002
0.255
(
(
µ
g/g)
10-6 M)e
µ
g/g)
×
10-6 M)e
1
g
Vmax(DO) [(
Km(DO)
µ
g/g)/h]
g/g)
g/g)
36.86f (0.81
×
×
×
10-3 M/h)e
±
3872f (9.8
×
10-3 M/h)e
) )
17.56 h-
′
3
3
(
µ
±
±
0.126
1.290
10- f (0.14
×
×
10-9 M)e
10-9 M)e
0.0012
±
0.0049f (7.8
×
10-9 M)e
10-6 M)e
3
3
KI(PE)
(
µ
10- f (1.8
0.305
37.01
1.116
0.062
0.116
±
1.459f (2.5
×
kDE hr-
±
±
±
±
13.03f
0.951f
1
kDH hr-
1
Km(PA)
Km(HV)
INFDO
Vmax(PA)
Vmax(HV)
k0DO (µ
CLDE (mL/h)
CLDH (mL/h)
µ
g/g
g/g
g/g)/hr
g/g)/hr
g/g)/hr
g/h)
×
×
×
×
10-6 M)e
10-6 M)e
0.170f (0.37
0.396f (0.64
×
10-6 M)e
×
10-6 M)e
µ
(µ
10-3 M/h)e
10-3 M/h)e
1733h (11.3
1532h (9.11
×
×
10-3 M/h)e
10-3 M/h)e
116.67 (0.761
×
10-3 M/hr)e
×
10-3 M/hr)e
(
(
µ
192.41 (1.14
15.915
9.567
′
µ
×
10-6 M/h)e
7.421h (40.74
142.1i
×
10-6 M/h)e
′
3.035i
−
0.174
(CLDP
15.78
1.305
0.0915
123.54i
125.67i
0.609i
Vmax(DO)
Vmax(PA)
Vmax(HV)
(
µ
g/h)
g/h)
)
1.44 mL/h)g
(µ
(µg/h)
SS valuesi
DA (50 mg/kg)
DA (25 mg/kg)
DOPAC (50 mg/kg)
DOPAC (25 mg/kg)
HVA (50 mg/kg)
HVA (25 mg/kg)
Total SS
1.611
0.448
3.772
1.151
0.410
0.085
7.477
84.45
1.651
0.251
2.935
1.448
0.164
0.218
6.668
86.10
−
−
−
−
−
−
−
−
AIC
a The values of the parameters [kDH, Km(HV), and Km(PA)] for HVA and DOPAC were fixed to the obtained values of the
[kDH, Km(HV), and Km(PA)] for HVA and DOPAC were estimated by fitting the data using FKDM. c The values of these parameters were obtained in the
d The values of these parameters were fixed to the values obtained from the -dopa study.17 e The values of these parameters were expressed using molar concentrations.
f The values of these parameters were estimated by fitting the data using the computer program FKDM. All values are expressed as the mean
SD of the estimated
parameters. g The values of these parameters were the metabolic constant (clearance) from DA to DOPAC in the -dopa study. h The values of these parameters were
calculated by eqs 8, 9, and 10 in the text, respectively. i The values of these parameters were calculated by the following equations, respectively: k0DO
INFDOVst,
CLDE kDEVst, CLDH kDHVst, Vmax(DO) Vmax(DO)′Vst, Vmax(PA) Vmax(PA)′Vst, Vmax(HV)
Vmax(HV)′Vst. j The values of sum of squares (SS) and Akaike’s information
criterion (AIC) on DA, DOPAC, and HVA data.
L
-dopa study.17 b The values of the parameters
L
-dopa study.17
L
±
L
)
)
)
)
)
)
lines in Figures 4 and 5 represent the calculated values. The
time courses of the striatum DOPAC concentration at 30 (50
mg/kg), 5, and 10 min (25 mg/kg) and the lag time of the
striatum HVA concentration could not be described by this
DA metabolism model. The parameters [Km(PA), kDH, and
between the striatum PEA concentration and DA, DOPAC,
and HVA concentrations was examined; these relationships
are shown in Figure 6. The solid and dotted lines represent
the calculated values of 50 and 25 mg/kg using the DA
metabolism model, respectively. The arrows on the solid lines
show the progress of time. Concerning the relationship
between the striatum DA concentration and the striatum PEA
concentration (Figure 6a), it was clarified that the anticlock-
wise hysteresis relationship between the alteration of the
striatum DA concentration (pharmacological response of PEA)
and the striatum PEA concentration is present after PEA
injection. This result indicated that the effect compartment
analysis34 or another assumption is necessary to quantita-
tively describe this anticlockwise hysteresis relationship. The
constructed DA metabolism model was able to describe the
time course of the striatum DA concentration after PEA
injection, indicating that the DA metabolism model is a useful
compartment model for the explanation of this anticlockwise
hysteresis relationship.
Figure 6b represents the relationship between the alteration
of the DOPAC concentration and the striatum PEA concen-
tration after PEA injection. In spite of the increase in the
striatum PEA concentration, the striatum DOPAC concentra-
tions were almost constant values (about 1.3 µg/g at 50 mg/
kg dosing and about 2.4 µg/g at 25 mg/kg dosing). This
response of the striatum DOPAC concentration seemed to be
an on-off response. It has been reported that the reduction
of the striatum DOPAC concentration after PEA injection is
caused by the competitive inhibition of PEA not only on the
reuptake of DA into DA neuronal terminals7,8 but also on the
K
m(HV)] for DOPAC and HVA were fixed to the obtained values
of the L-dopa study,17 and the other parameters [Vmax(PA)′ and
max(HV)′] were calculated by eqs 9 and 10; the values of these
V
parameters are listed in Table 3.
In order to examine the effects of PEA on the metabolism
rate from DA to HVA (kDH) and on the elimination rate of
DOPAC and HVA [Km(HV) and Km(PA)], and to confirm whether
the time courses of the striatum DOPAC and HVA concentra-
tions can be explained by the reestimated values of these
parameters [kDH, Km(HV), and Km(PA)], the analyses were
performed using eqs 5-10. The dotted lines in Figures 4 and
5 represent the calculated values. There was no significant
difference between the paths of the solid lines and the dotted
lines. In spite of the reestimation of these parameters, the
time courses of the striatum DOPAC and HVA concentrations
could not be explained, and there were notable differences
between the fixed and estimated values of the parameters
(Table 3). These results suggested that other assumptions
are necessary to explain the time courses of the striatum
DOPAC and HVA concentrations after PEA injection. The
values of the reestimated parameters, sum of square (SS), and
Akaike’s information criterion (AIC)33 are listed in Table 3.
In order to clarify the reasons for the differences between the
observed data and the calculated values of the striatum
DOPAC and HVA concentrations, the direct relationship
494 / Journal of Pharmaceutical Sciences
Vol. 86, No. 4, April 1997

Figure 6
s
Relationship between the striatum PEA concentration and the striatum DA, DOPAC, and HVA concentration after intravenous administration of PEA: (a)
3); (b) PEA vs DOPAC concentration (n 3); (c) PEA vs HVA concentration (n 3); , 50 mg/kg; , 25 mg/kg. The plotted points
SD. The solid (50 mg/kg) and dotted (25 mg/kg) lines represent the calculated values.
PEA vs DA concentration (n
)
)
)
b
O
represent the observed data. Each experimental point is shown as the mean
The allows on the solid lines show the progress of time.
±
metabolism from DA to DOPAC (the inhibition of PEA on
MAO-B activity) in the DA neuronal terminals.9 Moreover,
it has been well-recognized that PEA is rapidly and almost
quantitatively metabolized to PAA by MAO-B.6 Thus, the
MAO-B activity contributed to not only the metabolism from
DA to DOPAC but also the metabolism from PEA to PAA.
The discrepancy between the observed striatum DOPAC
concentrations and the calculation values using the DA
metabolism model might be caused by these complex effects
of PEA on the disposition of catecholamines in the striatum.
These results suggested that other assumptions such as the
separate evaluation of the effect of PEA on the reuptake of
DA into DA neuronal terminals and on MAO-B activity in the
DA neuronal terminals might be necessary to explain the time
course of the striatum DOPAC concentration after PEA
injection.
Conclusion
The time courses of the plasma and the striatum PEA
concentrations after PEA injection were well-explained by the
two-compartment model with nonlinear elimination kinetics
and the diffusion-limited model including a Michaelis-Menten
type transport system from plasma to the striatum and the
nonlinear elimination from the striatum, respectively. The
time courses of the striatum DA, DOPAC, and HVA concen-
trations after PEA injection were analyzed using the DA
metabolism model. The alteration of the striatum DA con-
centration after PEA injection could be described quantita-
tively by the assumption that the enhancement of the striatum
DA concentration is caused by the competitive inhibition of
PEA on the reuptake of DA into DA neuronal terminals.
However, the time courses of the striatum DOPAC and HVA
concentrations could not be described by this DA metabolism
model. From these results, the following circumstances were
clarified: (1) A quantitative relationship between PEA con-
centration and the enhancement of DA concentration in the
striatum is present after PEA injection. (2) Since there is no
quantitative relationship between the increase in DA concen-
tration and the reduction of DOPAC concentration in the
striatum, another assumption such as the separate evaluation
of the effect of PEA on the reuptake of DA into DA neuronal
terminals and on MAO-B activity in the DA neuronal termi-
nals might be necessary to explain the time course of the
striatum DOPAC concentration. (3) The assumption of the
metabolic pathway from DOPAC to HVA might be necessary
to explain the time courses of the striatum DOPAC and HVA
concentrations after PEA injection. Thus, although high doses
of PEA were used in the present study, the results obtained
contribute to the elucidation of the pharmacological mecha-
nism of PEA on the disposition of catecholamines in the
striatum.
Figure 6c represents the relationship between the alteration
of the HVA concentration and the striatum PEA concentration
after PEA injection. In spite of the increase in the striatum
PEA concentration, the striatum HVA concentrations did not
increase. This result indicated that here is no direct relation-
ship between the alteration of HVA concentration and that
of PEA concentration in the striatum. Westerink and Korf
showed that DA is predominantly metabolized to DOPAC and
that this metabolite is partly removed from the brain and
partly o-methylated to HVA.35 Westerink and Spaan dem-
onstrated that about 80% of HVA is formed from DOPAC and
20% from 3-methoxytyramine in rat striatum.36 These find-
ings indicated that the metabolic pathway from DOPAC to
HVA might be necessary to explain the time courses of the
striatum DOPAC and HVA concentrations after PEA injec-
tion. However, the ratio of the metabolism from DOPAC to
HVA was not measured in the present study. If the DA
metabolism model including the metabolism parameters from
DOPAC to HVA are constructed, this DA metabolism model
will be complicated and the estimation of these parameters
might be very difficult. Therefore, in this analysis, the
metabolism parameters from DOPAC to HVA were not
assumed.
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