- Email: [email protected]
Pharmacokinetics and toxicokinetics of d -serine in rats
Accepted Manuscript Title: Pharmacokinetics and toxicokinetics of D-serine in rats Authors: Hiroshi Hasegawa, Nami Masuda, Hiromi Natori, Yoshihiko Shinohara, Kimiyoshi Ichida PII: DOI: Reference:
S0731-7085(18)31200-7 https://doi.org/10.1016/j.jpba.2018.09.026 PBA 12219
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
23-5-2018 16-8-2018 13-9-2018
Please cite this article as: Hasegawa H, Masuda N, Natori H, Shinohara Y, Ichida K, Pharmacokinetics and toxicokinetics of D-serine in rats, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.09.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmacokinetics and toxicokinetics of D-serine in rats
Hiroshi Hasegawa, Nami Masuda, Hiromi Natori, Yoshihiko Shinohara, and Kimiyoshi Ichida
IP T
Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences
SC R
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
(Corresponding author)
A
Tokyo University of Pharmacy and Life Sciences
N
Department of Pathophysiology, School of Pharmacy,
U
Hiroshi Hasegawa, Ph.D.
(Phone) (+81)-426-76-5699
TE D
(Fax) (+81)-426-76-5686
M
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
(E-mail) [email protected]
Highlights
We investigated the relationship of D-serine kinetics with nephrotoxicity in
EP
CC
rats.
Following iv/po/ip D-serine, we measured plasma D-/L-serine with
A
GC-MS.
Histology revealed renal damage 24 after ip administration of D-serine at doses of 1.8–4.8 mmol/kg bw.
1
When Cmax of D-serine was >2 µmol/ml, plasma creatinine increased 24 h later.
Thus, Cmax of D-serine could be a good predictor of D-serine-induced
IP T
nephrotoxicity.
SC R
Abstract
In the mammalian brain, D-serine acts as a co-agonist at the glycine-binding site on
U
the N-methyl-D-aspartate receptor. Because plasma D-serine levels are significantly lower
N
in patients with schizophrenia than in healthy subjects, D-serine has been proposed as a
A
potential therapeutic agent for schizophrenia treatment. However,
D-serine
has a
nephrotoxic effect in rats at high doses. The purpose of this study was to investigate the
D-serine
D-serine
and nephrotoxicity in rats. We
intravenously (iv), orally (po), or intraperitoneally (ip) to male
TE D
administered
M
relationship between the plasma kinetics of
Wistar rats, and performed gas chromatography-mass spectrometry to measure the plasma concentrations of
D-
and L-serine. After iv administration (0.1 mmol/kg body
weight (bw)), plasma D-serine declined multiexponentially with an elimination t 1/2 of 108
was estimated to be 94 ± 27%. To evaluate the dose–response relationship of
CC
D-serine
EP
± 16 min, and the total clearance was 7.9 ± 0.9 ml/min/kg bw. The oral bioavailability of
D-serine-induced
kidney injury and the plasma kinetics of D-serine, we injected D-serine
A
into rats ip in doses ranging from 0.6 to 4.8 mmol/kg bw. Twenty-four hours after D-serine
administration, histological changes indicating renal damage were observed in
the kidneys of rats who received D-serine at doses of 1.8–4.8 mmol/kg bw; the severity of the tubular injury increased with increasing D-serine
D-serine
dose. When the Cmax value of
was approximately >2 µmol/ml, the plasma creatinine increased remarkably 24
h after D-serine administration. This suggests that the C max of D-serine could be a good
2
predictor of D-serine-induced nephrotoxicity.
Abbreviations: AUC, area under the plasma concentration–time curve; AUC0-last, AUC
from time 0 to the last point with a quantifiable plasma concentration; AUC ip; AUC after intraperitoneal administration; bw, body weight; CLtot, total plasma clearance; C max, maximal plasma concentration;
DAO,
D-Amino
acid oxidase; diMTPA-OMe,
bioavailability;
Fpo,
oral
bioavailability;
GC–MS,
gas
IP T
N,O-di-(+)-α-methoxy-α-trifluoromethylphenylacetyl methyl ester; Fip, intraperitoneal
chromatography-mass
spectrometry; ip, intraperitoneally; iv, intravenously; kabs, apparent absorption rate
SC R
constant; kel, apparent elimination rate constant; MRT, mean residence time; MTPA-Cl, (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl
chloride;
NMDA,
N-Methyl-D-aspartate; po, orally; SIM, selecting ion monitoring; t1/2, plasma elimination
N
U
half-life; tmax, time to reach C max.
1. Introduction
TE D
M
A
Keywords: D-serine; GC-MS; pharmacokinetics; toxicokinetics; nephrotoxicity.
The N-methyl-D-aspartate (NMDA) receptor, a type of glutamate receptor, plays
EP
important and indispensable roles in several higher brain functions such as learning, memory, and cognition [1]. NMDA receptor dysfunction has been hypothesized to play a
CC
role in the pathophysiology of schizophrenia [2]. D-Serine
is present in the mammalian brain [3], and serves as an endogenous
A
modulator of the glycine-binding site on NMDA receptors [4-7]. Serum D-serine levels are significantly lower in patients with schizophrenia than in healthy subjects [8, 9]. Since D-serine-mediated enhancement of NMDA receptor function may be beneficial in
3
the treatment of schizophrenia, several clinical trials have tested the effects of D-serine supplementation in schizophrenia [10-13]. In one of these trials, an oral D-serine dose of 30 mg/kg body weight (bw)/day (equivalent to 0.28 mmol/kg bw/day) significantly improved the positive, negative, and cognitive symptoms of schizophrenia. Although dose-dependent efficacy was observed at higher
doses, one subject receiving
at a dose of 120 mg/kg bw/day (1.1 mmol/kg bw/day) showed symptoms similar
IP T
D-serine
D-serine
to those associated with nephrotoxicity [11]. At the same dose, the plasma concentration D-serine
was elevated up to approximately 500 nmol/ml, which is 200 times the
SC R
of
physiological concentration.
Previous studies have revealed that that D-serine is nephrotoxic in rats. High doses of cause necrosis in renal proximal tubules, with proteinuria and glucosuria in rats
U
D-serine
D-serine
nephrotoxicity in humans remains unclear. To address
A
however, the potential
N
[14-19]. D-Serine is not nephrotoxic in mice, guinea pigs, rabbits, dogs, and hamsters;
concerns regarding the therapeutic usage of D-serine in humans, it is essential to clarify
D-serine
D-serine-induced
induces renal injury. To determine the dose dependency of
TE D
which
M
the mechanism underlying D-serine nephrotoxicity in rats and the plasma concentration at
nephrotoxicity in rats, Krug et al. [16] administered
D-serine
intraperitoneally (ip) and measured the consequent plasma concentrations of serine;
EP
however, the HPLC method they used could not separate D- from L-serine. Thus, minimal information is available on the plasma levels of D-serine that are associated with tubular D-serine
in rats
CC
necrosis in rats. In addition, no data exist on the plasma kinetics of
following oral administration; such data may provide information of D-serine kinetics in
A
humans.
Recently, we developed a method for the stereoselective determination of serine
enantiomers in biological fluids by using gas chromatography–mass spectrometry (GC-MS) with selecting ion monitoring (SIM) [20]. This method is based on a stable isotope dilution method, using stable isotope labeled
4
DL-serine
as an analytical standard,
and a diastereomeric method, using (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride (MTPA-Cl) as a chiral derivatizing reagent. The objective of the present study was to investigate the relationship between the plasma kinetics of D-serine and nephrotoxicity in rats.
IP T
2. Materials and methods
D-Serine
SC R
2.1. Chemicals
was purchased from Peptide Institute (Osaka, Japan). L-Serine was purchased
from Wako Pure Chemicals (Osaka, Japan).
DL-[2,3,3-
2
H3]Serine (DL-[2H3]serine, 98
U
atom % 2H) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA).
N
MTPA-Cl and 10% HCl in methanol were purchased from Tokyo Kasei (Tokyo, Japan).
A
A strong cation-exchange solid-phase extraction column BondElut SCX (H + form, size 1 ml/100 mg) was purchased from Agilent Technologies (Lake Forest, CA, USA).
M
Chloroform stabilized with amylene was purchased from Kanto Chemical (Tokyo, Japan).
further purification.
EP
2.2. Animals
TE D
All other chemicals and solvents were of analytical reagent grade and were used without
The experimental protocols were approved by the Institutional Animal Care
CC
Committee of Tokyo University of Pharmacy and Life Sciences (P14-57, P15-57). Male Wistar rats were purchased from SLC Japan (Hamamatsu, Japan). The rats were housed
A
in stainless-steel cages in an air-conditioned room maintained at 23 ± 1°C and 55 ± 5% humidity with a 12 h dark/light cycle. All rats were acclimated for at least 7 days prior to treatment. They were allowed free access to water and food (CE-2, Clea Japan, Tokyo, Japan), but were fasted for 16 h before dosing. Rats received D-serine dissolved in saline intravenously (iv; 0.1 mmol/kg bw) via the
5
femoral vein, orally (po; 0.6 mmol/kg bw) by gavage, or ip (0.6, 1.2, 2.4, and 4.8 mmol/kg bw). Heparinized blood samples (150 l) were obtained from the jugular vein 10 min before and 15, 20, 30, 60, 90, 120, 180, 240, 300, and 360 min after administration under anesthesia by sodium pentobarbital (50 mg/kg bw). Additional blood samples were
IP T
collected at 3, 5, and 10 min after iv and ip administration. The blood samples were centrifuged at 1000 × g for 10 min, and the plasma was stored at -80°C until analysis.
SC R
Rats administered D-serine ip were placed in individual metabolic cages and urine
was collected 6–24 h after D-serine administration. The urine was stored at -80°C until analysis. After urine collection, the rats were killed by exsanguination under anesthesia
U
by sodium pentobarbital. Blood samples from the heart were collected into heparinized
N
tubes, and the plasma was separated by centrifugation at 1000 × g for 10 min. The
A
plasma samples were submitted for biochemical analysis, and the right kidney was
TE D
2.3. Sample preparation for GC-MS
M
removed to evaluate the damage histologically.
Plasma concentrations of
D-
and L-serine were determined by the isotope dilution
method as described previously [20]. Briefly,
DL-[
2
H3]serine (9.98 nmol in 50 l) was
EP
added to plasma samples (10 or 20 l) as an analytical internal standard. The plasma sample was deproteinized with 10% trichloroacetic acid, purified by cation exchange
CC
chromatography using BondElut SCX cartridge, and derivatized with hydrochloric acid in methanol to form methyl ester (-OMe), followed by subsequent N,O-diacylation with
A
MTPA-Cl to form N,O-di-(+)-α-methoxy-α-trifluoromethylphenylacetyl methyl ester (diMTPA-OMe) derivative. All samples were then subjected to GC-MS-SIM analysis.
2.4. GC-MS-SIM GC-MS-SIM analysis was performed using a Shimadzu (Kyoto, Japan) QP2010
6
quadrupole GC-MS equipped with a data-processing system. The operating conditions were the same as those described in a previous publication [20]. Briefly, the GC column was a methylsilicone bonded-phase fused-silica capillary column SPB-1 (15 m x 0.25 mm i.d., 0.25 µm film thickness; Supelco, Bellefonte, PA, USA), and the mass spectrometer was operated in chemical ionization mode, with isobutane as the reagent
IP T
gas. SIM was performed on the quasimolecular-ions of the diMTPA-OMe derivatives of serine (m/z 552) and [2H3]serine (m/z 555). The diMTPA-OMe derivatives of
and
underwent baseline separation within 9 min and eluted in that order on GC. The
SC R
L-serine
D-
lower limit of quantitation for the method was approximately 100 fmol per injection. The intra- and inter-day precision for
D-serine
was < 5% and < 2%, respectively, and for
A
2.5. Biochemical markers against renal injury
N
U
L-serine, < 3% and < 2%, respectively.
Plasma creatinine was determined using HPLC with a cation exchange Supelcosil
M
LC-SCX column (250 x 4.6 mm i.d., particle size 5 µm; Supelco, Bellefonte, PA) under
TE D
the following conditions: mobile phase, sodium phosphate buffer (pH 5.1; 50 mM)-acetonitrile (3:2, v/v); flow rate, 1.3 ml/min; column temperature, ambient; detection, UV 234 nm. Creatinine was eluted at t R 10.4 min as a single peak.
EP
Urinary protein and glucose were measured with Nagahama Life Science Laboratory
CC
(Shiga, Japan) using Hitachi model 7180 Clinical Analyzer.
2.6. Histological analyses For light microscopy, kidney tissue samples were fixed by immersion in buffered
A
formalin (pH 7.4) and embedded in paraffin. For histological analysis, 3 m-thick sections were stained with hematoxylin and eosin, and were observed under an Olympus CX41 light microscope (× 200).
7
2.7. Data analysis Assuming that the plasma concentration of endogenous plasma concentration of administered
D-serine
D-serine
is constant, the
was estimated by subtracting the
endogenous D-serine concentration before administration from the net measurement at each point. The maximal plasma concentration (C max) and the time to reach C max (tmax)
IP T
were obtained from individual plasma concentration versus time data. Noncompartmental pharmacokinetic parameters were calculated using model-independent
analysis
SC R
performed with a macro-program MOMENT(EXCEL) [21] running on Microsoft Excel. The apparent elimination rate constant (k el) was determined from the linear regression slope of the terminal portion (last three to four quantifiable points) of the log plasma
U
concentration-time curve. Apparent terminal half-life was obtained using the following
N
formula: 0.693/k el. The apparent absorption rate constant (k abs) was determined using the
A
residual method. The area under the plasma concentration–time curve (AUC0-last) from time 0 to the last point with a quantifiable plasma concentration was calculated using the
M
trapezoidal rule. The extrapolation of AUC was based on the last plasma concentration
TE D
and the terminal slope. The total systemic plasma clearance (CL tot) was determined using the formula Dose/AUC0-∞. The oral bioavailability (F po) was calculated as follows: Fpo = (AUCpo/AUCiv) x (Doseiv/Dosepo)
EP
where AUCiv and AUC po are AUC estimates after intravenous and oral administration. Similarly, intraperitoneal bioavailability (F ip) was calculated using the following
CC
formula:
Fip = (AUCip/AUCiv) x (Doseiv/Doseip)
A
where AUCip are AUC estimates after intraperitoneal administration.
2.8. Statistical analysis Values are presented mean ± SD. Data were statistically analyzed with one-way ANOVA followed by Dunnett’s multiple comparison post hoc test using KaleidaGraph
8
version 4 (Synergy Software, Reading, PA, USA). A p-value < 0.05 was considered statistically significant.
3.1. Pharmacokinetics of D-serine after iv, po, and ip administration Following iv administration of
D-serine
IP T
3. Results
(0.1 mmol/kg bw) to rats (n = 6), we
SC R
measured the plasma concentration of D- and L-serine using the stable isotope dilution
GC-MS method described previously [20]. The representative SIM profiles obtained from the plasma samples obtained before and 5 min after administration are shown in Fig.
U
1A and 1B, respectively. No substances other than D- and L-serine, and the labeled D- and were found in their monitoring ions. The plasma concentration of endogenous
D-serine
before administration of exogenous D-serine was 1.9 ± 0.3 nmol/ml. Assuming
A
N
L-serine
that the plasma concentration of endogenous
D-serine
is constant, the plasma
concentration before administration from the net measurement at each point. Fig.
TE D
D-serine
M
concentration of the administered D-serine was estimated by subtracting the endogenous
2A shows the plasma concentration–time profiles of D-serine and L-serine. Administered D-serine
disappeared from the plasma multiexponentially, with a terminal elimination t1/2
EP
of 108 ± 16 min (Table 1). The CL tot was 7.9 ± 0.9 ml/min/kg bw. Plasma concentration of L-serine was almost constant during the 6-h period after administration.
CC
Fig. 2B shows the plasma concentration–time profiles of D-serine and L-serine after po administration of D-serine (0.6 mmol/kg bw) to rats (n = 5). Plasma concentration of
A
administered D-serine increased gradually to reach the maximal concentration (C max, 399 ± 92 nmol/ml) at 72 ± 16 min after administration, and then disappeared with an elimination t1/2 of 118 ± 13 min (Table 1), which was comparable to that for iv administration. The bioavailability (F po), estimated by dividing the AUC obtained after oral administration with the value obtained after intravenous administration, was 94 ±
9
27%. The plasma concentration of L-serine was almost constant during the 6-h period after administration. Following ip administration of D-serine (0.6 mmol/kg bw) to rats (n = 6), C max (610 ± 170 nmol/ml) at 23 ± 7 min was attained rapidly and then decreased with a t 1/2 value of 112 ± 24 min (Fig. 2C, Table 1). The bioavailability (F ip) was estimated to be 94 ± 12%. L-serine
was almost constant during the 6-h period after
IP T
Plasma concentration of
SC R
administration.
3.2. Toxicokinetics of D-serine
To evaluate the dose–response relationship of D-serine-induced kidney injury and the
U
plasma kinetics of D-serine, we injected D-serine ip into rats at doses ranging from 0.6 to D-serine
concentration. In
N
4.8 mmol/kg bw, and measured the alteration of plasma
A
addition, we determined the concentrations of plasma creatinine, and urinary protein and glucose as biomarkers for kidney function, and examined histopathological changes in
M
the kidneys.
TE D
Histopathological examination (Fig. 3) did not reveal any changes in the kidneys of rats 24 h after administration of D-serine at 0.6 and 1.2 mmol/kg bw. At doses above 1.8 mmol/kg bw, all rats showed proximal tubule injury; the severity of the tubular injury
EP
increased with increasing dose.
Following administration of
D-serine
in the range of 0.6 to 2.4 mmol/kg bw, we
CC
observed no changes in plasma creatinine during the first 6 h after administration (Table 2). At a dose of 4.8 mmol/kg bw, however, a slight but non-significant increase of
A
plasma creatinine was observed 6 h after administration (0.37 ± 0.11 mg/dl) compared to the plasma creatinine before administration of
D-serine
(0.22 ± 0.02 mg/dl). The
creatinine concentration 6 h after administration of 4.8 mmol/kg bw significantly higher than that in rats receiving 0.6 mmol/kg bw
D-serine
D-serine.
was
Plasma
creatinine increased significantly 24 h after administration of D-serine at 1.8, 2.4, and 4.8
10
mmol/kg bw compared to that after 0.6 mmol/kg bw D-serine (Table 2). There were no changes in urinary excretion of protein and glucose in rats receiving 0.6 and 1.2 mmol/kg bw
D-serine,
whereas above 1.8 mmol/kg bw their urinary biomarkers increased
proportionally with the dose. Urinary protein and glucose of rats receiving 4.8 mmol/kg bw
D-serine
increased 25- and 400-fold, respectively, as compared to that in rats
IP T
receiving 0.6 mmol/kg bw D-serine.
Fig. 4A shows the plasma concentration–time profiles of D-serine in rats administered
SC R
five different doses of D-serine ip. Although the concentration–time curves for all five
doses are apparently parallel, the CLtot (assuming a value of Fip of 0.94) at doses in the range of 1.8 to 4.8 mmol/kg bw were significantly lower than that at 0.6 mmol/kg bw
D-serine
administration, and C max and CLtot of
N
creatinine 24 h after
U
(Table 3). Fig. 4B and 4C show the relationship between plasma concentration of
administration, respectively. When the C max value of
D-serine
D-serine
after ip
was greater than
A
approximately 2 mol/ml, plasma creatinine increased significantly. In contrast, the CLtot
M
value of D-serine was not a reliable predictor of
EP
4. Discussion
nephrotoxicity (Fig.
TE D
4C).
D-serine-induced
The most important advantage of stable isotope dilution analysis using a stable
CC
isotopically labeled analog as an internal standard is that the labeled analog behaves in an almost identical manner to the analyte throughout all steps in the extraction,
A
derivatization, and chromatographic procedures. In this study, commercially available DL-[
2
H3]serine, which mimics
D-
and L-serine in plasma, was chosen as the internal
standard. Therefore, if the extraction and/or derivatization processes were incomplete, the measurement of serine species would not be affected because the 2H3-labeled internal standard would also undergo losses to the same extent that the D- and L-serine did.
11
As shown in Fig. 1, baseline separation between the peaks for the diMTPA-OMe derivatives of
D-serine
and L-serine was achieved with resolution factor of 5.09, and
eluted in that order. In addition to its selectivity, the present method also had high sensitivity. The value of lower limit of quantitation of
D-serine
with the present
GC-MS-SIM system was 100 fmol/injection (0.05 nmol/ml plasma), which made it
IP T
possible to determine the intrinsic D-serine even though the amount of D-serine is only 1% that of L-serine. Endogenous D-serine in plasma was 1.9 ± 0.3 nmol/ml, which was
D-Amino
SC R
consistent with the results of previous studies [22-25].
acid oxidase (DAO), which catalyzes the oxidative deamination of neutral
and basic D-amino acids including D-serine, is expressed in the kidney, liver, and brain in
U
rats [26]. In the present study, the po bioavailability of D-serine (94%) was quantitatively D-serine
N
almost identical to the ip bioavailability. This indicates that almost all the
pass metabolism of
D-serine
A
administered po was absorbed from the gastrointestinal tract. In addition, the low first can be due to low DAO activity in the liver. After po
M
administration of D-serine, the values of C max divided by dose (707 ± 154 g bw/l) in rats
TE D
were similar to those in mice (654 ± 45 g bw/l) [27] but higher than those in humans (477 ± 109 g bw/l) [11]. The biochemical characterization of rat DAO has shown that it differs from human DAO. The most significant difference is in substrate affinity: the K m value
EP
and the intrinsic clearance (K cat/Km) for D-serine is 40- and 20-fold lower, respectively, for rat DAO than for human DAO [28]. The lower activity of DAO in rats than in
CC
humans could explain why the plasma concentration of D-serine in rats is observed to be higher than that in humans. D-amino
acids are converted into the corresponding
L-enantiomers
via a
A
Several
two-step process. The initial step is oxidative deamination by DAO to form 2-oxo acids. In the next step, the 2-oxo acids are stereospecifically reaminated by transaminase to form L-amino acids. We have previously shown that D-leucine [29], D-methionine [30], and D-selenomethionine [31] are converted into the corresponding L-enantiomers in rats.
12
D-Serine
is also considered to be metabolized by DAO to 3-hydroxypyruvic acid,
followed by conversion into L-serine by serine-pyruvate aminotransferase. However, the transaminase activity of serine-pyruvate aminotransferase in the rat liver is lower with 3-hydroxypyruvic acid and L-alanine as substrates than with serine and pyruvic acid [32]. In addition, 3-hydroxypyruvic acid is metabolized to glycoaldehyde, which is a substrate
IP T
for glucose synthesis. If the administered D-serine is converted into L-serine, the plasma concentration of L-serine will increase transitorily. However, in the present study, plasma
SC R
concentration of L-serine remained mostly constant during the 6-h post-administration period, suggesting that chiral conversion of D-serine into the L-enantiomer does not occur in rats.
U
Orozco-Ibarra et al. [18] showed that administration of D-serine (400 mg/kg bw = 3.8
N
mmol/kg bw) induced a significant time-dependent increase in plasma creatinine. In the
A
present study, plasma creatinine tended to increase 6 h after administration of D-serine at a dose of 4.8 mmol/kg bw. Since plasma creatinine only changes after a significant loss
administration. Plasma creatinine levels of rats receiving 1.8 and 2.4 mmol/kg
TE D
D-serine
M
of kidney function, this finding suggests that kidney damage occurs within 6 h of
bw D-serine were not altered at 6 h compared to baseline levels, but the levels at 24 h were significantly higher than that after administration of 0.6 mmol/kg bw D-serine. At
EP
24 h after ip administration, D-serine at doses of 0.6 to 1.2 mmol/kg bw had no effect on the urinary excretion of protein and glucose. D-Serine at 1.8 to 4.8 mmol/kg bw induced
CC
an increase in urinary excretion of protein and glucose, and tubular damage. These results are consistent with the findings of earlier studies [17, 33]. D-serine
at doses ranging from 1.8 to 4.8 mmol/kg bw were
A
The CLtot values of
slightly but significantly decreased compared to that at 0.6 mmol/kg bw. The delayed elimination might be due to nephrotoxicity caused by metabolism processes. However, D-serine-induced
CLtot of
D-serine
D-serine
was not a predictor of
nephrotoxicity. When the C max value of
13
or the saturation of
D-serine
exceeded
approximately 2 mol/ml, plasma creatinine increased remarkably. This result suggested that the Cmax of
D-serine
is a reliable predictor of
D-serine-induced
nephrotoxicity.
Kantrowitz et al. [11] showed that a relationship between the po dose of D-serine and the Cmax levels in humans was linear. Even if the extent of D-serine-induced nephrotoxicity in humans was similar to that in rats, the dose required for nephrotoxicity would be
Whether the metabolism of
D-serine
IP T
approximately 450 mg/kg bw (4.2 mmol/kg bw).
by DAO is a key stage in the mechanism
that
D-serine
D-Serine
SC R
underlying the toxicity of D-serine is certainly of interest. Maekawa et al. [17] showed does not cause nephrotoxicity in mutant rats that lack DAO activity.
is metabolized by DAO to 3-hydroxypyruvic acid, ammonia, and hydrogen
U
peroxide. Since ammonia is not considered a significantly nephrotoxic substance except
N
at very high concentrations, hydrogen peroxide and the concomitant reactive oxygen
A
species are likely to be the toxic metabolites of D-serine. This hypothesis is supported by the results of a study by Krug et al. [16], wherein co-injection of D-serine and reduced
M
glutathione, which captures reactive oxygen species, prevented
D-serine-induced
is not involved in
TE D
aminoaciduria. In contrast, Orozco-Ibarra et al. [18] have suggested that oxidative stress D-serine-induced
nephrotoxicity. Chung et al. [34] examined the
cytotoxic effect of DAO-generated D-serine metabolites in several cell-lines, and found
EP
that not only hydrogen peroxide but also 3-hydroxypyruvic acid induced cell death in astroglial cells. However, the causal role of 3-hydroxypyruvic acid in rat renal damage in
CC
vivo should be carefully considered. Because there are no reliable methods for measuring 3-hydroxypyruvic acid, no information is currently available on the formation of
A
3-hydroxypyruvic acid from
D-serine
in rat kidneys in vivo. Detailed studies on the
pathological effects of 3-hydroxypyruvic acid are necessary to clarify its role in D-serine-induced
nephrotoxicity. Compared to human and rat DAO [28, 35], little is
known about the kinetic properties of DAO in mouse, which is not affected by D-serine at high doses. To clarify whether
D-serine
is nephrotoxic to humans, it is important to
14
investigate the species-specific differences between the enzymatic properties of DAO in humans, rats, and mice.
5. Conclusion In summary, in the present study, we elucidated the plasma kinetics of
D-serine
at
after ip
IP T
therapeutic and high doses in rats. When the C max value of
D-serine
administration exceeded approximately 2 mol/ml, the plasma creatinine increased D-serine
is a reliable
SC R
remarkably 24 h after administration, suggesting that C max of predictor of D-serine-induced nephrotoxicity. Declarations of interest
N
U
None
A
References
M
[1] J.E. Chatterton, M. Awobuluyi, L.S. Premkumar, H. Takahashi, M. Talantova, Y. Shin, J. Cui, S. Tu, K.A. Sevarino, N. Nakanishi, G. Tong, S.A. Lipton, D. Zhang,
TE D
Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits, Nature 415 (2002) 793-798.
[2] J.T. Coyle, G. Tsai, D. Goff, Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia, Ann. N. Y. Acad. Sci. 1003 (2003) 318-327.
EP
[3] A. Hashimoto, T. Nishikawa, T. Hayashi, N. Fujii, K. Harada, T. Oka, K. Takahashi, The presence of free D-serine in rat brain, FEBS Lett. 296 (1992) 33-36. [4] J.P. Mothet, A.T. Parent, H. Wolosker, R.O. Brady, Jr., D.J. Linden, C.D. Ferris, M.A.
CC
Rogawski, S.H. Snyder,
D-Serine
is an endogenous ligand for the glycine site of the
N-methyl-D-aspartate receptor, Proc. Natl. Acad. Sci. USA. 97 (2000) 4926-4931.
A
[5] E.R. Stevens, M. Esguerra, P.M. Kim, E.A. Newman, S.H. Snyder, K.R. Zahs, R.F. Miller, D-Serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors, Proc. Natl. Acad. Sci. USA. 100 (2003) 6789-6794. [6] H. Wolosker, NMDA receptor regulation by D-serine: new findings and perspectives, Mol. Neurobiol. 36 (2007) 152-164.
15
[7] H. Wolosker, K.N. Sheth, M. Takahashi, J.P. Mothet, R.O. Brady, Jr., C.D. Ferris, S.H. Snyder, Purification of serine racemase: biosynthesis of the neuromodulator D-serine,
Proc. Natl. Acad. Sci. USA. 96 (1999) 721-725.
[8] K. Hashimoto, T. Fukushima, E. Shimizu, N. Komatsu, H. Watanabe, N. Shinoda, M. Nakazato, C. Kumakiri, S. Okada, H. Hasegawa, K. Imai, M. Iyo, Decreased serum levels of
D-serine
in patients with schizophrenia: evidence in support of the
Psychiatry 60 (2003) 572-576. [9] S.E. Cho, K.S. Na, S.J. Cho, S.G. Kang, Low
D-serine
IP T
N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia, Arch. Gen. levels in schizophrenia: A
SC R
systematic review and meta-analysis, Neurosci. Lett. 634 (2016) 42-51.
[10] U. Heresco-Levy, D.C. Javitt, R. Ebstein, A. Vass, P. Lichtenberg, G. Bar, S. Catinari, M. Ermilov, D-Serine efficacy as add-on pharmacotherapy to risperidone and olanzapine for treatment-refractory schizophrenia, Biol. Psychiatry 57 (2005) 577-585.
U
[11] J.T. Kantrowitz, A.K. Malhotra, B. Cornblatt, G. Silipo, A. Balla, R.F. Suckow, C. D-serine
N
D'Souza, J. Saksa, S.W. Woods, D.C. Javitt, High dose
in the treatment of
schizophrenia, Schizophr. Res. 121 (2010) 125-130.
A
[12] G. Tsai, P. Yang, L.C. Chung, N. Lange, J.T. Coyle,
D-Serine
added to
M
antipsychotics for the treatment of schizophrenia, Biol. Psychiatry 44 (1998) 1081-1089. [13] G.E. Tsai, P. Yang, L.C. Chung, I.C. Tsai, C.W. Tsai, J.T. Coyle, D-Serine added to clozapine for the treatment of schizophrenia, Am. J. Psychiatry 156 (1999) 1822-1825. D-serine
TE D
[14] F.A. Carone, C.E. Ganote,
nephrotoxicity. The nature of proteinuria,
glucosuria, and aminoaciduria in acute tubular necrosis, Arch. Pathol. 99 (1975) 658-662.
[15] C.E. Ganote, D.R. Peterson, F.A. Carone, The nature of
D-serine-induced
EP
nephrotoxicity, Am. J. Pathol. 77 (1974) 269-282. [16] A.W. Krug, K. Volker, W.H. Dantzler, S. Silbernagl, Why is D-serine nephrotoxic
CC
and alpha-aminoisobutyric acid protective?, Am. J. Physiol. Renal. Physiol. 293 (2007) F382-390.
A
[17] M. Maekawa, T. Okamura, N. Kasai, Y. Hori, K.H. Summer, R. Konno, D-amino-acid
oxidase is involved in
D-serine-induced
nephrotoxicity, Chem. Res.
Toxicol. 18 (2005) 1678-1682. [18]
M.
Orozco-Ibarra,
O.N.
Medina-Campos,
D.J.
Sánchez-González,
C.M.
Martínez-Martínez, E. Floriano-Sánchez, A. Santamaría, V. Ramirez, N.A. Bobadilla, J. Pedraza-Chaverri, Evaluation of oxidative stress in Toxicology 229 (2007) 123-135.
16
D-serine
induced nephrotoxicity,
[19] R.E. Williams, M. Jacobsen, E.A. Lock, 1H NMR pattern recognition and
31
P NMR
studies with D-serine in rat urine and kidney, time- and dose-related metabolic effects, Chem. Res. Toxicol. 16 (2003) 1207-1216. [20] H. Hasegawa, Y. Shinohara, N. Masuda, T. Hashimoto, K. Ichida, Simultaneous determination of serine enantiomers in plasma using Mosher's reagent and stable isotope dilution gas chromatography-mass spectrometry, J. Mass Spectrom. 46 (2010) 502-507.
IP T
[21] K. Tabata, K. Yamaoka, A. Kaibara, S. Suzuki, M. Terakawa, T. Hata, Moment analysis program available on Microsoft Excel, Xenobio. Metabol. Dispos. 14 (1999) 286-293.
SC R
[22] H. Brückner, A. Schieber, Ascertainment of D-amino acids in germ-free, gnotobiotic and normal laboratory rats, Biomed. Chromatogr. 15 (2001) 257-262.
[23] A. Hashimoto, T. Nishikawa, T. Oka, K. Takahashi, T. Hayashi, Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid
U
chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and
N
o-phthaldialdehyde, J. Chromatogr. A 582 (1992) 41-48.
[24] S. Karakawa, K. Shimbo, N. Yamada, T. Mizukoshi, H. Miyano, M. Mita, W.
A
Lindner, K. Hamase, Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine
M
using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues, J. Pharm. Biomed. Anal. 115 (2015) 123-129. [25] Y. Miyoshi, K. Hamase, T. Okamura, R. Konno, N. Kasai, Y. Tojo, K. Zaitsu,
TE D
Simultaneous two-dimensional HPLC determination of free D-serine and D-alanine in the brain and periphery of mutant rats lacking
D-amino-acid
oxidase, J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci. 879 (2011) 3184-3189. [26] R. Konno, Y. Yasumura, D-Amino-acid oxidase and its physiological function, Int. J.
EP
Biochem. 24 (1992) 519-524.
[27] R. Rais, A.G. Thomas, K. Wozniak, Y. Wu, H. Jaaro-Peled, A. Sawa, C.A. Strick,
CC
S.J. Engle, N.J. Brandon, C. Rojas, B.S. Slusher, T. Tsukamoto, Pharmacokinetics of oral D-serine
in
D-amino
acid oxidase knockout mice, Drug Metab. Dispos. 40 (2012)
A
2067-2073.
[28] L.F. Frattini, L. Piubelli, S. Sacchi, G. Molla, L. Pollegioni, Is rat an appropriate animal model to study the involvement of D-serine catabolism in schizophrenia? Insights from characterization of D-amino acid oxidase, FEBS J 278 (2011) 4362-4373. [29] H. Hasegawa, T. Matsukawa, Y. Shinohara, T. Hashimoto, Assessment of the metabolic chiral inversion of D-leucine in rat by gas chromatography-mass spectrometry combined with a stable isotope dilution analysis, Drug Metab. Dispos. 28 (2000)
17
920-924. [30] H. Hasegawa, Y. Shinohara, K. Akahane, T. Hashimoto, Direct detection and evaluation of conversion of
D-methionine
into L-methionine in rats by stable isotope
methodology, J. Nutr. 135 (2005) 2001-2005. [31] T. Matsukawa, H. Hasegawa, H. Goto, Y. Shinohara, A. Shinohara, Y. Omori, K. K.
Yokoyama,
D-selenomethionine
Evaluation
of
the
metabolic
chiral
inversion
of
in rats by stable isotope dilution gas chromatography-mass
IP T
Ichida,
spectrometry, J. Pharm. Biomed. Anal. 116 (2015) 59-64.
[32] T. Noguchi, Y. Takada, R. Kido, Characteristics of hepatic serine-pyruvate
SC R
aminotransferase in different mammalian species, Biochem. J. 161 (1977) 609-614.
[33] F.A. Carone, S. Nakamura, B. Goldman, Urinary loss of glucose, phosphate, and protein by diffusion into proximal straight tubules injured by D-serine and maleic acid, Lab. Invest. 52 (1985) 605-610.
U
[34] S.P. Chung, K. Sogabe, H.K. Park, Y. Song, K. Ono, R.M. Abou El-Magd, Y. produced from
D-serine
by astroglial
acid oxidase, J. Biochem. 148 (2010)
A
743-753.
D-amino
N
Shishido, K. Yorita, T. Sakai, K. Fukui, Potential cytotoxic effect of hydroxypyruvate
M
[35] G. Molla, S. Sacchi, M. Bernasconi, M.S. Pilone, K. Fukui, L. Pollegioni,
EP
Figure captions
TE D
Characterization of human D-amino acid oxidase, FEBS Lett. 580 (2006) 2358-2364.
Fig. 1 Representative selecting ion monitoring profiles for serine enantiomers in plasma
CC
(A) before and (B) 5 min after intravenous administration of D-serine (100 mol/kg bw) to a rat. Masses at m/z 552 and 555 were monitored for diMTPA-OMe derivatives of
A
serine and [2H3]serine enantiomers, respectively. The retention times of the diMTPA-OMe derivatives of D- and L-serine were 7.82 and 7.93 min, respectively. Fig. 2 Plasma concentration–time profiles for D-serine and L-serine in rats. Rats received D-serine
(A) intravenously (0.1 mmol/kg bw, n = 6), (B) orally (0.6 mmol/kg bw, n = 5),
and (C) intraperitoneally (0.6 mmol/kg bw, n = 6). (●) D-serine, (○) L-serine.
18
Fig. 3 Hematoxylin and eosin-stained kidney sections from rats treated with D-serine-treated
at doses of 0.6 (○), 1.2 (◻), 1.8 (▲), 2.4 (▼), and 4.8 ( ◆) mmol/kg bw,
obtained 24 h after D-serine administration. Fig. 4 D-Serine kinetics after ip administration of D-serine at doses of 0.6 (○; n = 5), 1.2
IP T
(◻; n=8), 1.8 (▲; n = 4), 2.4 (▼; n = 5), and 4.8 (◆; n = 5) mmol/kg bw. (A) Plasma concentration–time profiles for D-serine. (B) Relationship between C max of D-serine and
A
CC
EP
TE D
M
A
N
U
SC R
plasma creatinine. (C) Relationship between CL tot of D-serine and plasma creatinine.
19
20
EP
CC
A TE D
IP T
SC R
U
N
A
M
21
EP
CC
A TE D
IP T
SC R
U
N
A
M
22
EP
CC
A TE D
IP T
SC R
U
N
A
M
I N U SC R
Table 1 Pharmacokinetic parameters of D-serine in Wistar rats after administration of D-serine
(mmol/kg bw) (min) (min) (min µmol/ml) (ml/min/kg bw) (min) (nmol/ml) (min-1) (min-1) (%)
CC E
PT
ED
M
Dose t1/2 MRT AUC CLtot tmax Cmax kabs kel (x 10-3) Bioavailability
A
Dosing route
Intravenous (n=6) 0.1 108 ± 16 94 ± 21 13.0 ± 1.6 7.9 ± 0.9
6.8 ± 1.8
Oral (n=5) 0.6 118 ± 13 207 ± 25 72.3 ± 19.6 8.3 ± 2.2 72 ± 16 399 ± 92 0.042 ± 0.018 6.3 ± 1.1 94 ± 27
Intraperitoneal (n=5) 0.6 112 ± 24 136 ± 27 72.4 ± 4.1 8.3 ± 0.5 23 ± 7 610 ± 170 0.134 ± 0.032 6.5 ± 1.3 94 ± 12
Values are expressed as mean ± SD.
A
AUC, area under the plasma concentration–time curve; bw, body weight; CLtot, total plasma clearance; Cmax, maximal plasma concentration; kabs, apparent absorption rate constant; kel, apparent elimination rate constant; MRT, mean residence time; t1/2, plasma elimination half-life; tmax, time to reach Cmax.
23
I N U SC R
Table 2 Plasma creatinine and urinary excretion of protein and glucose after intraperitoneal administration of D-serine n
(mmol/kg bw)
Glucose
(mg/mg Cre) 0.56 ± 0.20
(mg/mg Cre) 0.23 ± 0.07
0.92
± 0.18
0.97
± 0.73
0.23
± 0.03
1.2
5
0.23
± 0.03
0.20
± 0.03
0.23
± 0.05
1.8
4
0.28
± 0.04
0.31
± 0.05
1.13
± 0.48***†††
4.7
± 1.8***
26.1
± 11.5***
6
0.23
± 0.02
0.27
± 0.04
1.40
± 0.40***†††
7.3
± 3.9***
47.5
± 34.4***
5
0.22
± 0.02
0.37
± 0.11*
1.69
± 0.23***†††
14.5
± 2.6***
86.5
± 6.4***
PT
ED
M
5
p < 0.05, *** p < 0.001 vs 0.6 mmol/kg bw value p < 0.001 vs. predose value
CC E
†††
Protein
0.6
4.8
0.19
24 h ± 0.03
Urine
6h 0.22 ± 0.04
2.4
*
Predose
Plasma creatinine (mg/dl) Postdose
A
D-Serine
A
Cre, creatinine
24
I N U SC R
Table 3 Kinetic parameters of D-serine in Wistar rats after intraperitoneal administration of D-serine (mmol/kg bw)
0.6 (n =5) 23 ± 7
1.2 (n = 6) 21 ± 5
1.8 (n = 4) 28 ± 13
2.4 (n = 5) 15 ± 4
4.8 (n = 5) 28 ± 4
0.6 ± 0.2
1.6 ± 0.3
2.3 ± 0.2
4.2 ± 0.2
7.3 ± 0.3
112 ± 24
104 ± 13
138 ± 14
134 ± 23
146 ± 22*
A
Dose
(min)
Cmax
(µmol/ml)
t1/2
(min)
MRT
(min)
136 ± 27
112 ± 16
152 ± 31
122 ± 16
122 ± 15
AUC
(min µmol/ml)
72.4 ± 4.1
155.9 ± 13.1
295.6 ± 43.9
376.3 ± 45.5
714.2 ± 66.3
(ml/min/kg bw)
8.3 ± 0.5
7.7 ± 0.6
6.4 ± 0.7**
6.7 ± 0.6**
ED
PT
CC E
CLtot
M
tmax
6.1 ± 1.0***
kabs
(min-1)
0.134 ± 0.032
0.147 ± 0.038
0.117 ± 0.083
0.133 ± 0.052
0.068 ± 0.015
kel (x 10-3)
(min-1)
6.5 ± 1.3
6.9 ± 0.9
5.1 ± 0.5
5.4 ± 0.9
4.8 ± 0.8*
A
Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. *p<0.05
**
p<0.01 and
***
p<0.001 vs. 0.6 mmol/kg bw
value. AUC, area under the plasma concentration–time curve; CLtot, total plasma clearance; Cmax, maximal plasma concentration; kabs, apparent absorption rate constant; kel, apparent elimination rate constant; MRT, mean residence time; t1/2, plasma elimination half-life; tmax, time to reach Cmax.
25
S0731-7085(18)31200-7 https://doi.org/10.1016/j.jpba.2018.09.026 PBA 12219
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
23-5-2018 16-8-2018 13-9-2018
Please cite this article as: Hasegawa H, Masuda N, Natori H, Shinohara Y, Ichida K, Pharmacokinetics and toxicokinetics of D-serine in rats, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.09.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmacokinetics and toxicokinetics of D-serine in rats
Hiroshi Hasegawa, Nami Masuda, Hiromi Natori, Yoshihiko Shinohara, and Kimiyoshi Ichida
IP T
Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences
SC R
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
(Corresponding author)
A
Tokyo University of Pharmacy and Life Sciences
N
Department of Pathophysiology, School of Pharmacy,
U
Hiroshi Hasegawa, Ph.D.
(Phone) (+81)-426-76-5699
TE D
(Fax) (+81)-426-76-5686
M
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
(E-mail) [email protected]
Highlights
We investigated the relationship of D-serine kinetics with nephrotoxicity in
EP
CC
rats.
Following iv/po/ip D-serine, we measured plasma D-/L-serine with
A
GC-MS.
Histology revealed renal damage 24 after ip administration of D-serine at doses of 1.8–4.8 mmol/kg bw.
1
When Cmax of D-serine was >2 µmol/ml, plasma creatinine increased 24 h later.
Thus, Cmax of D-serine could be a good predictor of D-serine-induced
IP T
nephrotoxicity.
SC R
Abstract
In the mammalian brain, D-serine acts as a co-agonist at the glycine-binding site on
U
the N-methyl-D-aspartate receptor. Because plasma D-serine levels are significantly lower
N
in patients with schizophrenia than in healthy subjects, D-serine has been proposed as a
A
potential therapeutic agent for schizophrenia treatment. However,
D-serine
has a
nephrotoxic effect in rats at high doses. The purpose of this study was to investigate the
D-serine
D-serine
and nephrotoxicity in rats. We
intravenously (iv), orally (po), or intraperitoneally (ip) to male
TE D
administered
M
relationship between the plasma kinetics of
Wistar rats, and performed gas chromatography-mass spectrometry to measure the plasma concentrations of
D-
and L-serine. After iv administration (0.1 mmol/kg body
weight (bw)), plasma D-serine declined multiexponentially with an elimination t 1/2 of 108
was estimated to be 94 ± 27%. To evaluate the dose–response relationship of
CC
D-serine
EP
± 16 min, and the total clearance was 7.9 ± 0.9 ml/min/kg bw. The oral bioavailability of
D-serine-induced
kidney injury and the plasma kinetics of D-serine, we injected D-serine
A
into rats ip in doses ranging from 0.6 to 4.8 mmol/kg bw. Twenty-four hours after D-serine
administration, histological changes indicating renal damage were observed in
the kidneys of rats who received D-serine at doses of 1.8–4.8 mmol/kg bw; the severity of the tubular injury increased with increasing D-serine
D-serine
dose. When the Cmax value of
was approximately >2 µmol/ml, the plasma creatinine increased remarkably 24
h after D-serine administration. This suggests that the C max of D-serine could be a good
2
predictor of D-serine-induced nephrotoxicity.
Abbreviations: AUC, area under the plasma concentration–time curve; AUC0-last, AUC
from time 0 to the last point with a quantifiable plasma concentration; AUC ip; AUC after intraperitoneal administration; bw, body weight; CLtot, total plasma clearance; C max, maximal plasma concentration;
DAO,
D-Amino
acid oxidase; diMTPA-OMe,
bioavailability;
Fpo,
oral
bioavailability;
GC–MS,
gas
IP T
N,O-di-(+)-α-methoxy-α-trifluoromethylphenylacetyl methyl ester; Fip, intraperitoneal
chromatography-mass
spectrometry; ip, intraperitoneally; iv, intravenously; kabs, apparent absorption rate
SC R
constant; kel, apparent elimination rate constant; MRT, mean residence time; MTPA-Cl, (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl
chloride;
NMDA,
N-Methyl-D-aspartate; po, orally; SIM, selecting ion monitoring; t1/2, plasma elimination
N
U
half-life; tmax, time to reach C max.
1. Introduction
TE D
M
A
Keywords: D-serine; GC-MS; pharmacokinetics; toxicokinetics; nephrotoxicity.
The N-methyl-D-aspartate (NMDA) receptor, a type of glutamate receptor, plays
EP
important and indispensable roles in several higher brain functions such as learning, memory, and cognition [1]. NMDA receptor dysfunction has been hypothesized to play a
CC
role in the pathophysiology of schizophrenia [2]. D-Serine
is present in the mammalian brain [3], and serves as an endogenous
A
modulator of the glycine-binding site on NMDA receptors [4-7]. Serum D-serine levels are significantly lower in patients with schizophrenia than in healthy subjects [8, 9]. Since D-serine-mediated enhancement of NMDA receptor function may be beneficial in
3
the treatment of schizophrenia, several clinical trials have tested the effects of D-serine supplementation in schizophrenia [10-13]. In one of these trials, an oral D-serine dose of 30 mg/kg body weight (bw)/day (equivalent to 0.28 mmol/kg bw/day) significantly improved the positive, negative, and cognitive symptoms of schizophrenia. Although dose-dependent efficacy was observed at higher
doses, one subject receiving
at a dose of 120 mg/kg bw/day (1.1 mmol/kg bw/day) showed symptoms similar
IP T
D-serine
D-serine
to those associated with nephrotoxicity [11]. At the same dose, the plasma concentration D-serine
was elevated up to approximately 500 nmol/ml, which is 200 times the
SC R
of
physiological concentration.
Previous studies have revealed that that D-serine is nephrotoxic in rats. High doses of cause necrosis in renal proximal tubules, with proteinuria and glucosuria in rats
U
D-serine
D-serine
nephrotoxicity in humans remains unclear. To address
A
however, the potential
N
[14-19]. D-Serine is not nephrotoxic in mice, guinea pigs, rabbits, dogs, and hamsters;
concerns regarding the therapeutic usage of D-serine in humans, it is essential to clarify
D-serine
D-serine-induced
induces renal injury. To determine the dose dependency of
TE D
which
M
the mechanism underlying D-serine nephrotoxicity in rats and the plasma concentration at
nephrotoxicity in rats, Krug et al. [16] administered
D-serine
intraperitoneally (ip) and measured the consequent plasma concentrations of serine;
EP
however, the HPLC method they used could not separate D- from L-serine. Thus, minimal information is available on the plasma levels of D-serine that are associated with tubular D-serine
in rats
CC
necrosis in rats. In addition, no data exist on the plasma kinetics of
following oral administration; such data may provide information of D-serine kinetics in
A
humans.
Recently, we developed a method for the stereoselective determination of serine
enantiomers in biological fluids by using gas chromatography–mass spectrometry (GC-MS) with selecting ion monitoring (SIM) [20]. This method is based on a stable isotope dilution method, using stable isotope labeled
4
DL-serine
as an analytical standard,
and a diastereomeric method, using (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride (MTPA-Cl) as a chiral derivatizing reagent. The objective of the present study was to investigate the relationship between the plasma kinetics of D-serine and nephrotoxicity in rats.
IP T
2. Materials and methods
D-Serine
SC R
2.1. Chemicals
was purchased from Peptide Institute (Osaka, Japan). L-Serine was purchased
from Wako Pure Chemicals (Osaka, Japan).
DL-[2,3,3-
2
H3]Serine (DL-[2H3]serine, 98
U
atom % 2H) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA).
N
MTPA-Cl and 10% HCl in methanol were purchased from Tokyo Kasei (Tokyo, Japan).
A
A strong cation-exchange solid-phase extraction column BondElut SCX (H + form, size 1 ml/100 mg) was purchased from Agilent Technologies (Lake Forest, CA, USA).
M
Chloroform stabilized with amylene was purchased from Kanto Chemical (Tokyo, Japan).
further purification.
EP
2.2. Animals
TE D
All other chemicals and solvents were of analytical reagent grade and were used without
The experimental protocols were approved by the Institutional Animal Care
CC
Committee of Tokyo University of Pharmacy and Life Sciences (P14-57, P15-57). Male Wistar rats were purchased from SLC Japan (Hamamatsu, Japan). The rats were housed
A
in stainless-steel cages in an air-conditioned room maintained at 23 ± 1°C and 55 ± 5% humidity with a 12 h dark/light cycle. All rats were acclimated for at least 7 days prior to treatment. They were allowed free access to water and food (CE-2, Clea Japan, Tokyo, Japan), but were fasted for 16 h before dosing. Rats received D-serine dissolved in saline intravenously (iv; 0.1 mmol/kg bw) via the
5
femoral vein, orally (po; 0.6 mmol/kg bw) by gavage, or ip (0.6, 1.2, 2.4, and 4.8 mmol/kg bw). Heparinized blood samples (150 l) were obtained from the jugular vein 10 min before and 15, 20, 30, 60, 90, 120, 180, 240, 300, and 360 min after administration under anesthesia by sodium pentobarbital (50 mg/kg bw). Additional blood samples were
IP T
collected at 3, 5, and 10 min after iv and ip administration. The blood samples were centrifuged at 1000 × g for 10 min, and the plasma was stored at -80°C until analysis.
SC R
Rats administered D-serine ip were placed in individual metabolic cages and urine
was collected 6–24 h after D-serine administration. The urine was stored at -80°C until analysis. After urine collection, the rats were killed by exsanguination under anesthesia
U
by sodium pentobarbital. Blood samples from the heart were collected into heparinized
N
tubes, and the plasma was separated by centrifugation at 1000 × g for 10 min. The
A
plasma samples were submitted for biochemical analysis, and the right kidney was
TE D
2.3. Sample preparation for GC-MS
M
removed to evaluate the damage histologically.
Plasma concentrations of
D-
and L-serine were determined by the isotope dilution
method as described previously [20]. Briefly,
DL-[
2
H3]serine (9.98 nmol in 50 l) was
EP
added to plasma samples (10 or 20 l) as an analytical internal standard. The plasma sample was deproteinized with 10% trichloroacetic acid, purified by cation exchange
CC
chromatography using BondElut SCX cartridge, and derivatized with hydrochloric acid in methanol to form methyl ester (-OMe), followed by subsequent N,O-diacylation with
A
MTPA-Cl to form N,O-di-(+)-α-methoxy-α-trifluoromethylphenylacetyl methyl ester (diMTPA-OMe) derivative. All samples were then subjected to GC-MS-SIM analysis.
2.4. GC-MS-SIM GC-MS-SIM analysis was performed using a Shimadzu (Kyoto, Japan) QP2010
6
quadrupole GC-MS equipped with a data-processing system. The operating conditions were the same as those described in a previous publication [20]. Briefly, the GC column was a methylsilicone bonded-phase fused-silica capillary column SPB-1 (15 m x 0.25 mm i.d., 0.25 µm film thickness; Supelco, Bellefonte, PA, USA), and the mass spectrometer was operated in chemical ionization mode, with isobutane as the reagent
IP T
gas. SIM was performed on the quasimolecular-ions of the diMTPA-OMe derivatives of serine (m/z 552) and [2H3]serine (m/z 555). The diMTPA-OMe derivatives of
and
underwent baseline separation within 9 min and eluted in that order on GC. The
SC R
L-serine
D-
lower limit of quantitation for the method was approximately 100 fmol per injection. The intra- and inter-day precision for
D-serine
was < 5% and < 2%, respectively, and for
A
2.5. Biochemical markers against renal injury
N
U
L-serine, < 3% and < 2%, respectively.
Plasma creatinine was determined using HPLC with a cation exchange Supelcosil
M
LC-SCX column (250 x 4.6 mm i.d., particle size 5 µm; Supelco, Bellefonte, PA) under
TE D
the following conditions: mobile phase, sodium phosphate buffer (pH 5.1; 50 mM)-acetonitrile (3:2, v/v); flow rate, 1.3 ml/min; column temperature, ambient; detection, UV 234 nm. Creatinine was eluted at t R 10.4 min as a single peak.
EP
Urinary protein and glucose were measured with Nagahama Life Science Laboratory
CC
(Shiga, Japan) using Hitachi model 7180 Clinical Analyzer.
2.6. Histological analyses For light microscopy, kidney tissue samples were fixed by immersion in buffered
A
formalin (pH 7.4) and embedded in paraffin. For histological analysis, 3 m-thick sections were stained with hematoxylin and eosin, and were observed under an Olympus CX41 light microscope (× 200).
7
2.7. Data analysis Assuming that the plasma concentration of endogenous plasma concentration of administered
D-serine
D-serine
is constant, the
was estimated by subtracting the
endogenous D-serine concentration before administration from the net measurement at each point. The maximal plasma concentration (C max) and the time to reach C max (tmax)
IP T
were obtained from individual plasma concentration versus time data. Noncompartmental pharmacokinetic parameters were calculated using model-independent
analysis
SC R
performed with a macro-program MOMENT(EXCEL) [21] running on Microsoft Excel. The apparent elimination rate constant (k el) was determined from the linear regression slope of the terminal portion (last three to four quantifiable points) of the log plasma
U
concentration-time curve. Apparent terminal half-life was obtained using the following
N
formula: 0.693/k el. The apparent absorption rate constant (k abs) was determined using the
A
residual method. The area under the plasma concentration–time curve (AUC0-last) from time 0 to the last point with a quantifiable plasma concentration was calculated using the
M
trapezoidal rule. The extrapolation of AUC was based on the last plasma concentration
TE D
and the terminal slope. The total systemic plasma clearance (CL tot) was determined using the formula Dose/AUC0-∞. The oral bioavailability (F po) was calculated as follows: Fpo = (AUCpo/AUCiv) x (Doseiv/Dosepo)
EP
where AUCiv and AUC po are AUC estimates after intravenous and oral administration. Similarly, intraperitoneal bioavailability (F ip) was calculated using the following
CC
formula:
Fip = (AUCip/AUCiv) x (Doseiv/Doseip)
A
where AUCip are AUC estimates after intraperitoneal administration.
2.8. Statistical analysis Values are presented mean ± SD. Data were statistically analyzed with one-way ANOVA followed by Dunnett’s multiple comparison post hoc test using KaleidaGraph
8
version 4 (Synergy Software, Reading, PA, USA). A p-value < 0.05 was considered statistically significant.
3.1. Pharmacokinetics of D-serine after iv, po, and ip administration Following iv administration of
D-serine
IP T
3. Results
(0.1 mmol/kg bw) to rats (n = 6), we
SC R
measured the plasma concentration of D- and L-serine using the stable isotope dilution
GC-MS method described previously [20]. The representative SIM profiles obtained from the plasma samples obtained before and 5 min after administration are shown in Fig.
U
1A and 1B, respectively. No substances other than D- and L-serine, and the labeled D- and were found in their monitoring ions. The plasma concentration of endogenous
D-serine
before administration of exogenous D-serine was 1.9 ± 0.3 nmol/ml. Assuming
A
N
L-serine
that the plasma concentration of endogenous
D-serine
is constant, the plasma
concentration before administration from the net measurement at each point. Fig.
TE D
D-serine
M
concentration of the administered D-serine was estimated by subtracting the endogenous
2A shows the plasma concentration–time profiles of D-serine and L-serine. Administered D-serine
disappeared from the plasma multiexponentially, with a terminal elimination t1/2
EP
of 108 ± 16 min (Table 1). The CL tot was 7.9 ± 0.9 ml/min/kg bw. Plasma concentration of L-serine was almost constant during the 6-h period after administration.
CC
Fig. 2B shows the plasma concentration–time profiles of D-serine and L-serine after po administration of D-serine (0.6 mmol/kg bw) to rats (n = 5). Plasma concentration of
A
administered D-serine increased gradually to reach the maximal concentration (C max, 399 ± 92 nmol/ml) at 72 ± 16 min after administration, and then disappeared with an elimination t1/2 of 118 ± 13 min (Table 1), which was comparable to that for iv administration. The bioavailability (F po), estimated by dividing the AUC obtained after oral administration with the value obtained after intravenous administration, was 94 ±
9
27%. The plasma concentration of L-serine was almost constant during the 6-h period after administration. Following ip administration of D-serine (0.6 mmol/kg bw) to rats (n = 6), C max (610 ± 170 nmol/ml) at 23 ± 7 min was attained rapidly and then decreased with a t 1/2 value of 112 ± 24 min (Fig. 2C, Table 1). The bioavailability (F ip) was estimated to be 94 ± 12%. L-serine
was almost constant during the 6-h period after
IP T
Plasma concentration of
SC R
administration.
3.2. Toxicokinetics of D-serine
To evaluate the dose–response relationship of D-serine-induced kidney injury and the
U
plasma kinetics of D-serine, we injected D-serine ip into rats at doses ranging from 0.6 to D-serine
concentration. In
N
4.8 mmol/kg bw, and measured the alteration of plasma
A
addition, we determined the concentrations of plasma creatinine, and urinary protein and glucose as biomarkers for kidney function, and examined histopathological changes in
M
the kidneys.
TE D
Histopathological examination (Fig. 3) did not reveal any changes in the kidneys of rats 24 h after administration of D-serine at 0.6 and 1.2 mmol/kg bw. At doses above 1.8 mmol/kg bw, all rats showed proximal tubule injury; the severity of the tubular injury
EP
increased with increasing dose.
Following administration of
D-serine
in the range of 0.6 to 2.4 mmol/kg bw, we
CC
observed no changes in plasma creatinine during the first 6 h after administration (Table 2). At a dose of 4.8 mmol/kg bw, however, a slight but non-significant increase of
A
plasma creatinine was observed 6 h after administration (0.37 ± 0.11 mg/dl) compared to the plasma creatinine before administration of
D-serine
(0.22 ± 0.02 mg/dl). The
creatinine concentration 6 h after administration of 4.8 mmol/kg bw significantly higher than that in rats receiving 0.6 mmol/kg bw
D-serine
D-serine.
was
Plasma
creatinine increased significantly 24 h after administration of D-serine at 1.8, 2.4, and 4.8
10
mmol/kg bw compared to that after 0.6 mmol/kg bw D-serine (Table 2). There were no changes in urinary excretion of protein and glucose in rats receiving 0.6 and 1.2 mmol/kg bw
D-serine,
whereas above 1.8 mmol/kg bw their urinary biomarkers increased
proportionally with the dose. Urinary protein and glucose of rats receiving 4.8 mmol/kg bw
D-serine
increased 25- and 400-fold, respectively, as compared to that in rats
IP T
receiving 0.6 mmol/kg bw D-serine.
Fig. 4A shows the plasma concentration–time profiles of D-serine in rats administered
SC R
five different doses of D-serine ip. Although the concentration–time curves for all five
doses are apparently parallel, the CLtot (assuming a value of Fip of 0.94) at doses in the range of 1.8 to 4.8 mmol/kg bw were significantly lower than that at 0.6 mmol/kg bw
D-serine
administration, and C max and CLtot of
N
creatinine 24 h after
U
(Table 3). Fig. 4B and 4C show the relationship between plasma concentration of
administration, respectively. When the C max value of
D-serine
D-serine
after ip
was greater than
A
approximately 2 mol/ml, plasma creatinine increased significantly. In contrast, the CLtot
M
value of D-serine was not a reliable predictor of
EP
4. Discussion
nephrotoxicity (Fig.
TE D
4C).
D-serine-induced
The most important advantage of stable isotope dilution analysis using a stable
CC
isotopically labeled analog as an internal standard is that the labeled analog behaves in an almost identical manner to the analyte throughout all steps in the extraction,
A
derivatization, and chromatographic procedures. In this study, commercially available DL-[
2
H3]serine, which mimics
D-
and L-serine in plasma, was chosen as the internal
standard. Therefore, if the extraction and/or derivatization processes were incomplete, the measurement of serine species would not be affected because the 2H3-labeled internal standard would also undergo losses to the same extent that the D- and L-serine did.
11
As shown in Fig. 1, baseline separation between the peaks for the diMTPA-OMe derivatives of
D-serine
and L-serine was achieved with resolution factor of 5.09, and
eluted in that order. In addition to its selectivity, the present method also had high sensitivity. The value of lower limit of quantitation of
D-serine
with the present
GC-MS-SIM system was 100 fmol/injection (0.05 nmol/ml plasma), which made it
IP T
possible to determine the intrinsic D-serine even though the amount of D-serine is only 1% that of L-serine. Endogenous D-serine in plasma was 1.9 ± 0.3 nmol/ml, which was
D-Amino
SC R
consistent with the results of previous studies [22-25].
acid oxidase (DAO), which catalyzes the oxidative deamination of neutral
and basic D-amino acids including D-serine, is expressed in the kidney, liver, and brain in
U
rats [26]. In the present study, the po bioavailability of D-serine (94%) was quantitatively D-serine
N
almost identical to the ip bioavailability. This indicates that almost all the
pass metabolism of
D-serine
A
administered po was absorbed from the gastrointestinal tract. In addition, the low first can be due to low DAO activity in the liver. After po
M
administration of D-serine, the values of C max divided by dose (707 ± 154 g bw/l) in rats
TE D
were similar to those in mice (654 ± 45 g bw/l) [27] but higher than those in humans (477 ± 109 g bw/l) [11]. The biochemical characterization of rat DAO has shown that it differs from human DAO. The most significant difference is in substrate affinity: the K m value
EP
and the intrinsic clearance (K cat/Km) for D-serine is 40- and 20-fold lower, respectively, for rat DAO than for human DAO [28]. The lower activity of DAO in rats than in
CC
humans could explain why the plasma concentration of D-serine in rats is observed to be higher than that in humans. D-amino
acids are converted into the corresponding
L-enantiomers
via a
A
Several
two-step process. The initial step is oxidative deamination by DAO to form 2-oxo acids. In the next step, the 2-oxo acids are stereospecifically reaminated by transaminase to form L-amino acids. We have previously shown that D-leucine [29], D-methionine [30], and D-selenomethionine [31] are converted into the corresponding L-enantiomers in rats.
12
D-Serine
is also considered to be metabolized by DAO to 3-hydroxypyruvic acid,
followed by conversion into L-serine by serine-pyruvate aminotransferase. However, the transaminase activity of serine-pyruvate aminotransferase in the rat liver is lower with 3-hydroxypyruvic acid and L-alanine as substrates than with serine and pyruvic acid [32]. In addition, 3-hydroxypyruvic acid is metabolized to glycoaldehyde, which is a substrate
IP T
for glucose synthesis. If the administered D-serine is converted into L-serine, the plasma concentration of L-serine will increase transitorily. However, in the present study, plasma
SC R
concentration of L-serine remained mostly constant during the 6-h post-administration period, suggesting that chiral conversion of D-serine into the L-enantiomer does not occur in rats.
U
Orozco-Ibarra et al. [18] showed that administration of D-serine (400 mg/kg bw = 3.8
N
mmol/kg bw) induced a significant time-dependent increase in plasma creatinine. In the
A
present study, plasma creatinine tended to increase 6 h after administration of D-serine at a dose of 4.8 mmol/kg bw. Since plasma creatinine only changes after a significant loss
administration. Plasma creatinine levels of rats receiving 1.8 and 2.4 mmol/kg
TE D
D-serine
M
of kidney function, this finding suggests that kidney damage occurs within 6 h of
bw D-serine were not altered at 6 h compared to baseline levels, but the levels at 24 h were significantly higher than that after administration of 0.6 mmol/kg bw D-serine. At
EP
24 h after ip administration, D-serine at doses of 0.6 to 1.2 mmol/kg bw had no effect on the urinary excretion of protein and glucose. D-Serine at 1.8 to 4.8 mmol/kg bw induced
CC
an increase in urinary excretion of protein and glucose, and tubular damage. These results are consistent with the findings of earlier studies [17, 33]. D-serine
at doses ranging from 1.8 to 4.8 mmol/kg bw were
A
The CLtot values of
slightly but significantly decreased compared to that at 0.6 mmol/kg bw. The delayed elimination might be due to nephrotoxicity caused by metabolism processes. However, D-serine-induced
CLtot of
D-serine
D-serine
was not a predictor of
nephrotoxicity. When the C max value of
13
or the saturation of
D-serine
exceeded
approximately 2 mol/ml, plasma creatinine increased remarkably. This result suggested that the Cmax of
D-serine
is a reliable predictor of
D-serine-induced
nephrotoxicity.
Kantrowitz et al. [11] showed that a relationship between the po dose of D-serine and the Cmax levels in humans was linear. Even if the extent of D-serine-induced nephrotoxicity in humans was similar to that in rats, the dose required for nephrotoxicity would be
Whether the metabolism of
D-serine
IP T
approximately 450 mg/kg bw (4.2 mmol/kg bw).
by DAO is a key stage in the mechanism
that
D-serine
D-Serine
SC R
underlying the toxicity of D-serine is certainly of interest. Maekawa et al. [17] showed does not cause nephrotoxicity in mutant rats that lack DAO activity.
is metabolized by DAO to 3-hydroxypyruvic acid, ammonia, and hydrogen
U
peroxide. Since ammonia is not considered a significantly nephrotoxic substance except
N
at very high concentrations, hydrogen peroxide and the concomitant reactive oxygen
A
species are likely to be the toxic metabolites of D-serine. This hypothesis is supported by the results of a study by Krug et al. [16], wherein co-injection of D-serine and reduced
M
glutathione, which captures reactive oxygen species, prevented
D-serine-induced
is not involved in
TE D
aminoaciduria. In contrast, Orozco-Ibarra et al. [18] have suggested that oxidative stress D-serine-induced
nephrotoxicity. Chung et al. [34] examined the
cytotoxic effect of DAO-generated D-serine metabolites in several cell-lines, and found
EP
that not only hydrogen peroxide but also 3-hydroxypyruvic acid induced cell death in astroglial cells. However, the causal role of 3-hydroxypyruvic acid in rat renal damage in
CC
vivo should be carefully considered. Because there are no reliable methods for measuring 3-hydroxypyruvic acid, no information is currently available on the formation of
A
3-hydroxypyruvic acid from
D-serine
in rat kidneys in vivo. Detailed studies on the
pathological effects of 3-hydroxypyruvic acid are necessary to clarify its role in D-serine-induced
nephrotoxicity. Compared to human and rat DAO [28, 35], little is
known about the kinetic properties of DAO in mouse, which is not affected by D-serine at high doses. To clarify whether
D-serine
is nephrotoxic to humans, it is important to
14
investigate the species-specific differences between the enzymatic properties of DAO in humans, rats, and mice.
5. Conclusion In summary, in the present study, we elucidated the plasma kinetics of
D-serine
at
after ip
IP T
therapeutic and high doses in rats. When the C max value of
D-serine
administration exceeded approximately 2 mol/ml, the plasma creatinine increased D-serine
is a reliable
SC R
remarkably 24 h after administration, suggesting that C max of predictor of D-serine-induced nephrotoxicity. Declarations of interest
N
U
None
A
References
M
[1] J.E. Chatterton, M. Awobuluyi, L.S. Premkumar, H. Takahashi, M. Talantova, Y. Shin, J. Cui, S. Tu, K.A. Sevarino, N. Nakanishi, G. Tong, S.A. Lipton, D. Zhang,
TE D
Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits, Nature 415 (2002) 793-798.
[2] J.T. Coyle, G. Tsai, D. Goff, Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia, Ann. N. Y. Acad. Sci. 1003 (2003) 318-327.
EP
[3] A. Hashimoto, T. Nishikawa, T. Hayashi, N. Fujii, K. Harada, T. Oka, K. Takahashi, The presence of free D-serine in rat brain, FEBS Lett. 296 (1992) 33-36. [4] J.P. Mothet, A.T. Parent, H. Wolosker, R.O. Brady, Jr., D.J. Linden, C.D. Ferris, M.A.
CC
Rogawski, S.H. Snyder,
D-Serine
is an endogenous ligand for the glycine site of the
N-methyl-D-aspartate receptor, Proc. Natl. Acad. Sci. USA. 97 (2000) 4926-4931.
A
[5] E.R. Stevens, M. Esguerra, P.M. Kim, E.A. Newman, S.H. Snyder, K.R. Zahs, R.F. Miller, D-Serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors, Proc. Natl. Acad. Sci. USA. 100 (2003) 6789-6794. [6] H. Wolosker, NMDA receptor regulation by D-serine: new findings and perspectives, Mol. Neurobiol. 36 (2007) 152-164.
15
[7] H. Wolosker, K.N. Sheth, M. Takahashi, J.P. Mothet, R.O. Brady, Jr., C.D. Ferris, S.H. Snyder, Purification of serine racemase: biosynthesis of the neuromodulator D-serine,
Proc. Natl. Acad. Sci. USA. 96 (1999) 721-725.
[8] K. Hashimoto, T. Fukushima, E. Shimizu, N. Komatsu, H. Watanabe, N. Shinoda, M. Nakazato, C. Kumakiri, S. Okada, H. Hasegawa, K. Imai, M. Iyo, Decreased serum levels of
D-serine
in patients with schizophrenia: evidence in support of the
Psychiatry 60 (2003) 572-576. [9] S.E. Cho, K.S. Na, S.J. Cho, S.G. Kang, Low
D-serine
IP T
N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia, Arch. Gen. levels in schizophrenia: A
SC R
systematic review and meta-analysis, Neurosci. Lett. 634 (2016) 42-51.
[10] U. Heresco-Levy, D.C. Javitt, R. Ebstein, A. Vass, P. Lichtenberg, G. Bar, S. Catinari, M. Ermilov, D-Serine efficacy as add-on pharmacotherapy to risperidone and olanzapine for treatment-refractory schizophrenia, Biol. Psychiatry 57 (2005) 577-585.
U
[11] J.T. Kantrowitz, A.K. Malhotra, B. Cornblatt, G. Silipo, A. Balla, R.F. Suckow, C. D-serine
N
D'Souza, J. Saksa, S.W. Woods, D.C. Javitt, High dose
in the treatment of
schizophrenia, Schizophr. Res. 121 (2010) 125-130.
A
[12] G. Tsai, P. Yang, L.C. Chung, N. Lange, J.T. Coyle,
D-Serine
added to
M
antipsychotics for the treatment of schizophrenia, Biol. Psychiatry 44 (1998) 1081-1089. [13] G.E. Tsai, P. Yang, L.C. Chung, I.C. Tsai, C.W. Tsai, J.T. Coyle, D-Serine added to clozapine for the treatment of schizophrenia, Am. J. Psychiatry 156 (1999) 1822-1825. D-serine
TE D
[14] F.A. Carone, C.E. Ganote,
nephrotoxicity. The nature of proteinuria,
glucosuria, and aminoaciduria in acute tubular necrosis, Arch. Pathol. 99 (1975) 658-662.
[15] C.E. Ganote, D.R. Peterson, F.A. Carone, The nature of
D-serine-induced
EP
nephrotoxicity, Am. J. Pathol. 77 (1974) 269-282. [16] A.W. Krug, K. Volker, W.H. Dantzler, S. Silbernagl, Why is D-serine nephrotoxic
CC
and alpha-aminoisobutyric acid protective?, Am. J. Physiol. Renal. Physiol. 293 (2007) F382-390.
A
[17] M. Maekawa, T. Okamura, N. Kasai, Y. Hori, K.H. Summer, R. Konno, D-amino-acid
oxidase is involved in
D-serine-induced
nephrotoxicity, Chem. Res.
Toxicol. 18 (2005) 1678-1682. [18]
M.
Orozco-Ibarra,
O.N.
Medina-Campos,
D.J.
Sánchez-González,
C.M.
Martínez-Martínez, E. Floriano-Sánchez, A. Santamaría, V. Ramirez, N.A. Bobadilla, J. Pedraza-Chaverri, Evaluation of oxidative stress in Toxicology 229 (2007) 123-135.
16
D-serine
induced nephrotoxicity,
[19] R.E. Williams, M. Jacobsen, E.A. Lock, 1H NMR pattern recognition and
31
P NMR
studies with D-serine in rat urine and kidney, time- and dose-related metabolic effects, Chem. Res. Toxicol. 16 (2003) 1207-1216. [20] H. Hasegawa, Y. Shinohara, N. Masuda, T. Hashimoto, K. Ichida, Simultaneous determination of serine enantiomers in plasma using Mosher's reagent and stable isotope dilution gas chromatography-mass spectrometry, J. Mass Spectrom. 46 (2010) 502-507.
IP T
[21] K. Tabata, K. Yamaoka, A. Kaibara, S. Suzuki, M. Terakawa, T. Hata, Moment analysis program available on Microsoft Excel, Xenobio. Metabol. Dispos. 14 (1999) 286-293.
SC R
[22] H. Brückner, A. Schieber, Ascertainment of D-amino acids in germ-free, gnotobiotic and normal laboratory rats, Biomed. Chromatogr. 15 (2001) 257-262.
[23] A. Hashimoto, T. Nishikawa, T. Oka, K. Takahashi, T. Hayashi, Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid
U
chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and
N
o-phthaldialdehyde, J. Chromatogr. A 582 (1992) 41-48.
[24] S. Karakawa, K. Shimbo, N. Yamada, T. Mizukoshi, H. Miyano, M. Mita, W.
A
Lindner, K. Hamase, Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine
M
using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues, J. Pharm. Biomed. Anal. 115 (2015) 123-129. [25] Y. Miyoshi, K. Hamase, T. Okamura, R. Konno, N. Kasai, Y. Tojo, K. Zaitsu,
TE D
Simultaneous two-dimensional HPLC determination of free D-serine and D-alanine in the brain and periphery of mutant rats lacking
D-amino-acid
oxidase, J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci. 879 (2011) 3184-3189. [26] R. Konno, Y. Yasumura, D-Amino-acid oxidase and its physiological function, Int. J.
EP
Biochem. 24 (1992) 519-524.
[27] R. Rais, A.G. Thomas, K. Wozniak, Y. Wu, H. Jaaro-Peled, A. Sawa, C.A. Strick,
CC
S.J. Engle, N.J. Brandon, C. Rojas, B.S. Slusher, T. Tsukamoto, Pharmacokinetics of oral D-serine
in
D-amino
acid oxidase knockout mice, Drug Metab. Dispos. 40 (2012)
A
2067-2073.
[28] L.F. Frattini, L. Piubelli, S. Sacchi, G. Molla, L. Pollegioni, Is rat an appropriate animal model to study the involvement of D-serine catabolism in schizophrenia? Insights from characterization of D-amino acid oxidase, FEBS J 278 (2011) 4362-4373. [29] H. Hasegawa, T. Matsukawa, Y. Shinohara, T. Hashimoto, Assessment of the metabolic chiral inversion of D-leucine in rat by gas chromatography-mass spectrometry combined with a stable isotope dilution analysis, Drug Metab. Dispos. 28 (2000)
17
920-924. [30] H. Hasegawa, Y. Shinohara, K. Akahane, T. Hashimoto, Direct detection and evaluation of conversion of
D-methionine
into L-methionine in rats by stable isotope
methodology, J. Nutr. 135 (2005) 2001-2005. [31] T. Matsukawa, H. Hasegawa, H. Goto, Y. Shinohara, A. Shinohara, Y. Omori, K. K.
Yokoyama,
D-selenomethionine
Evaluation
of
the
metabolic
chiral
inversion
of
in rats by stable isotope dilution gas chromatography-mass
IP T
Ichida,
spectrometry, J. Pharm. Biomed. Anal. 116 (2015) 59-64.
[32] T. Noguchi, Y. Takada, R. Kido, Characteristics of hepatic serine-pyruvate
SC R
aminotransferase in different mammalian species, Biochem. J. 161 (1977) 609-614.
[33] F.A. Carone, S. Nakamura, B. Goldman, Urinary loss of glucose, phosphate, and protein by diffusion into proximal straight tubules injured by D-serine and maleic acid, Lab. Invest. 52 (1985) 605-610.
U
[34] S.P. Chung, K. Sogabe, H.K. Park, Y. Song, K. Ono, R.M. Abou El-Magd, Y. produced from
D-serine
by astroglial
acid oxidase, J. Biochem. 148 (2010)
A
743-753.
D-amino
N
Shishido, K. Yorita, T. Sakai, K. Fukui, Potential cytotoxic effect of hydroxypyruvate
M
[35] G. Molla, S. Sacchi, M. Bernasconi, M.S. Pilone, K. Fukui, L. Pollegioni,
EP
Figure captions
TE D
Characterization of human D-amino acid oxidase, FEBS Lett. 580 (2006) 2358-2364.
Fig. 1 Representative selecting ion monitoring profiles for serine enantiomers in plasma
CC
(A) before and (B) 5 min after intravenous administration of D-serine (100 mol/kg bw) to a rat. Masses at m/z 552 and 555 were monitored for diMTPA-OMe derivatives of
A
serine and [2H3]serine enantiomers, respectively. The retention times of the diMTPA-OMe derivatives of D- and L-serine were 7.82 and 7.93 min, respectively. Fig. 2 Plasma concentration–time profiles for D-serine and L-serine in rats. Rats received D-serine
(A) intravenously (0.1 mmol/kg bw, n = 6), (B) orally (0.6 mmol/kg bw, n = 5),
and (C) intraperitoneally (0.6 mmol/kg bw, n = 6). (●) D-serine, (○) L-serine.
18
Fig. 3 Hematoxylin and eosin-stained kidney sections from rats treated with D-serine-treated
at doses of 0.6 (○), 1.2 (◻), 1.8 (▲), 2.4 (▼), and 4.8 ( ◆) mmol/kg bw,
obtained 24 h after D-serine administration. Fig. 4 D-Serine kinetics after ip administration of D-serine at doses of 0.6 (○; n = 5), 1.2
IP T
(◻; n=8), 1.8 (▲; n = 4), 2.4 (▼; n = 5), and 4.8 (◆; n = 5) mmol/kg bw. (A) Plasma concentration–time profiles for D-serine. (B) Relationship between C max of D-serine and
A
CC
EP
TE D
M
A
N
U
SC R
plasma creatinine. (C) Relationship between CL tot of D-serine and plasma creatinine.
19
20
EP
CC
A TE D
IP T
SC R
U
N
A
M
21
EP
CC
A TE D
IP T
SC R
U
N
A
M
22
EP
CC
A TE D
IP T
SC R
U
N
A
M
I N U SC R
Table 1 Pharmacokinetic parameters of D-serine in Wistar rats after administration of D-serine
(mmol/kg bw) (min) (min) (min µmol/ml) (ml/min/kg bw) (min) (nmol/ml) (min-1) (min-1) (%)
CC E
PT
ED
M
Dose t1/2 MRT AUC CLtot tmax Cmax kabs kel (x 10-3) Bioavailability
A
Dosing route
Intravenous (n=6) 0.1 108 ± 16 94 ± 21 13.0 ± 1.6 7.9 ± 0.9
6.8 ± 1.8
Oral (n=5) 0.6 118 ± 13 207 ± 25 72.3 ± 19.6 8.3 ± 2.2 72 ± 16 399 ± 92 0.042 ± 0.018 6.3 ± 1.1 94 ± 27
Intraperitoneal (n=5) 0.6 112 ± 24 136 ± 27 72.4 ± 4.1 8.3 ± 0.5 23 ± 7 610 ± 170 0.134 ± 0.032 6.5 ± 1.3 94 ± 12
Values are expressed as mean ± SD.
A
AUC, area under the plasma concentration–time curve; bw, body weight; CLtot, total plasma clearance; Cmax, maximal plasma concentration; kabs, apparent absorption rate constant; kel, apparent elimination rate constant; MRT, mean residence time; t1/2, plasma elimination half-life; tmax, time to reach Cmax.
23
I N U SC R
Table 2 Plasma creatinine and urinary excretion of protein and glucose after intraperitoneal administration of D-serine n
(mmol/kg bw)
Glucose
(mg/mg Cre) 0.56 ± 0.20
(mg/mg Cre) 0.23 ± 0.07
0.92
± 0.18
0.97
± 0.73
0.23
± 0.03
1.2
5
0.23
± 0.03
0.20
± 0.03
0.23
± 0.05
1.8
4
0.28
± 0.04
0.31
± 0.05
1.13
± 0.48***†††
4.7
± 1.8***
26.1
± 11.5***
6
0.23
± 0.02
0.27
± 0.04
1.40
± 0.40***†††
7.3
± 3.9***
47.5
± 34.4***
5
0.22
± 0.02
0.37
± 0.11*
1.69
± 0.23***†††
14.5
± 2.6***
86.5
± 6.4***
PT
ED
M
5
p < 0.05, *** p < 0.001 vs 0.6 mmol/kg bw value p < 0.001 vs. predose value
CC E
†††
Protein
0.6
4.8
0.19
24 h ± 0.03
Urine
6h 0.22 ± 0.04
2.4
*
Predose
Plasma creatinine (mg/dl) Postdose
A
D-Serine
A
Cre, creatinine
24
I N U SC R
Table 3 Kinetic parameters of D-serine in Wistar rats after intraperitoneal administration of D-serine (mmol/kg bw)
0.6 (n =5) 23 ± 7
1.2 (n = 6) 21 ± 5
1.8 (n = 4) 28 ± 13
2.4 (n = 5) 15 ± 4
4.8 (n = 5) 28 ± 4
0.6 ± 0.2
1.6 ± 0.3
2.3 ± 0.2
4.2 ± 0.2
7.3 ± 0.3
112 ± 24
104 ± 13
138 ± 14
134 ± 23
146 ± 22*
A
Dose
(min)
Cmax
(µmol/ml)
t1/2
(min)
MRT
(min)
136 ± 27
112 ± 16
152 ± 31
122 ± 16
122 ± 15
AUC
(min µmol/ml)
72.4 ± 4.1
155.9 ± 13.1
295.6 ± 43.9
376.3 ± 45.5
714.2 ± 66.3
(ml/min/kg bw)
8.3 ± 0.5
7.7 ± 0.6
6.4 ± 0.7**
6.7 ± 0.6**
ED
PT
CC E
CLtot
M
tmax
6.1 ± 1.0***
kabs
(min-1)
0.134 ± 0.032
0.147 ± 0.038
0.117 ± 0.083
0.133 ± 0.052
0.068 ± 0.015
kel (x 10-3)
(min-1)
6.5 ± 1.3
6.9 ± 0.9
5.1 ± 0.5
5.4 ± 0.9
4.8 ± 0.8*
A
Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. *p<0.05
**
p<0.01 and
***
p<0.001 vs. 0.6 mmol/kg bw
value. AUC, area under the plasma concentration–time curve; CLtot, total plasma clearance; Cmax, maximal plasma concentration; kabs, apparent absorption rate constant; kel, apparent elimination rate constant; MRT, mean residence time; t1/2, plasma elimination half-life; tmax, time to reach Cmax.
25