Co-Administration of a D-Amino Acid Oxidase Inhibitor Potentiates the Efficacy of D-Serine in Attenuating Prepulse Inhibition Deficits After Administration of Dizocilpine

Kenji Hashimoto, Yuko Fujita, Mao Horio, Shinsui Kunitachi, Masaomi Iyo, Dana Ferraris, and Takashi Tsukamoto

Background: D-Serine, an endogenous agonist of the N-methyl-D-aspartate (NMDA) receptors, is effective in the treatment of schizophre- nia. However, orally administered D-serine is metabolized substantially by D-amino acid oxidase (DAAO), diminishing its oral bioavailability. In this study, we examined the effects of oral D-serine administration with or without a DAAO inhibitor, 5-chloro-benzo[d]isoxazol-3-ol (CBIO), on the prepulse inhibition (PPI) deficits after administration of the NMDA receptor antagonist dizocilpine.

Methods: Vehicle or D-serine (30, 300, or 900 mg/kg) with or without CBIO (30 mg/kg) was orally administered to mice 1 hour before administration of dizocilpine (.1 mg/kg), and then the PPI of the acoustic startle response was measured. We measured the extracellular levels of D-serine in the frontal cortex after oral administration of D-serine with or without CBIO.

Results: Coadministration of CBIO with D-serine (30 mg/kg), but not D-serine (30 mg/kg) alone, significantly attenuated dizocilpine- induced PPI deficits. Furthermore, coadministration of CBIO significantly increased the extracellular levels of D-serine in the frontal cortex after administration of D-serine.

Conclusions: These findings suggest that coadministration of CBIO significantly enhanced the efficacy of D-serine in attenuating PPI deficits by administration of dizocilpine. Therefore, coadministration of D-serine and a DAAO inhibitor has therapeutic potential for the treatment of schizophrenia.

Key Words: Bioavailability, D-Amino acid oxidase, D-Serine, NMDA receptors, prepulse inhibition

Accumulating evidence suggests that a dysfunction in glutamatergic neurotransmission via the N-methyl-D-as- partate (NMDA) receptors might be involved in the pathophysiology of schizophrenia and that D-serine, an endog- enous agonist at the NMDA receptors, plays a role in the pathophysiology of schizophrenia (1–3). First, it has been re- ported that levels of D-serine in the serum or cerebrospinal fluid of patients with schizophrenia are lower than those of normal control subjects (4 –7). Second, Tsai et al. (8) reported that D-serine significantly improved schizophrenic symptoms when used as adjuvant to conventional antipsychotic drugs. A subse- quent study demonstrated that the addition of D-serine to ongoing treatment with risperidone or olanzapine was beneficial for reducing positive, negative, and cognitive symptoms of patients with treatment-refractory schizophrenia (9). Third, the mRNA expression and activity of D-amino acid oxidase (DAAO) (10), which can metabolize D-serine, is increased in the post- mortem brain of schizophrenic patients (11,12). Fourth, the G72 gene on chromosome 13q has been significantly associated with schizophrenia (13,14). The G72 gene has been designated a DAAO activator, because the G72 protein interacts physically with DAAO (13). A recent meta-analysis provided highly signif- icant evidence of an association between nucleotide variations in the G72/G30 region and schizophrenia (15). In consideration of these findings, it is likely that reductions in brain D-serine levels play a role in the pathophysiology of schizophrenia (1–3).

In animals, D-serine is believed to be metabolized substan- tially by DAAO, diminishing its oral bioavailability (16). These findings prompted us to identify small molecule DAAO inhibitors that can be coadministered with D-serine to minimize its metab- olism by DAAO. Recently, we reported that oral administration of a potent DAAO inhibitor, 5-chloro-benzo[d]isoxazol-3-ol (CBIO), in conjunction with D-serine could enhance the plasma and brain levels of D-serine in rats compared with the oral adminis- tration of D-serine alone (17).

In this study, we examined the effects of oral D-serine administration with or without CBIO on the prepulse inhibition (PPI) deficits in mice after administration of the NMDA receptor antagonist dizocilpine. We also measured the levels of D-serine in the brain and plasma after oral administration of D-serine with or without CBIO.

Methods and Materials

Animals

Male Slc:ddy mice (6 weeks old) weighing 25–30 g were purchased from SLC Japan (Hamamatsu, Shizuoka, Japan). The mice were housed in clear polycarbonate cages (22.5 × 33.8 × 14.0 cm) in groups of five or six per cage under a controlled 12-hour light-dark cycle (lights on from 7:00 AM to 7:00 PM), with room temperature at 23 ± 1°C and humidity at 55% ± 5%. The mice were given free access to water and food pellets. The experimental procedure was approved by the Animal Care and Use Committee of Chiba University.

Measurement of Acoustic Startle Reactivity and Prepulse Inhibition of Startle

The mice were tested for their acoustic startle reactivity (ASR) in a startle chamber (SR-LAB; San Diego Instruments, San Diego, California) using the standard methods described previou- sly (18). The test sessions were begun after an initial 10-min acclimation period in the chamber. The mice were subjected to one of six trials: 1) pulse alone, as a 40-msec broadband burst; a pulse (40-msec broadband burst) preceded by 100 msec with a 20-msec prepulse that was 2) 4 dB, 3) 8 dB, 4) 12 dB, or 5) 16 dB over background (65 dB); and 6) background only (no stimulus). The amount of prepulse inhibition (PPI) was expressed as the percentage decrease in the amplitude of the startle reactivity caused by presentation of the prepulse (% PPI).
D-Serine (30, 300, or 900 mg/kg; Sigma-Aldrich, St. Louis, Missouri) with or without CBIO (30 mg/kg) (16) or vehicle (.5% carbomethoxycellulose [CMC]; Wako Pure Chemical, Tokyo, Japan; 10 mL/kg) was administered 60 min (including the 10-min acclimation period) before the machine records, and dizocilpine (.1 mg/kg as a hydrogen maleate salt; Sigma-Aldrich) or saline (10 mL/kg) was administered subcutaneously 10 min (including the 10-min acclimation period) before the records. The PPI test lasted 20 min in total.

Measurement of D-Serine levels

Vehicle (10 mL/kg), D-Serine (30 or 900 mg/kg), D-serine (30 mg/kg) with CBIO (30 mg/kg), or CBIO (30 mg/kg) was administered 90 min before sacrifice. Levels of D-serine and L-serine in the frontal cortex and plasma were measured by high performance liquid chromatography (HPLC), as reported previ- ously (19).

Mice were anesthetized with sodium pentobarbital before the stereotaxic implantation of a probe into the left frontal cortex (+2.1 mm anteroposterior, +1.0 mm mediolateral from the bregma, and –1.2 mm dorsoventral from the dura). Probes were secured onto the skull using stainless steel screws and dental acrylic. Twenty-four hours after surgery, in vivo microdialysis was performed on conscious mice. Probes were perfused con- tinuously with artificial cerebrospinal fluid (147 mmol/L NaCl,4 mmol/L KCl, and 2.3 mmol/L CaCl2) at a rate of 2 µL/min. D-Serine (30 mg/kg) with or without CBIO (30 mg/kg) was orally administered. The dialysate was collected in 30-min fractions, and then levels of D-serine and L-serine were measured as described earlier.

Statistical Analysis

The data are presented as the mean ± standard error of the mean (SEM). The PPI data were analyzed by multivariate analysis of variance (MANOVA). When appropriate, group means at individual dB levels were compared by one-way analysis of variance (ANOVA), followed by Bonferroni/Dunn test. The results of D-serine levels were also analyzed by one-way ANOVA, followed by Bonferroni/Dunn test. Significance for the results was set at p < .05.

Results

Effects of D-Serine With or Without CBIO on PPI Deficits After a Single Administration of Dizocilpine

There was no significant effect on the acoustic startle re- sponse at 120 dB in the all group. Figure 1 shows the effects of D-serine (30, 300, or 900 mg/kg) with or without CBIO (30 mg/kg) on dizocilpine (.1 mg/kg)-induced PPI deficits in mice. The MANOVA analysis of all PPI data revealed that there was a significant effect (Wilks lambda = .429, p < .001). Subsequent ANOVA analysis revealed the significant differences at all dB groups (69, 73, 77, and 81 dB). A posteriori analysis indicated a significant (p < .001) difference in PPI deficits between the vehicle + vehicle group and vehicle + dizocilpine (.1 mg/kg) group (Figure 1). Higher doses (300 or 900 mg/kg) of D-serine alone significantly attenuated PPI deficits induced by dizocilpine (.1 mg/kg; Figure 1). However, the low dose (30 mg/kg) of D-serine alone did not alter PPI deficits induced by dizocilpine. Interestingly, coadministration of CBIO (30 mg/kg) with D-serine (30 mg/kg) significantly attenuated dizocilpine-induced PPI def- icits at 73 dB (p = .010), 77 dB (p = .009), and 81 dB (p < .001; Figure 1). In contrast, CBIO (30 mg/kg) alone did not alter PPI in normal and dizocilpine-treated mice (Figure 1).

Figure 1. The effect of D-serine with or without 5-chloro-benzo[d]isoxazol-3-ol (CBIO) on dizocilpine-induced prepulse inhibition (PPI) deficits in mice. Sixty minutes after oral administration of vehicle (10 mL/kg), D-serine (30, 300, or 900 mg/kg), D-serine (30 mg/kg) plus CBIO (30 mg/kg), or CBIO (30 mg/kg) alone, with dizocilpine (.1 mg/kg), or with saline (10 mL/kg) was administered subcutaneously to the mice. Each value is mean ± SEM (n = 13–17 per group). The number in parentheses in the legend is the dose (mg/kg) of drugs. *p < .05, **p < .01, ***p < .001 compared with the vehicle + dizocilpine–treated group.

Figure 2. The effects of D-serine with or without 5-chloro-benzo[d]isoxazol-3-ol (CBIO) on levels of D-serine and L-serine in the frontal cortex and plasma. (A–D) Ninety minutes after oral administration of vehicle (10 mL/kg), D-serine (30, 300, or 900 mg/kg), D-serine (30 mg/kg) plus CBIO (30 mg/kg), or CBIO (30 mg/kg) alone, mice were sacrificed. Levels of D-serine and L-serine were measured by high-pressure liquid chromatography. The values are the mean ± SEM of six or seven mice. *p < .05, **p < .01, ***p < .001 compared with the control group.

Effects of CBIO on D-Serine Levels in the Frontal Cortex and Plasma After Oral Administration of D-Serine

We measured the levels of D-serine in the frontal cortex and plasma 90 min after oral administration of D-serine (30 or 900 mg/kg) with or without CBIO (30 mg/kg). Levels of D-serine in the frontal cortex were significantly increased after a single administration of D-serine (900 mg/kg), but not D-serine (30 mg/kg) with or without CBIO (Figure 2A). Plasma levels of D-serine were significantly increased after a single administration of D-serine (30 or 900 mg/kg) or D-serine (30 mg/kg) with CBIO (Figure 2C). In contrast, CBIO (30 mg/kg) alone slightly in- creased the plasma levels of D-serine, although the difference was not significant (Figure 2A). Levels of L-serine were not altered in any of the groups (Figure 2B and 2D).

To explore the effects of CBIO on the extracellular levels of D-serine in the brain, we used an in vivo microdialysis technique to examine extracellular D-serine levels in the frontal cortex of conscious mice. Coadministration of D-serine (30 mg/kg) and CBIO (30 mg/kg) significantly increased the extracellular D- serine levels in the mouse frontal cortex compared with the group receiving D-serine (30 mg/kg) alone (Supplement 1). This data from the mouse brain is consistent with the previous results of rat brain (17), but the effects of CBIO in the mouse brain were higher than those in the rat brain (17).

Discussion

In this study, we found that coadministration of the DAAO inhibitor CBIO potentiated the bioavailability of D-serine in mice. Interestingly, we found that coadministration of CBIO (30 mg/ kg) significantly enhanced the efficacy of D-serine (30 mg/kg) in attenuating dizocilpine-induced PPI deficits, although D-serine (30 mg/kg) alone was not effective in this model.

Plasma levels of D-serine after a single oral administration of D-serine with CBIO were significantly higher than those after administration of D-serine alone, indicating the increased oral bioavailability by CBIO. Furthermore, an in vivo microdialysis study revealed that extracellular D-serine levels in the frontal cortex after administration of D-serine with CBIO were signifi- cantly higher than those after treatment with D-serine alone. Taken together, these results suggest that the ability of CBIO to enhance the effects of D-serine on PPI deficits may be due to increased D-serine levels in the brain. In addition, combination therapy with D-serine and a DAAO inhibitor could be used to reduce the dose of D-serine in humans, because the dose of D-serine used for the treatment of schizophrenic patients is high (30 – 60 mg/kg) (8,9).

In conclusion, coadministration of D-serine and a DAAO inhibitor would be a new approach for the treatment of schizophrenia.This study was supported in part by grants from the Minister of Education, Culture, Sports, Science, and Technology of Japan (to KH).The authors report no biomedical financial interests or po- tential conflicts of interest.Supplementary material cited in this article is available online.

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