D-serine and DAAO inhibitors as therapeutics

D-Serine in Neuropsychiatric Disorders: New Advances

D-Serine (DSR) is an endogenous amino acid involved in glia-synapse interactions that has unique neurotransmitter characteristics. DSR acts as obligatory coagonist at the glycine site associated with the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) and has a cardinal modulatory role in major NMDAR-dependent processes including NMDAR-mediated neurotransmission, neurotoxicity, synaptic plasticity, and cell migration. Since either over- or underfunction of NMDARs may be involved in the pathophysiology of neuropsychiatric disorders; the pharmacological manipulation of DSR signaling represents a major drug development target. A first generation of proof-of-concept animal and clinical studies suggest beneficial DSR effects in treatment-refractory schizophrenia, movement, depression, and anxiety disorders and for the improvement of cognitive performance. A related developing pharmacological strategy is the indirect modification of DSR synaptic levels by use of compounds that alter the function of main enzymes responsible for DSR production and degradation. Accumulating data indicate that, during the next decade, we will witness important advances in the understanding of DSR role that will further contribute to elucidating the causes of neuropsychiatric disorders and will be instrumental in the development of innovative treatments.

Schematic diagram of D-serine signaling at a glutamatergic synapse. NMDARsyn and NMDARexsyn = synaptic and extrasynaptic N-methyl-D-aspartate receptor; GMS = Glycine modulatory site; GLU = L-glutamate binding site; SR = serine racemase; ASC 1 and ASCT 2 = neutral amino acid transporters; DAAO = D-amino-acid oxidase; αKa = alpha-Keto acid; NH3 = ammonia; H2O2 = hydrogen peroxide.
Schematic diagram of D-serine signaling at a glutamatergic synapse. NMDARsyn and NMDARexsyn = synaptic and extrasynaptic N-methyl-D-aspartate receptor; GMS = Glycine modulatory site; GLU = L-glutamate binding site; SR = serine racemase; ASC 1 and ASCT 2 = neutral amino acid transporters; DAAO = D-amino-acid oxidase; αKa = alpha-Keto acid; NH3 = ammonia; H2O2 = hydrogen peroxide.

“Over the last 20 years, glutamatergic models of schizophrenia have become increasingly accepted as etiopathological models of this disorder, mainly based on the observation that the cyclohexylamine “dissociative anesthetics” phencyclidine (PCP) and ketamine induce schizophrenia-like positive and negative symptoms and cognitive deficits by blocking NMDAR neurotransmission. The PCP/NMDAR model implies that treatments which aimed at potentiating NMDAR function should be therapeutically beneficial. Furthermore, pharmacological manipulation of DSR signaling represents a particularly attractive candidate strategy since convergent lines of evidence suggest an involvement of dysfunctional DSR transmission in schizophrenia. Single polymorphisms for SR and DAAO have been linked to schizophrenia, in rodents genetic loss of DAAO activity reverses schizophrenia-like phenotypes and reduced DSR serum and cerebrospinal fluid (CSF) levels were documented in chronic schizophrenia patients. Moreover, supporting the hypo-NMDAR hypothesis of schizophrenia, DSR selectively blocks PCP-induced hyperactivity and stereotypic behavior.

A number of clinical studies have demonstrated that adjuvant DSR (30–120 mg/kg/day) added to ongoing treatment with non-clozapine antipsychotics results in significant symptom improvements in chronic schizophrenia patients. The most significant changes were registered in the negative symptom cluster. Nevertheless, two recent meta-analytic reviews indicate that additional dysfunction domains may be affected by DSR. S. P. Singh and V. Singh reported medium effect sizes of DSR for negative symptoms (standardized mean difference (SMD), −0.53) and total symptomatology (SMD, −0.40). Tsai and Lin found DSR effective for negative symptoms (effect size (ES), 0.48), cognitive symptoms (ES, 0.42), and total psychopathology (ES, 0.40).

Two schizophrenia treatment issues stemming from these findings are the potential use of DSR for improving cognition and as stand-alone pharmacotherapy in this disorder. In a preliminary four week open-label study, it was shown that high dose DSR (≥60 mg/kg/day) improves neurocognitive functions as measured by the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) battery. An additional controlled pilot investigation compared the effectiveness of DSR (3 g/day) versus high-dose olanzapine (30 mg/day) as antipsychotic monotherapy in 18 treatment-resistant schizophrenia patients. The primary LOCF analysis indicated a lack of efficacy of DSR as compared to high-dose olanzapine. However, DSR was not inferior to the prestudy antipsychotic drug treatment. Furthermore, among the patients who completed the nine study weeks, high dose olanzapine and DSR did not differ in their effectiveness, suggesting that a subgroup of patients may be successfully maintained on DSR.

In all clinical trials performed to date with DSR in schizophrenia, no significant adverse events have been observed at doses of ≤4 g/day. A potential concern with DSR use is nephrotoxicity which has been reported in one patient receiving 120 mg/kg/day and resolved following DSR discontinuation. This apparent paucity of side effects seems remarkable in view of the fact that both acute and chronic administration of 1-2 g DSR results in ≥100 times increases in DSR serum levels. Nevertheless, orally administered DSR is substantially metabolized by DAAO diminishing its bioavailability and necessitating the administration of gram level doses. In view of these limitations, the ideal dosage and mode of administration of DSR remain to be determined.”

DAAO inhibition with sodium benzoate

“Benzoic acid and its salts, including sodium benzoate, exist in many plants and are widely used as food preservatives. Sodium benzoate also acts as a DAAO inhibitor and has favorable effects in NMDAR-based models such as pain relief and glial cell death . The potential molecular mechanisms of action of sodium benzoate remain to be determined. Since DAAO activity is high in the adult brain cerebellum, it is possible that DSR cerebellar levels may be increased following sodium benzoate administration. Furthermore, recent findings suggest that sodium benzoate may upregulate brain-derived neurotrophic factor (BDNF) via protein kinase A- (PKA-)mediated activation of cAMP response element binding (CREB) protein.

In two controlled trials, the administration of up to 1g/day sodium benzoate proved beneficial for schizophrenia  and MCI or mild AD patients. Furthermore, partial remission within 6 weeks was reported with a major depressive disorder patient treated with 500mg/day sodium benzoate. These preliminary findings show promise for DAAO inhibition as a novel treatment approach. Nevertheless, at present, the therapeutic potential of DAAO inhibitors is still relatively unexplored and preclinical studies have primarily addressed the relevance of these compounds mainly for schizophrenia. Further research is warranted given that the few published studies characterizing novel DAAO inhibitors have yielded conflicting results.”

  • While D-serine is a NMDAR activator at the glycine site, D-serine also inhibits some NMDARs active at hyperpolarized potentials. [1]
  • In healthy subjects, acute administration of D-serine (2.1g) “reduces subjective feelings of sadness and anxiety and has procognitive effects that are overall opposed to the known effects of NMDAR antagonists. The findings are relevant to translational research of NMDAR function and the development of NMDAR-glycine site treatments for specific psychiatric entities.” [2]
  • Distribution of D-serine levels is not correlated with DAAO activity in the adult brain, although a high correlation between D-serine levels and NMDA receptor density has been detected. No changes in D-serine levels were detected in the forebrain of mice lacking DAAO, suggesting that DAAO is not likely to regulate D-serine levels in the forebrain, where NMDA receptors are abundant: “It seems that DAAO inhibitors do not affect D-serine levels in the forebrain of adult rodents. …treatment with a novel DAAO inhibitor CBIO did not alter the extracellular D-serine concentrations in the frontal cortex of adult rats and mice” [3]
  • D-serine-dependent NMDAR activity is involved in mediating the cellular and behavioral effects of 5-HT2AR activation. [4]
  • Changes in plasma D-serine, L-serine, and glycine levels in treatment-resistant schizophrenia before and after clozapine treatment have been studied, the results demonstrating “changes of D-/L-serine and glycine/L-serine ratio between before and after clozapine treatment, suggesting that the plasma D-/L-serine ratio and glycine/L-serine ratio could be markers of therapeutic efficacy or clinical state in treatment-resistant schizophrenia.” [5]
  • Prebiotic feeding increases D-serine levels [6]
  • D-serine increases hippocampal neurogenesis [7]

D-serine shows promise in the treatment of co-morbid substance abuse:

Availability of N-methyl-D-aspartate Receptor Co-agonists Affects Cocaine-induced Conditioned Place Preference and Locomotor Sensitization: Implications for Co-morbid Schizophrenia and Substance Abuse

D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats.

From Examine.com:

D-Serine administration, via affecting the NMDA glycine binding site and thus positively regulating signalling through NDMA receptors, is thought to be able to reduce symptoms of schizophrenia. This is somewhat supported by the fact that schizophrenia appears to be a D-serine deficiency state (role in cause or effect not established)

“Limited positive studies tend to note 17-30% improvements in negative symptoms of schizophrenia with 30mg/kg (2.12+/-0.6g overall) D-serine as assessed by the Positive and Negative Syndrome Scale (PNSS) which is a potency similar to 800mg/kg Glycine under similar research conditions. Studies that measure negative symptom progression over time note beneficial effects within 2 weeks of supplementation, which increases in potency over 6 weeks of observation and is more apparent with higher doses in the 60-120mg/kg range. When looking specifically at positive symptoms, 30mg/kg (2.12+/-0.6g overall) D-serine has noted significant improvement after 6 weeks although the trend noted at weeks 2 and 4 was not statistically significant. Improvements have been noted with 60-120mg/kg to a higher degree than 30mg/kg, and benefits on both positive and negative symptoms are correlated with serum exposure to D-serine.

2,000mg of D-serine daily for 16 weeks in schizophrenics in addition to standard anti-psychotic medication failed to find a significant benefit of supplementation over placebo, although the authors cautioned that the larger than normal placebo response could in part explain the results although D-serine has elsewhere failed to be any significantly different than placebo at the 30mg/kg or 2,000mg dosage.These studies do note that individuals get benefit albeit not consistently enough to reach statistical significance, and this paired with the correlation between D-serine in the blood and the benefits to symptoms suggest that the established variance in serum D-serine from oral therapy may underlie the null effects observed.”

D-Serine appears to be effective at reducing all symptoms of schizophrenia (mostly negative and cognitive, with less effects on positive) but the standard recommended dose of 30mg/kg seems unreliable. This may be due to a large variability in how much D-serine reaches the blood, and taking higher doses seems to be more reliable based on limited evidence

Like D-serine, D-aspartate is also a NMDAR coagonist at the glycine site

DAAO knockout mice display enhanced short-term memory but increased anxiety behaviours, without sleep or circadian rhythm disruption. [8]. Sodium benzoate (SB) is a moderate affinity – Kd of 2.2 μM (Ki  = 2.0 μM) – DAAO inhibitor [9]  and “DAAO inhibition increases NMDA receptor glycine-site occupancy by D-serine in vivo” A later study found a Kd = 7 μM and a Ki = 9 μM for benzoate inhibition of hDAAO.

“The effects of DAAO inhibitors outside the cerebellar circuitry may be limited by the localized expression of DAAO. Activity of DAAO inhibitors in animal models of schizophrenia, for example, has been reported to require coadministration of exogenous D-serine to demonstrate efficacy. On the other hand, effects of DAAO inhibitors have been described in animal models of chronic pain, neuropathic pain, memory, hippocampal LTP, and EEG.

NMDAR-mediated changes in synaptic plasticity require coincidence between its activating neurotransmitter and depolarization of its postsynaptic membrane. In synapses characterized by astrocytic processes closely juxtaposed, this dependence could be shifted according to the extent of D-serine released from glial cells as demonstrated in the supraoptic nucleus or in prefrontal cortex. Here we demonstrated that inhibiting DAAO restored the synaptic D-serine concentrations, thus effectively increasing glial control of NMDARs in this excitatory synapse and possibly in other synapses such as those in defined hippocampal networks.”

DAAO is found in highest concentrations in cerebellum and brainstem rather than in brain regions typically associated with NMDA hypoactivity in schizophrenia . In addition, whereas DAAO regulates intracellular D-serine concentrations, synaptic concentrations are primarily regulated by the alanine–serine–cysteine transporter which exhibits a high affinity for D-serine.

As detailed above, sodium benzoate has been studied as an adjunctive agent in the treatment of schizophrenia [10]:

“A recent clinical study demonstrated that sodium benzoate (SB), a prototype competitive d-amino acid oxidase inhibitor, was effective in the treatment of several symptoms, such as positive and negative symptoms, and cognitive impairment in medicated patients with schizophrenia.”

SB has beneficial effects on pre-pulse inhibition deficits and hyperlocomotion induced in mice after administration of phencyclidine: “Sodium benzoate induced antipsychotic effects in the PCP model of schizophrenia, although it did not increase D-serine levels in the brain.” [11]

D-amino acid oxidase is expressed in the ventral tegmental area and modulates cortical dopamine [12]:

“…injection into the VTA of sodium benzoate, a DAAO inhibitor, increases frontal cortex extracellular dopamine, as measured by in vivo microdialysis and high performance liquid chromatography. Combining sodium benzoate and D-serine did not enhance this effect, and injection of D-serine alone affected dopamine metabolites but not dopamine. These data show that DAO is expressed in the VTA, and suggest that it impacts on the mesocortical dopamine system.”

Consumption of sodium benzoate as a food preservative may be linked with hyperactivity in children [13]

Sodium benzoate increased volumes of thalamus, amygdala, and brainstem in a drug-naïve patient with major depression [14]

The combined effects of sodium benzoate and D-Serine in animal models of cognitive impairment have been studied, the authors concluding that “D-serine and sodium benzoate plays a crucial role in treating cognitive impairment with reduced nephrotoxicity of D-serine.” [15]

While an above study have failed to find differential modulation of DAergic activity with such combinations, co-administration of a DAAO inhibitor and D-serine has effectively counteracted PPI deficits induced by NMDA antagonists in animal models and is a promising therapeutic approach. [16]

On the contrary, “D-Amino-Acid Oxidase Inhibition Increases D-Serine Plasma Levels in Mouse But not in Monkey or Dog“:

“D-serine has been shown to improve positive, negative, and cognitive symptoms when used as add-on therapy for the treatment of schizophrenia. However, D-serine has to be administered at high doses to observe clinical effects. This is thought to be due to D-serine undergoing oxidation by D-amino-acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor could be a way to lower the D-serine dose required to treat schizophrenia. Early studies in rodents to evaluate this hypothesis showed that concomitant administration of structurally distinct DAAO inhibitors with D-serine enhanced plasma and brain D-serine levels in rodents compared with administration of D-serine alone. In the present work we used three potent DAAO inhibitors and confirmed previous results in mice. In a follow-up effort, we evaluated plasma D-serine levels in monkeys after oral administration of D-serine in the presence or absence of these DAAO inhibitors. Even though the compounds reached steady state plasma concentrations exceeding their Ki values by >60-fold, plasma D-serine levels remained the same as those in the absence of DAAO inhibitors. Similar results were obtained with dogs. In summary, in contrast to rodents, DAAO inhibition in monkeys and dogs did not influence the exposure to exogenously administered D-serine. Results could be due to differences in D-serine metabolism and/or clearance mechanisms and suggest that the role of DAAO in the metabolism of D-serine is different across species. These data provide caution regarding the utility of DAAO inhibition for patients with schizophrenia”

One patient with sub-therapeutic levels of clozapine, also taking therapeutic doses of aripiprazole failed to find benefit from 1g sodium benzoate added once daily. Replicating the 2013 study by using 500mg b.d. may be more effective. The same patient noted rapid improvements in cognition and mood when 1g sodium benzoate tds was added to 2g/day D-serine but this was not trialed for a long period. 500mg sodium benzoate b.d. and D-serine 1g b.d. was as effective as 2g D-serine b.d. but the subjective beneficial effects were minimal in both cases, even on prolonged administration. No subjective improvement in positive symptoms or cognition was noted but there was a slight improvement in negative symptoms.

Benzoic acid is rapidly absorbed from the gastrointestinal tract. The volume of distribution is small (estimated 0.14 L/kg in neonates). Benzoic acid is conjugated in the liver with glycine to form hippuric acid, the major metabolite. In healthy adults, up to 97% of the dose of sodium benzoate is excreted as hippuric acid in the urine within 4 hours. Benzoate exhibits nonlinear kinetics (prolonged elimination half-life) at higher doses.  Sodium benzoate ingestions of up to 20 to 60 g daily have been tolerated. [17]

A patent has recently been filed: Sorbic and Benzoic Acid and Derivatives Thereof Enhance the Activity of a Neuropharmaceutical 

D-Amino Acid Oxidase Inhibitors as a Novel Class of Drugs for Schizophrenia Therapy

Over the years, accumulating evidence has indicated that D-serine represents the endogenous ligand for the glycine modulatory binding site on the NR1 subunit of N-methyl-D-aspartate receptors in various brain areas. Cellular concentrations of D-serine are regulated by synthesis due to the enzyme serine racemase (isomerization reaction) and by degradation due to the same enzyme (elimination reaction) as well as by the FAD-containing flavoenzyme D-amino acid oxidase (DAAO, oxidative deamination reaction). Several findings have linked low levels of D-serine to schizophrenia: D-serine concentrations in serum and cerebrospinal fluid have been reported to be decreased in schizophrenia patients while human DAAO activity and expression are increased; oral administration of D-serine improved positive, negative, and cognitive symptoms of schizophrenia as add-on therapy to typical and atypical antipsychotics. This evidence indicates that increasing NMDA receptor function, perhaps by inhibiting DAAO-induced degradation of D-serine may alleviate symptoms in schizophrenic patients. Furthermore, it has been suggested that co-administration of D-serine with a human DAAO inhibitor may be a more effective means of increasing D-serine levels in the brain. Here, we present an overview of the current knowledge of the structure-function relationships in human DAAO and of the compounds recently developed to inhibit its activity (specifically the ones recently exploited for schizophrenia treatment).

Clinical trials currently recruiting:

Study to Evaluate Safety & Efficacy of NaBen as Add-on Treatment for Negative Symptoms of Schizophrenia in Adults

Adaptive Phase II Study to Evaluate the Safety & Efficacy of Sodium Benzoate as an Add-on Treatment for Schizophrenia in Adolescents

DAAOI-1 Treatment for Treatment-resistant Schizophrenia

Other DAAO inhibitors have been studied [18]

“A crucial question is whether there are potential therapeutic advantages in using selective DAAO inhibitors rather than directly administering D-Ser. The use of a DAAO inhibitor avoids the potential for nephrotoxicity which emerges from the renal oxidation of administered D-Serince kidney DAAO will be inhibited as well.”

A further well-known inhibitor of DAAO is represented by chlorpromazine (CPZ), an essential drug according to the World Health Organization list, commonly employed to treat schizophrenia. In the 1950s, CPZ was reported to be an in vitro and in vivo inhibitor of pkDAAO: it acts as a competitive inhibitor of the flavin cofactor, Ki= 23 uM. Interestingly, the inhibitory capacity of DAAO activity of a number of phenothiazine derivatives correlated with their relative clinical efficacy and potency in antipsychotic therapy. We recently demonstrated that CPZ binds to the apoprotein form ofhDAAO with a Kd = 5 uM, an interaction not affected by the presence of benzoate

5-methylpyrazole-3-carboxylic acid (AS057278) is a moderately good inhibitor of hDAAO in vitro (IC50 = 0.5-0.9 uM), also showing good selectivity. In fact, negligible inhibitory activity over the glycine site of the NMDAR, D-aspartate oxidase (DASPO, the enzyme active on acidic D-amino acids and D-Pro), and SR was observed up to 100 uM, and for the human ERG potassium channel, too. In rat AS057278 showed an intraveneous half-life of 5.6 h and low clearance; the compound is orally bioavailable (F = 41%) with a terminal half-life of 7.2 h and a tmax of = 1 h. AS057278 penetrates the brain in rats the brain and CSF concentrations were increased following i.v.administration – but the brain-to-plasma and CSF-to-plasma ratios were relatively low at 1 h after administration (0.052 and 0.018, respectively). Indeed, AS057278 produced a modest increase in the D-Ser/(D-Ser + L-Ser) ratio in the rat cortex and midbrain after i.v.administration of 10 mg/kg AS057278. By employing an animal model that is used to evaluate antipsychotic agents (i.e., the mouse PCP-induced prepulse inhibition, PPI, and startle inhibition response), AS057278 normalized the PPI similarly to the atypical antipsychotic clozapine (3 mg/kg p.o.), both administered acutely (80 mg/kg p.o.) or chronically (28 days, bid, 10 mg/kg i.v.). The chronic treatment with AS057278 also normalized PCP-induced hyperlocomotion in mice

6-Chloro benzo[d]isoxazol-3-ol (CBIO) was the most potent analogue described in this series: it competitively inhibited DAAO activity with an IC50 = 17-570 nM and a Ki= 100 nM. Oral administration to rats (30 mg/kg) increased plasma D-Ser concentration by 25%, an increase similar to that observed following D-Ser administration (30 mg/kg), but failed to increase extracellular cortical D-Ser concentrations when administered alone – a similar result on rat brain D-Ser was also reported. However, CBIO potentiated the effects of both D-Ser and D-Ala in attenuating MK-801 (dizocilpine)-induced PPI deficits in mice. One possible explanation for CBIO’s inability to enhance D-Ser levels in the brain is its poor blood-brain barrier permeability

4H-thieno[3,2-b]pyrrole-5-carboxylate showed the same IC50 value for both rDAAO and hDAAO. In rats, the pharmacokinetics were good, including oral bioavailability, a dose-dependent inhibition of kidney and brain DAAO activity – by approximately 96% and 87% (kidney) and 80% and 60% (cerebellar) at 1 h and 8 h, respectively – and increased plasma D-Ser. However, doses of compound 4 that inhibited DAAO activity in kidney by ~ 80% and in brain by ~ 65% failed to affect cortical levels of the neuromodulator, whereas D-Ser levels in plasma, CSF, and brain cortex were elevated to 220% (plasma), 175% (CSF), and 133% (cortex) of controls 8 h after administration. In rats this compound did not affect the amphetamine-induced psychomotor activity, nucleus accumbens dopamine release, or a MK-801-induced deficit in a novel object recognition paradigm, while D-Ser (1280 mg/kg s.c.) attenuated amphetamine-induced psychomotor activity and dopamine release and improved novel object recognition performance.The inhibitor decreased spontaneous locomotor activity, indicating some effect on behavior, albeit distinct from that of the D-amino acid; chronically administered 4H-thieno[3,2-b]pyrrole-5-carboxylate was proposed to have a role in improving behavior similar to that of D-Ser.

See also:

Glycinergic, NMDA and AMPA augmentation – a review

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