Procognitive effects of methyl 2-amino-3-methoxybenzoate (or daopine) may involve the dorsal striatal anthranilic acid pathway and mutimetabolite-multitarget pharmacology

Abstract

Multifunctional drug treatment is currently the most promising approach in neuropsychopharmacology to overcome complex disorders, such as schizophrenia. We previously showed that the natural protoalkaloid, methyl 2-amino-3-methoxybenzoate [or daopine (DAO)] has procognitive effects in animal models of schizophrenia. Because DAO is a metabolite of the anthranilic acid biosynthesis pathway in Nigella damascena plant seeds, we sought to find out if DAO exerts its procognitive effects via the 'anthranilic acid-brain-pathway-twin' and mutimetabolite-multitarget pharmacology. We explored the procognitive effects of DAO using the operant set shift task in a rat model of attentional flexibility deficits induced by L-kynurenine, the precursor of both kynurenic acid and anthranilic acid. HPLC and liquid chromatography-mass spectrometry was used to identify brain and plasma DAO metabolites and the effects of DAO on dorsal striatal anthranilic acid. DAO attenuated kynurenine-induced cognitive deficits. We identified for the first time the brain (DAO-1 and DAO-3) and plasma (DAO-1 and DAO-2) metabolites of DAO, which remarkably are all methylated derivatives of 3-hydroxyanthranilic acid (3-OHAA), an endogenous brain astrocytic metabolite of anthranilic acid playing a crucial role in cognition. In vitro , DAO-2 and DAO-3 significantly reduced oxidative activity, lipid peroxidation, inflammation, and amyloid β-42-aggregation, all of which represent processes that play an important protective role against cognitive dysfunction. The results strengthen our hypothesis that administering small molecules structurally related to anthranilic acid/3-OHAA, such as DAO, may provide a multitarget strategy for the prevention and treatment of cognitive deficits in schizophrenia, and more broadly, in other cognitive disorders, such as Alzheimer's disease.

Keywords: Wistar rat; anthranilic acid pathway; cognitive disorder; daopine; dopamine; multitarget; schizophrenia.

Fig. 1

Acute effects of DAO (10 mg/kg) on cognitive flexibility deficits induced by kynurenine (100 mg/kg). (a) Schematic representation of both extradimensional shift and reversal learning stages. (b) Number of sessions needed during the visual discrimination stage before the treatments. (c) Effects of DAO on the number of sessions needed during the extradimensional shift and reversal learning deficits presented in male Wistar rats induced pharmacologically by systemic injection of L-KYN, the direct precursor of KYNA that induces cognitive deficits. N = 8 rats per group. Data were expressed as a mean of trials ± SD. *P < 0.05, **P < 0.01, using two-way ANOVA repeated measures analysis. No significant effects between groups were observed during the visual discrimination learning stage before the treatments using one-way ANOVA repeated measures analysis. ANOVA, analysis of variance; DAO, daopine; L-KYN, L-kynurenine.

Fig. 2

Kinetics of dorsal striatal anthranilic acid and dopamine whole-tissue concentrations after an acute dose of DAO (10 mg/kg) in naive male Wistar rats for 8 h after subcutaneous injection. (a) Schematic representation of experimental design. (b) Percentage of dorsal striatal anthranilic acid and dopamine whole-tissue concentrations compared with basal concentrations. N = 5 rats per group. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 using ANOVA repeated measures analysis. ANOVA, analysis of variance; DAO, daopine; ECD, electrochemical detection.

Fig. 3

HPLC-UV chromatograms showing a blank plasma extract (a), plasma extract from Wistar rats (b), 60 min postsubcutaneous injection with DAO (100 mg/kg), and DAO standard (c). DAO, daopine; UV, ultraviolet.

Fig. 4

LC-MS chromatograms showing a total scan of the isolated concentrated peaks, 1 and 2 from HPLC-UV (a and c), and their MS analyses (b and d). DAO, daopine; ESI, electrospray ionization; ITMS, ion trap mass spectrometry; LC-MS, liquid chromatography–mass spectrometry; PDA, photodiode array detector; UV, ultraviolet.

Fig. 5

In-vitro activities of DAO and its metabolites. (a–c) Antioxidant effects of DAO and its metabolites, DAO-1, DAO-2, and DAO-3. (a) Scavenging activity of DPPH• radicals. (b) Scavenging activity of ABTS•+ radicals. ASC (vitamin C) was used as a positive control. (c) Inhibitory activity of 100 µM DAO-2 and DAO-3 against linoleic acid peroxidation (BHA was used as the positive control). (d) Effects of DAO-2 and DAO-3 on the proinflammatory mediators TNF-α, IL-1β, and IL-6 produced by LPS-stimulated (1 µg/ml) RAW 264.7 macrophage cell culture. (e) Anti-Aβ42 aggregation effects of DAO-2 and DAO-3 in vitro using the Congo red binding method. Data are expressed as mean percentage ± SD of three independent experiments (n = 3 each). ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); ASC, ascorbic acid; BHA, butylated hydroxyanisole; DAO, daopine; DPPH, 2,2′-diphenyl-1-picrylhydrazyl; IL, interleukin; LPS, 3–4,5-dimethylthiazol-2-yl)-2,5-diphenyl; TNF-α, tumor necrosis factor alpha.

Fig. 6

Kynurenine catabolic pathway showing relevant enzymes related directly or indirectly to the anthranilic acid pathway and nigella damascena seed- anthranilic acid-twin pathway, leading to structurally anthranilic acid/3-OHAA related protoalkaloids. While the brain (microglia)-anthranilic acid pathway gives rise to only 3-OHAA, multiple methylated 3-OHAA-related compounds (including DAO) are biosynthesized from the plant seed anthranilic acid pathway, yet with multitarget activities. The different possible targeted enzymes are presented inside the cycles. AA, anthranilic acid; AAMO, anthranilic acid monoxygenase; DAO, daopine; 3-HK-KYN, 3-hydroxykynurenine; KAT-II, kynurenine aminotransferase II; KMO, kynurenine monoxygenase; KYN, kynurenine; KYNA, kynurenic acid; KYNU, kynureninase; 3-OHAA, 3-hydroxyanthranilic acid; QA, quinolinic acid.