The Natural Protoalkaloid Methyl-2-Amino-3-Methoxybenzoate (MAM) Alleviates Positive as well as Cognitive Symptoms in Rat and Mouse Schizophrenia Models

The development of new antipsychotics with pro-cognitive properties and less side effects represents a priority in schizophrenia drug research. In this study, we present for the first time a preclinical exploration of the effects of the promising natural atypical antipsychotic Methyl-2-Amino-3-Methoxybenzoate (MAM), a brain-penetrable protoalkaloid from the seed of the plant Nigella damascena. Using animal models related to hyperdopaminergic activity, namely the pharmacogenetic apomorphine (D2/D1 receptor agonist)-susceptible (APO-SUS) rat model and pharmacologically induced mouse and rat models of schizophrenia, we found that MAM reduced gnawing stereotypy and climbing behaviours induced by dopaminergic agents. This predicts antipsychotic activity. In line, MAM antagonized apomorphine-induced c-Fos and NPAS4 mRNA levels in post-mortem brain nucleus accumbens and dorsolateral striatum of APO-SUS rats. Furthermore, phencyclidine (PCP, an NMDA receptor antagonist) and 2,5-Dimethoxy-4-iodoamphetamine (DOI, a 5HT2A/2C receptor agonist) induced prepulse inhibition deficits, reflecting the positive symptoms of schizophrenia, which were rescued by treatment with MAM and atypical antipsychotics alike. Post-mortem brain immunostaining revealed that MAM blocked the strong activation of both PCP- and DOI-induced c-Fos immunoreactivity in a number of cortical areas. Finally, during a 28-day subchronic treatment regime, MAM did not induce weight gain, hyperglycemia, hyperlipidemia or hepato- and nephrotoxic effects, side effects known to be induced by atypical antipsychotics. MAM also did not show any cataleptic effects. In conclusion, its brain penetrability, the apparent absence of preclinical side effects, and its ability to antagonize positive and cognitive symptoms associated with schizophrenia make MAM an exciting new antipsychotic drug that deserves clinical testing.


Apomorphine Induced Climbing
This test has been described by Costall et al. [1]; Peuch et al. [2]; Protais et al. [3].Mice that are administered an appropriate dose of apomorphine (a dopamine agonist) will climb the walls of a cage and remain at or near the top for 20-30 minutes (an average of 25 min).Untreated mice on the other hand will occasionally climb up and then climb down rapidly.The exaggerated climbing of apomorphine-treated mice can be antagonized by pretreatment with dopamine blocking agents (atypical antipsychotic, Olanzapine was used as a standard: 2.5 mg/kg).Mice were placed first into cylindrical cages (diameter, 12 cm; height, 14 cm with a wall consisting of metal bars of 0.2 cm diameter; with walls lined with 1 cm2 wire mesh) surmounted by a wire mesh size 3 mm for 1 h before the experiments to adjust to the new environment.The vehicle itself [DMSO/Tween-80/Saline 2.5% 2.5%/95%: v/v] or MAM (3 and 10 mg/kg) were administered subcutaneously.After 15 minutes, apomorphine HCl was administered subcutaneously into the neck at 1.0 mg/kg, upon which the mice could climb.Mice were scored for different climbing time scores.Three behaviours were taken into account: full climbing (four paws holding the wall), partial climbing (front paws holding the wall) and no climbing (four paws on the floor).

Assessment of Catalepsy
The test was adapted from Sanberg et al. [4] and Hoffman and Donovan, [5].Catalepsy is defined as a reduced ability to initiate movement and a failure to achieve correct posture.Typical Aps such as Haloperidol and some of the atypical Aps (at a higher doses) induce catalepsy in mice, predictive of extrapyramidal side-effects in humans.Mice were handled 5 days before the test.On test day, mice were transferred into individual cages and left undisturbed for at least 1h.Catalepsy was measured by a bar test in which, a 1-cm-diameter metal bar was fixed horizontally 4 cm above the floor.Mice were first pretreated subcutaneously with saline, 30 min before vehicle [DMSO/Tween-80/Saline 2.5% 2.5%/95%: v/v, s.c], MAM (10 and 100 mg/kg, s.c), or haloperidol (1 mg/kg) and 1 h later, catalepsy was assessed.The forepaws of each mouse were placed gently on the bar and the time in seconds until the mouse took both paws off the bar was recorded, with a maximum cutoff of 180s.

Subchronic Hepato-nephrotoxicity Study
Study Design: A total of 32 adult male Swiss albino mice were used in the sub-chronic hepato-nephrotoxicity study.Mice were randomly divided into 4 groups of 7 mice.Two control groups received respectively normal saline (0.9% NaCl) solution (Group 1) or vehicle [DMSO/Tween-80/Saline 2.5% 2.5%/95%: v/v] (Group 2).Two groups (3 and 4) received respectively 10 mg/kg and 100 mg/kg (dissolved in vehicle) doses of MAM.All treatments were administered subcutaneously in a volume of 5 mL/Kg body weight once daily for 28 days at 10:00 am.The animals were observed for general symptoms of toxicity, external symptoms and mortality.All animals were sacrificed by decapitation under anesthesia 24 h after the last exposure.Blood was collected in heparinized tubes.The criteria of necropsy applied for all animals were based on color changes, size differences, and missing or mislocated organs according to Parkinson et al. [6].The kidney and liver were removed, cleaned with saline solution, weighed, and preserved in 10% formalin for histopathology examinations.The experiment was conducted according to the protocols described by OECD Guideline 407 [7] with minor modifications.Blood Biochemistry: Plasma was isolated from blood collected in heparinized tubes after centrifugation at 3000 rpm/min for 5 min.Following, biochemical parameters: creatinine, urea, bilirubin, aspartate transaminase (AST), alanine aminotransferase (ALT), glucose, and cholesterol were analyzed using a Beckman automatic analyzer.

Histopathological Study
Fixed liver and kidney tissues were transferred to 70% ethanol.They were then processed using a graded ethanol series (70 to 95%), cleared in xylene, and embedded in paraffin.The paraffin sections were cut into 5 μM-thick slices using a microtome (Leica RM2025 rotary microtome) and stained with hematoxylin and eosin.A Leica (DM2000 LED) light microscope was used to scan the slides for conventional morphological evaluation.

Table 1S .
Body weight (in grams), relative liver weight (RLW) and relative kidney weight (RKW) after 28 days subcutaneous injection of MAM (10 and 100 mg/kg) to Swiss albino mice.