Structure-activity relationship of methylene blue and its analogues as lead compounds for novel antidepressant development

Abstract

Antidepressants target serotonergic and/or catecholaminergic responses in an attempt at treating depression yet are at best 65% effective. New antidepressants, as well as new targets for antidepressant action, are thus urgently needed. The nitric oxide (NO)-cGMP cascade is implicated in the pathophysiology of depression. Methylene blue (MB) is a tricyclic compound that is structurally dissimilar to any known antidepressant, inhibits NO synthase (NOS) and guanylate cyclase and demonstrates significant antidepressive-like activity in rodents. The structural-activity relationship of MB and selected analogues will be studied as potential new lead antidepressant compounds using the acute and chronic forced swim test (FST), and compared to imipramine. Those analogues that demonstrate antidepressant-like activity will be studied with respect to their potential to inhibit monoamine oxidase (MAO) -A and –B in vitro, as well as modifying the NO-cGMP pathway in rat hippocampus following sub-chronic treatment. MB analogues include methylene green (MG), methylene violet (MV), thionin acetate (TA), phenothiazine (PHE), tacrine (TAC) and acriflavine (ACR). MB and MB analogues were tested over a dosage range of 0.5-60 mg/kg in the acute FST study and compared to saline and imipramine. Swimming was analysed with respect to swimming and climbing behaviour to provide an indication of serotonergic and catecholaminergic properties. All the analogues, including imipramine, were tested for their inhibitory action on MAO-A (and B if required) by spectrofluorometric assay using human recombinant MAO. Those analogues with efficacy in the acute FST were tested in the chronic FST following 7 days treatment at the most effective dose identified in the acute FST protocol and compared to imipramine. Hippocampi were removed for analysis of nitrogen oxides, a surrogate marker of NOS activity. IMI significantly reduced immobility in the acute FST, as did MB and MG, without effects on locomotor activity, thus indicating substantial antidepressant-like activity. ACR, TAC, MV, THI and PHE failed in this regard, while ACR and TAC significantly reduced locomotor activity. MB, MG and imipramine increased climbing behaviour in the acute FST, indicating catecholaminergic potentiation. MB (IC50=0.073 μm), MG (IC50=0.169 μm) and ACR (IC50= 0.43 μm) significantly inhibited MAO-A with moderate inhibition of MAO-B, while IMI and the other analogues were ineffective. In the chronic FST, MG was as effective as imipramine, while MB was more effective than imipramine in reversing immobility, with limited locomotor effects. Interestingly, MB and MG increased swimming behaviour during chronic treatment, indicative of bolstering serotonergic neurotransmission, while imipramine again increased climbing behaviour. Neither imipramine, MG or MB had notable effects on hippocampal NOS. The antidepressant activity of MB and MG involves actions on MAO than on NOS, although MB is a more effective antidepressant than imipramine. Of the various analogues tested, only MG presents with antidepressant-like activity. Both MB and MG are charged entities that have unique structural characteristics, including a dimethylamine substituent at C-3 and C-7, which appears to be a requirement for antidepressant activity. These attributes provide important clues for novel antidepressant drug development.