A safe lithium mimetic for bipolar disorder

Nisha Singh, Amy C Halliday, Justyn M Thomas, Olga V Kuznetsova, Rhiannon Baldwin, Esther C Y Woon, Parvinder K Aley, Ivi Antoniadou, Trevor Sharp, Sridhar R Vasudevan, Grant C Churchill, Nisha Singh, Amy C Halliday, Justyn M Thomas, Olga V Kuznetsova, Rhiannon Baldwin, Esther C Y Woon, Parvinder K Aley, Ivi Antoniadou, Trevor Sharp, Sridhar R Vasudevan, Grant C Churchill

Abstract

Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, therapeutic target of lithium remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself.

Figures

Fig. 1. Ebselen inhibits inositol monophosphatase in…
Fig. 1. Ebselen inhibits inositol monophosphatase in vitro
(a) Chemical structures. For the ebselen conjugate, R is glutathione or another ebselen molecule. (b) Concentration–inhibition relationships for ebselen and known inhibitors of IMPase. Assay used expressed human IMPase. (c) Concentration–inhibition relationships for ebselen on glycogen synthase kinase 3ß. (d,e) Effect of ebselen on the enzyme kinetics of IMPase shown as a Michaelis-Menten plot (d) or a Lineweaver-Burk plot (e). (f) Effect of dilution on inhibition of IMPase by ebselen (20 μM before and 0.2 μM after dilution). (g) Effect of recovery time after dilution on inhibition of IMPase by ebselen. (h) Mass spectroscopy under mild denaturing and non-denaturing conditions of IMPase incubated with ebselen or L-690,330 (100 μM each). (i-k) Effect of mutation cysteine-218 to alanine on inhibition of IMPase by ebselen assessed by concentration-inhibition curves (i), Michaelis-Menton kinetics (ebselen 50 μM) (j) and Vmax (k). Analyzed by a pre-planned t-test, n=6. (l) Concentration-response relationships for the sulfur analogue of ebselen and dibenzyldiselenide on IMPase. (m,n) Effect of disulfide reducing agents on inhibition of IMPase by ebselen (50 μM) with either post-incubation with glutathione (GSH, 1 mM) or dithiothreitol (50 mM) (m) or pre-incubation with 5 mM and 250 mM, respectively (n). All error bars represent standard error of the means.
Fig. 2. Ebselen permeates the blood-brain barrier…
Fig. 2. Ebselen permeates the blood-brain barrier and inhibits endogenous inositol monophosphatase in mouse brain
(a) Schematic illustrating the experimental protocol for assessing IMPase in ex vivo and brain homogenate experiments. Ebselen was injected at 10 mg/kg. (b,c) Concentration-inhibition relationships for novel and known inhibitors of mouse IMPase expressed in bacteria (b) or present in homogenates from mouse brain (c). (d) Michaelis-Menten plots showing the effect of injected ebselen on IMPase in ex vivo brain homogenate. Statistical significance was determined by a global fit of the Michaelis-Menten equation to the entire data set. (e) Effect of ebselen on the Vmax of IMPase over time after injection, analysed by pre-planned paired t-test between the treatment and control, n=5-6. All error bars represent standard error of the means.
Fig. 3. Ebselen induces lithium-like behaviour
Fig. 3. Ebselen induces lithium-like behaviour
(a) Ebselen attenuates the head-twitch response induced by the 5-HT2A agonist DOI (2 mg/kg), analyzed by pre-planned, one-tailed t-tests, n=6. (b, c) Ebselen (10 mg/kg) attenuates the increase in the immediate-early gene Arc mRNA induced by DOI (2 mg/kg) in two cortical regions. Analyzed by pre-planned, one-tailed t-tests, n=5-6. (d, f) Experimental protocols for assessing the effect of ebselen on behaviour in the open field test during exploratory activity (rearing, d) and amphetamine-induced hyperactivity (mobility, f). (e) Effect of ebselen (10 mg/kg) on rearing over time, analyzed by pre-planned paired t-tests, n=5-6. (g) Effect of ebselen on mobility during amphetamine-induced hyperactivity, analyzed by pre-planned paired t-test, n=6-8. All error bars represent standard error of the means.
Fig. 4. The pharmacological effects of ebselen…
Fig. 4. The pharmacological effects of ebselen are mediated by inositol depletion
(a) Schematic outlining the experimental protocol used to investigate inositol reversal of behaviours induced by ebselen. Drugs were injected as follows: 1 μL of 0.5 M inositol, 5 mg/kg ebselen and 2 mg/kg amphetamine. (b) Effect of inositol on the ability of ebselen to attenuate rearing, analysed by pre-planned paired t-tests between ebselen and control, n=4-6. (c) Effect of inositol on the ability of ebselen to attenuate amphetamine-induced hyperactivity (mobility) analyzed by pre-planned paired t-tests between ebselen and control, n=5-6. (d) Proton NMR spectra of authentic inositol and brain extracts from mice injected intraperitoneally with either ebselen (10 mg/kg) or hydroxypropyl ß-cyclodextrin(4% w/v, Control). (e), Effect of ebselen (10 mg/kg) on inositol levels in mouse brain. Inositol was quantified by integration of the C1 and C3 peaks (indicated by the bar in d), and analyzed by a pre-planned one-way t-test between ebselen and control, n=4. All error bars represent standard error of the means.

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