Combined treatment with diazepam and allopregnanolone reverses tetramethylenedisulfotetramine (TETS)-induced calcium dysregulation in cultured neurons and protects TETS-intoxicated mice against lethal seizures

Abstract

Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca2+ oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca2+ dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 μM) 20 min after TETS challenge normalizes synchronous Ca2+ oscillations when added in combination but not when added singly. Similarly, doses (0.03–0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.

Influence of tetramethylenedisulfotetramine on synchronous calcium oscillations at distinct developmental stages of hippocampal neuronal cultures

“…7E, Table 1). The observed TETS response on HN cultures at 14 DIV is consistent with previous studies (Cao et al, 2012a, Bruun et al, 2015). By comparison, HNG culture at 14 DIV responded to TETS challenge in a manner similar to that observed on 6 DIV (Fig.…”

“…In 14 DIV HN, we also observed a sustained [Ca 2+ ] i overloading which can be abolished by the NMDA receptor antagonist, MK-801 (Cao et al, 2012a). The results of recent studies indicate that application of MK-801 or ketamine more effectively prevents TETS-induced lethality compared to the prevention on TETS-induced tonic-clonic seizures suggesting the Phase I response contributes to the delayed pathology of TETS which has been observed in experimental and clinical studies (Cheng et al, 2002, Deng et al, 2012, Zolkowska et al, 2012, Vito et al, 2014, Bruun et al, 2015). In our in vitro study, HN/HNG responded to TETS differentially with distinct efficacy on the amplification of SCO amplitude.…”

“…Importantly, seizurogenic chemicals that engage distinct receptor targets were found to produce distinct changes in Ca 2+ dynamics and SCO patterns. Analysis of in vitro SCO patterns may serve not only as valuable rapid screening tool for identifying and classifying excitotoxicity of potential seizurogenic agents, but may also serve as a valuable means of high throughput discovery of anticonvulsants (Cao et al 2012a; Brunn et al, 2015). In this regard, new and innovative approaches and analytical tools are emerging for measurement and analysis of SCO patterns produced by dissociated neuronal networks and intact brain slices(Hongo et al, 2015, Jang and Nam, 2015, Patel et al, 2015).…”

First‐in‐man allopregnanolone use in super‐refractory status epilepticus

“…Tolerance and physical dependence after prolonged exposure to barbiturates and benzodiazepines results in a need for increased doses of these toxic drugs and there is a high propensity for seizures recurrence 15. Allopregnanolone has limited systemic side effects,16 and is highly effective in terminating ongoing status epilepticus in animal models 9, 17. These cases provided critical translational evidence of the activity of allopregnanolone in patients with SRSE, that led directly to the treatment of additional patients, including two children 12.…”

Behavioral intoxication following voluntary oral ingestion of tetramethylenedisulfotetramine: Dose-dependent onset, severity, survival, and recovery

“…There is currently no standard treatment for TETS poisoning in the United States, but a common Chinese protocol for TETS poisoning involves gastric lavage and high-volume or charcoal-filter hemoperfusion to remove as much TETS from the body as possible [5, 12]. Several potential drug treatments have also been identified for TETS poisoning, including diazepam [13–16], ketamine [13], MK-801 [13, 15], high-dosage γ-aminobutyric acid (GABA) [17], allopregnanolone [14, 17], and sodium dimercaptopropane sulfonate (Na-DMPS) [17]. However, no drug treatment (single or combinatorial) has been completely effective at preventing behavioral intoxication and delayed lethality following TETS poisoning, possibly due to the mechanism by which TETS toxicity occurs.…”