2DG and Glycolysis as Therapeutic Targets for Status Epilepticus

Abstract

2-deoxy-D-glucose (2DG) is a glucose analogue differing from glucose only by removal of an oxygen atom at the 2 position, which prevents the isomerization of glucose-6-phosphate to fructose-6-phosphate, and thereby reversibly inhibits glycolysis. PET studies of regional brain glucose utilization positron-emitting 18F-2DG demonstrate that brain regions generating seizures have diminished glucose utilization during interictal conditions, but rapidly transition to markedly increased glucose delivery and utilization during seizures, particularly in status epilepticus (SE). 2DG has acute antiseizure actions in multiple in vivo and in vitro seizure models, including models of SE induced by the chemoconvulsants pilocarpine and kainic acid, suggesting that focal enhanced delivery of 2DG to ictal brain circuits is a potential novel anticonvulsant intervention for treatment of SE.

Continuous or rapidly recurring episodes of neuronal synchrony underlying the clinical condition of status epilepticus (SE) produce extreme energy demands and metabolic stress at cellular, brain circuit, and network levels. As the electrographic and clinical manifestations of SE evolving into refractory and super-refractory SE persist in as many as ~30% of patients despite currently available interventions with antiseizure medications and anesthesia [1–3], metabolic intervention to modify the cellular metabolic processes driving seizures and contributing to seizure-induced neuronal damage is a compelling potential therapeutic opportunity.

While the human brain represents ~ 2% of total body weight, in states of normal physiological and neural activity the brain consumes about ~25% of available nutrient energy. About 75% of brain nutrient use in normal physiological conditions is required for maintenance of ionic gradients and normal synaptic activity [4]. Glucose is the major source of energy and primary metabolic substrate in the adult brain supporting sensory perception, cognition, locomotion, learning, memory formation, and the broad range of behaviors required for successful environmental adaptation. Lactate and monocarboxylic acids such as acetoacetate and β – hydroxy-butyrate may also contribute to brain energy supply and metabolism, particularly during development, but the brain requires a continuous supply of glucose and O₂ for normal function. In metabolically stressful pathological conditions such as seizures and hypoglycemia [5], the supply of glucose is rate-limiting for brain metabolism.

The critical importance of delivery of glucose and O₂ as essential substrates for brain metabolism is regulated by neurovascular coupling, which precisely controls local blood flow and delivery of glucose and O₂ at the neuronal and brain circuit level based on local levels of neural activity [6]. The precisely regulated matching of blood flow delivery with neural activity and cellular energy needs in specific brain circuits is referred to as cerebral autoregulation, and depends on “neurovascular” coupling mediated by specific, locally connected neurons, astrocytes, and vascular endothelial cells. Blood flow and delivery of glucose and O₂ by neurovascular coupling is regulated with remarkable spatial and temporal precision at levels of microns and mSec in local brain circuits. This precise local regulation in settings of intense neural activity such as SE offers a potential therapeutic intervention target.

The close correspondence of neural activity and the rate of focal brain glycolysis in specific brain regions as a consequence of neurovascular coupling and cerebral autoregulation was initially demonstrated in rodents by autoradiography and in humans by positron emission tomography (PET) using radiolabeled 2-deoxy-D-glucose (2DG) [7,8]. 2DG is a glucose analogue differing from glucose only by removal of an oxygen atom at the 2 position. Both glucose and 2DG undergo uptake into brain via glucose transporters in response to cellular energy demand and neural activity. After uptake, both glucose and 2DG undergo phosphorylation at the 6-position by the enzyme glucose-6-kinase, yielding glucose-6-phosphate (glucose-6P) or 2-deoxy-D-glucose-6-phosphate (2DG-6P), respectively. Glucose-6P then undergoes isomerization to fructose-6P by phosphoglucose isomerase (PGI). Due to the removal of oxygen at the 2-position, 2DG-6P cannot undergo isomerization, reversibly blocking subsequent steps of glycolysis. With reversible isomerization block of the glycolytic pathway, radiolabeled 2DG accumulates in brain regions as a function of local neural activity, enabling measurement and imaging of local glucose utilization by ³H-2DG or ¹⁴C-2DG autoradiography, or positron-emitting ¹⁸F- 2-deoxy-D-glucose (¹⁸F-2DG PET). The concentrations of radiolabeled 2DG producing isomerization block of the glycolytic pathway sufficient for autoradiographic or PET imaging is in the femtomolar range.

¹⁸F-2DG PET imaging studies in patients with epilepsy have confirmed that synchronous activity and repetitive EEG discharges during acute seizures and SE dramatically increase local glucose utilization in neural circuitry. While patients with temporal lobe epilepsy during interical periods commonly demonstrate glucose hypometabolism in regions of brain circuitry identified as seizure onset zones, multiple PET studies serendipitously obtained during acute ictal episodes and SE confirm that glucose utilization dramatically increases in these regions in association with increased neural activity during seizures and SE [9,10]. A recent ¹⁸F-2DG PET study in patients with lateralized periodic discharges (LPDs) demonstrated focal areas of increased glucose utilization in brain regions at sites generating LPDs which monotonically correlated with LPD frequency in those areas [11]. Taken together, ¹⁸F-2DG PET studies in patients with acute seizures, SE, and LPDs unequivocally demonstrate increased glucose uptake and utilization in response to increased energy needs in brain regions generating synchronous and ictal activity, confirming that neural activity underlying these pathological states is significantly dependent on glycolytic metabolism. Human ¹⁸F-2DG PET studies and ³H-2DG or ¹⁸C-2DG autoradiographic studies in rodents thus suggest that metabolic interventions including glycolytic inhibition have the potential to modify cellular processes underlying seizures, SE, and seizure-induced neuronal damage.

The potential of glycolytic inhibition as a target for treatment of SE is suggested by evidence that 2DG also has antiseizure effects. While femtomolar concentrations of 2DG are sufficient to produce isomerization block at the PGI step of the glycolytic pathway and reversible glycolytic inhibition enabling measurement of glucose utilization and imaging in PET and autoradiographic studies, higher concentrations of 2DG administered in both in-vitro and in-vivo experimental models of seizures and epilepsy have additionally demonstrated pronounced anticonvulsant and antiseizure actions. 2DG concentrations of 5–10mM in hippocampal slices reduce increases in spontaneous excitatory postsynaptic currents (sEPSCs) observed in hippocampal CA3 neurons exposed to elevated [K⁺]_o [12, 13] and epileptic field bursts in CA3 evoked by 7.5mM [K⁺]_o, 50–100μM 4AP, and 10μM bicuculline [12]. The actions of 2DG are presynaptic as indicated by reduction in the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) [14].

The acute antiseizure actions of 2DG observed in-vitro in hippocampal slice preparations have been confirmed by evidence of dose-dependent acute anticonvulsant and antiseizure effects in multiple experimental in vivo models of seizures and epilepsy. These confirmatory studies include seizures evoked by 6 Hz corneal stimulation in mice (doses of 75—300 mg/kg i.p.; ED₅₀ = 79.7 mg/kg) , audiogenic seizures evoked in Frings mice (doses of 220—250 mg/kg i.p.; ED₅₀ = 206.4 mg/kg), and seizures evoked by pentylentetrazol (PTZ) in rats (at 30 min after doses of 200—400 mg/kg i.p.) [12].

2DG also has in vivo chronic ‘‘disease-modifying’’ antiepileptic effects against progressive effects of repeated seizures evoked by kindling. Doses of 37.5—250 mg/kg 30 min prior to kindling stimulation result in 2-fold slowing of progression of repeated seizures evoked by perforant path or olfactory bulb kindling (12, 15]. The “disease-modifying” antiepileptic effects against kindling progression were observed when 2DG was administered immediately after, and approximately 10 min after, evoked seizures [16, 17]. The “disease-modifying” antiepileptic effects with post-seizure administration are most likely due to enhanced post-ictal delivery of 2DG into brain regions with continuing increased energy demand even after the seizure, as ¹⁸F-2DG PET has demonstrated increased glucose utilization persisting for about 15 minutes after seizures. This novel “post-seizure” therapeutic effect of 2DG as a consequence of neurovascular coupling and autoregulation of cerebral blood flow in response to regional metabolic and energetic needs implies a potential opportunity for ‘‘post-seizure’’ anticonvulsant administration for the treatment of seizure clusters and SE.

The anticonvulsant and antiseizure actions of 2DG observed in models of acute, isolated, brief seizures have also been documented in experimental chemoconvulsant-evoked models of SE in adult mice and both developing and adult rats. Administration of 2DG (250 mg/kg i.p.) in adult rats followed by SE induced 30 minutes later by kainic acid (10 mg/kg, i.p.) or pilocarpine (300 mg/kg i.p.) delayed the latency to onset of seizures, and reduced seizure severity and duration [18]. Administration of 2DG in adult rats followed by SE induced 30 minutes later by pilocarpine (320 mg/kg i.p.) demonstrated dose-dependent anticonvulsant effects with delayed latency to onset of seizures (doses of 250, 500 mg/kg i.p.), reduced seizure severity (doses of 250, 500 mg/kg i.p.), and reduced seizure duration (doses of 100, 250, and 500 mg/kg i.p.) [19]. In developing rat pups (postnatal days 10–17) treated with pilocarpine (300 mg/kg i.p.), 2DG administered at doses of 50, 100, or 500 mg/kg i.p. after the onset of clinical seizures evolving into SE dose-dependently stopped behavioral and electrographic seizures, with onset of seizure suppression occurring more rapidly with higher doses [20].

Preclinical toxicity studies have demonstrated that chronic administration of 2DG for 28 days induces dose-dependent, fully reversible cardiac myocyte vacuolation with features of reversible autophagy in rats [21]. However, acute oral dosing of less than 2 weeks did not reveal any cardiac or other toxicity. Other routine preclinical genotoxic and other studies have not demonstrated any other toxicities. As this reversible toxicity is observed only after 2 weeks of daily administration, single or limited repeated doses of 2DG for treatment of SE appear to have a favorable safety, tolerability, and toxicity profile.

2DG has been administered to more than 100 people in studies unrelated to seizures, including in human clinical cancer trials as a pre-treatment to sensitize cancer cells for susceptibility to radiation or chemotherapy treatment [22, 23]. It has been administered intravenously in normal volunteers to study the acute effects of glucoprivation on hunger responses and cerebral blood flow [24, 25]. In these studies acute dosing of 2DG was usually well-tolerated without any consistent side effects including single IV doses of 60 mg/kg [24] and acute oral dosing up to 250 mg/kg [22]. Oral doses of > 60 mg/kg daily for a week in every other week cycles prolonged the QTc interval in cancer patients [23]. These studies suggest that 2DG could be safely tolerated as an acute IV dose in the treatment of status epilepticus.

Conclusions and clinical implications

2DG is currently in preclinical development as a potential treatment for SE. The impetus for this development program is the well-established dependence of synchronized neural circuitry generating ictal events on glycolytic metabolism, the remarkable focally enhanced delivery of 2DG to synchronized circuitry with high metabolic demand, and substantial evidence that 2DG has activity-dependent acute antiseizure effects as well as chronic “disease-modifying” antiepileptic actions against consequences and progression of repeated seizures. The efficacy and safety of brief reversible inhibition of glycolysis as demonstrated by 2DG suggests that glycolytic inhibition is a potentially promising therapeutic target for all forms of seizures, including SE.

With a novel activity-dependent metabolic antiseizure mechanism of action, minimal acute toxicity at anticipated doses, and cardiac toxicity unlikely with single or limited repeated dosing, 2DG offers a potentially novel approach for intervention compared to all available anticonvulsants. In regard to potential application in treatment of SE, 2DG has had no sedating effects in preclinical studies in animals and in human clinical trials as a potential adjuvant treatment for cancer, and thus also has potential as a first non-sedating treatment for SE.