Spinal cord injury pharmacotherapy: Current research & development and competitive commercial landscape as of 2015.

CONTEXT: Current treatment of spinal cord injury (SCI) focuses on cord stabilization to prevent further injury, rehabilitation, management of non-motor symptoms, and prevention of complications. Currently, no approved treatments are available, and limited treatment options exist for symptoms and complications associated with chronic SCI. This review describes the pharmacotherapy landscape in SCI from both commercial and research and development (R&D) standpoints through March 2015. METHODS: Information about specific compounds has been obtained through drug pipeline monographs in the Pharmaprojects® (Citeline, Inc., New York, New York, USA) drug database (current as of a search on May 30, 2014), websites of individual companies with compounds in development for SCI (current as of March 24, 2015), and a literature search of published R&D studies to validate the Pharmaprojects® source for selected compounds (current as of March 24, 2015). RESULTS: Types of studies conducted and outcomes measured in earlier phases of development are described for compounds in clinical development Currently four primary mechanisms are under investigation and may yield promising therapeutic targets: 1) neuronal regeneration; 2) neuroprotection (including anti-inflammation); 3) axonal reconnection; and 4) neuromodulation and signal enhancement. Many other compounds are no longer under investigation for SCI are mentioned; however, in most cases, the reason for terminating their development is not clear. CONCLUSION: There is urgent need to develop disease-modifying therapy for SCI, yet the commercial landscape remains small and highly fragmented with a paucity of novel late-stage compounds in R&D.

Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy.

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5'-bromo-2'deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.

Differential dependence of axo-dendritic and axo-somatic GABAergic synapses on GABAA receptors containing the alpha1 subunit in Purkinje cells.

Synapse formation and maintenance require extensive transsynaptic interactions involving multiple signal transduction pathways. In the cerebellum, Purkinje cells (PCs) receive GABAergic, axo-dendritic synapses from stellate cells and axo-somatic synapses from basket cells, both with GABAA receptors containing the alpha1 subunit. Here, we investigated the effects of a targeted deletion of the alpha1 subunit gene on GABAergic synaptogenesis in PCs, using electrophysiology and immunoelectron microscopy. Whole-cell patch-clamp recordings in acute slices revealed that PCs from alpha1(0/0) mice lack spontaneous and evoked IPSCs, demonstrating that assembly of functional GABAA receptors requires the alpha1 subunit. Ultrastructurally, stellate cell synapses on PC dendrites were reduced by 75%, whereas basket cell synapses on the soma were not affected, despite the lack of GABAA-mediated synaptic transmission. Most strikingly, GABAergic terminals were retained in the molecular layer of adult alpha1(0/0) mice and formed heterologous synapses with PC spines characterized by a well differentiated asymmetric postsynaptic density. These synapses lacked presynaptic glutamatergic markers and postsynaptic AMPA-type glutamate receptors but contained delta2-glutamate receptors. During postnatal development, initial steps of GABAergic synapse formation were qualitatively normal, and heterologous synapses appeared in parallel with maturation of dendritic spines. These results suggest that synapse formation in the cerebellum is governed by neurotransmitter-independent mechanisms. However, in the absence of GABAA-mediated transmission, GABAergic terminals in the molecular layer apparently become responsive to synaptogenic signals from PC spines and form stable heterologous synapses. In contrast, maintenance of axo-somatic GABAergic synapses does not depend on functional GABAA receptors, suggesting differential regulation in distinct subcellular compartments.

Compensatory alteration of inhibitory synaptic circuits in cerebellum and thalamus of gamma-aminobutyric acid type A receptor alpha1 subunit knockout mice.

Targeted deletion of the alpha1 subunit gene results in a profound loss of gamma-aminobutyric acid type A (GABA(A)) receptors in adult mouse brain but has only moderate behavioral consequences. Mutant mice exhibit several adaptations in GABA(A) receptor subunit expression, as measured by Western blotting. By using immunohistochemistry, we investigated here whether these adaptations serve to replace the missing alpha1 subunit or represent compensatory changes in neurons that normally express these subunits. We focused on cerebellum and thalamus and distinguished postsynaptic GABA(A) receptor clusters by their colocalization with gephyrin. In the molecular layer of the cerebellum, alpha1 subunit clusters colocalized with gephyrin disappeared from Purkinje cell dendrites of mutant mice, whereas alpha3 subunit/gephyrin clusters, presumably located on dendrites of Golgi interneurons, increased sevenfold, suggesting profound network reorganization in the absence of the alpha1 subunit. In thalamus, a prominent increase in alpha3 and alpha4 subunit immunoreactivity was evident, but without change in regional distribution. In the ventrobasal complex, which contains primarily postsynaptic alpha1- and extrasynaptic alpha4-GABA(A) receptors, the loss of alpha1 subunit was accompanied by disruption of gamma2 subunit and gephyrin clustering, in spite of the increased alpha4 subunit expression. However, in the reticular nucleus, which lacks alpha1-GABA(A) receptors in wild-type mice, postsynaptic alpha3/gamma2/gephyrin clusters were unaffected. These results demonstrate that adaptive responses in the brain of alpha1(0/0) mice involve reorganization of GABAergic circuits and not merely replacement of the missing alpha1 subunit by another receptor subtype. In addition, clustering of gephyrin at synaptic sites in cerebellum and thalamus appears to be dependent on expression of a GABA(A) receptor subtype localized postsynaptically.

Disruption of the neurogenic potential of the dentate gyrus in a mouse model of temporal lobe epilepsy with focal seizures.

Adult hippocampal neurogenesis is enhanced in response to multiple stimuli including seizures. However, the relationship between neurogenesis and the development of temporal lobe epilepsy (TLE) remains unclear. Unilateral intrahippocampal injection of kainate in adult mice models the morphological characteristics (e.g. neuronal loss, gliosis, granule cell dispersion and hypertrophy) and occurrence of chronic, spontaneous recurrent partial seizures observed in human TLE. We investigated the influence of a kainate-induced epileptogenic focus on hippocampal neurogenesis, comparing neural stem cell proliferation following status epilepticus and spontaneous recurrent partial seizures. Cell proliferation in the subgranular zone was transiently increased bilaterally after kainate treatment. As a result, neurogenesis was stimulated in the contralateral dentate gyrus. In contrast, the epileptic hippocampus exhibited a strongly reduced neurogenic potential, even after onset of spontaneous recurrent partial seizures, possibly due to an alteration of the neurogenic niche in the subgranular zone. These results show that neurogenesis does not contribute to the formation of the epileptic focus and may be affected when dispersion of dentate gyrus granule cells occurs. Therefore, in patients with TLE, hippocampal sclerosis and granule cell dispersion may play a significant role in disrupting the potential for hippocampal neurogenesis.

Alpha 1 subunit-containing GABA type A receptors in forebrain contribute to the effect of inhaled anesthetics on conditioned fear.

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA(A)-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA(A)-Rs and individual GABA(A)-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA(A)-Rs that harbor the alpha1 subunit. To test this, we used global knockout mice that completely lack the alpha1 subunit and forebrain-specific, conditional knockout mice that lack the alpha1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that alpha1-containing GABA(A)-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.

Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice.

Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that gamma-aminobutyric acidA (GABA(A)) receptor alpha1-/- mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABA(A) receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABA(A) receptor alpha1-/- mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.

NPI-031G (puerarin) reduces anxiogenic effects of alcohol withdrawal or benzodiazepine inverse or 5-HT2C agonists.

Because extracts of kudzu have been used as a hangover remedy in China for many centuries, we tested the ability of NPI-031G (puerarin), an isoflavone isolated from kudzu, to counteract anxiogenic effects associated with withdrawal from chronic alcohol exposure. NPI-O31G (50 and 150 mg/kg ip) significantly increased the social interaction and locomotor activity reduced by withdrawal from 17 days of alcohol (7%) diet. The effects of NPI-031G resembled those of the benzodiazepine antagonist, flumazenil (5 mg/kg), and the 5-HT(2C) antagonist, SB 242084 (1 mg/kg). In a separate study, control rats were pretreated with NPI-031G (30 min) and then given the anxiogenic compounds DMCM, a benzodiazepine inverse agonist, or Ro 600175, a 5-HT(2C) agonist. NPI-031G significantly counteracted the reduction in social interaction induced by either compound. To identify a potential mechanism of action of NPI-031G, synaptoneurosomes were isolated from the cerebral cortex of untreated rats and chloride uptake assays were carried out. NPI-031G did not have any effect on the stimulation of chloride uptake by muscimol, a GABA(A) agonist. However, it reduced the potentiation of muscimol-stimulated chloride uptake by flunitrazepam, a benzodiazepine agonist, at a concentration of 100 microM. A reduction in [3H]flunitrazepam binding was also seen at this concentration. These findings are consistent with NPI-031G being a weak benzodiazepine site antagonist.