The efficacy of a TrkB monoclonal antibody agonist in preserving the auditory nerve in deafened guinea pigs.

The auditory nerve typically degenerates following loss of cochlear hair cells or synapses. In the case of hair cell loss neural degeneration hinders restoration of hearing through a cochlear implant, and in the case of synaptopathy suprathreshold hearing is affected, potentially degrading speech perception in noise. It has been established that neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) can mitigate auditory nerve degeneration. Several potential BDNF mimetics have also been investigated for neurotrophic effects in the cochlea. A recent in vitro study showed favorable effects of M3, a TrkB monoclonal antibody agonist, when compared with BDNF. In the present study we set out to examine the effect of M3 on auditory nerve preservation in vivo. Thirty-one guinea pigs were bilaterally deafened, and unilaterally treated with a single 3-µl dose of 7 mg/ml, 0.7 mg/ml M3 or vehicle-only by means of a small gelatin sponge two weeks later. During the experiment and analyses the experimenters were blinded to the three treatment groups. Four weeks after treatment, we assessed the treatment effect (1) histologically, by quantifying survival of SGCs and their peripheral processes (PPs); and (2) electrophysiologically, with two different paradigms of electrically evoked compound action potential (eCAP) recordings shown to be indicative of neural health: single-pulse stimulation with varying inter-phase gap (IPG), and pulse-train stimulation with varying inter-pulse interval. We observed a consistent and significant preservative effect of M3 on SGC survival in the lower basal turn (approximately 40% more survival than in the untreated contralateral cochlea), but also in the upper middle and lower apical turn of the cochlea. This effect was similar for the two treatment groups. Survival of PPs showed a trend similar to that of the SGCs, but was only significantly higher for the highest dose of M3. The protective effect of M3 on SGCs was not reflected in any of the eCAP measures: no statistically significant differences were observed between groups in IPG effect nor between the M3 treatment groups and the control group using the pulse-train stimulation paradigm. In short, while a clear effect of M3 was observed on SGC survival, this was not clearly translated into functional preservation.

A neurotrophic approach to treating hearing loss: Translation from animal models to clinical proof-of-concept.

Currently, there are no approved medicines available for the treatment of hearing loss. However, research over the past two decades has contributed to a growing understanding of the pathological mechanisms in the cochlea that result in hearing difficulties. The concept that a loss of the synapses connecting inner hair cells with the auditory nerve (cochlear synaptopathy) contributes to hearing loss has gained considerable attention. Both animal and human post-mortem studies support the idea that these synapses (ribbon synapses) are highly vulnerable to noise, ototoxicity, and the aging process. Their degeneration has been suggested as an important factor in the speech-in-noise difficulties commonly experienced by those suffering with hearing loss. Neurotrophins such as brain derived neurotrophic factor (BDNF) have the potential to restore these synapses and provide improved hearing function. OTO-413 is a sustained exposure formulation of BDNF suitable for intratympanic administration that in preclinical models has shown the ability to restore ribbon synapses and provide functional hearing benefit. A phase 1/2 clinical trial with OTO-413 has provided initial proof-of-concept for improved speech-in-noise hearing performance in subjects with hearing loss. Key considerations for the design of this clinical study, including aspects of the speech-in-noise assessments, are discussed.

Hearing loss: The final frontier of pharmacology.

Despite a prevalence greater than cancer or diabetes, there are no currently approved drugs for the treatment of hearing loss. Research over the past two decades has led to a vastly improved understanding of the cellular and molecular mechanisms in the cochlea that lead to hearing deficits and the advent of novel strategies to combat them. Combined with innovative methods that enable local drug delivery to the inner ear, these insights have paved the way for promising therapies that are now under clinical investigation. In this review, we will outline this renaissance of cochlear biology and drug development, focusing on noise, age-related, and chemotherapy-induced hearing dysfunction.

Therapeutic Neuroprotection by an Engineered Neurotrophin that Selectively Activates Tropomyosin Receptor Kinase (Trk) Family Neurotrophin Receptors but Not the p75 Neurotrophin Receptor.

The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

BDNF, NT-3 and Trk receptor agonist monoclonal antibodies promote neuron survival, neurite extension, and synapse restoration in rat cochlea ex vivo models relevant for hidden hearing loss.

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.

Inhibitors of the Neutral Amino Acid Transporters ASCT1 and ASCT2 Are Effective in In Vivo Models of Schizophrenia and Visual Dysfunction.

The N-methyl-d-aspartate receptor coagonist d-serine is a substrate for the neutral amino acid transporters ASCT1 and ASCT2, which may regulate its extracellular levels in the central nervous system (CNS). We tested inhibitors of ASCT1 and ASCT2 for their effects in rodent models of schizophrenia and visual dysfunction, which had previously been shown to be responsive to d-serine. L-4-fluorophenylglycine (L-4FPG), L-4-hydroxyPG (L-4OHPG), and L-4-chloroPG (L-4ClPG) all showed high plasma bioavailability when administered systemically to rats and mice. L-4FPG showed good brain penetration with brain/plasma ratios of 0.7-1.4; however, values for L-4OHPG and L-4ClPG were lower. Systemically administered L-4FPG potently reduced amphetamine-induced hyperlocomotion in mice, whereas L-4OHPG was 100-fold less effective and L-4ClPG inactive at the doses tested. L-4FPG and L-4OHPG did not impair visual acuity in naive rats, and acute systemic administration of L-4FPG significantly improved the deficit in contrast sensitivity in blue light-treated rats caused by retinal degeneration. The ability of L-4FPG to penetrate the brain makes this compound a useful tool to further evaluate the function of ASCT1 and ASCT2 transporters in the CNS.

Phenylglycine analogs are inhibitors of the neutral amino acid transporters ASCT1 and ASCT2 and enhance NMDA receptor-mediated LTP in rat visual cortex slices.

The N-methyl-d-aspartate receptor (NMDA) co-agonist d-serine is a substrate for the neutral amino acid transporters ASCT1 (SLC1A4) and ASCT2 (SLC1A5). We identified l-phenylglycine (PG) and its analogs as inhibitors of ASCT1 and ASCT2. PG analogs were shown to be non-substrate inhibitors of ASCT1 and ASCT2 with a range of activities relative to other amino acid transport systems, including sodium-dependent glutamate transporters, the sodium-independent d-serine transporter asc-1 and system L. L-4-chloroPG was the most potent and selective ASCT1/2 inhibitor identified. The PG analogs facilitated theta-burst induced long-term potentiation in rat visual cortex slices in a manner that was dependent on extracellular d-serine. For structurally-related PG analogs, there was an excellent correlation between ASCT1/2 transport inhibition and enhancement of LTP which was not the case for inhibition of asc-1 or system L. The ability of PG analogs to enhance LTP is likely due to inhibition of d-serine transport by ASCT1/2, leading to elevated extracellular levels of d-serine and increased NMDA receptor activity. These results suggest that ASCT1/2 may play an important role in regulating extracellular d-serine and NMDA receptor-mediated physiological effects and that ASCT1/2 inhibitors have the potential for therapeutic benefit.

Restoration of visual performance by d-serine in models of inner and outer retinal dysfunction assessed using sweep VEP measurements in the conscious rat and rabbit.

The NMDA subtype of glutamate receptor and its co-agonist d-serine play a key role in synaptic function in the central nervous system (CNS), including visual cortex and retina. In retinal diseases such as glaucoma and macular degeneration, a loss of vision arises from malfunction of retinal cells, resulting in a glutamate hypofunctional state along the visual pathway in the affected parts of the visual field. An effective strategy to remedy this loss of function might be to increase extracellular levels of d-serine and thereby boost synaptic NMDA receptor-mediated visual transmission and/or plasticity to compensate for the impairment. We tested this idea in brain slices of visual cortex exhibiting long-term potentiation, and in rodent models of visual dysfunction caused by retinal insults at a time when the injury had stabilized to look for neuroenhancement effects. An essential aspect of the in vivo studies involved adapting sweep VEP technology to conscious rats and rabbits and combining it with intracortical recording while the animals were actively attending to visual information. Using this technology allowed us to establish complete contrast sensitivity function curves. We found that systemic d-serine dose-dependently rescued the contrast sensitivity impairment in rats with blue light-induced visual dysfunction. In rabbits with inner retinal dysfunction, both systemic and intravitreal routes of d-serine provided a rescue of visual function. In sum, we show that co-agonist stimulation of the NMDA receptor via administration of exogenous d-serine might be an effective therapeutic strategy to enhance visual performance and compensate for the loss of vision resulting from retinal disease.

D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures.

N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity.

Activity of the enantiomers of erythro-3-hydroxyaspartate at glutamate transporters and NMDA receptors.

The enantiomers of erythro-3-hydroxyaspartate were tested for activity at glutamate transporters and NMDA receptors. Both enantiomers inhibited glutamate transporters in rat hippocampal crude synaptosomes and elicited substrate-like activity at excitatory amino acid transporter 1, 2, and 3 as measured by voltage clamp in the Xenopus oocyte expression system. The enantiomers had similar affinities, but the D-enantiomer showed a lower maximal effect at excitatory amino acid transporter 1, 2, and 3 than the L-enantiomer. Surprisingly, D-erythro-3-hydroxyaspartate was a potent NMDA receptor agonist with an EC50 value in rat hippocampal neurons of 320 nM, whereas the L-enantiomer was 100-fold less potent. L-erythro-3-hydroxyaspartate showed activity at both glutamate transporters and NMDA receptors at concentrations that are reported to inhibit serine racemase, indicating a lack of selectivity. This enantiomeric pair may assist in shedding further light on the structural requirements for substrate activity at glutamate transporters and for agonist activity at NMDA receptors. The erythro enantiomers of 3-hydroxyaspartate had interesting and surprising effects on glutamate neurotransmitter systems. L-erythro-3-hydroxyaspartate had activity at both glutamate transporters (EAAT1/2/3) and NMDA receptors. D-erythro-3-hydroxyaspartate acted on EAATs, but was also identified as a highly potent NMDA receptor agonist. These enantiomers shed further light on the structural requirements for activity at EAATs and NMDA receptors.

Effects of 3-aminoglutarate, a "silent" false transmitter for glutamate neurons, on synaptic transmission and epileptiform activity.

Pharmacological tools that interact with the mechanisms that regulate vesicular filling and release of the neurotransmitter L-glutamate would be of enormous value. In this study, we provide physiological evidence that the glutamate analog, 3-aminoglutarate (3-AG), acts as a false transmitter to reduce presynaptic glutamate release. 3-AG inhibits glutamate-mediated neurotransmission both in primary neuronal cultures and in brain slices with more intact neural circuits. When assayed with the low affinity glutamate receptor antagonist γ-DGG, we demonstrate that 3-AG significantly reduces the synaptic cleft glutamate concentration, suggesting that 3-AG may act as a false transmitter to compete with glutamate during vesicle filling. Furthermore, using three different epileptic models (Mg(2+)-free, 4-AP, and high K(+)), we demonstrate that 3-AG is capable of suppressing epileptiform activity both before and after its induction. Our studies, along with those of the companion paper by Foster et al. (2015) indicate that 3-AG is a "silent" false transmitter for glutamate neurons that is a useful pharmacological tool to probe the mechanisms governing vesicular storage and release of glutamate under both physiological and pathophysiological conditions. 3-AG may have potential therapeutic value in conditions where the glutamate neurotransmitter system is pathologically overactive.

3-Aminoglutarate is a "silent" false transmitter for glutamate neurons.

Understanding the storage and release of the excitatory neurotransmitter, L-glutamate by synaptic vesicles has lagged behind receptor characterizations due to a lack of pharmacological agents. We report that the glutamate analog, 3-aminoglutarate (3-AG) is a "silent" false transmitter for glutamate neurons that may be a useful tool to study storage and release mechanisms. Like L-glutamate itself, 3-AG is a high-affinity substrate for both the plasma membrane (EAATs) and vesicular (vGLUT) glutamate transporters. As such, EAATs facilitate 3-AG entry into neuronal cytoplasm allowing 3-AG to compete with L-glutamate for transport into vesicles thus reducing glutamate content. In a synaptosomal preparation, 3-AG inhibited calcium-dependent endogenous L-glutamate release. Unlike L-glutamate, 3-AG had low affinity for both ionotropic (NMDA and AMPA) and G-protein coupled (mGlu1-8) receptors. Consequently, 3-AG behaves as a "silent" false transmitter that may be used in physiological experiments to probe synaptic vesicle storage and release mechanisms for L-glutamate. The companion paper by Wu et al. (2015) describes initial experiments that explore the effects of 3-AG on glutamate synaptic transmission under physiological and pathophysiological conditions.

Characterization of thien-2-yl 1S,2R-milnacipran analogues as potent norepinephrine/serotonin transporter inhibitors for the treatment of neuropathic pain.

Thien-2-yl 1S,2R-milnacipran analogues were synthesized and characterized as norepinephrine/serotonin transporter inhibitors. These compounds possessed higher potencies than 1S,2R-milnacipran (2R-1) while maintaining low molecular weight and moderate lipophilicity, which are the important features for the pharmacological and pharmacokinetic characteristics of milnacipran (1). Thus, compound 5c exhibited IC50 values of 2.3 and 32 nM, respectively, at NET and SERT, which were more than 10-fold better than those of 1 (NET IC50 = 77 nM, SERT IC50 = 420 nM). Moreover, 5c achieved the same efficacy as 1, but with much lower doses, in a rodent spinal nerve ligation pain model. In addition, 5c displayed desirable pharmacokinetic properties in several species, including high oral availability and significant brain penetration.

Studies on the structure-activity relationship of bicifadine analogs as monoamine transporter inhibitors.

Compounds with various activities and selectivities were discovered through structure-activity relationship studies of bicifadine analogs as monoamine transporter inhibitors. The norepinephrine-selective 2-thienyl compound S-6j was efficacious in a rodent pain model.

Identification of 1S,2R-milnacipran analogs as potent norepinephrine and serotonin transporter inhibitors.

A series of milnacipran analogs were synthesized and studied as monoamine transporter inhibitors, and several potent compounds with moderate lipophilicity were identified from the 1S,2R-isomers. Thus, 15l exhibited IC(50) values of 1.7nM at NET and 25nM at SERT, which were, respectively, 20- and 13-fold more potent than 1S,2R-milnacipran 1-II.

Studies on a series of milnacipran analogs containing a heteroaromatic group as potent norepinephrine and serotonin transporter inhibitors.

A series of milnacipran analogs containing a heteroaromatic group were synthesized and studied as monoamine transporter inhibitors. Many compounds exhibited higher potency than milnacipran at NET and NET/SERT with no significant change in lipophilicity. For example, compound R-26f was about 10-fold more potent than milnacipran with IC(50) values of 8.7 and 26nM at NET and SERT, respectively.

Pharmacological and pharmacokinetic characterization of 2-piperazine-alpha-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists.

A series of 2-piperazine-alpha-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compounds 8-12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists. These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations. Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model. After intraperitoneal (ip) administration of 1mg/kg dose, mice treated with 10d had significantly more food intake and weight gain than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d was dosed orally at 20mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in the treatment of cancer cachexia.

Design and synthesis of 3-arylpyrrolidine-2-carboxamide derivatives as melanocortin-4 receptor ligands.

Based on 3-phenylpropionamides, a series of 3-arylpyrrolidine-2-carboxamide derivatives was designed and synthesized to study the effect of cyclizations as melanocortin-4 receptor ligands. It was found that the 2R,3R-pyrrolidine isomer possessed the most potent affinity among the four stereoisomers.

Studies on the SAR and pharmacophore of milnacipran derivatives as monoamine transporter inhibitors.

Derivatives of milnacipran were synthesized and studied as monoamine transporter inhibitors. Potent analogs were discovered at NET (9k) and at both NET and SERT (9s and 9u). A pharmacophore model was established based on the conformational analysis of milnacipran in aqueous solution using NMR techniques and was consistent with the SAR results.

Identification and characterization of pyrrolidine diastereoisomers as potent functional agonists and antagonists of the human melanocortin-4 receptor.

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 13b displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-pyrrolidine 13b-1 possessed a Ki of 1.0 nM and an EC50 of 3.8 nM, while its 3R,4S-isomer 13b-2 exhibited a Ki of 4.7 and an IC50 of 64 nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 13b-1 also demonstrated efficacy in a diet-induced obesity model in rats.