Brian D. Klein

Developing novel antiepileptic drugs: characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models.

SummaryThe endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs, NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active after intravenous, intraperitoneal, and subcutaneous administration, and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved antiepileptic drugs.

Pharmacokinetic-Pharmacodynamic Relationships of (2S,3S)-Valnoctamide and Its Stereoisomer (2R,3S)-Valnoctamide in Rodent Models of Epilepsy

AbstractPurpose. Racemic valnoctamide (VCD) is a central nervous system- active drug commercially available in Europe. VCD possesses two chiral centers and, therefore, it exists as a mixture of four stereoisomers. The purpose of this study was to evaluate the anticonvulsant activity of two VCD stereoisomers in comparison with VCD (racemate), valpromide (VPD), and valproic acid (VPA) and to study their pharmacokinetic-pharmacodynamic relationships. Methods. The ability of racemic VCD, (2S,3S)-VCD, (2R,3S)-VCD and VPD to block partial seizures was studied in the 6Hz psychomotor seizure model in mice and in the hippocampal kindled rat. The ability of (2S,3S)-VCD and (2R,3S)-VCD to prevent generalized seizures was evaluated in the maximum electroshock (MES) and subcutaneous metrazole (sc Met) seizure tests. The PK of (2S,3S)-VCD, (2R,3S)-VCD, and VPD was studied in the mice utilized in the 6Hz model. Results. All of the tested compounds were effective in the models tested. No significant difference in ED50 values was observed but the plasma and brain EC50 values of (2R,3S)-VCD in the 6Hz model at 32 mA stimulation were 2-fold higher than the EC50 values of (2S,3S)-VCD. An excellent pharmacokinetic-pharmacodynamic correlation was found between the plasma and brain concentrations of the VCD stereoisomers and their anticonvulsant effect in mice. Stereoselectivity was observed in clearance, volume of distribution, and in brain-to-plasma AUC ratio at a dose of 25 mg/kg, but the difference disappeared at higher doses as the clearance of the stereoisomers decreased and their half-life increased. For (2R,3S)-VCD the brain-to-plasma AUC ratio doubled at the tested dose range, while it remained constant for (2S,3S)-VCD. Conclusions. Racemic VCD, VPD, (2R,3S)-VCD, and (2S,3S)-VCD are effective anticonvulsants in animal models of partial seizures and are more potent than VPA. The more favorable brain penetration of (2S,3S)-VCD and its lower EC50 value in the 6Hz test provides one advantage over (2R,3S)-VCD as a new antiepileptic drug.

c-Fos immunohistochemical mapping of the audiogenic seizure network and tonotopic neuronal hyperexcitability in the inferior colliculus of the Frings mouse.

The Frings mouse is a model of audiogenic seizure (AGS) susceptibility. The genetic locus responsible for the AGS phenotype in the Frings mouse has been named monogenic audiogenic seizure-susceptible (MASS1). MASS1 is unique in that it is one of only two identified seizure loci that are not associated with an ion channel mutation. Furthermore, Frings mice display a robust AGS phenotype demonstrating very high and prolonged susceptibility to sound-induced tonic extension seizures. The purpose of this investigation was to use c-Fos immunohistochemistry to map the brain structures involved in the Frings AGS and to examine neuronal hyperexcitability in the inferior colliculus, the brain structure that is recognized as the site of AGS initiation. AGS mapping revealed that intense seizure-induced neuronal activation was mostly limited to structures involved in a brainstem seizure network, including the external and dorsal nuclei of the inferior colliculus, as observed in other AGS rodents. Acoustically induced c-Fos expression in the central nucleus of the inferior colliculus to sub-AGS threshold tone stimulations displayed a greater level of neuronal activation in AGS-susceptible Frings, DBA/2J and noise-primed C57BL/6J mice compared to AGS-resistant C57BL/6J and CF1 mice. The AGS-susceptible mice also displayed c-Fos immunoreactivity that was more focused within the tonotopic response domain of the inferior colliculus compared to AGS-resistant mice. Furthermore, Frings mice displayed significantly greater tonotopic hyper-responsiveness compared to other AGS-susceptible mice.

Efficacy and tolerability of the galanin analog NAX 5055 in the multiple-hit rat model of symptomatic infantile spasms

Infantile spasms are seizures manifesting in infantile epileptic encephalopathies that are associated with poor epilepsy and cognitive outcomes. The current therapies are not always effective or are associated with serious side effects. Early cessation of spasms has been proposed to improve long-term outcomes. To identify new therapies for infantile spasms with rapid suppression of spasms, we are using the multiple-hit rat model of infantile spasms, which is a model of refractory infantile spasms. Here, we are testing the efficacy and tolerability of a single dose of the galanin receptor 1 preferring analog, NAX 5055, in the multiple-hit model of spasms. To induce the model, postnatal day 3 (PN3) male Sprague-Dawley rats underwent right intracerebral infusions of doxorubicin and lipopolysaccharide; p-chlorophenylalanine was then injected intraperitoneally (i.p.) at PN5. After the onset of spasms at PN4, 11-14 rats/group were injected i.p. with either NAX 5055 (0.5, 1, 2, or 4mg/kg) or vehicle. Video monitoring for spasms included a 1h pre-injection period, followed by 5h of recording post-injection, and two 2h sessions on PN5. The study was conducted in a randomized, blinded manner. Neurodevelopmental reflexes were assessed daily as well as at 2h after injection. Respiratory function, heart rate, pulse distension, oximetry and blood glucose were measured 4h after injection. The relative expression of GalR1 and GalR2 mRNA over β-actin in the cerebral cortex and hippocampus was determined with real time reverse transcription polymerase chain reaction. There was no acute effect of NAX 5055 on spasm frequency after the single dose of NAX 5055 (n=11-13 rats/group, following exclusions). Neurodevelopmental reflexes, vital signs, blood glucose measured 4h post-injection, and survival were not affected. A reduction in pulse and breath distention of unclear clinical significance was observed with the 7mg/kg NAX 5055 dose. GalR1 mRNA was present in the cerebral cortex and hippocampus of PN4 and adult rats. The hippocampal - but not the cortical - GalR1 mRNA expression was significantly lower in PN4 pups than in adults. GalR1 mRNA was also at least 20 times less abundant in the PN4 cortex than GalR2 mRNA. In conclusion, a single dose of NAX 5055 has no acute efficacy on spasms or toxicity in the multiple hit rat model of medically refractory infantile spasms. Our findings cannot exclude the possibility that repetitive NAX 5055 administration may show efficacy on spasms. The higher expression of GalR2 in the PN4 cortex suggests that GalR2-preferring analogs may be of interest to test for efficacy on spasms.

Cytokine and intracellular signaling regulation of tissue factor expression in astrocytes

There is evidence that inflammatory cytokines such as IL-1beta, TNFalpha, and IL-6 are involved in the pathogenesis of cerebrovascular disorders including stroke. One action of cytokines that contributes to diseases in peripheral tissues is upregulation of the procoagulant receptor tissue factor (TF). In the CNS, astrocytes are the primary cells that express TF; although little is known about how TF is regulated in these cells. Experiments were performed to evaluate the effect of cytokine treatment on TF activity in primary cultures of murine cortical astrocytes and in the human astrocytoma cell line (CCF). IL-1beta treatment induced a 2.5-fold increase in TF activity in the primary astrocytes and a 3-fold induction in the astrocytoma cells. TNFalpha treatment induced a 2.5-fold increase in TF activity in both the primary astrocytes and astrocytoma cells. IL-6 upregulated TF activity 2-fold in primary astrocytes, however, it had no effect on TF activity in the astrocytoma cells. The signaling pathways regulating TF expression in these cells were examined by using staurosporine, a broad spectrum inhibitor of serine-threonine protein kinases, and by examining the effects of intermediates in the sphingomyelin signaling pathway. Staurosporine inhibited IL-1beta-induced TF activity in the primary astrocytes but did not effect IL-1beta- or TNFalpha-induced TF activity in the astrocytoma cells. TF activity in the astrocytoma cells was upregulated 1.5-fold over constitutive levels by a ceramide analogue or the enzyme sphingomyelinase, however the ceramide analogue had no effect on TF activity in the primary astrocytes. These results suggest inflammatory cytokines can upregulate TF activity in astrocytes and the astrocytoma CCF cell line although the two cell types appear to utilize different signaling pathways to mediate TF expression. Further studies will be important to more completely define the signaling regulation of TF in astrocytes since alterations in brain TF levels may play a key role in CNS pathophysiology.

Introduction of lipidization–cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities

The neuropeptides galanin (GAL), neuropeptide Y (NPY) or neurotensin (NT) exhibit anticonvulsant activities mediated by their respective receptors in the brain. To transform these peptides into potential neurotherapeutics, their systemic bioavailability and metabolic stability must be improved. Our recent studies with GAL analogs suggested that an introduction of lipoamino acids in the context of oligo‐Lys residues (lipidization–cationization motif) significantly increases their penetration into the brain, yielding potent antiepileptic compounds. Here, we describe an extension of this strategy to NPY and NT. Rationally designed analogs of NPY and NT containing the lipidization–cationization motif were chemically synthesized and their physicochemical and pharmacological properties were characterized. The analogs NPY‐BBB2 and NT‐BBB1 exhibited increased serum stability, possessed log D > 1.1, retained high affinities toward their native receptors and produced potent antiseizure activities in animal models of epilepsy following intraperitoneal administration. Our results suggest that the combination of lipidization and cationization may be an effective strategy for improving systemic bioavailability and metabolic stability of various neuroactive peptides. Copyright

Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n-octanol/water partitioning coefficient (logD). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.

The National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP)

For over 40 years, the National Institute of Neurological Disorders and Stroke/National Institutes of Health-funded Anticonvulsant Screening Program has provided a preclinical screening service for participants world-wide that helped identify/characterize new antiseizure compounds, a number of which advanced to the market for the treatment of epilepsy. The newly-renamed Epilepsy Therapy Screening Program (ETSP) has a refocused mission to identify novel agents which will help address the considerable remaining unmet medical needs in epilepsy. These include identifying antiseizure agents for treatment-resistant epilepsy, as well as anti-epileptogenic agents that will prevent the development of epilepsy or disease-modifying agents that will ameliorate or even cure established epilepsy and its comorbidities. This manuscript provides an overview of the ETSP’s efforts aimed at identifying the next generation of therapeutic agents to further reduce the suffering from and burden of epilepsy.

Auditory deficits associated with the frings mgr1 (mass1) mutation in mice.

The gene responsible for the audiogenic seizure (AGS) phenotype in Frings mice, which was identified and originally designated Mass1, is now referred to as Mgr1. Although the function of the gene product is not known, the expression pattern suggests a role in the developing CNS. Hearing impairment is often observed in AGS-susceptible rodents and is thought to contribute to the pathology of AGS. We therefore hypothesized that the Frings mouse exhibits early-onset hearing impairment and that the Frings Mgr1 mutation is responsible for the hearing impairment phenotype that leads to the development of AGS susceptibility. Auditory brainstem response (ABR) thresholds were used to evaluate auditory function in mice carrying the Frings Mgr1 allele and were compared with other AGS-susceptible and -resistant mice. ABR testing demonstrated that mice possessing the Frings Mgr1 allele exhibit a mild to moderate level of hearing impairment that is present during the days following hearing onset. Furthermore, the hearing impairment resulting from the Frings Mgr1 allele is relatively stable, which explains the long duration of AGS susceptibility exhibited by Frings mice compared with other AGS-susceptible mice.

Analgesic Properties of a Peripherally Acting and GalR2 Receptor–Preferring Galanin Analog in Inflammatory, Neuropathic, and Acute Pain Models

There are ongoing efforts to develop pain therapeutics with novel mechanisms of action that avoid common side effects associated with other analgesics. The anticonvulsant neuropeptide galanin is a potent regulator of neuronal excitability and has a well established role in pain modulation, making it a potential target for novel therapies. Our previous efforts focused on improving blood-brain-barrier penetration and enhancing the metabolic stability of galanin analogs to protect against seizures. More recently, we designed peripherally acting galanin analogs that reduce pain-related behaviors by acting in the periphery and exhibit preferential binding toward galanin receptor (GalR)2 over GalR1. In this study, we report preclinical studies of a monodisperse oligoethylene glycol–containing galanin analog, NAX 409-9 (previously reported as GalR2-dPEG24), in rodent analgesic and safety models. Results obtained with NAX 409-9 in these tests were compared with the representative analgesics gabapentin, ibuprofen, acetylsalicylic acid, acetaminophen, and morphine. In mice that received intraplantar carrageenan, NAX 409-9 increased paw withdrawal latency with an ED50 of 6.6 mg/kg i.p. NAX 409-9 also increased the paw withdrawal threshold to mechanical stimulation following partial sciatic nerve ligation in rats (2 mg/kg). Conversely, NAX 409-9 had no effect in the tail flick or hot plate assays (up to 24 mg/kg). Importantly, NAX 409-9 did not negatively affect gastrointestinal motility (4–20 mg/kg), respiratory rate (40–80 mg/kg), or bleed time (20 mg/kg). These studies illustrate that this nonbrain-penetrating galanin analog reduces pain behaviors in several models and does not produce some of the dose-limiting toxicities associated with other analgesics.