ST-2560, a selective inhibitor of the NaV1.7 sodium channel, affects nocifensive and cardiovascular reflexes in non-human primates.

BACKGROUND AND PURPOSE: The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. EXPERIMENTAL APPROACH: Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. KEY RESULTS: ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. CONCLUSIONS AND IMPLICATIONS: Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.

IL-12-assisted immunization against Listeria monocytogenes using replication-restricted VSV-based vectors.

Co-administration of IL-12 with vaccine immunogens has proven to be an effective strategy for eliciting potent Th1-biased immunity. Unfortunately, the use of IL-12 as a vaccine component has been limited because it is unstable at ambient temperatures, expensive to produce, and toxic when administered at excessive dosages. Using reverse genetics, we created a recombinant replication-restricted vesicular stomatitis virus that expresses large quantities of an IL-12 fusion protein (VSVDeltaG-IL12F), but can only establish a single round of infection because the genome does not encode the viral glycoprotein (G protein) that is required for viral entry into host cells. Here, we report that immunization of mice with a poorly immunogenic listerial antigen preparation (LMAg) in combination with VSVDeltaG-IL12F elicits potent T cell- and B cell-mediated responses that confer protective listerial immunity.

Recombinant replication-restricted VSV as an expression vector for murine cytokines.

Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus that rapidly and efficiently shuts down the production of host cell-encoded proteins and utilizes the cell's protein production machinery to express high levels of virally encoded proteins. In an effort to take advantage of this characteristic of VSV, we have employed a reverse genetics system to create recombinant forms of VSV encoding a variety of murine cytokines. Previous studies have revealed that cells infected with recombinant VSV that lack expression of the surface glycoprotein (G protein), designated deltaG-VSV, more efficiently express and secrete recombinant proteins than do recombinant "wild-type" VSV. Therefore, murine cytokine-expressing recombinants were produced as deltaG viruses. Propagation of these deltaG viruses in cells that transiently express G protein in vitro results in G-complemented virions that can infect cells, shut down host protein synthesis, and express at high levels each virally encoded protein (including the designated cytokine). We assessed the ability of each deltaG-VSV construct to express recombinant cytokine by infecting BHK cells and then monitoring/measuring the production of the desired cytokine. When possible, the bioactivity of the cytokine products was also measured. The results presented here reveal that large quantities of bioactive cytokines can be produced rapidly and inexpensively using deltaG-VSV as a protein expression system.

Adjuvanticity of an IL-12 fusion protein expressed by recombinant deltaG-vesicular stomatitis virus.

The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described. Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection. In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-deltaG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F. VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored. The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12. Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity. These studies demonstrated that VSVdeltaG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVdeltaG-IL12F as a vector for delivery of IL-12F in vivo.