Elsevier

Neuropharmacology

Volume 118, 15 May 2017, Pages 223-232
Neuropharmacology

A novel therapeutic with two SNAP-25 inactivating proteases shows long-lasting anti-hyperalgesic activity in a rat model of neuropathic pain

https://doi.org/10.1016/j.neuropharm.2017.03.026Get rights and content

Highlights

  • A novel protein was generated that efficaciously alleviates neuropathic pain in rats.

  • This genetically-engineered chimera of botulinum neurotoxins contains two proteases.

  • It enters sensory nerves and cleaves a SNARE essential for releasing pain mediators.

  • One local injection relieves pain for ∼2 weeks, far longer than common analgesics.

Abstract

A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex – a potent, specific and prolonged inhibitor of neuro-exocytosis – gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for ∼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.

Introduction

Major healthcare and economic problems arise from ∼20% of the population suffering from chronic pain (Volkow and McLellan, 2016), with 21% of such patients experiencing depression and 19% losing their jobs as a result of their pain (Breivik et al., 2006). Conventional analgesics (typically opiates) and anti-inflammatory drugs (steroidal and non-steroidal) are short-lived, usually exert adverse effects or may be associated with tolerance/dependence, and some patients are non-responsive (Volkow and McLellan, 2016). Due to severe side-effects or lack of efficacy, 23% of patients stop taking medication for their pain (Breivik et al., 2006). Clearly, there is an urgent unmet need for effective, long-lasting, well-tolerated and non-addictive analgesics to overcome these limitations. Nerve/tissue damage is a major cause of chronic pain due to abnormal signalling from peripheral nociceptors that elevates the release of excitatory transmitters and neuropeptides, as well as increasing the surface trafficking of signal transducing channels (Ellis and Bennett, 2013). This, together with increased secretion of pro-inflammatory cytokines, leads to neuronal hyper-sensitisation and consequential neuropathic pain (Basbaum et al., 2009). Lowering such over-excitability should be achievable by inhibiting the release of pain mediators and lowering the elevated level of transient receptor potential (TRP) channels on the plasmalemma of pain-sensing nerves (Meng et al., 2016). Such an intervention could provide an attractive alternative to current drugs that mainly act centrally.

Notably, botulinum neurotoxin type A (BoNT/A)-haemagglutinin complex from Clostridium botulinum is a selective, prolonged inhibitor of transmitter release, and has been extensively used for treating the hyper-activity of neurons innervating muscles or secretory glands (Naumann et al., 2013, Thenganatt and Fahn, 2012). Encouragingly, the toxin complex relieves chronic headache in a restricted number of patients (reviewed by (Diener et al., 2011, Whitcup et al., 2014)), and has shown promise for the treatment of certain neuropathic pain conditions in clinical research investigations (Jabbari, 2015, Oh and Chung, 2015, Whitcup et al., 2014). Thus, it is notable that BoNT/A inhibits regulated exocytosis and alters the surface trafficking of cation channel transducers in sensory neurons (Meng et al., 2016, Zhang et al., 2016). Each of the 7 serotypes of BoNT possesses a Zn2+-dependent light chain (LC) protease that is linked via a disulphide and non-covalent bonds to a heavy chain (HC); the latter binds neuronal ecto-acceptors and leads to endocytotic uptake (Dolly et al., 1984, Meng et al., 2013). Then, LC is translocated via the N-terminal half of HC (HN domain) into the cytoplasm (Pirazzini et al., 2015) where it cleaves and inactivates soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), which are essential for the exocytotic release of transmitters and pain mediators (Sudhof, 2014). Despite the advantageous long duration of action of LC/A, its limited effectiveness against pain may arise from it being unable to block capsaicin-evoked neuronal release of a representative peptide, CGRP (calcitonin gene-related peptide) (Meng et al., 2007). Although BoNT/A cleaves off 9 C-terminal residues from SNAP-25, the ability of the product (SNAP-25A) to form stable SNARE complexes with other partners might support CGRP secretion elicited by a large and sustained elevation of [Ca2+]i arising from activation of the TRP vanilloid 1 receptor (TRPV1) by capsaicin (Meng et al., 2009, Meng et al., 2014). Attaching BoNT/E protease (LC/E), which instead truncates 26 residues from SNAP-25, to a protease-inactive mutant of /A (BoTIM/A) generated LC/E-BoTIM/A; this enters sensory neurons, produces SNAP-25E and reduces capsaicin-evoked CGRP release (Meng et al., 2009, Wang et al., 2011).

Herein, two active SNAP-25 proteases were incorporated into a new fusion toxin, LC/E-BoNT/A. This protein showed greater solubility and stability than LC/E-BoTIM/A, and retained biological activity. Accordingly, a single intra-plantar (IPL) injection of LC/E-BoNT/A attenuated for up to 2 weeks both mechanical and cold hyper-sensitivities induced in a rat spared nerve injury (SNI) model of peripheral neuropathic pain (Decosterd and Woolf, 2000). It proved more efficacious than BoNT/A or repeated systemic administration of pregabalin, a clinically-used short-acting pain modulator (Verma et al., 2014), and its benefit was prolonged by a second injection. A single administration of LC/E-BoNT/A at a time when the pain behaviour had become fully established still proved effective.

Section snippets

Animals and study approval

Adult male Sprague-Dawley rats for behavioural tests were purchased from Envigo (formerly Harlan, UK) and weighed 200–250 g at the time of surgery. The rats (for neuronal cultures), and Tuck-Ordinary mice were bred in an approved Bio-Resource Unit at Dublin City University. Animals were group-housed in individually ventilated cages, with food and water available ad libitum. Room temperature was maintained between 18 and 22 °C and relative humidity between 40 and 60%; a 12 h light/12 h dark

A chimera of BoNT/A and /E engineered to possess two SNAP-25 cleaving proteases

A LC/E gene was ligated to that for BoNT/A and inserted into a pET29a vector to yield a plasmid encoding the composite toxin, LC/E-BoNT/A, with a His6 tag attached for IMAC. The construct encoded two thrombin consensus sites (Fig. 1A) so that this protease could simultaneously convert the pro-form SC to the active DC of the expressed protein, and remove its His6. The sequence-verified construct was expressed in E. coli at a yield >2 mg/L of culture and partially purified by IMAC; SDS-PAGE

Discussion

It is well accepted now that BoNT/A not only blocks the release of acetylcholine but also other neurotransmitters (e.g. ATP, pain-related neuropeptides) from bladder (Lawrence et al., 2010) and sensory neurons (Meng et al., 2007). In cultured sensory neurons from rodent trigeminal ganglia, BoNT/A cleaves off 9 C-terminal residues of SNAP-25 (yielding SNAP-25A) and dose-dependently inhibits the release of CGRP elicited by K+-depolarisation or bradykinin in vitro (Meng et al., 2007). However,

Summary

In summary, this new biotherapeutic outperformed clinically-used pregabalin or BoNT/A in attenuating nocifensive behaviour in the SNI model, whilst leaving normal somato-sensation unaltered in sham animals.

Author contributions and acknowledgements

The project was conceived by JOD, JM and JW, protein engineering and purification of the therapeutic proteins were carried out by JW. In vitro characterisation of the fusion protein was completed by JM. OPE purified samples for pain experiments, which were performed by LC, TZ and JN. The manuscript was written by JW, OM, TZ, LC, JN, JM and JOD. This study was supported by grants from the Commercialization Fund of Enterprise Ireland (to JOD, CF/2014/4355), a Principle Investigator (JOD,

Conflict of interest statement

The authors declare that they have no conflict of interest.

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    These authors contributed equally to this work.

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