Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog

Abstract

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS₁ and NTS₂ receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8–13) derivative denoted JMV2009, in which the Pro¹⁰ residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS₁ activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS₁ and NTS₂, improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptive-related behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerve-injured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.

Introduction

Today, opioids remain at the forefront of the pharmacological treatment of chronic pain (de Leon-Casasola, 2013; Gomes et al., 2014). However, opioid therapy is associated with a wide range of side effects, such as constipation, nausea, drowsiness, dizziness, sleep disturbance, dry mouth, respiratory depression and tolerance that negatively affects patients’ well-being and even discourages them for continuing their medications (Benyamin et al., 2008; Bruneau et al., 2018; Devulder et al., 2005; McNicol et al., 2003). In the ongoing opioid crisis, involving drug misuse, abuse, overdose and death (Coyle et al., 2018), there is therefore an urgent need for new, effective and safe non-opioid analgesics to improve the care of patients suffering from chronic pain (Besserer-Offroy and Sarret, 2019).

Among the alternative to opioids, neurotensin (NT) receptors recently emerged as attractive targets to develop new painkillers (Perez de Vega et al., 2018). Through its binding with two G protein-coupled receptors (GPCRs), referred to as NTS₁ and NTS₂, the tridecapeptide NT(1–13) was indeed found to induce a potent opioid-independent analgesia in a variety of pain paradigms following systemic or central administration (Dobner, 2006; Feng et al., 2014; Kleczkowska and Lipkowski, 2013; Sarret and Cavelier, 2018). Accordingly, it was pharmacologically demonstrated that the antinociceptive responses induced by NT and its analogs, designed and synthesized based on the minimal biologically active C-terminal region (Arg⁸-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³), could not be reversed by the opioid antagonists, naloxone and naltrexone (Behbehani and Pert, 1984; Bredeloux et al., 2006; Osbahr et al., 1981; Wustrow et al., 1995). Importantly, NT was also reported to provide stronger pain relief than morphine on a molecular basis (al-Rodhan et al., 1991; Nemeroff et al., 1979).

In the last two decades, peptide therapeutics have gained increased interest from the biomedical and pharmaceutical industry, filling the gap between the two main classes of available market drugs, small molecules and biologics (Fosgerau and Hoffmann, 2015; Kaspar and Reichert, 2013). Peptides are definitely attractive drug candidates due to their high potency/efficacy, target specificity and safety/tolerability profile. Despite offering many advantages, their extensive use as therapeutics is, however, limited by their short half-life and rapid plasma clearance, which leads to low bioavailability (Fosgerau and Hoffmann, 2015; McGonigle, 2012). Several strategies have therefore been developed to overcome the undesirable physicochemical properties of therapeutic peptides. These approaches successfully applied to the C-terminal NT(8–13) hexapeptide include backbone and side-chain modifications, such as d-amino acid and non-natural amino acid substitutions, incorporation of reduced bonds, lactam bridged mimetics or peptide-peptoid hybrids, N- and C-terminal modifications as well as introduction of conformational constraints via cyclization (Bittermann et al., 2004; Boules et al., 2010; Bredeloux et al., 2008; Einsiedel et al., 2011; Fanelli et al., 2015; Hughes et al., 2010; Labbé-Jullié et al., 1994; Sarret and Cavelier, 2018; Sousbie et al., 2018; Tyler-McMahon et al., 2000; Wustrow et al., 1995).

According to recent structure-activity relationship (SAR) studies, structural modifications with unnatural amino acids were effective in producing NT(8–13) analogs with improved metabolic stability, receptor selectivity and in vivo activity (Boules et al., 2010; Fanelli et al., 2015; Hapău et al., 2016; Hughes et al., 2010; Magafa et al., 2019; Tokumura et al., 1990; Tyler-McMahon et al., 2000). In that respect, non-natural silylated amino acids are of great interest for their ability to modulate the physicochemical properties of bioactive peptides (Cavelier et al., 2002, 2004; Dalkas et al., 2010; Fanelli et al., 2015, 2017; Remond et al., 2016). Herein, we report the synthesis and in vitro/in vivo characterization of a new NT(8–13) analog, named JMV2009, which binds to both NTS₁ and NTS₂ receptors. In this compound, the Pro¹⁰ residue was substituted by a silicon-containing unnatural amino acid proline surrogate, denoted silaproline (Sip), which has been shown to retain peptide conformation (Cavelier et al., 2002, 2006; Remond et al., 2017; Vivet et al., 2000). This Sip-containing NT(8–13) analog was further screened in a battery of pain models to assess its putative analgesic properties and evaluate for its ability to induce adverse effects associated with chronic opioid therapy, such as constipation or analgesic tolerance.