Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia

Highlights

• Incorporation of reduced amide bond, silaproline and TMSAla in NT(8−13) resulted in JMV5296, a 25-fold NTS2-selective analog.

• Combination of these three chemical modifications increased resistance toward proteases having a plasma stability over 20 h.

• Intrathecal injection of JMV5296 induced potent antinociception in acute, tonic and chronic inflammatory pain models.

• Central delivery of JMV5296 had no impact on body temperature.

Abstract

The endogenous tridecapeptide neurotensin (NT) has emerged as an important inhibitory modulator of pain transmission, exerting its analgesic action through the activation of the G protein-coupled receptors, NTS1 and NTS2. Whereas both NT receptors mediate the analgesic effects of NT, NTS1 activation also produces hypotension and hypothermia, which may represent obstacles for the development of new pain medications. In the present study, we implemented various chemical strategies to improve the metabolic stability of the biologically active fragment NT(8−13) and assessed their NTS1/NTS2 relative binding affinities. We then determined their ability to reduce the nociceptive behaviors in acute, tonic, and chronic pain models and to modulate blood pressure and body temperature. To this end, we synthesized a series of NT(8−13) analogs carrying a reduced amide bond at Lys⁸-Lys⁹ and harboring site-selective modifications with unnatural amino acids, such as silaproline (Sip) and trimethylsilylalanine (TMSAla). Incorporation of Sip and TMSAla respectively in positions 10 and 13 of NT(8−13) combined with the Lys⁸-Lys⁹ reduced amine bond (JMV5296) greatly prolonged the plasma half-life time over 20 h. These modifications also led to a 25-fold peptide selectivity toward NTS2. More importantly, central delivery of JMV5296 was able to induce a strong antinociceptive effect in acute (tail-flick), tonic (formalin), and chronic inflammatory (CFA) pain models without inducing hypothermia. Altogether, these results demonstrate that the chemically-modified NT(8−13) analog JMV5296 exhibits a better therapeutic profile and may thus represent a promising avenue to guide the development of new stable NT agonists and improve pain management.

Introduction

Chronic pain is an important public health problem affecting more than 20 % of the worldwide population [1]. Opioid drugs are extensively used in the treatment of chronic pain despite a long list of undesired effects and their limited long-term efficacy to relieve pain for many patients [2,3]. Even with the growing awareness of the risks associated with opioid misuse, overdose and addiction, opioid use is still rising, thus leading to the current opioid crisis in North America [4,5]. Thus, the societal and economic burden, healthcare costs and high prevalence of chronic pain encourage researchers to seek for new pain medications with an increased benefit/risk ratio [6,7]. Among the current development strategies, drugs targeting non-opioid G protein-coupled receptors (GPCR) represent a promising therapeutic avenue in pain research [7].

The development of peptide-based therapeutics is undergoing an exciting revival in the last decade, when compared to small molecule drugs [8,9]. Peptides often offer high target selectivity and specificity as well as enhanced efficacy, safety and tolerability profiles. However, naturally occurring peptides are often not directly suitable for clinical use due to low oral bioavailability, poor blood-brain barrier (BBB) penetration, and short half-life in physiological fluids related to their poor resistance to proteolytic degradation [8,10].

Among the promising alternatives to opioid pain medications, neurotensin (NT) receptors emerge as attractive targets for the treatment of pain [7,11]. Neurotensin (NT) is a small neuropeptide of 13 amino acids (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) [12] known to mediate its physiological effects through its binding to two receptors that belongs to 7TMRs superfamily, namely NTS1 and NTS2 [13]. Peripherally, NT acts as a hormone in the cardiovascular system where it induces a drop in blood pressure [14,15], controls appetite [16] and regulates gastrointestinal motility [17]. When administered directly into the central nervous system (CNS), NT is known to play a role in the regulation of anxiety [18,19] as well as in dopaminergic (DAergic)-associated diseases, such as schizophrenia, drug abuse, and Parkinson’s disease [[20], [21], [22]]. NT and its analogs also produce persistent hypothermia [20,23] and analgesia [[24], [25], [26]]. Both NTS1 and NTS2 receptors are present in CNS regions involved in nociceptive transmission and pain modulation, such as the spinal dorsal horn, periaqueductal gray (PAG), rostroventral medulla (RVM), dorsal raphe nucleus, raphe magnus and pallidus [25,[27], [28], [29]]. Moreover, the neurotensinergic system is gaining further interests for pain relief as NT-induced analgesia is not altered by the administration of the opioid antagonists’ naloxone and naltrexone, thereby supporting that NT receptor activation mediates its antinociception action independently of the opioid system [30].

Amino acids forming the C-terminal moiety of the native NT peptide, at position 8–13 (Arg-Arg-Pro-Tyr-Ile-Leu), were identified as the minimal sequence for NT receptor binding and biological activity [31,32]. Therefore, compound synthesis and structure-activity relationship studies have allowed the development of new NT(8−13) selective analogs targeting either NTS1 or NTS2 receptors and permitted the discrimination of their respective physiological effects [33,34]. While analgesia in acute, tonic, and chronic pain paradigms was demonstrated to be mediated by the activation of both receptors [24,25,28,[34], [35], [36]], only NTS1 activation was associated with hypotension [14,37] and hypothermia [38].

These adverse effects (hypothermia and hypotension) combined with the low metabolic stability of the native NT peptide preclude its therapeutic use as non-opioid pain-relieving medications. Therefore, chemical modifications of the backbone and the incorporation of unnatural amino acids are necessary to optimize the pharmacological properties of newly synthesized NT(8−13) drug candidates. In the present study, we evaluate the impact of site-selective chemical modifications of NT(8−13) to improve the peptide metabolic stability and its analgesic efficacy in different experimental pain models as well as to reduce the unwanted effects triggered by NTS1 activation.