In vitro and in vivo pharmacological characterization of the novel NK1 receptor selective antagonist Netupitant

doi:10.1016/j.peptides.2012.06.010

Peptides

Volume 37, Issue 1, September 2012, Pages 86-97

https://doi.org/10.1016/j.peptides.2012.06.010 Get rights and content

Abstract

The novel NK₁ receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK₁ cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK_B 8.87). In cells expressing NK₂ or NK₃ receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK_B 7.85) and, in functional washout experiments, displayed persistent (up to 5 h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1–10 mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK₁ agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID₅₀ 1.5 mg/kg) or orally (ID₅₀ 0.5 mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK₁ antagonist. Thus this molecule can be useful for investigating the NK₁ receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK₁ receptors has been demonstrated valuable for patients.

Highlights

► Netupitant behaves in vitro as a highly potent and selective NK₁ receptor antagonist. ► In bioassay washout experiments, Netupitant displayed persistent (up to 5 h) antagonist effects. ► Netupitant antagonizes nociceptive responses to intrathecal substance P in mice. ► Netupitant prevents foot tapping induced by a NK₁ agonist in gerbils showing long lasting effects. ► Netupitant is as a brain penetrant, orally active, potent and selective NK₁ antagonist.

Introduction

Tachykinins, i.e. substance P (SP), neurokinin A (NKA) and B (NKB), are small peptides characterized by the C-terminal sequence Phe-X-Gly-Leu-Met-NH₂. Tachykinins are able to bind and activate three different G-protein coupled receptors the NK₁, NK₂, and NK₃ [25]. Tachykinins are widely distributed in the central and peripheral nervous systems where they act as neurotransmitters. Several important biological functions are controlled by these peptides; for instance the SP/NK₁ system has been implicated in the regulation of pain and migraine, nausea and vomiting, mood and anxiety levels, alcoholism and inflammatory conditions of the gastrointestinal tract [23]. Despite this large range of potential indications, up to now NK₁ ligands are marketed only as antiemetics. In particular the NK₁ receptor antagonist Aprepitant [22] has been approved for the control of chemotherapy-induced and postoperative nausea and vomiting [24].

The aim of the present study was the in vitro and in vivo pharmacological characterization of the novel NK₁ receptor ligand Netupitant (2-(3,5-Bis-trifluoromethyl-phenyl)-N-methyl-N-[6-(4-methyl-piperazin-1-yl)-4-o-tolyl-pyridin-3-yl]-isobutyramide, Fig. 1) [14]. The actions of Netupitant were compared to those evoked by Aprepitant, used as a standard reference drug. In vitro studies were performed at recombinant human and rat NK receptors stably expressed in CHO and HEK293 cells, respectively, measuring intracellular calcium concentrations and at native guinea pig NK₁ receptors expressed in the ileum measuring SP-induced contractions. In vivo studies were performed in mice using the scratching, biting, and licking (SBL) test and in gerbils using the foot tapping assay.

Section snippets

Calcium mobilization studies in cells expressing human or rat tachykinin receptors

Cells stably expressing NK receptors were a generous gift from the laboratories of Prof. T. Costa (ISS, Rome, IT, HEK293 cells stably expressing the rat NK₁ receptor), Prof. C. Rojas (Johns Hopkins University School of Medicine, Baltimore, US, CHO cells stably expressing the human NK₁ receptor), and Prof. T.W. Schwartz (University of Copenhagen, DK, CHO cells expressing the human NK₂ or NK₃ receptors). CHO cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l

Calcium mobilization experiments in cells expressing the human and rat recombinant NK receptors

The first series of experiments was performed by testing the effects of the naturally occurring tachykinins SP, NKA and NKB in CHO cells expressing the three human NK receptors and in HEK293 cells expressing the rat NK₁ receptor (Fig. 2). SP and NKA produced a concentration-dependent mobilization of intracellular calcium in CHOhNK₁ cells with similar maximal effects approximately corresponding to 200% over the basal levels. NKB was inactive up to 1 μM. SP and NKA displayed high potency with the

Discussion

In the present study the pharmacological profile of the novel NK₁ receptor ligand Netupitant has been investigated in vitro and in vivo. Netupitant behaved as a pure and selective NK₁ receptor antagonist showing high potency at the human receptor and lower potency at the rat protein. The standard NK₁ receptor antagonist Aprepitant displayed a similar pharmacological profile. Both compounds behaved as pure and potent antagonists in the guinea pig ileum and their effects were only slightly

Disclosure statement

SC and CP are employed by Helsinn Healthcare SA, that is the exclusive licensee of the Netupitant (INN) patent EP 1035115 and of all the corresponding patents as extended worldwide.

Conflict of interest

All the other authors declare that there is no actual or potential conflict of interest related to this study.

Acknowledgment

This study was supported by an Helsinn Healthcare SA grant to GC.

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In vitro and in vivo pharmacological characterization of the novel NK1 receptor selective antagonist Netupitant

Abstract

Highlights

Introduction

Section snippets

Calcium mobilization studies in cells expressing human or rat tachykinin receptors

Calcium mobilization experiments in cells expressing the human and rat recombinant NK receptors

Discussion

Disclosure statement

Conflict of interest

Acknowledgment

Biochem Biophys Res Commun

Eur J Pharmacol

Eur J Pharmacol

Neuropharmacology

Neurosci Lett

Bioorg Med Chem Lett

Eur J Pharmacol

Brain Res

Neuropharmacology

Biochem Pharmacol

Support Cancer Ther

Trends Pharmacol Sci

Pain

Peptides

Eur J Pharmacol

Peptides

Eur J Pharmacol

Neuropharmacology

In vitro and in vivo pharmacological characterization of the novel NK₁ receptor selective antagonist Netupitant