Neurotensin Analogues Research Articles

Preclinical evaluation of a new technetium-99m labeled neurotensin analogue for NTSR1 targeted radionuclide imaging

Neurotensin is a regulatory peptide that can act as a growth factor on different types of normal and cancerous cells. Binding of Neurotensin to relevant receptors leads to cell proliferation, survival, migration and invasion by changing intracellular enzyme activity. Therefore, the design of a neurotensin-based radiopeptide plays an important role in targeted imaging or therapy of neurotensin receptor-positive tumors. A [Lys8]-neurotensin (7–13) peptide was synthesized and attached to HYNIC as a chelator via a linker. The labeling procedure was carried out at 100 °C for 10 min using 99mTc as a radionuclide and EDDA/tricine as coligands. Stability of the labeled peptide in human serum was determined using RTLC and HPLC methods. The receptor binding internalization was studied using HT-29 colon carcinoma cells, and tissue biodistribution was evaluated in mice bearing CT-26 tumors. The [99mTc]Tc-Tricine/EDDA/HYNIC-GABA-[Lys8]-neurotensin (7–13) peptide demonstrated a labeling yield of over 98 %, a specific activity of 37.00 GBq/µmol, high stability in human serum, a nanomolar range of Kd, and a tumor uptake of 0.36 ± 0.15 % ID/g at 1-h post-injection. These results suggest that the labeled peptide is a suitable imaging agent for neurotensin receptor-positive tumors.

Neurotensin agonist PD149163 modulates lipopolysaccharide induced inflammation and oxidative stress in the female reproductive system of mice

Inflammation-mediated reproductive health problems in females have become an emerging concern. The present investigation was aimed to elucidate the efficacy of the PD149163, agonist of the type I neurotensin receptor, in preventing/ameliorating the lipopolysaccharide (LPS) induced inflammation of the female reproductive system of the mice. Female Swiss Albino Mice (8 weeks old) were maintained in three groups (6/group): Group I as Control, Group II and Group III were exposed to intraperitoneal (i.p) LPS (1 mg/kg bw) for 5 days followed by treatment with PD149163 (100 μg/kg BW i.p.) to Group III (LPS + PD) for 28 days. After termination of the experiment on 29th day, plasma levels of inflammatory cytokines, LH, FSH, estradiol, corticosterone, oxidative stress effects in the ovary and histopathological study of the ovary and uterine horn were done. LPS-induced inflammation of the ovary and uterine horn was ameliorated/prevented by PD149163 as reflected in the reduced histopathological scores, significant elevation of the plasma anti-inflammatory cytokine IL-10 and decrease of the pro inflammatory cytokines TNF-α and IL-6. Significant decrease of lipid peroxide, increase of antioxidant defense enzymes, Superoxide dismutase and Catalase in the ovary indicated reduction of oxidative stress. The plasma levels of the reproduction related hormones and corticosterone were restored. PD149163 acts as an anti-inflammatory and anti-oxidative agent in modulation of inflammation in the female reproductive system (ovary & uterine horn). These findings suggest that the therapeutic potential of the analogs of neurotensin including PD149163 should be explored for the treatment of the female reproductive health issues.

Side-Chain Modified [99mTc]Tc-DT1 Mimics: A Comparative Study in NTS1R-Positive Models

Radiolabeled neurotensin analogs have been developed as candidates for theranostic use against neurotensin subtype 1 receptor (NTS1R)-expressing cancer. However, their fast degradation by two major peptidases, neprilysin (NEP) and angiotensin-converting enzyme (ACE), has hitherto limited clinical success. We have recently shown that palmitoylation at the ε-amine of Lys7 in [99mTc]Tc-[Lys7]DT1 (DT1, N4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized [99mTc]Tc-DT9 analog, displaying high uptake in human pancreatic cancer AsPC-1 xenografts but unfavorable pharmacokinetics in mice. Aiming to improve the in vivo stability of [99mTc]Tc-DT1 without compromising pharmacokinetics, we now introduce three new [99mTc]Tc-DT1 mimics, carrying different pendant groups at the ε-amine of Lys7: MPBA (4-(4-methylphenyl)butyric acid)—[99mTc]Tc-DT10; MPBA via a PEG4-linker—[99mTc]Tc-DT11; or a hydrophilic PEG6 chain—[99mTc]Tc-DT12. The impact of these modifications on receptor affinity and internalization was studied in NTS1R-positive cells. The effects on stability and AsPC-1 tumor uptake were assessed in mice without or during NEP/ACE inhibition. Unlike [99mTc]Tc-DT10, the longer-chain modified [99mTc]Tc-DT11 and [99mTc]Tc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to [99mTc]Tc-DT1. [99mTc]Tc-DT11 was found to achieve the highest AsPC-1 tumor values and good pharmacokinetics, either without or during NEP inhibition, qualifying for further validation in patients with NTS1R-positive tumors using SPECT/CT.

Toward Stability Enhancement of NTS1R-Targeted Radioligands: Structural Interventions on [99mTc]Tc-DT1.

The neurotensin subtype 1 receptor (NTS1R) is overexpressed in a number of human tumors, thereby representing a valid target for cancer theranostics with radiolabeled neurotensin (NT) analogs like [99mTc]Tc-DT1 (DT1, N4-Gly7-NT(8-13)). Thus far, the fast degradation of intravenously injected NT-radioligands by neprilysin (NEP) and angiotensin-converting enzyme (ACE) has compromised their clinical applicability. Aiming at metabolic stability enhancements, we herein introduce (i) DT7 ([DAsn14]DT1) and (ii) DT8 ([β-Homoleucine13]DT1), modified at the C-terminus, along with (iii) DT9 ([(palmitoyl)Lys7]DT1), carrying an albumin-binding domain (ABD) at Lys7. The biological profiles of the new [99mTc]Tc-radioligands were compared with [99mTc]Tc-DT1, using NTS1R-expressing AsPC-1 cells and mice models without or during NEP/ACE inhibition. The radioligands showed enhanced in vivo stability vs. [99mTc]Tc-DT1, with [99mTc]Tc-DT9 displaying full resistance to both peptidases. Furthermore, [99mTc]Tc-DT9 achieved the highest cell internalization and tumor uptake even without NEP/ACE-inhibition but with unfavorably high background radioactivity levels. Hence, unlike C-terminal modification, the introduction of a pendant ABD group in the linker turned out to be the most promising strategy toward metabolic stability, cell uptake, and tumor accumulation of [99mTc]Tc-DT1 mimics. To improve the observed suboptimal pharmacokinetics of [99mTc]Tc-DT9, the replacement of palmitoyl on Lys7 by other ABD groups is currently being pursued.

Bradykinin and Neurotensin Analogues as Potential Compounds in Colon Cancer Therapy.

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential.

Neurotensin(8–13) analogs as dual NTS1 and NTS2 receptor ligands with enhanced effects on a mouse model of Parkinson's disease

The modulatory interactions between neurotensin (NT) and the dopaminergic neurotransmitter system in the brain suggest that NT may be associated with the progression of Parkinson's disease (PD). NT exerts its neurophysiological effects by interactions with the human NT receptors type 1 (hNTS1) and 2 (hNTS2). Therefore, both receptor subtypes are promising targets for the development of novel NT-based analogs for the treatment of PD. In this study, we used a virtually guided molecular modeling approach to predict the activity of NT(8–13) analogs by investigating the docking models of ligands designed for binding to the human NTS1 and NTS2 receptors. The importance of the residues at positions 8 and/or 9 for hNTS1 and hNTS2 receptor binding affinity was experimentally confirmed by radioligand binding assays. Further in vitro ADME profiling and in vivo studies revealed that, compared to the parent peptide NT(8–13), compound 10 exhibited improved stability and BBB permeability combined with a significant enhancement of the motor function and memory in a mouse model of PD. The herein reported NTS1/NTS2 dual-specific NT(8–13) analogs represent an attractive tool for the development of therapeutic strategies against PD and potentially other CNS disorders.

Design, Synthesis, and Biological Evaluation of the First Radio-Metalated Neurotensin Analogue Targeting Neurotensin Receptor 2.

Neurotensin receptor 2 (NTS2) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS2 overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS2. JMV 7488 (DOTA-(βAla)2-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with 68Ga and 111In, and investigated in vitro on HT-29 cells and MCF-7 cells, respectively, and in vivo on HT-29 xenografts. [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 were quite hydrophilic (logD7.4 = -3.1 ± 0.2 and -2.7 ± 0.2, respectively, p < 0.0001). Saturation binding studies showed good affinity toward NTS2 (K D = 38 ± 17 nM for [68Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; K D = 36 ± 4 nM for [111In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS1 binding up to 500 nM). On cell-based evaluation, [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 showed high and fast NTS2-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [111In]In-JMV 7488, respectively, along with low NTS2-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [68Ga]Ga-JMV 7488 on HT-29 and increased for [111In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS2 agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS2. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [68Ga]Ga-JMV 7488 uptake although the mechanism was not NTS2-mediated.

Opening the amino acid toolbox for peptide-based NTS2-selective ligands as promising lead compounds for pain management.

Chronic pain is one of the most critical health issues worldwide. Despite considerable efforts to find therapeutic alternatives, opioid drugs remain the gold standard for pain management. The administration of μ-opioid receptor (MOR) agonists is associated with detrimental and limiting adverse effects. Overall, these adverse effects strongly overshadow the effectiveness of opioid therapy. In this context, the development of neurotensin (NT) ligands has shown to be a promising approach for the management of chronic and acute pain. NT exerts its opioid-independent analgesic effects through the binding of two G protein-coupled receptors (GPCRs), NTS1 and NTS2. In the last decades, modified NT analogues have been proven to provide potent analgesia in vivo. However, selective NTS1 and nonselective NTS1/NTS2 ligands cause antinociception associated with hypothermia and hypotension, whereas selective NTS2 ligands induce analgesia without altering the body temperature and blood pressure. In light of this, various structure-activity relationship (SAR) studies provided findings addressing the binding affinity of ligands towards NTS2. Herein, we comprehensively review peptide-based NTS2-selective ligands as a robust alternative for future pain management. Particular emphasis is placed on SAR studies governing the desired selectivity and associated in vivo results.

Development of a Neurotensin-Derived 68Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS1 Receptor-Expressing Tumors.

Simple SummaryCancer diagnostics based on molecular imaging techniques such as positron emission tomography (PET) requires radiolabeled tracers, which are taken up by tumors. As the neurotensin receptor type 1 (NTS1R) is present in certain malignant tumors, radiolabeled NTS1R ligands can serve as molecular tools for tumor imaging. A straightforward approach for developing NTS1R PET ligands would be the preparation of fluorine-18 or gallium-68 labeled analogs of the peptide neurotensin. However, as neurotensin derivatives are prone to enzymatic cleavage, structural modifications are needed to prevent peptide degradation while retaining NTS1R affinity. Applying a new strategy for peptide stabilization, it is possible to develop a peptidic gallium-68 labeled NTS1R PET ligand with high in vivo stability and high NTS1R affinity. Investigations of the PET ligand in mice with subcutaneous NTS1R-positive tumors revealed the NTS1R-mediated visualization of the tumor. Future developments, such as NTS1R PET ligands with improved biodistribution, will benefit from these results.Overexpression of the neurotensin receptor type 1 (NTS1R), a peptide receptor located at the plasma membrane, has been reported for a variety of malignant tumors. Thus, targeting the NTS1R with 18F- or 68Ga-labeled ligands is considered a straightforward approach towards in vivo imaging of NTS1R-expressing tumors via positron emission tomography (PET). The development of suitable peptidic NTS1R PET ligands derived from neurotensin is challenging due to proteolytic degradation. In this study, we prepared a series of NTS1R PET ligands based on the C-terminal fragment of neurotensin (NT(8–13), Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) by attachment of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an Nω-carbamoylated arginine side chain. Insertion of Ga3+ in the DOTA chelator gave potential PET ligands that were evaluated concerning NTS1R affinity (range of Ki values: 1.2–21 nM) and plasma stability. Four candidates were labeled with 68Ga3+ and used for biodistribution studies in HT-29 tumor-bearing mice. [68Ga]UR-LS130 ([68Ga]56), containing an N-terminal methyl group and a β,β-dimethylated tyrosine instead of Tyr11, showed the highest in vivo stability and afforded a tumor-to-muscle ratio of 16 at 45 min p.i. Likewise, dynamic PET scans enabled a clear tumor visualization. The accumulation of [68Ga]56 in the tumor was NTS1R-mediated, as proven by blocking studies.

Neurotensin analogs by fluoroglycosylation at Nω-carbamoylated arginines for PET imaging of NTS1-positive tumors

Since neurotensin (NT) receptors of subtype-1 (NTS1) are expressed by different types of malignant tumors, such as pancreatic adenocarcinoma, colorectal and prostate carcinoma, they represent an interesting target for tumor imaging by positron emission tomography (PET) and endoradiotherapy. Previously reported neurotensin-derived NTS1 ligands for PET were radiolabeled by modification and prelongation of the N-terminus of NT(8–13) peptide analogs. In this study, we demonstrate that modifying Arg8 or Arg9 by Nω-carbamoylation and subsequent fluoroglycosylation provides a suitable approach for the development of NT(8–13) analogs as PET imaging agents. The Nω-carbamoylated and fluoroglycosylated NT(8–13) analogs retained high NTS1 affinity in the one-digit nanomolar range as well as high metabolic stability in vitro. In vivo, the radioligand [18F]21 demonstrated favorable biokinetics in HT-29 tumor-bearing mice with high tumor uptake and high retention, predominantly renal clearance, and fast wash-out from blood and other non-target tissues. Therefore, [18F]21 has the potential to be used as molecular probe for the imaging of NTS1-expressing tumors by PET.

Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception.

Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltage-gated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.

Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8–13) analog. The Tyr11 residue of NT(8–13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.

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

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.

Design, Structural Optimization, and Characterization of the First Selective Macrocyclic Neurotensin Receptor Type 2 Non-opioid Analgesic.

Neurotensin (NT) receptor type 2 (NTS2) represents an attractive target for the development of new NT-based analgesics. Here, we report the synthesis and functional in vivo characterization of the first constrained NTS2-selective macrocyclic NT analog. While most chemical optimization studies rely on the NT(8-13) fragment, we focused on NT(7-12) as a scaffold to design NTS2-selective macrocyclic peptides. Replacement of Ile12 by Leu, and Pro7/Pro10 by allylglycine residues followed by cyclization via ring-closing metathesis led to macrocycle 4, which exhibits good affinity for NTS2 (50 nM), high selectivity over NTS1 (>100 μM), and improved stability compared to NT(8-13). In vivo profiling in rats reveals that macrocycle 4 produces potent analgesia in three distinct rodent pain models, without causing the undesired effects associated with NTS1 activation. We further provide evidence of its non-opioid antinociceptive activity, therefore highlighting the strong therapeutic potential of NTS2-selective analogs for the management of acute and chronic pain.

Silicon-Containing Neurotensin Analogues as Radiopharmaceuticals for NTS1-Positive Tumors Imaging.

Several independent studies have demonstrated the overexpression of NTS1 in various malignancies, which make this receptor of interest for imaging and therapy. To date, radiolabeled neurotensin analogues suffer from low plasmatic stability and thus insufficient availability for high uptake in tumors. We report the development of 68Ga-radiolabeled neurotensin analogues with improved radiopharmaceutical properties through the introduction of the silicon-containing amino acid trimethylsilylalanine (TMSAla). Among the series of novel radiolabeled neurotensin analogues, [68Ga]Ga-JMV6659 exhibits high hydrophilicity (log D7.4 = -3.41 ± 0.14), affinity in the low nanomolar range toward NTS1 (Kd = 6.29 ± 1.37 nM), good selectivity (Kd NTS1/Kd NTS2 = 35.9), and high NTS1-mediated internalization. It has lower efflux and prolonged plasmatic half-life in human plasma as compared to the reference compound ([68Ga]Ga-JMV6661 bearing the minimum active fragment of neurotensin and the same linker and chelate as other analogues). In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV6659 uptake reached 7.8 ± 0.54 %ID/g 2 h post injection. Uptake was decreased to 1.38 ± 0.71 %ID/g with injection of excess of non-radioactive neurotensin. Radiation dose as extrapolated to human was estimated as 2.35 ± 0.6 mSv for a standard injected activity of 100MBq. [68Ga]Ga-JMV6659 was identified as a promising lead compound suitable for PET imaging of NTS1-expressing tumors.

Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8-13) analog.

Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8–13) (NT(8–13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8–13). We first examined the binding affinities of this novel NT(8–13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article “Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog” published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.

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

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.

Key-Protease Inhibition Regimens Promote Tumor Targeting of Neurotensin Radioligands.

Neurotensin subtype 1 receptors (NTS1R) represent attractive molecular targets for directing radiolabeled neurotensin (NT) analogs to tumor lesions for diagnostic and therapeutic purposes. This approach has been largely undermined by the rapid in vivo degradation of linear NT-based radioligands. Herein, we aim to increase the tumor targeting of three 99mTc-labeled NT analogs by the in-situ inhibition of two key proteases involved in their catabolism. DT1 ([N4-Gly7]NT(7-13)), DT5 ([N4-βAla7,Dab9]NT(7-13)), and DT6 ([N4-βAla7,Dab9,Tle12]]NT(7-13)) were labeled with 99mTc. Their profiles were investigated in NTS1R-positive colon adenocarcinoma WiDr cells and mice treated or not with the neprilysin (NEP)-inhibitor phosphoramidon (PA) and/or the angiotensin converting enzyme (ACE)-inhibitor lisinopril (Lis). Structural modifications led to the partial stabilization of 99mTc-DT6 in peripheral mice blood (55.1 ± 3.9% intact), whereas 99mTc-DT1 and 99mTc-DT5 were totally degraded within 5 min. Coinjection of PA and/or Lis significantly stabilized all three analogs, leading to a remarkable enhancement of tumor uptake for 99mTc-DT1 and 99mTc-DT5, but was less effective in the case of poorly internalizing 99mTc-DT6. In conclusion, NEP and/or ACE inhibition represents a powerful tool to improve tumor targeting and the overall pharmacokinetics of NT-based radioligands, and warrants further validation in the field of NTS1R-targeted tumor imaging and therapy.

Insightful Backbone Modifications Preventing Proteolytic Degradation of Neurotensin Analogs Improve NTS1-Induced Protective Hypothermia.

Therapeutic hypothermia represents a brain-protective strategy for multiple emergency situations, such as stroke or traumatic injury. Neurotensin (NT), which exerts its effects through activation of two G protein-coupled receptors, namely NTS1 and NTS2, induces a strong and long-lasting decrease in core body temperature after its central administration. Growing evidence demonstrates that NTS1 is the receptor subtype mediating the hypothermic action of NT. As such, potent NTS1 agonists designed on the basis of the minimal C-terminal NT(8-13) bioactive fragment have been shown to produce mild hypothermia and exert neuroprotective effects under various clinically relevant conditions. The high susceptibility of NT(8-13) to protease degradation (half-life <2 min) represents, however, a serious limitation for its use in pharmacological therapy. In light of this, we report here a structure-activity relationship study in which pairs of NT(8-13) analogs have been developed, based on the incorporation of a reduced Lys8-Lys9 bond. To further stabilize the peptide bonds, a panel of backbone modifications was also inserted along the peptide sequence, including Sip10, D-Trp11, Dmt11, Tle12, and TMSAla13. Our results revealed that the combination of appropriate chemical modifications leads to compounds exhibiting improved resistance to proteolytic cleavages (>24 h; 16). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla13 (4) and Sip10 (6) or the di-substitution Lys11 - TMSAla13 (12), D-Trp11-TMSAla13 (14), and Dmt11-Tle12 (16) produced sustained hypothermic effects (−3°C for at least 1 h). Importantly, we observed that hypothermia was mainly driven by the increased stability of the NT(8-13) derivatives, instead of the high binding-affinity at NTS1. Altogether, these results reveal the importance of the reduced amine bond in optimizing the metabolic properties of the NT(8-13) peptide and support the development of stable NTS1 agonists as first drug candidate in neuroprotective hypothermia.

Potential use of radiolabelled neurotensin in PET imaging and therapy of patients with pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related death in both men and women. Neurotensin receptors are overexpressed in different malignancies, above all pancreatic cancer. On the other hand, neurotensin receptor expression in inflammation is quite low. This fact can probably solve the most important problem of F-FDG PET imaging - distinguishing malignant and inflammatory processes. The first therapeutic injection of radiolabelled neurotensin in human with pancreatic cancer has been successfully performed. Animal experiments are also very close to the first in human injection of radiolabelled neurotensin for diagnostic purposes. The purpose of this article is to provide an overview of radiolabelled neurotensin analogues that can be used in imaging and therapy in patients with pancreatic ductal adenocarcinoma.