BNT12, a novel hybrid peptide of opioid and neurotensin pharmacophores, produces potent central antinociception with limited side effects.

The development of multitarget opioid drugs has emerged as an attractive approach for innovative pain management with reduced side effects. In the present study, a novel hybrid peptide BNT12 containing the opioid and neurotensin (NT)-like fragments was synthesized and pharmacologically characterized. In acute radiant heat paw withdrawal test, intracerebroventricular (i.c.v.) administration of BNT12 produced potent antinociception in mice. The central antinociceptive activity of BNT12 was mainly mediated by µ-, δ-opioid receptor, neurotensin receptor type 1 (NTSR1) and 2 (NTSR2), supporting a multifunctional agonism of BNT12 in the functional assays. BNT12 also exhibited significant antinociceptive effects in spared nerve injury (SNI)-neuropathic pain, complete Freund's adjuvant (CFA)-induced inflammatory pain, acetic acid-induced visceral and formalin-induced pain after i.c.v. administration. Furthermore, BNT12 exhibited substantial reduction of acute antinociceptive tolerance, shifted the dose-response curve to the right by only 1.3-fold. It is noteworthy that BNT12 showed insignificant chronic antinociceptive tolerance at the supraspinal level. In addition, BNT12 exhibited reduced or no opioid-like side effects on conditioned place preference (CPP) response, naloxone-precipitated withdrawal response, acute hyperlocomotion, motor coordination, gastrointestinal transit, and cardiovascular responses. The present investigation demonstrated that the novel hybrid peptide BNT12 might serve as a promising analgesic candidate with limited opioid-like side effects.

Clinical use of progesterone in human sperm preparation media for increasing IVF success.

RESEARCH QUESTION: Can the addition of progesterone and neurotensin, molecular agents found in the female reproductive tract, after sperm washing increase the fertilization potential of human spermatozoa? DESIGN: (i) Cohort study of 24 men. Spermatozoa selected by swim-up were incubated in either progesterone or neurotensin (0.1-100 µM) for 1-4 h, and hyperactive motility and binding to hyaluronan (0.1-100 µM) were assessed. The effect of progesterone 10 µM on sperm function was assessed in a blinded manner, including: hyperactive motility, binding to hyaluronan, tyrosine phosphorylation, acrosome reaction and oxidative DNA damage. (i) Embryo safety testing [one-cell mouse embryo assay (MEA), endotoxin and sterility counts (n = 3)] in preclinical embryo models of IVF (murine and porcine, n = 7 each model) and a small preliminary human study (n = 4) of couples undergoing standard IVF with oocytes inseminated with spermatozoa ± 10 µM progesterone. RESULTS: Progesterone 10 µM increased sperm binding to hyaluronan, hyperactive motility and tyrosine phosphorylation (all P < 0.05). Neurotensin had no effect (P > 0.05). Progesterone 10 µM in human embryo culture media passed embryo safety testing (MEA, endotoxin concentration and sterility plate count). In preclinical models of IVF, the exposure of spermatozoa to progesterone 10 µM and oocytes to progesterone 1 µM was not detrimental, and increased the fertilization rate in mice and the blastocyst cell number in mice and pigs (all P ≤ 0.03). In humans, every transferred blastocyst that had been produced from spermatozoa exposed to progesterone resulted in a live birth. CONCLUSION: The addition of progesterone to sperm preparation media shows promise as an adjunct to current methods for increasing fertilization potential. Randomized controlled trials are required to determine the clinical utility of progesterone for improving IVF outcomes.

Neurotensin counteracts hair growth inhibition induced by chronic restraint stress.

Stress has been considered as a potential trigger for hair loss through the neuroendocrine-hair follicle (HF) axis. Neurotensin (NTS), a neuropeptide, is known to be dysregulated in the inflammatory-associated skin diseases. However, the precise role of NTS in stress-induced hair loss is unclear. To investigate the function and potential mechanisms of NTS in stress-induced hair growth inhibition, we initially detected the expression of neurotensin receptor (Ntsr) and NTS in the skin tissues of stressed mice by RNA-sequencing and ELISA. We found chronic restraint stress (CRS) significantly decreased the expression of both NTS and Ntsr in the skin tissues of mice. Intracutaneous injection of NTS effectively counteracted CRS-induced inhibition of hair growth in mice. Furthermore, NTS regulated a total of 1093 genes expression in human dermal papilla cells (HDPC), with 591 genes being up-regulated and 502 genes being down-regulated. GO analysis showed DNA replication, cell cycle, integral component of plasma membrane and angiogenesis-associated genes were significantly regulated by NTS. KEGG enrichment demonstrated that NTS also regulated genes related to the Hippo signalling pathway, axon guidance, cytokine-cytokine receptor interaction and Wnt signalling pathway in HDPC. Our results not only uncovered the potential effects of NTS on stress-induced hair growth inhibition but also provided an understanding of the mechanisms at the gene transcriptional level.

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.

Neurotensin receptor 1-biased ligand attenuates neurotensin-mediated excitation of ventral tegmental area dopamine neurons and dopamine release in the nucleus accumbens.

Strong expression of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) in ventral tegmental area (VTA) dopamine (DA) neurons and terminals makes it an attractive target to modulate DA neuron activity and normalize DA-related pathologies. Recent studies have identified a novel class of NTR1 ligand that shows promising effects in preclinical models of addiction. A lead molecule, SBI-0654553 (SBI-553), can act as a positive allosteric modulator of NTR1 ß-arrestin recruitment while simultaneously antagonizing NTR1 Gq protein signaling. Using cell-attached recordings from mouse VTA DA neurons we discovered that, unlike neurotensin (NT), SBI-553 did not independently increase spontaneous firing. Instead, SBI-553 blocked the NT-mediated increase in firing. SBI-553 also antagonized the effects of NT on dopamine D2 auto-receptor signaling, potentially through its inhibitory effects on G-protein signaling. We also measured DA release directly, using fast-scan cyclic voltammetry in the nucleus accumbens and observed antagonist effects of SBI-553 on an NT-induced increase in DA release. Further, in vivo administration of SBI-553 did not notably change basal or cocaine-evoked DA release measured in NAc using fiber photometry. Overall, these results indicate that SBI-553 blunts NT's effects on spontaneous DA neuron firing, D2 auto-receptor function, and DA release, without independently affecting these measures. In the presence of NT, SBI-553 has an inhibitory effect on mesolimbic DA activity, which could contribute to its efficacy in animal models of psychostimulant use.

Neurotensin modulation of lipopolysaccharide induced inflammation of gut-liver axis: Evaluation using neurotensin receptor agonist and antagonist.

Lipopolysaccharide (LPS), a toxic component of the cell wall of Gram-negative bacteria, is a potent immune stressor. LPS-induced inflammation of the gut-liver axis is well demonstrated. Neurotensin (NTS), a tri-decapeptide present in the gastrointestinal tract, has anti-inflammatory, anti-oxidative, and growth-promoting properties. This study elucidated the efficacy of PD149163, the type I NTS receptor agonist (NTS1) in the modulation of LPS-induced inflammation of the gut-liver axis of mice. Young-adult female mice (Age: 8 weeks; BW: 25 ± 2.5 g) were maintained in six groups (6/group); Group I as control and Group II, III & IV were exposed to LPS (1 mg/kg BW/Day; i.p.) for five days. LPS pre-exposed Group III and Group IV mice were treated with NTS1 agonist PD149163 (100 µg/kg BW i.p.) and antagonist SR48692 (0.5 mg/kg BW i.p.) respectively for 28 days. Group V and Group VI mice were exposed to only PD149163 and only SR48692 respectively with the doses as mentioned above for 28 days. Group I and LPS-exposed Group II mice were also maintained four weeks without further treatment. Histopathology revealed LPS-induced inflammation of the gut and liver. Significant elevation of plasma TNF-α and IL-6 and serum ALT and AST reflected as biomarkers of inflammation. Oxidative stress on both organs was distinct from decreased glutathione reductase and increased lipid peroxidation. PD149163 but not SR48692 ameliorated LPS-induced inflammation in both gut and liver counteracting inflammatory responses and oxidative stress. The use of NTS agonists including PD149163 could be exploited for therapeutic intervention of inflammatory diseases including that of the gut-liver axis.

Intravenous administration of xenin-25 accelerates cyclic ruminal contractions in healthy conscious sheep.

The present study aimed to determine the effect and mode of action of the intravenous injection of xenin-25 on cyclic contractions of the rumen in healthy conscious sheep and mode of its action. Clinically healthy male sheep were equipped with a rumen cannula by surgery under anesthesia, and ruminal contractions were recorded with manometry in conscious animals after the recovery period. Intravenous xenin-25 injection induced a cluster of premature ruminal phasic contractions in a dose-dependent manner between 0.03 and 1 nmol/kg, and the change at the highest dose was statistically significant. In contrast, intravenous neurotensin injection inhibited the amplitude of cyclic rumen contractions. The xenin-25 effect was not significantly altered by prior injection of the neurotensin receptor subtype-1 antagonist SR 48692 at 30 and 100 nmol/kg. After euthanasia the ruminal muscles were excised for in vitro experiments. A single xenin-25 application (0.3-10 µM) to the longitudinal and circular muscle strips of the rumen did not induce any change in tension or electric field stimulation-induced phasic contractions of the muscle strips. These results demonstrated that circulating xenin-25 stimulates rumen contractions by acting on sites except the intramural intrinsic nerve plexus or smooth muscles of the rumen, implying that xenin-25 acts on the gastric center and/or cholinergic efferent nerve innervated to the ovine rumen.

Neurotensin and xenin stimulates pancreatic exocrine secretion through the peripheral cholinergic nerves in conscious sheep.

The present study aimed to clarify the effects of neurotensin and xenin on pancreatic exocrine secretion in conscious sheep and their mechanism of actions. The animals were equipped with two silastic cannulae in the common bile duct to separately collect pancreatic fluid and bile, and a silastic cannula in the proximal duodenum to continuously return the mixed fluids. NT and xenin were intravenously injected at range of 0.01-3.0 nmol/kg during the phase I of duodenal migrating motor complex. A single intravenous NT injection significantly and dose-dependently increased pancreatic fluid, protein, and bicarbonate outputs. The effect of NT at 1 nmol/kg was completely inhibited by a background intravenous infusion of atropine methyl nitrate at a dose of 10 nmol/kg/min, however, the effect was not altered by a prior injection of the neurotensin receptor subtype (NTR)-1 antagonist SR 48692 at 60 nmol/kg. Moreover, a single intravenous xenin-25 injection significantly and dose-dependently increased pancreatic fluid and protein output, whereas the effect of xenin-25 did not clearly show dose-dependence. The prior SR 48692 injection at 30 nmol/kg did not significantly alter the effects of xenin-25 at 0.3 nmol/kg, while the atropine infusion significantly inhibited the increase in fluid secretion. Under the atropine infusion, xenin-25 at 0.3 nmol/kg did not increase protein and bicarbonate outputs, whereas the inhibitory effect of the atropine was not significant compared to that of the single injection of xenin-25. A single intravenous injection of NTR-2 agonist levocabastine at 0.1-3 nmol/kg did not alter pancreatic exocrine secretion. These results suggest that both NT and xenin-25 effectively stimulates pancreatic exocrine secretion through the peripheral cholinergic system in sheep and that NTR-2 is not involved in the regulation of pancreatic exocrine secretion, however, we did not precisely determine the role of NTR-1 in the actions of both the peptides on pancreatic exocrine secretion.

Minimum biological domain of xenin-25 required to induce anion secretion in the rat ileum.

Xenin-25 has a variety of physiological functions in the gastrointestinal tract, including ion transport and motility. Xenin-25 and neurotensin show sequence homology, especially near their C-terminal regions. The sequence similarity between xenin-25 and neurotensin indicates that the effects of xenin-25 is mediated by the neurotensin receptor but some biological actions of xenin-25 are independent. We have previously reported that xenin-25 modulates intestinal ion transport and colonic smooth muscle activity. However, minimal biological domain of xenin-25 to induce ion transport was not clear. To improve the mechanistic understanding of xenin-25 and to gain additional insights into the functions of xenin-25, the present study was designed to determine the minimal biological domain of xenin-25 required for ion transport in the rat ileum using various truncated xenin fragments and analogues in an Ussing chamber system. The present results demonstrate that the minimum biological domain of xenin-25 to induce Cl-/HCO3- secretion in the ileum contains the C-terminal pentapeptide. Furthermore, Arg at position 21 is important to retain the biological activity of xenin-25 and induces Cl-/HCO3- secretion in the rat ileum.

Protective effect of neurotensin receptor-1 agonist PD 149163 against lipopolysaccharide-induced gut toxicity in mice.

The interaction between neuroendocrine and immune components of the gut maintains the organism's physical and psychological health. Its disruption may reflect in disease conditions such as inflammatory bowel disease (IBD) and mental illness. The lipopolysaccharide (LPS) disrupts the endocrine-immune homeostasis resulting in gut toxicity. The Neurotensin receptor-1 (NTR-1) agonist PD 149163 (PD) acts as an atypical antipsychotic drug in psychiatric illness, but its role in modulating gut pathophysiology remains unknown. Therefore, the aim of the present study was to evaluate the protective effect of PD against LPS-induced gut toxicity. Swiss albino female mice (12 weeks) were divided into six groups (n = 6/group): (I) Control, (II) LPS (1 mg/kg, for 5 days), (III) LPS (1 mg/kg, for 5 days)+PD low (100 µg/kg, for 21 days), (IV) LPS (1 mg/kg, for 5 days)+PD high (300 µg/kg, for 21 days), (V) PD low (100 µg/kg, for 21 days), and (VI) PD high (300 µg/kg, for 21 days). Drugs were given intraperitoneal in the morning. PD administration prevented the LPS-induced gut inflammation observed in damage of epithelial barrier, disruption of goblet cells, and condensation of lamina propria (LP). The LPS-induced oxidative stress characterized by decreased superoxide dismutase (SOD) activity and increased lipid hydroperoxide (LOOH) (p < 0.001 for both), and enhanced interleukine-6 (IL-6) & tumor necrosis factor-α (TNF-α) (p < 0.001 for both) as well as immunointensity of NT (p < 0.01) and NTR-1 (p < 0.05) were reversed and normalized to control after PD treatment. Thus, the anti-inflammatory, anti-oxidative, and cell proliferation properties of PD modulate the gut toxicity in LPS-challenged mice.

TH1902, a new docetaxel-peptide conjugate for the treatment of sortilin-positive triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of cancers which lacks the expression and/or amplification of targetable biomarkers (ie, estrogen receptor, progestrogen receptor, and human epidermal growth factor receptor 2), and is often associated with the worse disease-specific outcomes than other breast cancer subtypes. Here, we report that high expression of the sortilin (SORT1) receptor correlates with the decreased survival in TNBC patients, and more importantly in those bearing lymph node metastases. By exploiting SORT1 function in ligand internalization, a new anticancer treatment strategy was designed to target SORT1-positive TNBC-derived cells both in vitro and in two in vivo tumor xenografts models. A peptide (TH19P01), which requires SORT1 for internalization and to which many anticancer drugs could be conjugated, was developed. In vitro, while the TH19P01 peptide itself did not exert any antiproliferative or apoptotic effects, the docetaxel-TH19P01 conjugate (TH1902) exerted potent antiproliferative and antimigratory activities when tested on TNBC-derived MDA-MB-231 cells. TH1902 triggered faster and more potent apoptotic cell death than did unconjugated docetaxel. The apoptotic and antimigratory effects of TH1902 were both reversed by two SORT1 ligands, neurotensin and progranulin, and on siRNA-mediated silencing of SORT1. TH1902 also altered microtubule polymerization and triggered the downregulation of the anti-apoptotic Bcl-xL biomarker. In vivo, both i.p. and i.v. administrations of TH1902 led to greater tumor regression in two MDA-MB-231 and HCC-70 murine xenograft models than did docetaxel, without inducing neutropenia. Altogether, the data demonstrates the high in vivo efficacy and safety of TH1902 against TNBC through a SORT1 receptor-mediated mechanism. This property allows for selective treatment of SORT1-positive TNBC and makes TH1902 a promising avenue for personalized therapy with the potential of improving the therapeutic window of cytotoxic anticancer drugs such as docetaxel.

Diverse intracellular signaling pathways mediate the effects of neurotensin on the excitability of type II neurons in the rat dorsolateral bed nucleus of the stria terminalis.

We examined the effects of neurotensin (NTS) on the excitability of type II neurons in the rat dorsolateral bed nucleus of the stria terminalis (dlBNST) using whole-cell patch-clamp electrophysiology. Bath-application of NTS depolarized type II dlBNST neurons. Analyses of the steady-state I-V relationships implied that the depolarizing effect of NTS is due to potassium conductance blocking. The depolarizing effect of NTS was abolished in the presence of a PLC inhibitor, but not affected by a protein kinase C inhibitor. In the presence of a CaMKII inhibitor, NTS showed depolarizing effects via the increase in non-selective cation conductance in addition to the decrease in potassium conductance. Unexpectedly, in the presence of a PKA inhibitor, NTS hyperpolarized type II dlBNST neurons. These results reveal that diverse signaling pathways mediate the effects of NTS on the excitability of type II dlBNST neurons. The elevation of intracellular Ca2+ levels via the inositol phosphate-mediated signaling activates both Ca2+-dependent adenylate cyclase (AC) and CaMKII. Activation of the AC-cAMP-PKA pathway exerts depolarizing effects on type II dlBNST neurons by decreasing potassium conductance and increasing non-selective cation conductance, whereas activation of the CaMKII pathway exerts hyperpolarizing effects on dlBNST neurons by decreasing non-selective cation conductance.

Neurotensin is an anti-thermogenic peptide produced by lymphatic endothelial cells.

The lymphatic vasculature plays important roles in the physiology of the organs in which it resides, though a clear mechanistic understanding of how this crosstalk is mediated is lacking. Here, we performed single-cell transcriptional profiling of human and mouse adipose tissue and found that lymphatic endothelial cells highly express neurotensin (NTS/Nts). Nts expression is reduced by cold and norepinephrine in an α-adrenergic-dependent manner, suggesting a role in adipose thermogenesis. Indeed, NTS treatment of brown adipose tissue explants reduced expression of thermogenic genes. Furthermore, adenoviral-mediated overexpression and knockdown or knockout of NTS in vivo reduced and enhanced cold tolerance, respectively, an effect that is mediated by NTSR2 and ERK signaling. Inhibition of NTSR2 promoted energy expenditure and improved metabolic function in obese mice. These data establish a link between adipose tissue lymphatics and adipocytes with potential therapeutic implications.

The effect of Xenin25 on spontaneous circular muscle contractions of rat distal colon in vitro.

Xenin25 has a variety of physiological functions in the Gastrointestinal (GI) tract, including ion transport and motility. However, the motility responses in the colon induced by Xenin25 remain poorly understood. Therefore, the effect of Xenin25 on the spontaneous circular muscle contractions of the rat distal colon was investigated using organ bath chambers and immunohistochemistry. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. This inhibitory effect of Xenin25 was significantly suppressed by TTX but not by atropine. The inhibitory time (the duration of inhibition) caused by Xenin25 was shortened by the NTSR1 antagonist SR48692, the NK1R antagonist CP96345, the VPAC2 receptor antagonist PG99-465, the nitric oxide-sensitive guanylate-cyclase inhibitor ODQ, and the Ca2+ -dependent K+ channel blocker apamin. The higher frequency of postinhibitory spontaneous contractions induced by Xenin25 was also attenuated by ODQ and apamin. SP-, NOS-, and VIP-immunoreactive neurons were detected in the myenteric plexus (MP) of the rat distal colon. Small subsets of the SP-positive neurons were also Calbindin positive. Most of the VIP-positive neurons were also NOS positive, and small subsets of the NK1R-positive neurons were also VIP positive. Based on the present results, we propose the following mechanism. Xenin25 activates neuronal NTSR1 on the SP neurons of IPANs, and transmitters from the VIP and apamin-sensitive NO neurons synergistically inhibit the spontaneous circular muscle contractions via NK1R. Subsequently, the postinhibitory spontaneous contractions are induced by the offset of apamin-sensitive NO neuron activation via the interstitial cells of Cajal. In addition, Xenin25 also activates the muscular NTSR1 to induce relaxation. Thus, Xenin25 is considered to be an important modulator of post prandial circular muscle contraction of distal colon since the release of Xenin25 from enteroendocrine cells is stimulated by food intake.

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.

Neurolysin substrates bradykinin, neurotensin and substance P enhance brain microvascular permeability in a human in vitro model.

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of several acute neurodegenerative disorders, including stroke. Numerous studies suggest the involvement of bradykinin (BK), neurotensin (NT) and substance P (SP) in BBB impairment and oedema formation after stroke; however, there is paucity of data in regard to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The present study aimed to evaluate the direct effects of BK, NT and SP on the permeability of BBB in an in vitro model based on human induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. The combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model indicating potentiation of their action. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors. Additional mechanistic studies indicate that the short-term effects of these peptides are not mediated through alteration of tight-junction proteins claudin-5 and occludin, but likely involve redistribution of F-actin and secretion of vascular endothelial growth factor. This is the first experimental study to document the increased permeability of the BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognised actions of BK on BBB. Lastly, we demonstrate that peptidase neurolysin can neutralise the effects of these peptides on BBB, suggesting potential therapeutic implications.

Identification and in vivo characterization of a brain-penetrating nanobody.

BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.

Natural α,ß-unsaturated lactones inhibit neuropeptide-induced mast cell activation in an in vitro model of neurogenic inflammation.

INTRODUCTION: Mast cells are involved in not only inducing, but also maintaining neurogenic inflammation and neuropathic pain. In previous work, we have demonstrated that dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit rat peritoneal and human LAD2 mast cell degranulation induced by compound 48/80 and calcium ionophore A23187. However, the effect of these molecules on neuropeptide-induced mast cell activation has not been studied so far. OBJECTIVE: The aim of this study was to determine whether dehydroleucodine, xanthatin, and 3-benzyloxymethyl-5H-furan-2-one inhibit neuropeptide-induced mast cell activation. METHODS: This work is based on in vitro simulation of a neurogenic inflammation scenario involving neuropeptides and mast cells, to subsequently analyze potential therapeutic strategies for neuropathic pain. RESULTS: Neuromedin-N did not stimulate mast cell serotonin release but substance P and neurotensin did induce serotonin release from peritoneal mast cells in a dose-dependent manner. Mast cell serotonin release induced by substance P and neurotensin was inhibited by dehydroleucodine and xanthatin, but not by 3-benzyloxymethyl-5H-furan-2-one. The inhibitory potency of dehydroleucodine and xanthatin was higher than that obtained with the reference compounds, ketotifen and sodium chromoglycate, when mast cells were preincubated with dehydroleucodine before substance P incubation, and with dehydroleucodine or xanthatin before neurotensin incubation. CONCLUSIONS: These results are the first strong evidence supporting the hypothesis that dehydroleucodine and xanthatin inhibit substance P- and neurotensin-induced serotonin release from rat peritoneal mast cells. Our findings suggest, additionally, that these α,ß-unsaturated lactones could be of value in future pharmacological research related to inappropriate mast cell activation conditions such as neurogenic inflammation and neuropathic pain.

Effect of neurotensin on cultured mouse preimplantation embryos.

Previously, we revealed that neurotensin (NTS) derived from the oviduct and uterus can function during fertilization. However, little is known about NTS actions on the pre-implantation embryo after fertilization. Here, we found that pro-Nts mRNA is expressed in the oviduct and uterus during when preimplantation embryos develop and an increase in mRNA level in the uterus is induced by human chorionic gonadotropin (hCG) treatment. Expression of mRNA for two NTS receptors, Ntr1 and Ntr3, was found throughout these stages, whereas Ntr2 mRNA was not detected, suggesting that NTS signaling occurred through NTR1 and NTR3. Supplementation of 1, 10, 100 or 1000 nM NTS to embryo culture medium after fertilization showed that 100 nM NTS significantly improved the blastocyst formation. In comparison, the total number of cells and inner cell mass ratio of blastocysts was not significant different between the 0 nM and 100 nM NTS treatment groups. These results indicate that NTS has a positive effect upon preimplantation embryo development in vitro.

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.