Antinociceptive effects of intrathecal Neuropeptide B/W receptor 1 agonists in mouse acute nociception, peripheral neuropathy, and inflammatory pain models

Antinociceptive effects of intrathecal Neuropeptide B/W receptor 1 agonists in mouse acute nociception, peripheral neuropathy, and inflammatory pain models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antinociceptive effects of intrathecal Neuropeptide B/W receptor 1 agonists in mouse acute nociception, peripheral neuropathy, and inflammatory pain models Yuma T. Ortiz, Thuy Nguyen, Jenny L. Wilkerson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6559172/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Pharmacological Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background The Neuropeptide B/W Receptor 1 (NPBWR1) system, including its two endogenous ligands, Neuropeptides B and W (NPB and NPW), has garnered interest as potential target to develop novel analgesics. Behavioral studies were typically conducted with exogenously administered endogenous ligands. In this study, we examined truncated NPB-23 and its peptidomimetic RTIBW-16 in a panel of antinociceptive assays including the hot plate, carrageenan-induced inflammatory, and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN) pain assays. Methods Male and female C57BL/6 mice underwent testing in the hot plate acute nociception assay. After a minimum one-week washout, mice were enrolled in the carrageenan inflammatory pain model, receiving intraplanar carrageenan (0.3% carrageenan in a 20 µL). Separate mouse cohorts received a cycle of intraperitoneal paclitaxel injections (cumulative dose 32 mg/kg). The von Frey assay was utilized to assess CIPN and carrageenan-induced allodynia. Results NPB-23 and RTIBW-16 dose-dependently produced thermal antinociception, attenuated CIPN allodynia and carrageenan-induced allodynia with some differences regarding onset time, potency and duration of action. In the hot plate assay, RTIBW-16 showed earlier onset but shorter duration of action than NPB-23 with similar maximum peak effects. Both compounds were statistically equipotent in the reversal of mechanical allodynia induced by either paclitaxel or carrageenan. RTIBW-16 maintained a longer duration of action than NPB-23 in CIPN assay. Conclusions Both NPBWR1 agonists alleviated thermal and inflammatory pain. Notably, we demonstrated for the first time that NPBWR1 agonists exhibited analgesic effect in the CIPN model. Our findings highlight NPBWR1 as a promising target for developing analgesics with novel mechanisms. Neuropeptide B/W Receptor 1 Neuropeptide B peptidomimetic pain neuropathic pain hot plate latency inflammatory pain Figures Figure 1 Figure 2 Figure 3 Introduction Pathological pain encompasses chronic pain conditions such as neuropathic pain, inflammatory pain, and centralized pain syndromes [ 1 – 4 ]. Unlike acute pain, which serves as a protective mechanism, chronic pathological pain persists beyond normal healing and often arises from maladaptive changes in the nervous system [ 5 – 7 ]. It affects over 30% of the population worldwide, with a notable impact on quality of life, mental health, and daily functioning [ 8 ]. The global burden is profound, contributing to increased healthcare utilization, loss of productivity, and economic costs exceeding billions of dollars annually [ 8 ]. Pathological pain is intricately linked to conditions like diabetes, cancer, and autoimmune diseases, further amplifying its prevalence and impact [ 1 , 9 , 10 ]. Despite advances in pain management, opioids remain the only treatment for chronic moderate to severe pain. While effective, their use is restricted by significant limitations, including dependence, tolerance, and respiratory depression. The complexity of pain mechanisms and individual variability further challenge the development of effective treatments, emphasizing the urgent need for targeted research and innovation in therapeutic approaches [ 8 ]. The neuropeptides B and W (NPB and NPW) are endogenous ligands of the Neuropeptide B/W Receptors 1 and 2 (NPBWR1 and NPBWR2) [ 11 ]. Together, they have been implied to play a role in pain signaling pathways [ 12 , 13 ]. NPBWR1 is expressed in brain and peripheral organs of both humans and rodents whereas NPBWR2 is not found in rodents [ 14 ]. Within the central nervous system (CNS), NPBWR1 expression was found in cerebellum, prefrontal cortex, hippocampus, amygdala, and spinal cord [ 12 – 16 ]. In the peripheral nervous system, NPBWR1 has been identified on both human and rat myelinating Schwann cells under basal conditions and was found to be overexpressed in neuropathic conditions [ 17 ]. A recent study utilizing immunohistochemistry demonstrated that NPBWR1 and µ opioid receptor are colocalized in the superficial dorsal horn of rat spinal cord [ 18 ]. In preclinical animal studies, mice lacking NPB developed hyperalgesia in both the formalin and acetic acid inflammatory pain models but responded normally in acute hot plate nociceptive pain and non-inflammatory pain models [ 19 ]. Exogenous intracerebroventricular (i.c.v.) administration of synthetic NPB produced thermal antinociception and decreased paw licking in rat carrageenan and formalin models, respectively [ 20 ]. Intrathecal (i.t.), but not i.c.v., administration of exogenous NPW and NPB reversed mechanical allodynia, or light touch sensitivity in the rat partial nerve ligation neuropathic pain model [ 21 ]. In another study that utilized rats intrathecal NPB and NPW attenuated formalin-induced nociceptive behaviors as well as carrageenan-induced mechanical allodynia but did not alter carrageenan-induced thermal hyperalgesia or acute hot plate nociception [ 22 ]. In a rat formalin test, i.c.v. injection of NPW into different brain regions revealed that its analgesic effects were mediated through the activation of the descending pain modulatory system [ 23 ]. Morphine co-administered with either NPB or NPW synergistically alleviated both inflammatory and neuropathic pain as well as significantly reduced morphine-induced conditioned place preference and constipation [ 18 ]. So far, behavioral studies have mostly been conducted in rats and relied on exogenous administration of endogenous ligands. NPB is a 29 amino acid peptide with a bromine at the C-6 position of the indole moiety in the N-terminal tryptophan (Trp) [ 11 , 20 , 24 ]. The truncated NPB-23 with the N-terminal 23 amino acid residues and no bromine at the N-terminal Trp was found to exhibit equipotent in vitro NPBWR1 agonist activity [ 24 , 25 ]. NPW exists in two functional peptide forms consisting of 23 and 30 amino acid residues [ 24 , 26 ]. NPB exhibits a higher affinity for NPBWR1, while NPW binds to both NPBWR1 and NPBWR2 with similar affinities [ 26 ]. Our group and others have studied the structure-activity relationship of NPB and identified that the pharmacophore for NPBWR1’s agonist activity consists of two epitopes, N-terminal WYK and C-terminal GRAAGLL [ 25 , 27 ]. From this understanding, we developed RTIBW-16, a NPB peptidomimetic with a shorter sequence of 13 amino acid residues exhibited similar in vitro potencies while exhibiting improved metabolic stability in rat plasma [ 27 ]. In the current study we sought to evaluate the effects of i.t. administration of NPB-23, a truncated human NPB without a bromine at the N-terminal Trp residue and RTIBW-16 using a panel of mouse pain models including the hot plate assay for acute nociception, a chemotherapy-induced peripheral neuropathy (CIPN) and a carrageenan-induced inflammatory pain models. The hot plate assay is a widely used tool for screening novel analgesics [ 28 – 30 ]. CIPN is a clinically relevant that mimics the debilitating sensory and pain symptoms experienced by cancer patients undergoing chemotherapy, providing a valuable tool for developing therapies to alleviate this common side effect [ 1 , 5 , 10 ]. The carrageenan-induced pain model is a standard tool for evaluating potential analgesics for conditions like arthritis and other inflammatory disorders [ 20 , 22 , 31 ]. By profiling across various pain models, we aim to investigate the roles of NPBWR1 agonists in acute nociception, neuropathic and inflammatory pain. Materials and Methods Animals All studies were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Texas Tech University Health Science Center Institutional Animal Care and Use Committee (IACUC number 21034). A total of 44 adult male and 44 adult female (22–29 g upon arrival) C57BL/6 (Jackson Laboratories, Bar Harbor, ME) mice were used in this study. Mice were split into cohorts (n = 8/group) and used for either the chemotherapy-induced peripheral neuropathy model or the hotplate latency assay with later use in the carrageenan paw inflammation model. Housing facilities for mice were maintained at a consistent temperature (20–22 o C)-, humidity (55% ± 10%)-, and light-schedule (12 hr light/dark; lights on at 0700) which were approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Food and Water were available ad libitum for all mice. Drugs and Dosing NPB-23 (Trp-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-NH 2 ) and RTIBW-16 ([desaminoTrp 1 ]-Tyr-Lys-Ava-Ava-Ava-Gly-Arg-Ala-Ala-Gly-Leu-Leu-NH 2 , compound 30 in Ref. 27) were synthesized as previously reported [ 27 ]. RTIBW-16 and NPB-23 were administered via i.t. injections between L4 and L5 vertebrae with an injection volume of 5 µL. The dosing of these compounds was based on previous literature [ 21 , 22 , 27 ]. Paclitaxel was purchased from Bio-Techne (Minneapolis, MN) and was administered in a vehicle solution containing a 1:1:18 ratio of ethanol (100%), polyethylene glycol monooleyl ether (Tokyo Chemical Industry America; Portland, OR), and saline (0.9% w/v NaCl) as previously described [ 22 , 24 ]. A dosing cycle of paclitaxel consisted of a total of four intraperitoneal (i.p.) paclitaxel injections (8 mg/kg per injection, given every other day [ 29 , 32 ]. Carrageenan was purchased from Sigma-Aldrich (St. Louis, MO) and made into a 0.3% w/v solution in saline for injections [ 33 ]. Intrathecal Injections Intrathecal injections were conducted as previously described [ 34 – 36 ]; injections occurred between the L4 and L5 vertebrae. Briefly, an ‘injection catheter’ made from a 27-gauge needle and polyethylene (PE) 20 tubing was connected to a 10 µl Hamilton syringe. Injections occurred over 5 sec and were generally accompanied by a hind paw twitch response. A 100% motor recovery rate was observed. Acute Nociception Hot Plate Assay Acute nociception measurements were collected via the hot plate latency assay as previously described [ 37 ]. Mice were placed on a heated (52 o C) enclosed Hot Plate Analgesia Meter (Columbus Instruments, Columbus, OH). The latency to jump/lick/shake/flick a hind paw was recorded. A 30 second cut-off was used to prevent tissue damage. Latencies were recorded prior to administration of compounds and at each time point over the 24-hour study (0.5, 1, 1.5, 2, 3, 4, 6, 24 hr time points). Latency measurements were taken by observers blinded to treatment conditions. Mice were given a 1-week washout period between test days. Paclitaxel CIPN Model To assess mechanical allodynia within CIPN, mice were habituated to the von Frey testing environment for four consecutive days in 30-minute sessions prior to the first testing session. Baselines were measured as previously described using von Frey monofilaments (North Coast Medical; Morgan Hills, CA) to establish responses to light mechanical touch before paclitaxel or vehicle administration [ 24 ]. During testing mice were placed atop a wire mesh screen with spaces 0.5 mm apart. Mice were singly placed beneath an inverted wire mesh basket (8 cm diameter, 15 cm height) and allowed to habituate to the apparatus for 30 minutes before testing. The von Frey assay utilizes a series of calibrated monofilaments (0.4–4.0 g stimulus intensity), applied to the left and right plantar surface of the hind paws utilizing the “up-down” method [ 32 ]. Monofilament application was done in a manner avoiding sequential application to the same paw (i.e., left, right, left, right, as opposed to left, left, left, right). Lifting, licking, or shaking of the tested paw upon filament application was considered a response. Five responses out of five monofilament stimulations were coded as the minimum force required to elicit responses within the von Frey assay. Mice were tested with experimental compounds starting the day after finishing paclitaxel treatment and were given at least a 1-week washout period between test days. As mechanical allodynia in this model lasts through 3 months [ 32 ], animals experienced 5–7 test days. Reported measurements from the von Frey assay are mean response thresholds collected from both the left and right hind paw of each subject as paclitaxel produces bilateral allodynia of similar magnitude in both hind paws. Responses from the von Frey assay were measured over a 24-hour time course (1, 2, 3, 4, 6, 24 hr timepoints). Mechanical allodynia assessments were conducted by observers blinded to treatment conditions. Carrageenan Inflammatory Pain Model After use in the hot plate latency assay, the same cohort was then used for the carrageen model of inflammatory pain. Mice were given at least a one-week washout period between experimental use. Mice were habituated to the von Frey apparatus and were assessed using the same “up-down” method as described earlier [ 38 ]. The carrageenan inflammatory pain model was induced as previously described [ 33 ]. Briefly, an intraplantar injection of 0.3% w/v carrageenan in a 20 µL volume using a 27-gauge needle was administered into the right hind paw. Carrageenan was injected into the right hind paw. The left paw served as an internal control for each subject. The change in paw thickness from baseline was assessed with digital caliper measurements. Paw thickness and von Frey responses were measured over a 24-hour time course (1, 2, 3, 4, 6, 24 hr timepoints), with paw thickness measurements occurring immediately after the von Frey assay at each time point. Mechanical allodynia assessments and paw thickness measurements were conducted by observers blinded to treatment conditions. Intraplantar administration of carrageenan and digital caliper measurement of paw edema were conducted with mice restrained utilizing a plexiglass mouse restrainer (Stoelting Co., Wood Dale, IL) Data analysis Hot plate data were converted to percent maximum possible effect (% MPE) with the following equation: ([experimental test value – baseline value) / (30 sec – baseline value)] * 100). Behavioral data were analyzed using a repeated measures two-way analysis of variance (ANOVA) for RTIBW-16 and NPB-23 treatment time course analysis with time as one factor and compound dosing as a second factor [ 35 ]. Individual treatment effects were analyzed using repeated measures one-way ANOVA. Dunnett’s test was used for post hoc analysis following a significant ANOVA result. Two tailed, paired t-tests were conducted to compare the magnitude of baseline von Frey measurements to their respective pre-paclitaxel and pre-carrageenan measurements. A p value of < 0.05 was considered statistically significant. As no significant sex effect was observed, male and female data were collapsed. If the mean effect of treatment did not produce a 50% or greater effect, an effective dose (ED 50 ) value was not generated. When the mean effect of a drug in an experiment was greater than 50%, the ED 50 values and corresponding 95% confidence limits were calculated using linear regression, where slopes were allowed to vary [ 39 ]. The computer program GraphPad Prism version 10.3 (GraphPad Software Inc.; San Diego, CA) was used in the statistical analyses. Data are plotted as mean ± the standard error of the mean. Results Intrathecal RTIBW-16 and NPB-23 produced acute antinociception Intrathecal vehicle administration did not alter response latencies ( p = 0.268) in the hot plate assay. At 56 and 100 µg, both RTIBW-16 (Fig. 1 A) and NPB-23 (Fig. 1 B) produced significant antinociceptive effect as early as 0.5 hour and peaked at 1.5 hours after administration. At 100 µg ( F (2.17, 15.18) = 11.73, p < 0.001) and 56 µg ( F (2.08, 14.56) = 34.01, p < 0.001) of RTIBW-16 produced significant antinociceptive effects through 2 hours after administration. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time ( F (49, 343) = 7.68, p < 0.001). Similarly, 100 µg ( F (1.44, 10.06) = 28.51, p < 0.001) and 56 µg ( F (1.70, 11.87) = 44.08, p < 0.001) NPB-23 produced significant antinociception up to 6 hours following administration. A two-way ANOVA of NPB-23 confirmed a significant interaction between treatment and time ( F (35, 245) = 9.61, p < 0.001). At 1 hour timepoint, the calculated ED 50 values of RTIBW-16 and NPB-23 were 43.86 (25.09–73.21) and 18.36 (10.17–33.15) µg respectively (Table 1 ). Generally, in the hot plate assay, RTIBW-16 showed greater antinociceptive effect earlier at 0.5 hour timepoint but shorter duration of action than NPB-23. Both ligands produced similar maximum antinociceptive effects at their peak time of 1.5 hours. Table 1 Calculated ED 50 (µg) values for the effects of RTIBW-16 and NPB-23 as shown in Figs. 1 through 4 at 1 hour following treatment. Experiment Treatment ED 50 (µg) Hot Plate Latency RTIBW-16 42.86 (25.09–73.21) NPB-23 18.36 (10.17–33.15) Paclitaxel CIPN von Frey RTIBW-16 57.79 (53.13–95.06) NPB-23 45.06 (33.91–59.88) Carrageenan von Frey RTIBW-16 29.47 (21.71–40.00) NPB-23 19.92 (13.52–29.35) Sample sizes are described in each figure legend. Values in parentheses are 95% confidence intervals. Intrathecal RTIBW-16 and NPB-23 Reversed Paclitaxel-Induced Mechanical Allodynia A separate cohort of mice were utilized to examine RTIBW-16 (Fig. 2 A) and NPB-23 (Fig. 2 B) in a model of paclitaxel-induced peripheral neuropathy. Following paclitaxel administration, mice displayed significant ( p < 0.001) mechanical allodynia compared to pre-paclitaxel baselines. Vehicle treatment did not alter mechanical allodynia thresholds. Both ligands produced maximum effects at 1 hour. RTIBW-16 significantly attenuated CIPN mechanical allodynia from 1 to 3 hours post administration. Specifically,100 µg ( F (2.07, 31.1) = 80.8, p < 0.001), 56 µg ( F (2.8, 42.04) = 209.1, p < 0.001), and 10 µg ( F (2.38, 35.62) = 86.24, p < 0.001) produced dose-related mechanical allodynia reversal. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the CIPN model ( F (5.56, 83.43) = 28.5, p < 0.001). NPB-23 significantly attenuated mechanical allodynia for 2 hours following 100 µg ( F (1.33, 19.89) = 54.57, p < 0.001) and 56 µg ( F (1.97, 29.54) = 59.64, p < 0.001) administration. A two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the CIPN model ( F (3.94, 59.16) = 23.09, p < 0.001). At 1 hour timepoint, the calculated ED 50 values of RTIBW-16 and NPB-23 were 57.79 (53.13–95.06) and 45.06 (33.91–59.88) µg respectively (Table 1 ). At 1 hour timepoint, NPB-23 was able to reverse the CIPN mechanical allodynia back to the baseline. RTIBW-16 maintained a longer duration of action than NPB-23. Both ligands were statistically equipotent in the reversal of mechanical allodynia. Intrathecal RTIBW-16 and NPB-23 Blocked Carrageenan-Induced Mechanical Allodynia and Edema Intraplantar administration of carrageenan produced significant ( p < 0.0001) mechanical allodynia (Fig. 3 ) in vehicle treated mice compared to their pre-carrageenan baselines. Vehicle treatment did not alter mechanical allodynia thresholds throughout the time course tested, including 1 hour ( p = 0.815), 2 hours ( p = 0.271), and 3 hours ( p = 0.271) following treatment. RTIBW-16 (Fig. 3 A) significantly attenuated mechanical allodynia at the 100 µg ( F (2.36, 16.54) = 57.46, p < 0.001) and 56 µg ( F (2.26, 15.83) = 42.61, p < 0.001) doses. Lower RTIBW-16 doses of 10 µg ( F (2.93, 20.51) = 74.5, p < 0.001) and 5.6 µg ( F (3.3, 23.12) = 35.31, p < 0.001) produced delayed-onset partial mechanical allodynia attenuation 3 hours after administration. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the attenuation of mechanical allodynia in the carrageenan model ( F (4.03, 28.18) = 9.23, p < 0.001). NPB-23 (Fig. 3 B) also attenuated mechanical allodynia in a dose- and time-dependent manner similar to RTIBW-16. The doses of 100 µg ( F (3.62, 25.34) = 24.02, p < 0.001) and 56 µg ( F (2.75, 19.26) = 27.76, p < 0.001) NPB-23 produced robust mechanical allodynia blockade in the carrageenan inflammatory pain model, Lower NPB-23 doses of 10 µg ( F (2.01, 14.05) = 66.11, p < 0.001) and 5.6 µg ( F (3.71, 25.95) = 107, p < 0.001) produced delayed-onset partial attenuation of mechanical allodynia 2 hours after administration. A two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the attenuation of mechanical allodynia in the carrageenan model ( F (5.30, 37.07) = 7.15, p < 0.001). At the 1 hour timepoint, the calculated ED 50 values of RTIBW-16 and NPB-23 were 29.47 (21.71–40.00) and 19.92 (13.52–29.37) µg respectively (Table 1 ). In the carrageenan inflammatory pain model RTIBW-16 and NPB-23 were statistically equipotent in the reversal of mechanical allodynia. Intraplantar carrageenan produced edema of the right hind paw as indicated by initial measurements at the 1 hour time point (Fig. 3 C, D). Both RTIBW-16 ( F (1.27, 8.86) = 5.47, p = 0.039) and NPB-23 ( F (1.59, 11.16) = 241.6, p < 0.001) significantly reduced the magnitude of initial swelling as compared to vehicle treatment (Fig. 2 C, D) although NPB-23 was more effective in reducing carrageenan-induced edema. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the attenuation of paw edema in the carrageenan model ( F (1.8, 12.61) = 10.51, p = 0.002). Likewise, a two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the attenuation of paw edema in the carrageenan model ( F (1.93, 13.52) = 42.65, p < 0.001). Discussion Here we demonstrated that intrathecal administration of the truncated NPB-23 and its peptidomimetic RTIBW-16 displayed similar antinociceptive profiles as compared to naturally occurring ligands [ 19 , 21 , 22 ]. As all previous studies using NPB or NPW were conducted in rats [ 18 , 20 – 23 ], the observation of antinociceptive effects of NPBWR1 agonists in mice in this study together with the hyperalgesic effect seen in NPB-deficient mice in response to inflammatory pain support the conserved role of NPBWR1 in pain processing across species [ 19 ]. Both RTIBW-16 and NPB-23 demonstrated antinociceptive effects in a panel of hot plate assay, CIPN, and carrageenan-induced inflammatory pain. They both produced maximum antinociceptive effect in the hot plate assay and exhibited equipotency in reversing mechanical allodynia in the CIPN and carrageenan-induced inflammatory pain models. However, they differed in onset, and duration of action. In the hot plate assay, RTIBW-16 exhibited a stronger antinociceptive effect at the earlier time point (0.5 hour), but with a shorter duration of action compared to NPB-23. In the CIPN assay, RTIBW-16 demonstrated a longer duration of action compared to NPB-23. While both RTIBW-16 and NPB-23 were statistically equipotent in the reversal of mechanical allodynia, NPB-23 was more effective in reducing carrageenan-induced edema. The variation in efficacy may stem from differences in the signaling pathways that drive the observed behaviors. Acute hot plate nociception occurs via nociceptive transmission and is mediated predominately via neuronal A δ and C fibers [ 40 , 41 ]. CIPN mechanical allodynia arises from neurotoxicity and leads to peripheral nerve hyperexcitability, central sensitization, and enhanced synaptic efficacy at A β fibers [ 42 , 43 ]. Although converging on similar downstream mechanisms as CIPN, carrageenan-induced mechanical allodynia arises from peripheral immune cell activation [ 1 , 39 ]. The variations in their antinociceptive profiles are likely attributable to differences in their pharmacokinetic properties, which can influence factors such as onset and duration of action. In conclusion, our studies demonstrate that NPBWR1 agonists exhibit robust antinociceptive activities across a range of pain models including acute thermal pain, inflammatory pain and neuropathic pain. The results underscore their potential as novel therapeutic agents for pain management. Notably, this is the first report of NPBWR1 agonists showing efficacy CIPN model, expanding the understanding of NBPWR1’s role in neuropathic pain. Future efforts should focus on the development of stable peptidomimetics to provide more potent and reliable tools for research and therapeutic use. These improved compounds would complement existing methods, such as naturally occurring compounds and genetic knockout or transgenic strategies, which are currently employed to explore the pharmacological profile of the NPBWR1. Additionally, mechanistic studies will be essential to elucidate the pathways underlying their analgesic effects, further advancing the field of pain research and drug development. Abbreviations % MPE percentage maximum possible effect ANOVA analysis of variance CIPN chemotherapy–induced peripheral neuropathy CNS central nervous system ED effective dose i.c.v. intracerebroventricular i.p. intraperitoneal i.t. intrathecal NPB Neuropeptide B NPW Neuropeptide W NPBWR NPB Neuropeptide B/W Receptor PE polyethylene Declarations Conflict of Interest Statement On behalf of all authors, the corresponding author states that there is no conflict of interest. Conducted experiments Ortiz Contributed new reagents or analytic tools Nguyen Performed data analysis Ortiz, Nguyen, Wilkerson Wrote or contributed to the writing of the manuscript Ortiz, Nguyen, Wilkerson Funding Statement This work was supported by Texas Tech University Health Sciences Center Department of Pharmaceutical Sciences startup funding (J.L.W.) and National Institute on Drug Abuse (Grant DA058800 to T.N.). Author Contribution Author ContributionsParticipated in research design: Y.O., T.N., J.L.W.Conducted experiments: Y.O.Contributed new reagents or analytic tools: T.N.Performed data analysis: Y.O., T.N., J.L.W.Wrote or contributed to the writing of the manuscript: Y.O., T.N., J.L.W. Data Availability The data that support the findings of this study are openly available in the Open Science Framework repository at DOI 10.17605/OSF.IO/EP9FS . References Donvito G, Nass SR, Wilkerson JL, Curry ZA, Schurman LD, Kinsey SG, et al. 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Anti-hyperalgesic effects of intrathecally administered neuropeptide W-23, and neuropeptide B, in tests of inflammatory pain in rats. Brain Res. 2005;1045:97–106. Nakamura S, Nonaka T, Yoshida K, Yamada T, Yamamoto T, Neuropeptide W. an endogenous NPBW1 and NPBW2 ligand, produced an analgesic effect via activation of the descending pain modulatory system during a rat formalin test. Mol Pain. 2021;17:1744806921992187. Fujii R, Yoshida H, Fukusumi S, Habata Y, Hosoya M, Kawamata Y, et al. Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7. J Biol Chem. 2002;277:34010–6. Kanesaka M, Matsuda M, Hirano A, Tanaka K, Kanatani A, Tokita S. Development of a potent and selective GPR7 (NPBW1) agonist: a systematic structure-activity study of neuropeptide B. J Pept Sci. 2007;13:379–85. Shimomura Y, Harada M, Goto M, Sugo T, Matsumoto Y, Abe M, et al. Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8. J Biol Chem. 2002;277:35826–32. Nguyen T, Decker AM, Snyder RW, Tonetti EC, Gamage TF, Zhang Y. Neuropeptide B/W receptor 1 peptidomimetic agonists: Structure-activity relationships and plasma stability. Eur J Med Chem. 2022;231:114149. Wilson-Poe AR, Lau BK, Vaughan CW. Repeated morphine treatment alters cannabinoid modulation of GABAergic synaptic transmission within the rat periaqueductal grey. Br J Pharmacol. 2015;172:681–90. Ortiz YT, Bilbrey JA, Felix JS, Kienegger EA, Mottinelli M, Mukhopadhyay S, et al. Cannabidiol and mitragynine exhibit differential interactive effects in the attenuation of paclitaxel-induced mechanical allodynia, acute antinociception, and schedule-controlled responding in mice. Pharmacol Rep. 2023;75:937–50. Obeng S, Leon F, Patel A, Zuarth Gonzalez JD, Chaves Da Silva L, Restrepo LF, et al. Interactive Effects of micro-Opioid and Adrenergic-alpha (2) Receptor Agonists in Rats: Pharmacological Investigation of the Primary Kratom Alkaloid Mitragynine and Its Metabolite 7-Hydroxymitragynine. J Pharmacol Exp Ther. 2022;383:182–98. Morris CJ. Carrageenan-induced paw edema in the rat and mouse. Methods Mol Biol. 2003;225:115–21. Toma W, Kyte SL, Bagdas D, Alkhlaif Y, Alsharari SD, Lichtman AH, et al. Effects of paclitaxel on the development of neuropathy and affective behaviors in the mouse. Neuropharmacology. 2017;117:305–15. Wilkerson JL, Ghosh S, Mustafa M, Abdullah RA, Niphakis MJ, Cabrera R, et al. The endocannabinoid hydrolysis inhibitor SA-57: Intrinsic antinociceptive effects, augmented morphine-induced antinociception, and attenuated heroin seeking behavior in mice. Neuropharmacology. 2017;114:156–67. Wilkerson JL, Ghosh S, Bagdas D, Mason BL, Crowe MS, Hsu KL, et al. Diacylglycerol lipase beta inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain. Br J Pharmacol. 2016;173:1678–92. Wilkerson JL, Alberti LB, Kerwin AA, Ledent CA, Thakur GA, Makriyannis A, et al. Peripheral versus central mechanisms of the cannabinoid type 2 receptor agonist AM1710 in a mouse model of neuropathic pain. Brain Behav. 2020;10:e01850. Wilkerson JL, Alberti LB, Thakur GA, Makriyannis A, Milligan ED. Peripherally administered cannabinoid receptor 2 (CB(2)R) agonists lose anti-allodynic effects in TRPV1 knockout mice, while intrathecal administration leads to anti-allodynia and reduced GFAP, CCL2 and TRPV1 expression in the dorsal spinal cord and DRG. Brain Res. 2022;1774:147721. Ignatowska-Jankowska B, Wilkerson JL, Mustafa M, Abdullah R, Niphakis M, Wiley JL, et al. Selective monoacylglycerol lipase inhibitors: antinociceptive versus cannabimimetic effects in mice. J Pharmacol Exp Ther. 2015;353:424–32. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55–63. Wu WP, Hao JX, Halldner-Henriksson L, Xu XJ, Jacobson MA, Wiesenfeld-Hallin Z, et al. Decreased inflammatory pain due to reduced carrageenan-induced inflammation in mice lacking adenosine A3 receptors. Neuroscience. 2002;114:523–7. Eddy NB, Leimbach D. Synthetic Analgesics. II. Dithienylbutenyl- and Dithienylbutylamines. J Pharmacol Exp Ther. 1953;107:385–93. Sapio MR, Iadarola MJ, LaPaglia DM, Lehky T, Thurm AE, Danley KM, et al. Haploinsufficiency of the brain-derived neurotrophic factor gene is associated with reduced pain sensitivity. Pain. 2019;160:1070–81. Staff NP, Grisold A, Grisold W, Windebank A. J. Chemotherapy-induced peripheral neuropathy: A current review. Ann Neurol. 2017;81:772–81. Milligan ED, Watkins LR. Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci. 2009;10:23–36. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Pharmacological Reports → Version 1 posted Editorial decision: Revision requested 23 May, 2025 Reviews received at journal 22 May, 2025 Reviewers agreed at journal 16 May, 2025 Reviews received at journal 14 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers invited by journal 14 May, 2025 Editor assigned by journal 02 May, 2025 Submission checks completed at journal 02 May, 2025 First submitted to journal 29 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6559172","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":457239738,"identity":"a22e7867-2baa-4f66-a228-716471bd84eb","order_by":0,"name":"Yuma T. Ortiz","email":"","orcid":"","institution":"Texas Tech University Health Sciences Center","correspondingAuthor":false,"prefix":"","firstName":"Yuma","middleName":"T.","lastName":"Ortiz","suffix":""},{"id":457239739,"identity":"ab9df966-bba4-4240-9d93-034d975aacea","order_by":1,"name":"Thuy Nguyen","email":"","orcid":"","institution":"RTI International","correspondingAuthor":false,"prefix":"","firstName":"Thuy","middleName":"","lastName":"Nguyen","suffix":""},{"id":457239740,"identity":"cd120a31-f500-43b3-8886-725eb2fd983c","order_by":2,"name":"Jenny L. Wilkerson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYBAC9gYwdZiHjwciIAfEbBDmAexaGGFa2KBajHnYiNTCANOS2ENQS3vvscc8DIdl2HgOH/v4o+JO+n75HrOHP2oY5PhuJGDX0nMu3ZiH4TYPG29b8myeM89ye9h4zI15jjEYS+LSMiPHTBqshZ/HmJmx7TBIi5k00L2JG3Bpmf8GpoX/M+PPtsPpwIAwk/zZwFCPS4vgDB6Qlv9Ah/UwM/C2HU4AaZHgbWBIMMChRZonx0xyjsFzoMJjxsw8Zw4b9hxLK5PmOSZhOPPMA6xa+NjPmEm8qThsz8+T/JjxR8Vhefbmw9skf9TYyPMdx24LCDDxGGAKSuBUDg6CH3ilR8EoGAWjYMQDAPnrUxr1MvQWAAAAAElFTkSuQmCC","orcid":"","institution":"Texas Tech University Health Sciences Center","correspondingAuthor":true,"prefix":"","firstName":"Jenny","middleName":"L.","lastName":"Wilkerson","suffix":""}],"badges":[],"createdAt":"2025-04-29 19:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6559172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6559172/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43440-025-00761-2","type":"published","date":"2025-07-31T16:05:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82907887,"identity":"39980c2d-4b59-4e57-8a03-7a12b245e5d8","added_by":"auto","created_at":"2025-05-16 14:38:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntrathecal RTIBW-16 and NPB-23 produce dose- and time-dependent antinociception in the hot plate assay. \u003c/strong\u003eRTIBW-16 (A) produced antinociception in the hot plate latency assay at the doses of 100 (blue, hollow triangle) and 56 μg (blue, filled triangle) through 2 hrs after administration. Similarly, NPB-23 (B) at doses of 100 (red, hollow triangle) and 56 μg (red, filled triangle) produced antinociception through 6 hrs after administration. Abscissae: time points in hours; ordinates: stimulus intensity to illicit paw withdrawal. Filled data points (black) indicate significance from vehicle measurements (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). Data reflect mean ± SEM, n = 8.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6559172/v1/b37fa400ca0d9d5c6a103050.jpeg"},{"id":82907885,"identity":"92f99c79-0f02-4410-9531-caee483fd214","added_by":"auto","created_at":"2025-05-16 14:38:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntrathecal RTIBW-16 and NPB-23 produce dose- and time-dependent antinociception in the paclitaxel CIPN model.\u003c/strong\u003e In the CIPN model RTIBW-16 \u0026nbsp;(A) reversed mechanical allodynia for up to 3 hours following treatment with the 100 (blue, hollow triangle) and 56 μg (blue, filled triangle) doses. NPB-23 (B) produced antinociceptive effects at similar dosing (100 μg and 56 μg, red hollow and filled triangles, respectively) in the CIPN model up to 2 hrs following treatment. Abscissae: time points in hours; ordinates: stimulus intensity to illicit paw withdrawal. Filled data points (black) indicate significance from vehicle measurements (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). Data reflect mean ± SEM, n = 8.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6559172/v1/779b30d009157d6f824c89ef.png"},{"id":82907888,"identity":"6aae07ee-4f9a-4659-be4f-3ddf99bcf265","added_by":"auto","created_at":"2025-05-16 14:38:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntrathecal RTIBW-16 and NPB-23 decrease mechanical allodynia and inflammation in a model of carrageenan-induced inflammatory pain.\u003c/strong\u003e RTIBW-16 (A) at doses of 100 μg (blue, hollow triangle) and 56 μg (blue, filled triangle) fully attenuated peak carrageenan-induced mechanical allodynia. NPB-23 (B) produced similar antinociceptive effects at similar dosing (100 μg and 56 μg, red hollow and filled triangles, respectively). RTIBW-16(C) and NPB-23 (D) treatment resulted in reduced initial swelling at the same time point of 1 hour. Abscissae: time points in hours; ordinates (A, B): stimulus intensity to illicit paw withdrawal; (C, D) change in paw thickness compared to baseline measurements. Filled data points (black) indicate significance from vehicle measurements (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). Data reflect mean ± SEM, n = 8.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6559172/v1/da4699ead7686b0bc5952c48.png"},{"id":88268250,"identity":"6b305fb3-3a4d-4e25-b98a-902aa7f54b6d","added_by":"auto","created_at":"2025-08-04 16:50:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1418836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6559172/v1/01707095-ad41-4811-90ea-03163e63d6a9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antinociceptive effects of intrathecal Neuropeptide B/W receptor 1 agonists in mouse acute nociception, peripheral neuropathy, and inflammatory pain models","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePathological pain encompasses chronic pain conditions such as neuropathic pain, inflammatory pain, and centralized pain syndromes [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Unlike acute pain, which serves as a protective mechanism, chronic pathological pain persists beyond normal healing and often arises from maladaptive changes in the nervous system [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It affects over 30% of the population worldwide, with a notable impact on quality of life, mental health, and daily functioning [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The global burden is profound, contributing to increased healthcare utilization, loss of productivity, and economic costs exceeding billions of dollars annually [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Pathological pain is intricately linked to conditions like diabetes, cancer, and autoimmune diseases, further amplifying its prevalence and impact [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite advances in pain management, opioids remain the only treatment for chronic moderate to severe pain. While effective, their use is restricted by significant limitations, including dependence, tolerance, and respiratory depression. The complexity of pain mechanisms and individual variability further challenge the development of effective treatments, emphasizing the urgent need for targeted research and innovation in therapeutic approaches [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe neuropeptides B and W (NPB and NPW) are endogenous ligands of the Neuropeptide B/W Receptors 1 and 2 (NPBWR1 and NPBWR2) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Together, they have been implied to play a role in pain signaling pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. NPBWR1 is expressed in brain and peripheral organs of both humans and rodents whereas NPBWR2 is not found in rodents [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Within the central nervous system (CNS), NPBWR1 expression was found in cerebellum, prefrontal cortex, hippocampus, amygdala, and spinal cord [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the peripheral nervous system, NPBWR1 has been identified on both human and rat myelinating Schwann cells under basal conditions and was found to be overexpressed in neuropathic conditions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A recent study utilizing immunohistochemistry demonstrated that NPBWR1 and \u0026micro; opioid receptor are colocalized in the superficial dorsal horn of rat spinal cord [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn preclinical animal studies, mice lacking NPB developed hyperalgesia in both the formalin and acetic acid inflammatory pain models but responded normally in acute hot plate nociceptive pain and non-inflammatory pain models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Exogenous intracerebroventricular (i.c.v.) administration of synthetic NPB produced thermal antinociception and decreased paw licking in rat carrageenan and formalin models, respectively [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Intrathecal (i.t.), but not i.c.v., administration of exogenous NPW and NPB reversed mechanical allodynia, or light touch sensitivity in the rat partial nerve ligation neuropathic pain model [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In another study that utilized rats intrathecal NPB and NPW attenuated formalin-induced nociceptive behaviors as well as carrageenan-induced mechanical allodynia but did not alter carrageenan-induced thermal hyperalgesia or acute hot plate nociception [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a rat formalin test, i.c.v. injection of NPW into different brain regions revealed that its analgesic effects were mediated through the activation of the descending pain modulatory system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Morphine co-administered with either NPB or NPW synergistically alleviated both inflammatory and neuropathic pain as well as significantly reduced morphine-induced conditioned place preference and constipation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSo far, behavioral studies have mostly been conducted in rats and relied on exogenous administration of endogenous ligands. NPB is a 29 amino acid peptide with a bromine at the C-6 position of the indole moiety in the N-terminal tryptophan (Trp) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The truncated NPB-23 with the N-terminal 23 amino acid residues and no bromine at the N-terminal Trp was found to exhibit equipotent in vitro NPBWR1 agonist activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. NPW exists in two functional peptide forms consisting of 23 and 30 amino acid residues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. NPB exhibits a higher affinity for NPBWR1, while NPW binds to both NPBWR1 and NPBWR2 with similar affinities [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our group and others have studied the structure-activity relationship of NPB and identified that the pharmacophore for NPBWR1\u0026rsquo;s agonist activity consists of two epitopes, N-terminal WYK and C-terminal GRAAGLL [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. From this understanding, we developed RTIBW-16, a NPB peptidomimetic with a shorter sequence of 13 amino acid residues exhibited similar in vitro potencies while exhibiting improved metabolic stability in rat plasma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study we sought to evaluate the effects of i.t. administration of NPB-23, a truncated human NPB without a bromine at the N-terminal Trp residue and RTIBW-16 using a panel of mouse pain models including the hot plate assay for acute nociception, a chemotherapy-induced peripheral neuropathy (CIPN) and a carrageenan-induced inflammatory pain models. The hot plate assay is a widely used tool for screening novel analgesics [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. CIPN is a clinically relevant that mimics the debilitating sensory and pain symptoms experienced by cancer patients undergoing chemotherapy, providing a valuable tool for developing therapies to alleviate this common side effect [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The carrageenan-induced pain model is a standard tool for evaluating potential analgesics for conditions like arthritis and other inflammatory disorders [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. By profiling across various pain models, we aim to investigate the roles of NPBWR1 agonists in acute nociception, neuropathic and inflammatory pain.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All studies were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Texas Tech University Health Science Center Institutional Animal Care and Use Committee (IACUC number 21034). A total of 44 adult male and 44 adult female (22\u0026ndash;29 g upon arrival) C57BL/6 (Jackson Laboratories, Bar Harbor, ME) mice were used in this study. Mice were split into cohorts (n\u0026thinsp;=\u0026thinsp;8/group) and used for either the chemotherapy-induced peripheral neuropathy model or the hotplate latency assay with later use in the carrageenan paw inflammation model. Housing facilities for mice were maintained at a consistent temperature (20\u0026ndash;22 \u003csup\u003eo\u003c/sup\u003eC)-, humidity (55% \u0026plusmn; 10%)-, and light-schedule (12 hr light/dark; lights on at 0700) which were approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Food and Water were available \u003cem\u003ead libitum\u003c/em\u003e for all mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrugs and Dosing\u003c/h3\u003e\n\u003cp\u003eNPB-23 (Trp-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-NH\u003csub\u003e2\u003c/sub\u003e) and RTIBW-16 ([desaminoTrp\u003csup\u003e1\u003c/sup\u003e]-Tyr-Lys-Ava-Ava-Ava-Gly-Arg-Ala-Ala-Gly-Leu-Leu-NH\u003csub\u003e2\u003c/sub\u003e, compound \u003cb\u003e30\u003c/b\u003e in Ref. 27) were synthesized as previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. RTIBW-16 and NPB-23 were administered via i.t. injections between L4 and L5 vertebrae with an injection volume of 5 \u0026micro;L. The dosing of these compounds was based on previous literature [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePaclitaxel was purchased from Bio-Techne (Minneapolis, MN) and was administered in a vehicle solution containing a 1:1:18 ratio of ethanol (100%), polyethylene glycol monooleyl ether (Tokyo Chemical Industry America; Portland, OR), and saline (0.9% w/v NaCl) as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A dosing cycle of paclitaxel consisted of a total of four intraperitoneal (i.p.) paclitaxel injections (8 mg/kg per injection, given every other day [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Carrageenan was purchased from Sigma-Aldrich (St. Louis, MO) and made into a 0.3% w/v solution in saline for injections [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIntrathecal Injections\u003c/h3\u003e\n\u003cp\u003eIntrathecal injections were conducted as previously described [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; injections occurred between the L4 and L5 vertebrae. Briefly, an \u0026lsquo;injection catheter\u0026rsquo; made from a 27-gauge needle and polyethylene (PE) 20 tubing was connected to a 10 \u0026micro;l Hamilton syringe. Injections occurred over 5 sec and were generally accompanied by a hind paw twitch response. A 100% motor recovery rate was observed.\u003c/p\u003e\n\u003ch3\u003eAcute Nociception Hot Plate Assay\u003c/h3\u003e\n\u003cp\u003eAcute nociception measurements were collected via the hot plate latency assay as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Mice were placed on a heated (52 \u003csup\u003eo\u003c/sup\u003eC) enclosed Hot Plate Analgesia Meter (Columbus Instruments, Columbus, OH). The latency to jump/lick/shake/flick a hind paw was recorded. A 30 second cut-off was used to prevent tissue damage. Latencies were recorded prior to administration of compounds and at each time point over the 24-hour study (0.5, 1, 1.5, 2, 3, 4, 6, 24 hr time points). Latency measurements were taken by observers blinded to treatment conditions. Mice were given a 1-week washout period between test days.\u003c/p\u003e\n\u003ch3\u003ePaclitaxel CIPN Model\u003c/h3\u003e\n\u003cp\u003eTo assess mechanical allodynia within CIPN, mice were habituated to the von Frey testing environment for four consecutive days in 30-minute sessions prior to the first testing session. Baselines were measured as previously described using von Frey monofilaments (North Coast Medical; Morgan Hills, CA) to establish responses to light mechanical touch before paclitaxel or vehicle administration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. During testing mice were placed atop a wire mesh screen with spaces 0.5 mm apart. Mice were singly placed beneath an inverted wire mesh basket (8 cm diameter, 15 cm height) and allowed to habituate to the apparatus for 30 minutes before testing. The von Frey assay utilizes a series of calibrated monofilaments (0.4\u0026ndash;4.0 g stimulus intensity), applied to the left and right plantar surface of the hind paws utilizing the \u0026ldquo;up-down\u0026rdquo; method [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Monofilament application was done in a manner avoiding sequential application to the same paw (i.e., left, right, left, right, as opposed to left, left, left, right). Lifting, licking, or shaking of the tested paw upon filament application was considered a response. Five responses out of five monofilament stimulations were coded as the minimum force required to elicit responses within the von Frey assay. Mice were tested with experimental compounds starting the day after finishing paclitaxel treatment and were given at least a 1-week washout period between test days. As mechanical allodynia in this model lasts through 3 months [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], animals experienced 5\u0026ndash;7 test days. Reported measurements from the von Frey assay are mean response thresholds collected from both the left and right hind paw of each subject as paclitaxel produces bilateral allodynia of similar magnitude in both hind paws. Responses from the von Frey assay were measured over a 24-hour time course (1, 2, 3, 4, 6, 24 hr timepoints). Mechanical allodynia assessments were conducted by observers blinded to treatment conditions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCarrageenan Inflammatory Pain Model\u003c/h2\u003e \u003cp\u003eAfter use in the hot plate latency assay, the same cohort was then used for the carrageen model of inflammatory pain. Mice were given at least a one-week washout period between experimental use. Mice were habituated to the von Frey apparatus and were assessed using the same \u0026ldquo;up-down\u0026rdquo; method as described earlier [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The carrageenan inflammatory pain model was induced as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, an intraplantar injection of 0.3% w/v carrageenan in a 20 \u0026micro;L volume using a 27-gauge needle was administered into the right hind paw. Carrageenan was injected into the right hind paw. The left paw served as an internal control for each subject. The change in paw thickness from baseline was assessed with digital caliper measurements. Paw thickness and von Frey responses were measured over a 24-hour time course (1, 2, 3, 4, 6, 24 hr timepoints), with paw thickness measurements occurring immediately after the von Frey assay at each time point. Mechanical allodynia assessments and paw thickness measurements were conducted by observers blinded to treatment conditions. Intraplantar administration of carrageenan and digital caliper measurement of paw edema were conducted with mice restrained utilizing a plexiglass mouse restrainer (Stoelting Co., Wood Dale, IL)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eHot plate data were converted to percent maximum possible effect (% MPE) with the following equation: ([experimental test value \u0026ndash; baseline value) / (30 sec \u0026ndash; baseline value)] * 100). Behavioral data were analyzed using a repeated measures two-way analysis of variance (ANOVA) for RTIBW-16 and NPB-23 treatment time course analysis with time as one factor and compound dosing as a second factor [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Individual treatment effects were analyzed using repeated measures one-way ANOVA. Dunnett\u0026rsquo;s test was used for post hoc analysis following a significant ANOVA result. Two tailed, paired t-tests were conducted to compare the magnitude of baseline von Frey measurements to their respective pre-paclitaxel and pre-carrageenan measurements. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. As no significant sex effect was observed, male and female data were collapsed. If the mean effect of treatment did not produce a 50% or greater effect, an effective dose (ED\u003csub\u003e50\u003c/sub\u003e) value was not generated. When the mean effect of a drug in an experiment was greater than 50%, the ED\u003csub\u003e50\u003c/sub\u003e values and corresponding 95% confidence limits were calculated using linear regression, where slopes were allowed to vary [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The computer program GraphPad Prism version 10.3 (GraphPad Software Inc.; San Diego, CA) was used in the statistical analyses. Data are plotted as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error of the mean.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntrathecal RTIBW-16 and NPB-23 produced acute antinociception\u003c/h2\u003e \u003cp\u003eIntrathecal vehicle administration did not alter response latencies (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.268) in the hot plate assay. At 56 and 100 \u0026micro;g, both RTIBW-16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and NPB-23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) produced significant antinociceptive effect as early as 0.5 hour and peaked at 1.5 hours after administration. At 100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.17, 15.18)\u0026thinsp;=\u0026thinsp;11.73, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.08, 14.56)\u0026thinsp;=\u0026thinsp;34.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of RTIBW-16 produced significant antinociceptive effects through 2 hours after administration. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time (\u003cem\u003eF\u003c/em\u003e (49, 343)\u0026thinsp;=\u0026thinsp;7.68, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, 100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (1.44, 10.06)\u0026thinsp;=\u0026thinsp;28.51, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (1.70, 11.87)\u0026thinsp;=\u0026thinsp;44.08, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) NPB-23 produced significant antinociception up to 6 hours following administration. A two-way ANOVA of NPB-23 confirmed a significant interaction between treatment and time (\u003cem\u003eF\u003c/em\u003e (35, 245)\u0026thinsp;=\u0026thinsp;9.61, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 1 hour timepoint, the calculated ED\u003csub\u003e50\u003c/sub\u003e values of RTIBW-16 and NPB-23 were 43.86 (25.09\u0026ndash;73.21) and 18.36 (10.17\u0026ndash;33.15) \u0026micro;g respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Generally, in the hot plate assay, RTIBW-16 showed greater antinociceptive effect earlier at 0.5 hour timepoint but shorter duration of action than NPB-23. Both ligands produced similar maximum antinociceptive effects at their peak time of 1.5 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalculated ED\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g) values for the effects of RTIBW-16 and NPB-23 as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e through 4 at 1 hour following treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eED\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eHot Plate Latency\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRTIBW-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42.86 (25.09\u0026ndash;73.21)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPB-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.36 (10.17\u0026ndash;33.15)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ePaclitaxel CIPN von Frey\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRTIBW-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57.79 (53.13\u0026ndash;95.06)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPB-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45.06 (33.91\u0026ndash;59.88)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eCarrageenan von Frey\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRTIBW-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29.47 (21.71\u0026ndash;40.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPB-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.92 (13.52\u0026ndash;29.35)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSample sizes are described in each figure legend. Values in parentheses are 95% confidence intervals.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntrathecal RTIBW-16 and NPB-23 Reversed Paclitaxel-Induced Mechanical Allodynia\u003c/h2\u003e \u003cp\u003eA separate cohort of mice were utilized to examine RTIBW-16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and NPB-23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) in a model of paclitaxel-induced peripheral neuropathy. Following paclitaxel administration, mice displayed significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) mechanical allodynia compared to pre-paclitaxel baselines. Vehicle treatment did not alter mechanical allodynia thresholds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth ligands produced maximum effects at 1 hour. RTIBW-16 significantly attenuated CIPN mechanical allodynia from 1 to 3 hours post administration. Specifically,100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.07, 31.1)\u0026thinsp;=\u0026thinsp;80.8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.8, 42.04)\u0026thinsp;=\u0026thinsp;209.1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 10 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.38, 35.62)\u0026thinsp;=\u0026thinsp;86.24, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) produced dose-related mechanical allodynia reversal. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the CIPN model (\u003cem\u003eF\u003c/em\u003e (5.56, 83.43)\u0026thinsp;=\u0026thinsp;28.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). NPB-23 significantly attenuated mechanical allodynia for 2 hours following 100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (1.33, 19.89)\u0026thinsp;=\u0026thinsp;54.57, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (1.97, 29.54)\u0026thinsp;=\u0026thinsp;59.64, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) administration. A two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the CIPN model (\u003cem\u003eF\u003c/em\u003e (3.94, 59.16)\u0026thinsp;=\u0026thinsp;23.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 1 hour timepoint, the calculated ED\u003csub\u003e50\u003c/sub\u003e values of RTIBW-16 and NPB-23 were 57.79 (53.13\u0026ndash;95.06) and 45.06 (33.91\u0026ndash;59.88) \u0026micro;g respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At 1 hour timepoint, NPB-23 was able to reverse the CIPN mechanical allodynia back to the baseline. RTIBW-16 maintained a longer duration of action than NPB-23. Both ligands were statistically equipotent in the reversal of mechanical allodynia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIntrathecal RTIBW-16 and NPB-23 Blocked Carrageenan-Induced Mechanical Allodynia and Edema\u003c/h2\u003e \u003cp\u003eIntraplantar administration of carrageenan produced significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) mechanical allodynia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in vehicle treated mice compared to their pre-carrageenan baselines. Vehicle treatment did not alter mechanical allodynia thresholds throughout the time course tested, including 1 hour (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.815), 2 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.271), and 3 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.271) following treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRTIBW-16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) significantly attenuated mechanical allodynia at the 100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.36, 16.54)\u0026thinsp;=\u0026thinsp;57.46, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.26, 15.83)\u0026thinsp;=\u0026thinsp;42.61, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) doses. Lower RTIBW-16 doses of 10 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.93, 20.51)\u0026thinsp;=\u0026thinsp;74.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 5.6 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (3.3, 23.12)\u0026thinsp;=\u0026thinsp;35.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) produced delayed-onset partial mechanical allodynia attenuation 3 hours after administration. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the attenuation of mechanical allodynia in the carrageenan model (\u003cem\u003eF\u003c/em\u003e (4.03, 28.18)\u0026thinsp;=\u0026thinsp;9.23, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eNPB-23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) also attenuated mechanical allodynia in a dose- and time-dependent manner similar to RTIBW-16. The doses of 100 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (3.62, 25.34)\u0026thinsp;=\u0026thinsp;24.02, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 56 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.75, 19.26)\u0026thinsp;=\u0026thinsp;27.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) NPB-23 produced robust mechanical allodynia blockade in the carrageenan inflammatory pain model, Lower NPB-23 doses of 10 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (2.01, 14.05)\u0026thinsp;=\u0026thinsp;66.11, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 5.6 \u0026micro;g (\u003cem\u003eF\u003c/em\u003e (3.71, 25.95)\u0026thinsp;=\u0026thinsp;107, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) produced delayed-onset partial attenuation of mechanical allodynia 2 hours after administration. A two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the attenuation of mechanical allodynia in the carrageenan model (\u003cem\u003eF\u003c/em\u003e (5.30, 37.07)\u0026thinsp;=\u0026thinsp;7.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At the 1 hour timepoint, the calculated ED\u003csub\u003e50\u003c/sub\u003e values of RTIBW-16 and NPB-23 were 29.47 (21.71\u0026ndash;40.00) and 19.92 (13.52\u0026ndash;29.37) \u0026micro;g respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the carrageenan inflammatory pain model RTIBW-16 and NPB-23 were statistically equipotent in the reversal of mechanical allodynia.\u003c/p\u003e \u003cp\u003eIntraplantar carrageenan produced edema of the right hind paw as indicated by initial measurements at the 1 hour time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Both RTIBW-16 (\u003cem\u003eF\u003c/em\u003e (1.27, 8.86)\u0026thinsp;=\u0026thinsp;5.47, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) and NPB-23 (\u003cem\u003eF\u003c/em\u003e (1.59, 11.16)\u0026thinsp;=\u0026thinsp;241.6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) significantly reduced the magnitude of initial swelling as compared to vehicle treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D) although NPB-23 was more effective in reducing carrageenan-induced edema. A two-way ANOVA confirmed a significant interaction between RTIBW-16 treatment and time in the attenuation of paw edema in the carrageenan model (\u003cem\u003eF\u003c/em\u003e (1.8, 12.61)\u0026thinsp;=\u0026thinsp;10.51, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). Likewise, a two-way ANOVA confirmed a significant interaction between NPB-23 treatment and time in the attenuation of paw edema in the carrageenan model (\u003cem\u003eF\u003c/em\u003e (1.93, 13.52)\u0026thinsp;=\u0026thinsp;42.65, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we demonstrated that intrathecal administration of the truncated NPB-23 and its peptidomimetic RTIBW-16 displayed similar antinociceptive profiles as compared to naturally occurring ligands [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As all previous studies using NPB or NPW were conducted in rats [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the observation of antinociceptive effects of NPBWR1 agonists in mice in this study together with the hyperalgesic effect seen in NPB-deficient mice in response to inflammatory pain support the conserved role of NPBWR1 in pain processing across species [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth RTIBW-16 and NPB-23 demonstrated antinociceptive effects in a panel of hot plate assay, CIPN, and carrageenan-induced inflammatory pain. They both produced maximum antinociceptive effect in the hot plate assay and exhibited equipotency in reversing mechanical allodynia in the CIPN and carrageenan-induced inflammatory pain models. However, they differed in onset, and duration of action. In the hot plate assay, RTIBW-16 exhibited a stronger antinociceptive effect at the earlier time point (0.5 hour), but with a shorter duration of action compared to NPB-23. In the CIPN assay, RTIBW-16 demonstrated a longer duration of action compared to NPB-23. While both RTIBW-16 and NPB-23 were statistically equipotent in the reversal of mechanical allodynia, NPB-23 was more effective in reducing carrageenan-induced edema. The variation in efficacy may stem from differences in the signaling pathways that drive the observed behaviors. Acute hot plate nociception occurs via nociceptive transmission and is mediated predominately via neuronal A\u003cem\u003eδ\u003c/em\u003e and C fibers [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. CIPN mechanical allodynia arises from neurotoxicity and leads to peripheral nerve hyperexcitability, central sensitization, and enhanced synaptic efficacy at A\u003cem\u003eβ\u003c/em\u003e fibers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although converging on similar downstream mechanisms as CIPN, carrageenan-induced mechanical allodynia arises from peripheral immune cell activation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The variations in their antinociceptive profiles are likely attributable to differences in their pharmacokinetic properties, which can influence factors such as onset and duration of action.\u003c/p\u003e \u003cp\u003eIn conclusion, our studies demonstrate that NPBWR1 agonists exhibit robust antinociceptive activities across a range of pain models including acute thermal pain, inflammatory pain and neuropathic pain. The results underscore their potential as novel therapeutic agents for pain management. Notably, this is the first report of NPBWR1 agonists showing efficacy CIPN model, expanding the understanding of NBPWR1\u0026rsquo;s role in neuropathic pain. Future efforts should focus on the development of stable peptidomimetics to provide more potent and reliable tools for research and therapeutic use. These improved compounds would complement existing methods, such as naturally occurring compounds and genetic knockout or transgenic strategies, which are currently employed to explore the pharmacological profile of the NPBWR1. Additionally, mechanistic studies will be essential to elucidate the pathways underlying their analgesic effects, further advancing the field of pain research and drug development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e% MPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epercentage maximum possible effect\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCIPN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echemotherapy\u0026ndash;induced peripheral neuropathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecentral nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eED\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eeffective dose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.c.v.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintracerebroventricular\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.p.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.t.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintrathecal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeuropeptide B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeuropeptide W\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPBWR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNPB Neuropeptide B/W Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyethylene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\n\u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eConducted experiments\u003c/h2\u003e\n\u003cp\u003eOrtiz\u003c/p\u003e\n\u003ch2\u003eContributed new reagents or analytic tools\u003c/h2\u003e\n\u003cp\u003eNguyen\u003c/p\u003e\n\u003ch2\u003ePerformed data analysis\u003c/h2\u003e\n\u003cp\u003eOrtiz, Nguyen, Wilkerson\u003c/p\u003e\n\u003ch2\u003eWrote or contributed to the writing of the manuscript\u003c/h2\u003e\n\u003cp\u003eOrtiz, Nguyen, Wilkerson\u003c/p\u003e\n\u003ch2\u003eFunding Statement\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Texas Tech University Health Sciences Center Department of Pharmaceutical Sciences startup funding (J.L.W.) and National Institute on Drug Abuse (Grant DA058800 to T.N.).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAuthor ContributionsParticipated in research design: Y.O., T.N., J.L.W.Conducted experiments: Y.O.Contributed new reagents or analytic tools: T.N.Performed data analysis: Y.O., T.N., J.L.W.Wrote or contributed to the writing of the manuscript: Y.O., T.N., J.L.W.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are openly available in the Open Science Framework repository at DOI 10.17605/OSF.IO/EP9FS .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDonvito G, Nass SR, Wilkerson JL, Curry ZA, Schurman LD, Kinsey SG, et al. 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Neuropeptide B/W receptor 1 peptidomimetic agonists: Structure-activity relationships and plasma stability. Eur J Med Chem. 2022;231:114149.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson-Poe AR, Lau BK, Vaughan CW. Repeated morphine treatment alters cannabinoid modulation of GABAergic synaptic transmission within the rat periaqueductal grey. Br J Pharmacol. 2015;172:681\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtiz YT, Bilbrey JA, Felix JS, Kienegger EA, Mottinelli M, Mukhopadhyay S, et al. Cannabidiol and mitragynine exhibit differential interactive effects in the attenuation of paclitaxel-induced mechanical allodynia, acute antinociception, and schedule-controlled responding in mice. Pharmacol Rep. 2023;75:937\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObeng S, Leon F, Patel A, Zuarth Gonzalez JD, Chaves Da Silva L, Restrepo LF, et al. Interactive Effects of micro-Opioid and Adrenergic-alpha (2) Receptor Agonists in Rats: Pharmacological Investigation of the Primary Kratom Alkaloid Mitragynine and Its Metabolite 7-Hydroxymitragynine. J Pharmacol Exp Ther. 2022;383:182\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris CJ. Carrageenan-induced paw edema in the rat and mouse. Methods Mol Biol. 2003;225:115\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToma W, Kyte SL, Bagdas D, Alkhlaif Y, Alsharari SD, Lichtman AH, et al. Effects of paclitaxel on the development of neuropathy and affective behaviors in the mouse. Neuropharmacology. 2017;117:305\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkerson JL, Ghosh S, Mustafa M, Abdullah RA, Niphakis MJ, Cabrera R, et al. 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Nat Rev Neurosci. 2009;10:23\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neuropeptide B/W Receptor 1, Neuropeptide B, peptidomimetic, pain, neuropathic pain, hot plate latency, inflammatory pain","lastPublishedDoi":"10.21203/rs.3.rs-6559172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6559172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe Neuropeptide B/W Receptor 1 (NPBWR1) system, including its two endogenous ligands, Neuropeptides B and W (NPB and NPW), has garnered interest as potential target to develop novel analgesics. Behavioral studies were typically conducted with exogenously administered endogenous ligands. In this study, we examined truncated NPB-23 and its peptidomimetic RTIBW-16 in a panel of antinociceptive assays including the hot plate, carrageenan-induced inflammatory, and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN) pain assays.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMale and female C57BL/6 mice underwent testing in the hot plate acute nociception assay. After a minimum one-week washout, mice were enrolled in the carrageenan inflammatory pain model, receiving intraplanar carrageenan (0.3% carrageenan in a 20 \u0026micro;L). Separate mouse cohorts received a cycle of intraperitoneal paclitaxel injections (cumulative dose 32 mg/kg). The von Frey assay was utilized to assess CIPN and carrageenan-induced allodynia.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNPB-23 and RTIBW-16 dose-dependently produced thermal antinociception, attenuated CIPN allodynia and carrageenan-induced allodynia with some differences regarding onset time, potency and duration of action. In the hot plate assay, RTIBW-16 showed earlier onset but shorter duration of action than NPB-23 with similar maximum peak effects. Both compounds were statistically equipotent in the reversal of mechanical allodynia induced by either paclitaxel or carrageenan. RTIBW-16 maintained a longer duration of action than NPB-23 in CIPN assay.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBoth NPBWR1 agonists alleviated thermal and inflammatory pain. Notably, we demonstrated for the first time that NPBWR1 agonists exhibited analgesic effect in the CIPN model. Our findings highlight NPBWR1 as a promising target for developing analgesics with novel mechanisms.\u003c/p\u003e","manuscriptTitle":"Antinociceptive effects of intrathecal Neuropeptide B/W receptor 1 agonists in mouse acute nociception, peripheral neuropathy, and inflammatory pain models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 14:38:29","doi":"10.21203/rs.3.rs-6559172/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T13:17:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T02:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267813867194246968703087598168790494854","date":"2025-05-16T09:38:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T00:27:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204639794678020031278479372925464811892","date":"2025-05-14T23:54:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-14T09:30:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-02T07:40:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-02T06:59:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pharmacological Reports","date":"2025-04-29T19:28:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3405036b-b902-4e3f-995c-4360a929ff60","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:41:06+00:00","versionOfRecord":{"articleIdentity":"rs-6559172","link":"https://doi.org/10.1007/s43440-025-00761-2","journal":{"identity":"pharmacological-reports","isVorOnly":false,"title":"Pharmacological Reports"},"publishedOn":"2025-07-31 16:05:12","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2025-05-16 14:38:29","video":"","vorDoi":"10.1007/s43440-025-00761-2","vorDoiUrl":"https://doi.org/10.1007/s43440-025-00761-2","workflowStages":[]},"version":"v1","identity":"rs-6559172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6559172","identity":"rs-6559172","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}