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Sustained endosomal release of a neurokinin-1 receptor antagonist from nanostars provides long-lasting relief of chronic pain

Latorre, Rocco; Ramírez-Garcia, Paulina D; Hegron, Alan; Grace, James L; Retamal, Jeffri S; Shenoy, Priyank; Tran, Mai; Aurelio, Luigi; Flynn, Bernard; Poole, Daniel P; Klein-Cloud, Rafael; Jensen, Dane D; Davis, Thomas P; Schmidt, Brian L; Quinn, John F; Whittaker, Michael R; Veldhuis, Nicholas A; Bunnett, Nigel W
Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.
PMID: 35533442
ISSN: 1878-5905
CID: 5215272

Agonist that activates the µ-opioid receptor in acidified microenvironments inhibits colitis pain without side effects

Jiménez-Vargas, Nestor Nivardo; Yu, Yang; Jensen, Dane D; Bok, Diana Daeun; Wisdom, Matthew; Latorre, Rocco; Lopez, Cintya; Jaramillo-Polanco, Josue O; Degro, Claudius; Guzman-Rodriguez, Mabel; Tsang, Quentin; Snow, Zachary; Schmidt, Brian L; Reed, David E; Lomax, Alan Edward; Margolis, Kara Gross; Stein, Christoph; Bunnett, Nigel W; Vanner, Stephen J
OBJECTIVE:The effectiveness of µ-opioid receptor (MOPr) agonists for treatment of visceral pain is compromised by constipation, respiratory depression, sedation and addiction. We investigated whether a fentanyl analogue, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), which preferentially activates MOPr in acidified diseased tissues, would inhibit pain in a preclinical model of inflammatory bowel disease (IBD) without side effects in healthy tissues. DESIGN/METHODS:Antinociceptive actions of NFEPP and fentanyl were compared in control mice and mice with dextran sodium sulfate colitis by measuring visceromotor responses to colorectal distension. Patch clamp and extracellular recordings were used to assess nociceptor activation. Defecation, respiration and locomotion were assessed. Colonic migrating motor complexes were assessed by spatiotemporal mapping of isolated tissue. NFEPP-induced MOPr signalling and trafficking were studied in human embryonic kidney 293 cells. RESULTS:NFEPP inhibited visceromotor responses to colorectal distension in mice with colitis but not in control mice, consistent with acidification of the inflamed colon. Fentanyl inhibited responses in both groups. NFEPP inhibited the excitability of dorsal root ganglion neurons and suppressed mechanical sensitivity of colonic afferent fibres in acidified but not physiological conditions. Whereas fentanyl decreased defecation and caused respiratory depression and hyperactivity in mice with colitis, NFEPP was devoid of these effects. NFEPP did not affect colonic migrating motor complexes at physiological pH. NFEPP preferentially activated MOPr in acidified extracellular conditions to inhibit cAMP formation, recruit β-arrestins and evoke MOPr endocytosis. CONCLUSION/CONCLUSIONS:In a preclinical IBD model, NFEPP preferentially activates MOPr in acidified microenvironments of inflamed tissues to induce antinociception without causing respiratory depression, constipation and hyperactivity.
PMID: 33785555
ISSN: 1468-3288
CID: 4840882

Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment

Scheff, Nicole N; Wall, Ian M; Nicholson, Sam; Williams, Hannah; Chen, Elyssa; Tu, Nguyen H; Dolan, John C; Liu, Cheng Z; Janal, Malvin N; Bunnett, Nigel W; Schmidt, Brian L
Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.
PMID: 35260737
ISSN: 2045-2322
CID: 5183522

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain

Latorre, Rocco; Hegron, Alan; Peach, Chloe J; Teng, Shavonne; Tonello, Raquel; Retamal, Jeffri S; Klein-Cloud, Rafael; Bok, Diana; Jensen, Dane D; Gottesman-Katz, Lena; Rientjes, Jeanette; Veldhuis, Nicholas A; Poole, Daniel P; Schmidt, Brian L; Pothoulakis, Charalabos H; Rankin, Carl; Xie, Ying; Koon, Hon Wai; Bunnett, Nigel W
G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.
PMID: 35110404
ISSN: 1091-6490
CID: 5156302

Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice

De Logu, Francesco; Nassini, Romina; Hegron, Alan; Landini, Lorenzo; Jensen, Dane D; Latorre, Rocco; Ding, Julia; Marini, Matilde; Souza Monteiro de Araujo, Daniel; Ramírez-Garcia, Paulina; Whittaker, Michael; Retamal, Jeffri; Titiz, Mustafa; Innocenti, Alessandro; Davis, Thomas P; Veldhuis, Nicholas; Schmidt, Brian L; Bunnett, Nigel W; Geppetti, Pierangelo
Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.
PMID: 35115501
ISSN: 2041-1723
CID: 5156312

Contributions of bile acids to gastrointestinal physiology as receptor agonists and modifiers of ion channels

Keely, Stephen J; Urso, Andreacarola; Ilyaskin, Alexandr V; Korbmacher, Christoph; Bunnett, Nigel W; Poole, Daniel P; Carbone, Simona E
Bile acids (BAs) are known to be important regulators of intestinal motility and epithelial fluid and electrolyte transport. Over the past two decades, significant advances in identifying and characterizing the receptors, transporters, and ion channels targeted by BAs have led to exciting new insights into the molecular mechanisms involved in these processes. Our appreciation of BAs, their receptors, and BA-modulated ion channels as potential targets for the development of new approaches to treat intestinal motility and transport disorders is increasing. In the current review, we aim to summarize recent advances in our knowledge of the different BA receptors and BA-modulated ion channels present in the gastrointestinal system. We discuss how they regulate motility and epithelial transport, their roles in pathogenesis, and their therapeutic potential in a range of gastrointestinal diseases.
PMID: 34755536
ISSN: 1522-1547
CID: 5166832

Mice expressing fluorescent PAR(2) reveal that endocytosis mediates colonic inflammation and pain

Latorre, Rocco; Hegron, Alan; Peach, Chloe J.; Teng, Shavonne; Tonello, Raquel; Retamal, Jeffri S.; Klein-Cloud, Rafael; Bok, Diana; Jensen, Dane D.; Gottesman-Katz, Lena; Rientjes, Jeanette; Veldhuis, Nicholas A.; Poole, Daniel P.; Schmidt, Brian L.; Pothoulakis, Charalabos H.; Rankin, Carl; Xie, Ying; Koon, Hon Wai; Bunnett, Nigel W.
ISSN: 0027-8424
CID: 5207592

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models

Tu, Nguyen Huu; Inoue, Kenji; Chen, Elyssa; Anderson, Bethany M; Sawicki, Caroline M; Scheff, Nicole N; Tran, Hung D; Kim, Dong H; Alemu, Robel G; Yang, Lei; Dolan, John C; Liu, Cheng Z; Janal, Malvin N; Latorre, Rocco; Jensen, Dane D; Bunnett, Nigel W; Edgington-Mitchell, Laura E; Schmidt, Brian L
Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.
PMID: 34572924
ISSN: 2072-6694
CID: 5012742

Nanotechnology for Pain Management: Current and Future Therapeutic Interventions

Bhansali, Divya; Teng, Shavonne L; Lee, Caleb S; Schmidt, Brian L; Bunnett, Nigel W; Leong, Kam W
Pain is one of the most common medical conditions and affects more Americans than diabetes, heart disease, and cancer combined. Current pain treatments mainly rely on opioid analgesics and remain unsatisfactory. The life-threatening side effects and addictive properties of opioids demand new therapeutic approaches. Nanomedicine may be able to address these challenges as it allows for sensitive and targeted treatments without some of the burdens associated with current clinical pain therapies. This review discusses the physiology of pain, the current landscape of pain treatment, novel targets for pain treatment, and recent and ongoing efforts to effectively treat pain using nanotechnology-based approaches. We highl ight advances in nanoparticle-based drug delivery to reduce side effects, gene therapy to tackle the source of pain, and nanomaterials-based scavenging to proactively mediate pain signaling.
PMID: 34899962
ISSN: 1748-0132
CID: 5109602

An American Physiological Society cross-journal Call for Papers on "Inter-Organ Communication in Homeostasis and Disease" [Editorial]

Bodine, Sue C; Brooks, Heddwen L; Bunnett, Nigel W; Coller, Hilary A; Frey, Mark R; Joe, Bina; Kleyman, Thomas R; Lindsey, Merry L; Marette, André; Morty, Rory E; Ramírez, Jan-Marino; Thomsen, Morten B; Yosten, Gina L C
PMID: 34010064
ISSN: 1522-1504
CID: 4972992