Faculty Bibliography

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.

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.

OBJECTIVES/OBJECTIVE:Salivary hypofunction and xerostomia, are common side effects of radiotherapy, negatively impacting quality of life. The OraRad study presents results on the longitudinal impact of radiotherapy on salivary flow and patient-reported outcomes. PATIENTS AND METHODS/METHODS:Prospective, multicenter cohort study of 572 patients receiving curative-intent head and neck radiotherapy (RT). Stimulated salivary flow (SSF) rate and patient-reported outcomes were measured prior to RT and at 6- and 18-months post-RT. Linear mixed effects models examined the relationship between RT dose and change in salivary flow, and change in patient-reported outcomes. RESULTS:544 patients had baseline salivary flow measurement, with median (IQR) stimulated flow rate of 0.975 (0.648, 1.417) g/min. Average RT dose to parotid glands was associated with change in salivary flow post-RT (p < 0.001). Diminished flow to 37% of pre-RT level was observed at 6 months (median: 0.358, IQR: 0.188 to 0.640 g/min, n = 481) with partial recovery to 59% of pre-RT at 18 months (median: 0.575, IQR: 0.338 to 0.884 g/min, n = 422). Significant improvement in patient-reported swallowing, senses (taste and smell), mouth opening, dry mouth, and sticky saliva (p-values < 0.03) were observed between 6 and 18 months post-RT. Changes in swallowing, mouth opening, dry mouth, and sticky saliva were significantly associated with changes in salivary flow from baseline (p-values < 0.04). CONCLUSION/CONCLUSIONS:Salivary flow and patient-reported outcomes decreased as a result of RT, but demonstrated partial recovery during follow-up. Continued efforts are needed to improve post-RT salivary function to support quality of life.

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 Gα_q, Gα_i, 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.

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.

BACKGROUND:Patients with head and neck cancer (HNC) treated with radiation therapy (RT) are at risk for jaw osteoradionecrosis (ORN), which is largely characterized by the presence of exposed necrotic bone. This report describes the incidence and clinical course of and risk factors for exposed intraoral bone in the multicenter Observational Study of Dental Outcomes in Head and Neck Cancer Patients (OraRad) cohort. METHODS:Participants were evaluated before RT and at 6, 12, 18, and 24 months after RT. Exposed bone was characterized by location, sequestrum formation, and other associated features. The radiation dose to the affected area was determined, and the history of treatment for exposed bone was recorded. RESULTS:The study enrolled 572 participants; 35 (6.1%) were diagnosed with incident exposed bone at 6 (47% of reports), 12 (24%), 18 (20%), and 24 months (8%), with 60% being sequestrum and with 7 cases (20%) persisting for >6 months. The average maximum RT dose to the affected area of exposed bone was 5456 cGy (SD, 1768 cGy); the most frequent associated primary RT sites were the oropharynx (42.9%) and oral cavity (31.4%), and 76% of episodes occurred in the mandible. The diagnosis of ORN was confirmed in 18 participants for an incidence rate of 3.1% (18 of 572). Risk factors included pre-RT extractions (P = .008), a higher RT dose (P = .039), and tobacco use (P = .048). CONCLUSIONS:The 2-year incidence of exposed bone in the OraRad cohort was 6.1%; the incidence of confirmed ORN was 3.1%. Exposed bone after RT for HNC is relatively uncommon and, in most cases, is a short-term complication, not a recurring or persistent one.

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 Na_v1.8-positive neurons (Par₂Na_v1.8), 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.

ABSTRACT/UNASSIGNED:Migraine affects ∼15% of the world's population greatly diminishing their quality of life. Current preventative treatments are effective in only a subset of migraine patients, and while cannabinoids appear beneficial in alleviating migraine symptoms, central nervous system (CNS) side effects limit their widespread use. We developed peripherally-restricted cannabinoids (PRCBs) that relieve chronic pain symptoms of cancer and neuropathies, without appreciable CNS side effects or tolerance development. Here we determined PRCB effectiveness in alleviating hypersensitivity symptoms in mouse models of migraine and medication overuse headache (MOH). Chronic glyceryl trinitrate (GTN, 10 mg/kg) administration led to increased sensitivity to mechanical stimuli, and increased expression of phosphorylated protein kinase A (p-PKA), neuronal nitric oxide synthase (nNOS), and transient receptor potential ankyrin 1 (TRPA1) proteins in trigeminal ganglia. PRCB pretreatment, but not posttreatment, prevented behavioral and biochemical correlates of GTN-induced sensitization. Low pH- and allyl isothiocyanate-activated currents in acutely isolated trigeminal neurons were reversibly attenuated by PRCB application. Chronic GTN treatment significantly enhanced these currents. Chronic sumatriptan treatment also led to development of allodynia to mechanical and cold stimuli which was slowly reversible after sumatriptan discontinuation. Subsequent challenge with a previously ineffective low-dose GTN (0.1-0.3 mg/kg) revealed latent behavioral sensitization and increased expression of p-PKA, nNOS, and TRPA1 proteins in trigeminal ganglia. PRCB pretreatment prevented all behavioral and biochemical correlates of allodynia and latent sensitization. Importantly, chronic PRCB treatment alone did not produce any behavioral or biochemical signs of sensitization. These data validate peripheral cannabinoid receptors as potential therapeutic targets in migraine and MOH.

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.