Faculty Bibliography

OBJECTIVES/OBJECTIVE:To determine the relationships between the number and class of xerogenic medications on whole stimulated salivary flow rates and oral health-related quality of life (OH-QOL) measures in patients who received high-dose external beam radiation therapy (RT) for head and neck cancer (HNC). STUDY DESIGN/METHODS:Complete medication lists were generated using patient electronic health records from every attended study visit for 146 HNC patients. Whole stimulated salivary flow was measured before RT, and 6 and 18-months after RT. Ten single-item questions and two composite scales of swallowing problems and senses problems (taste and smell) were assessed at baseline and at 6-month intervals up to 24 months after RT. Linear mixed-effects models examined associations between the total number and class of medications and stimulated salivary flow and OH-QOL. RESULTS:There was no detected association between the total number of medications and stimulated salivary flow (p-value = .18). Only antidepressant usage was significantly associated with stimulated salivary flow (P = .006). Number of medications, narcotic analgesic, and antidepressant usage were significantly associated with a clinically meaningful decrease in OH-QOL. CONCLUSION/CONCLUSIONS:Antidepressants were associated with reduced stimulated salivary flow, but no cumulative negative effect on whole stimulated salivary flow was identified. Polypharmacy was associated with worse OH-QOL.

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of intracellular GPCR signaling are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically encoded biosensors targeted to the plasma membrane and early endosomes were used to analyze compartmentalized signaling of protease-activated receptor 2 (PAR₂); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to preferentially antagonize intracellular PAR₂ signaling of pain. PAR₂ agonists evoked sustained activation of PAR₂, Gαq, and β-arrestin-1 in early endosomes and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with targeted biosensors. Fluorescent dendrimer and core-shell polymeric NPs accumulated in endosomes of HEK293T cells, colonic epithelial cells, and nociceptors, detected by confocal microscopy. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR₂ modulator. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR₂, Gαq, and β-arrestin-1 activation in early endosomes and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, fluorescent dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs accumulated in neurons, sites of PAR₂ expression. Both NP formulations of AZ3451, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. These results provide evidence that PAR₂ endosomal signaling mediates pain and that nanomedicines that antagonize PAR₂ in endosomes effectively relieve pain. NP-mediated delivery may improve the efficacy of other GPCR antagonists for treatment of diverse diseases.

By improving the delivery and tumor retention of chemotherapeutics, nanomedicines hold potential for cancer treatment. The usefulness of nanoparticle (NP)-encapsulated analgesics for the cancer pain treatment is comparatively unexplored. We investigated whether NPs encapsulating olcegepant (OCP), an antagonist of the calcitonin receptor-like receptor (CLR) for the calcitonin gene-related peptide (CGRP), effectively relieved oral cancer pain in mice. Because persistent endosomal CLR signaling in Schwann cells mediates craniofacial pain, we reasoned that the predisposition of NPs to accumulate in endosomes could be leveraged to effectively relieve oral cancer pain. By expressing biosensors for activated CLR, Gα proteins and β-arrestins in HEK293T and Schwann cells, we found that CGRP activates CLR signaling first at the plasma membrane and then in early, late and recycling endosomes and the cis- and trans-Golgi apparatus. We synthesized biocompatible NPs encapsulating OCP and fluorophores by integrating hydrophobic ion pairing nanoformulation with Flash NanoPrecipitation. NPs slowly released OCP and accumulated in early endosomes, leading to sustained inhibition of endosomal CLR signaling in HEK293T and Schwann cells. Oral cancers were established in mice, which led to heightened pain-like responses. After intra-tumoral injection, NPs were retained in tumors for at least one week. OCP-loaded NPs almost completely reversed allodynia and hyperalgesia for a prolonged period, whereas unencapsulated OCP had small and transient effects. The NP accumulation in endosomal sites of pain signaling, the sustained release of antagonist, and the retention of NPs in tumors explain their beneficial actions. Thus, NP-encapsulation holds promise for the relief of painful cancers that are inadequately treated by opioids.

Analgesia by non-steroidal anti-inflammatory drugs (NSAIDs) is ascribed to inhibition of prostaglandin (PG) biosynthesis and ensuing inflammation. However, NSAIDs have life-threatening side effects, and inhibition of inflammation delays pain resolution. Decoupling the mechanisms underlying PG-evoked pain vs. protective inflammation would facilitate pain treatment. Herein, we reveal that selective silencing of the PGE₂ receptor 2 (EP2) in Schwann cells via adeno-associated viral vectors abrogates the indomethacin-sensitive component of pain-like responses in mice elicited by inflammatory stimuli without affecting inflammation. In human Schwann cells and in mice, EP2 activation and optogenetic stimulation of adenylyl cyclase evokes a plasma membrane-compartmentalized cyclic adenosine monophosphate (cAMP) signal that, via A-kinase anchor protein-associated protein kinase A, sustains inflammatory pain-like responses, but does not delay their resolution. Thus, an unforeseen and druggable EP2 receptor in Schwann cells, via specific cAMP nanodomains, encodes PGE₂-mediated persistent inflammatory pain but not PG-dependent protective inflammation.

OBJECTIVE:To compare salivary flow rates between females and males, before and after radiation therapy (RT) for head and neck cancer (HNC). METHODS:Prospective observational multicenter cohort study (OraRad). Stimulated whole salivary flow was measured before RT and at 6 and 18 months after RT. RESULTS:Mean (95% confidence interval) salivary flow in g/min before RT was 0.81 (0.71, 0.90) in females (n = 107) and 1.20 (1.15, 1.25) in males (n = 391) (p < 0.001); at 6 months was 0.34 (0.24, 0.44) in females and 0.50 (0.44, 0.55) in males (p = 0.01); at 18 months was 0.49 (0.38, 0.59) in females and 0.70 (0.64, 0.75) in males (p < 0.001). Median nadir salivary flow after RT was 0.22 in females and 0.35 in males (p < 0.001). A lower nadir salivary flow in females, but not males, was associated with an increased risk for tooth failure (p = 0.02). CONCLUSIONS:Females with HNC have lower stimulated whole salivary flow than males, before and after RT. Low salivary flow after RT may be a risk factor for tooth failure among females. The lower pre-RT salivary flow rates in females, combined with prior literature in other populations, indicates that, in general, females have lower stimulated salivary flow than males.

BACKGROUND:Oral cancer causes intense pain at the primary site, and such pain can impair oral functions. However, the underlying mechanisms for oral cancer pain are still not fully understood. In the present study, it is investigated whether programmed cell death protein 1 (PD-1) is involved in the development of oral cancer pain. METHODS:RMP1-14, a specific anti-PD-1 antibody, was injected into spinal trigeminal nucleus caudalis (Sp5C) and measured pain behaviors using von Frey filaments and dolognawmeter. Western blotting and immunofluorescence staining were performed to analyze the expression of PD-1 and tumor necrosis factor alpha (TNFα) in the Sp5C. RESULTS:It was observed that the PD-1 antibody significantly inhibited mechanical hypersensitivity and functional allodynia in our oral cancer pain mouse model. Moreover, we found that TNFα was highly upregulated in the Sp5C following the induction of oral cancer pain and that intra-Sp5C injection of the PD-1 antibody diminished the upregulation of TNFα. It was found that genetic deletion of TNFα or its receptor antagonism synergized the analgesic effect of PD-1 antibody on oral cancer pain. CONCLUSION/CONCLUSIONS:Our results suggest that PD-1 in the Sp5C contributes to oral cancer pain by altering TNFα signaling in the trigeminal nociceptive system, and PD-1 could be targeted to develop a novel approach for oral cancer pain management.

INTRODUCTION/UNASSIGNED:Patients with oral cancer often experience intense functional pain due to mechanical stimulation at the cancer site. The role of mechanosensitive ion channels in oral cancer pain, such as TRPV4, is not fully understood. OBJECTIVES/UNASSIGNED:Our objective was to investigate the role of Schwann cell TRPV4 in oral cancer pain. METHODS/UNASSIGNED:imaging, and patch-clamp electrophysiology. The effect of TRPV4 activation on Schwann cell responses to mechanical stimulation was evaluated using a piezo stimulator. Conditioned media (CM) from TRPV4-activated Schwann cells were injected into the mouse paw to evaluate the contribution of TRPV4 in Schwann cells to mechanical hypersensitivity. RESULTS/UNASSIGNED:responses and whole-cell membrane currents in human Schwann cells. Mechanoactivated currents in human Schwann cells were inhibited by the TRPV4 antagonist HC-067047. Schwann cell CM induced mechanical hypersensitivity in mice, which was blocked by pre-treatment with HC-067047. CONCLUSION/UNASSIGNED:TRPV4 activation plays a role in mediating mechanically induced pain of oral cancer.

Nerve growth factor (NGF) monoclonal antibodies inhibit chronic pain, yet failed to gain approval due to worsened joint damage in osteoarthritis patients. We report that neuropilin-1 (NRP1) is a coreceptor for NGF and tropomyosin-related kinase A (TrkA) pain signaling. NRP1 was coexpressed with TrkA in human and mouse nociceptors. NRP1 inhibitors suppressed NGF-stimulated excitation of human and mouse nociceptors and NGF-evoked nociception in mice. NRP1 knockdown inhibited NGF/TrkA signaling, whereas NRP1 overexpression enhanced signaling. NGF bound NRP1 with high affinity and interacted with and chaperoned TrkA from the biosynthetic pathway to the plasma membrane and endosomes, enhancing TrkA signaling. Molecular modeling suggested that the C-terminal R/KXXR/K NGF motif interacts with the extracellular "b" NRP1 domain within a plasma membrane NGF/TrkA/NRP1 of 2:2:2 stoichiometry. G α interacting protein C-terminus 1 (GIPC1), which scaffolds NRP1 and TrkA to myosin VI, colocalized in nociceptors with NRP1/TrkA. GIPC1 knockdown abrogated NGF-evoked excitation of nociceptors and pain-like behavior. Thus, NRP1 is a nociceptor-enriched coreceptor that facilitates NGF/TrkA pain signaling. NRP binds NGF and chaperones TrkA to the plasma membrane and signaling endosomes via the GIPC1 adaptor. NRP1 and GIPC1 antagonism in nociceptors offers a long-awaited nonopioid alternative to systemic antibody NGF sequestration for the treatment of chronic pain.