Tally M Largent-Milnes

Interests

Teaching

Biochemistry, pharmacology, neuroscience, physiology, drugs of abuse, pain mechanisms to undergraduate, graduate, and medical students. Integration of basic science with clinical cases for Clinical Reasoning to medical students and fellows (GME)

Research

Understand neuroscience, physiology, and pharmacology to enhance drug discover and delivery to better manage chronic pain

Courses

Scholarly Contributions

Journals/Publications

• Levine, A. A., Liktor-Busa, E., Balasubramanian, S., Palomino, S. M., Burtman, A. M., Couture, S. A., Lipinski, A. A., Langlais, P. R., & Largent-Milnes, T. M. (2023). Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome. International journal of molecular sciences, 25(1).
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  Mounting evidence supports the role of the endocannabinoid system in neurophysiology, including blood-brain barrier (BBB) function. Recent work has demonstrated that activation of endocannabinoid receptors can mitigate insults to the BBB during neurological disorders like traumatic brain injury, cortical spreading depression, and stroke. As alterations to the BBB are associated with worsening clinical outcomes in these conditions, studies herein sought to examine the impact of endocannabinoid depletion on BBB integrity. Barrier integrity was investigated in vitro via bEnd.3 cell monolayers to assess endocannabinoid synthesis, barrier function, calcium influx, junctional protein expression, and proteome-wide changes. Inhibition of 2-AG synthesis using DAGLα inhibition and siRNA inhibition of DAGLα led to loss of barrier integrity via altered expression of VE-cadherin, which could be partially rescued by exogenous application of 2-AG. Moreover, the deleterious effects of DAGLα inhibition on BBB integrity showed both calcium and PKC (protein kinase C)-dependency. These data indicate that disruption of 2-AG homeostasis in brain endothelial cells, in the absence of insult, is sufficient to disrupt BBB integrity thus supporting the role of the endocannabinoid system in neurovascular disorders.
• Liktor-Busa, E., Levine, A. A., Palomino, S. M., Singh, S., Wahl, J., Vanderah, T. W., Stella, N., & Largent-Milnes, T. M. (2023). ABHD6 and MAGL control 2-AG levels in the PAG and allodynia in a CSD-induced periorbital model of headache. Frontiers in pain research (Lausanne, Switzerland), 4, 1171188.
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  The high prevalence and severe symptoms of migraines in humans emphasizes the need to identify underlying mechanisms that can be targeted for therapeutic benefit. Clinical Endocannabinoid Deficiency (CED) posits that reduced endocannabinoid tone may contribute to migraine development and other neuropathic pain conditions. While strategies that increase levels of the endocannabinoid n-arachidonoylethanolamide have been tested, few studies have investigated targeting the levels of the more abundant endocannabinoid, 2-arachidonoylgycerol, as an effective migraine intervention.
• Martinez, C. I., Liktor-Busa, E., & Largent-Milnes, T. M. (2023). Molecular mechanisms of hormones implicated in migraine and the translational implication for transgender patients. Frontiers in pain research (Lausanne, Switzerland), 4, 1117842.
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  Migraine is a primary headache disorder recognized by the World Health Organization as one of the most poorly understood and debilitating neurological conditions impacting global disability. Chronic pain disorders are more frequently diagnosed among cisgender women than men, suggesting that female sex hormones could be responsible for mediating chronic pain, including migraine and/or that androgens can be protective. This review discusses the major gonadal hormones, estrogens, progesterone, and testosterone in the context of molecular mechanisms by which they play a role in migraine pathophysiology. In addition, the literature to date describing roles of minor sex hormones including prolactin, luteinizing hormone, follicular stimulating hormone, and gonadotropin releasing hormone in migraine are presented. Because transgender and gender non-conforming (trans*) individuals are an underserved patient population in which gender-affirming sex hormone replacement therapy (HRT) is often medically necessary to align biological sex with gender identity, results from cisgender patient populations are discussed in the context of these major and minor sex hormones on migraine incidence and management in trans* patients.
• Milnes, T. M., Langlais, P. R., Lipinski, A. A., Couture, S., Burtman, A. M., Palomino, S. M., Balasubramanian, S., Liktor-Busa, E., & Levine, A. A. (2023). Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome. Int J Mol Sci.
• Sulaiman, M. I., Alabsi, W., Szabo, L., Hay, M., Polt, R., Largent-Milnes, T. M., & Vanderah, T. W. (2023). PNA6, a Lactosyl Analogue of Angiotensin-(1-7), Reverses Pain Induced in Murine Models of Inflammation, Chemotherapy-Induced Peripheral Neuropathy, and Metastatic Bone Disease. International journal of molecular sciences, 24(19).
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  Pain is the most significant impairment and debilitating challenge for patients with bone metastasis. Therefore, the primary objective of current therapy is to mitigate and prevent the persistence of pain. Thus, cancer-induced bone pain is described as a multifaceted form of discomfort encompassing both inflammatory and neuropathic elements. We have developed a novel non-addictive pain therapeutic, PNA6, that is a derivative of the peptide Angiotensin-(1-7) and binds the Mas receptor to decrease inflammation-related cancer pain. In the present study, we provide evidence that PNA6 attenuates inflammatory, chemotherapy-induced peripheral neuropathy (CIPN) and cancer pain confined to the long bones, exhibiting longer-lasting efficacious therapeutic effects. PNA6, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-β-Lact)-amide, was successfully synthesized using solid phase peptide synthesis (SPPS). PNA6 significantly reversed inflammatory pain induced by 2% carrageenan in mice. A second murine model of platinum drug-induced painful peripheral neuropathy was established using oxaliplatin. Mice in the oxaliplatin-vehicle treatment groups demonstrated significant mechanical allodynia compared to the oxaliplatin-PNA6 treatment group mice. In a third study modeling a complex pain state, E0771 breast adenocarcinoma cells were implanted into the femur of female C57BL/6J wild-type mice to induce cancer-induced bone pain (CIBP). Both acute and chronic dosing of PNA6 significantly reduced the spontaneous pain behaviors associated with CIBP. These data suggest that PNA6 is a viable lead candidate for treating chronic inflammatory and complex neuropathic pain.
• Thompson, A. L., Grenald, S. A., Ciccone, H. A., Mohty, D., Smith, A. F., Coleman, D. L., Bahramnejad, E., De Leon, E., Kasper-Conella, L., Uhrlab, J. L., Margolis, D. S., Salvemini, D., Largent-Milnes, T. M., & Vanderah, T. W. (2023). Morphine-induced osteolysis and hypersensitivity is mediated through toll-like receptor-4 in a murine model of metastatic breast cancer. Pain, 164(11), 2463-2476.
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  The propensity for breast cancer to metastasize to bone is coupled to the most common complaint among breast cancer patients: bone pain. Classically, this type of pain is treated using escalating doses of opioids, which lack long-term efficacy due to analgesic tolerance, opioid-induced hypersensitivity, and have recently been linked to enhanced bone loss. To date, the molecular mechanisms underlying these adverse effects have not been fully explored. Using an immunocompetent murine model of metastatic breast cancer, we demonstrated that sustained morphine infusion induced a significant increase in osteolysis and hypersensitivity within the ipsilateral femur through the activation of toll-like receptor-4 (TLR4). Pharmacological blockade with TAK242 (resatorvid) as well as the use of a TLR4 genetic knockout ameliorated the chronic morphine-induced osteolysis and hypersensitivity. Genetic MOR knockout did not mitigate chronic morphine hypersensitivity or bone loss. In vitro studies using RAW264.7 murine macrophages precursor cells demonstrated morphine-enhanced osteoclastogenesis that was inhibited by the TLR4 antagonist. Together, these data indicate that morphine induces osteolysis and hypersensitivity that are mediated, in part, through a TLR4 receptor mechanism.
• Wiese, B. M., Alvarez Reyes, A., Vanderah, T. W., & Largent-Milnes, T. M. (2023). The endocannabinoid system and breathing. Frontiers in neuroscience, 17, 1126004.
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  Recent changes in cannabis accessibility have provided adjunct therapies for patients across numerous disease states and highlights the urgency in understanding how cannabinoids and the endocannabinoid (EC) system interact with other physiological structures. The EC system plays a critical and modulatory role in respiratory homeostasis and pulmonary functionality. Respiratory control begins in the brainstem without peripheral input, and coordinates the preBötzinger complex, a component of the ventral respiratory group that interacts with the dorsal respiratory group to synchronize burstlet activity and drive inspiration. An additional rhythm generator: the retrotrapezoid nucleus/parafacial respiratory group drives active expiration during conditions of exercise or high CO. Combined with the feedback information from the periphery: through chemo- and baroreceptors including the carotid bodies, the cranial nerves, stretch of the diaphragm and intercostal muscles, lung tissue, and immune cells, and the cranial nerves, our respiratory system can fine tune motor outputs that ensure we have the oxygen necessary to survive and can expel the CO waste we produce, and every aspect of this process can be influenced by the EC system. The expansion in cannabis access and potential therapeutic benefits, it is essential that investigations continue to uncover the underpinnings and mechanistic workings of the EC system. It is imperative to understand the impact cannabis, and exogenous cannabinoids have on these physiological systems, and how some of these compounds can mitigate respiratory depression when combined with opioids or other medicinal therapies. This review highlights the respiratory system from the perspective of central versus peripheral respiratory functionality and how these behaviors can be influenced by the EC system. This review will summarize the literature available on organic and synthetic cannabinoids in breathing and how that has shaped our understanding of the role of the EC system in respiratory homeostasis. Finally, we look at some potential future therapeutic applications the EC system has to offer for the treatment of respiratory diseases and a possible role in expanding the safety profile of opioid therapies while preventing future opioid overdose fatalities that result from respiratory arrest or persistent apnea.
• Bartolo, N. D., Reid, S. E., Krishnan, H. S., Haseki, A., Renganathan, M., Largent-Milnes, T. M., Norwood, B. A., Loggia, M. L., & Hooker, J. M. (2022). Radiocaine: An Imaging Marker of Neuropathic Injury. ACS chemical neuroscience.
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  Voltage-gated sodium channels (Nas) play a crucial electrical signaling role in neurons. Na-isoforms present in peripheral sensory neurons and dorsal root ganglia of the spinal cord are critically involved in pain perception and transmission. While these isoforms, particularly Na1.7, are implicated in neuropathic pain disorders, changes in the functional state and expression levels of these channels have not been extensively studied in vivo. Radiocaine, a fluorine-18 radiotracer based on the local anesthetic lidocaine, a non-selective Na blocker, has previously been used for cardiac Na1.5 imaging using positron-emission tomography (PET). In the present study, we used Radiocaine to visualize changes in neuronal Na expression after neuropathic injury. In rats that underwent unilateral spinal nerve ligation, PET/MR imaging demonstrated significantly higher uptake of Radiocaine into the injured sciatic nerve, as compared to the uninjured sciatic nerve, for up to 32 days post-surgery. Radiocaine, due to its high translational potential, may serve as a novel diagnostic tool for neuropathic pain conditions using PET imaging.
• Blackwell, J. A., Silva, J. F., Louis, E. M., Savu, A., Largent-Milnes, T. M., Brooks, H. L., & Pires, P. W. (2022). Cerebral arteriolar and neurovascular dysfunction after chemically induced menopause in mice. American journal of physiology. Heart and circulatory physiology, 323(5), H845-H860.
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  Cognitive decline is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of dementia, possibly because of reduced functional dilation in parenchymal arterioles. These vessels are bottlenecks of the cerebral microcirculation, and dysfunction can limit functional hyperemia in the brain. Large-conductance Ca-activated K channels (BK) are the final effectors of several pathways responsible for functional hyperemia, and their expression is modulated by estrogen. However, it remains unknown whether BK function is altered in cerebral parenchymal arterioles after menopause. Using a chemically induced model of menopause, the 4-vinylcyclohexene diepoxide (VCD) model, which depletes follicles while maintaining intact ovaries, we hypothesized that menopause would be associated with reduced functional vasodilatory responses in cerebral parenchymal arterioles of wild-type mice via reduced BK function. Using pressure myography of isolated parenchymal arterioles, we observed that menopause (Meno) induced a significant increase in spontaneous myogenic tone. Endothelial function, assessed as nitric oxide production and dilation after cholinergic stimulation or endothelium-dependent hyperpolarization pathways, was unaffected by Meno. BK function was significantly impaired in Meno compared with control, without changes in voltage-gated K channel activity. Cerebral functional hyperemia, measured by laser-speckle contrast imaging during whisker stimulation, was significantly blunted in Meno mice, without detectable changes in basal perfusion. However, behavioral testing identified no change in cognition. These findings suggest that menopause induces cerebral microvascular and neurovascular deficits. Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BK channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.
• Bruhns, R. P., Sulaiman, M. I., Gaub, M., Bae, E. H., Davidson Knapp, R. B., Larson, A. R., Smith, A., Coleman, D. L., Staatz, W. D., Sandweiss, A. J., Joseph, B., Hay, M., Largent-Milnes, T. M., & Vanderah, T. W. (2022). Angiotensin-(1-7) improves cognitive function and reduces inflammation in mice following mild traumatic brain injury. Frontiers in behavioral neuroscience, 16, 903980.
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  Traumatic brain injury (TBI) is a leading cause of disability in the US. Angiotensin 1-7 (Ang-1-7), an endogenous peptide, acts at the G protein coupled MAS1 receptors (MASR) to inhibit inflammatory mediators and decrease reactive oxygen species within the CNS. Few studies have identified whether Ang-(1-7) decreases cognitive impairment following closed TBI. This study examined the therapeutic effect of Ang-(1-7) on secondary injury observed in a murine model of mild TBI (mTBI) in a closed skull, single injury model.
• Kwak, E. A., Pan, C. C., Ramonett, A., Kumar, S., Cruz-Flores, P., Ahmed, T., Ortiz, H. R., Lochhead, J. J., Ellis, N. A., Mouneimne, G., Georgieva, T. G., Lee, Y. S., Vanderah, T. W., Largent-Milnes, T., Mohler, P. J., Hund, T. J., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). β-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature communications, 13(1), 1326.
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  Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report β-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific β-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, β-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, β-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.
• Kwak, E. A., Pan, C. C., Ramonett, A., Kumar, S., Cruz-Flores, P., Ahmed, T., Ortiz, H. R., Lochhead, J. L., Ellis, N., Mouneimne, G., Lee, Y. S., Vanderah, T. W., Milnes, T. M., Mohler, P. J., Hund, T. J., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). BIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature Communications.
• Lee, N. Y., Mythreye, K., Langlais, P. R., Kashatus, D. F., Milnes, T. M., Vanderah, T. W., Lee, Y. S., Ortiz, H. R., Flores, P. C., Ahmed, T., Kwak, E. A., & Ramonett, A. (2022). Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport. Molecular biology of the cell, 33(1), ar4.
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  Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.
• Martin, L. F., Cheng, K., Washington, S. M., Denton, M., Goel, V., Khandekar, M., Largent-Milnes, T. M., Patwardhan, A., & Ibrahim, M. M. (2022). Green Light Exposure Elicits Anti-inflammation, Endogenous Opioid Release and Dampens Synaptic Potentiation to Relieve Post-surgical Pain. The journal of pain.
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  Light therapy improves multiple conditions such as seasonal affective disorders, circadian rhythm dysregulations, and neurodegenerative diseases. However, little is known about its potential benefits in pain management. While current pharmacologic methods are effective in many cases, the associated side effects can limit their use. Non-pharmacological methods would minimize drug dependence, facilitating a reduction of the opioid burden. Green light therapy has been shown to be effective in reducing chronic pain in humans and rodents. However, its underlying mechanisms remain incompletely defined. In this study, we demonstrate that green light exposure reduced postsurgical hypersensitivity in rats. Moreover, this therapy potentiated the antinociceptive effects of morphine and ibuprofen on mechanical allodynia in male rats. Importantly, in female rats, GLED potentiated the antinociceptive effects of morphine but did not affect that of ibuprofen. We showed that green light increases endogenous opioid levels while lessening synaptic plasticity and neuroinflammation. Importantly, this study reveals new insights into how light exposure can affect neuroinflammation and plasticity in both genders. Clinical translation of these results could provide patients with improved pain control and decrease opioid consumption. Given the noninvasive nature of green light, this innovative therapy would be readily implementable in hospitals. Perspective: This study provides a potential additional therapy to decrease postsurgical pain. Given the safety, availability, and the efficacy of green light therapy, there is a significant potential for advancing the green light therapy to clinical trials and eventual translation to clinical settings.
• Milnes, T. M., Vanderah, T. W., Streicher, J. M., Langlais, P. R., Cottier, K. E., Almuslim, M., Palomino, S. M., Vivek, A., & Wahl, J. R. (2022). Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome. Biology of sex differences.
• Palomino, S. M., Levine, A. A., Wahl, J., Liktor-Busa, E., Streicher, J. M., & Largent-Milnes, T. M. (2022). Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression. Pharmaceutics, 14(8).
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  Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood-brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood-brain barrier. In this study, the possible role of HSP90 in CSD-associated blood-brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood-endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.
• Riegel, A. C., Vanderah, T. W., Milnes, T. M., Langlais, P. R., Smith, A., Majuta, L., Franca-Solomon, G., Barber, K. R., & Vizcarra, V. S. (2022). Targeting 5-HT2A receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model.. Neuroscience Letters.
• Riegel, A. C., Vanderah, T. W., Milnes, T. M., Langlais, P. R., Smith, A., Majuta, L., Franca-Solomon, G., Barber, K. R., & Vizcarra, V. S. (2022). Targeting 5-HT2A receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model. . Neuroscience Letters.
• Vizcarra, V. S., Barber, K. R., Franca-Solomon, G., Majuta, L., Smith, A., Langlais, P. R., Largent-Milnes, T. M., Vanderah, T. W., & Riegel, A. C. (2022). Targeting 5-HT receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model. Neuroscience letters, 789, 136864.
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  Chronic pain remains a disabling disease with limited therapeutic options. Pyramidal neurons in the prefrontal cortex (PFC) express excitatory G-coupled 5-HT receptors (5-HTR) and their effector system, the inhibitory Kv7 ion channel. While recent publications show these cells innervate brainstem regions important for regulating pain, the cellular mechanisms underlying the transition to chronic pain are not well understood. The present study examined whether local blockade of 5-HTR or enhanced Kv7 ion channel activity in the PFC would attenuate mechanical allodynia associated with spared nerve injury (SNI) in rats. Following SNI, we show that inhibition of PFC 5-HTRs with M100907 or opening of PFC Kv7 channels with retigabine reduced mechanical allodynia. Parallel proteomic and RNAScope experiments evaluated 5-HTR/Kv7 channel protein and mRNA. Our results support the role of 5-HTRs and Kv7 channels in the PFC in the maintenance of chronic pain.
• Wahl, J. R., Vivek, A., Palomino, S. M., Almuslim, M., Cottier, K. E., Langlais, P. R., Streicher, J. M., Vanderah, T. W., Liktor-Busa, E., & Largent-Milnes, T. M. (2022). Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome. Pharmaceutics, 14(7).
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  Pathologies of the blood-brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood-brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic "checkpoint" of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation.
• Blawn, K. T., Kellohen, K. L., Galloway, E. A., Verkhovsky, V. G., Wahl, J., Vivek, A., Barker, N. K., Cottier, K. E., Vallecillo, T. G., Langlais, P. R., Liktor-Busa, E., Vanderah, T. W., & Milnes, T. M. (2021). Sex hormones regulate NHE1 functional expression and brain endothelial proteome to control paracellular integrity of the blood endothelial barrier. Biology of Sex Differences.
• Blawn, K. T., Kellohen, K. L., Galloway, E. A., Wahl, J., Vivek, A., Verkhovsky, V. G., Barker, N. K., Cottier, K. E., Vallecillo, T. G., Langlais, P. R., Liktor-Busa, E., Vanderah, T. W., & Largent-Milnes, T. M. (2021). Sex hormones regulate NHE1 functional expression and brain endothelial proteome to control paracellular integrity of the blood endothelial barrier. Brain research, 1763, 147448.
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  Sex hormones have been implicated in pH regulation of numerous physiological systems. One consistent factor of these studies is the sodium-hydrogen exchanger 1 (NHE1). NHE1 has been associated with pH homeostasis at epithelial barriers. Hormone fluctuations have been implicated in protection and risk for breaches in blood brain barrier (BBB)/blood endothelial barrier (BEB) integrity. Few studies, however, have investigated BBB/BEB integrity in neurological disorders in the context of sex-hormone regulation of pH homeostasis.
• Larsen, E. G., Cho, T. S., McBride, M. L., Feng, J., Manivannan, B., Madura, C., Klein, N. E., Wright, E. B., Wickstead, E. S., Garcia-Verdugo, H. D., Jarvis, C., Khanna, R., Hu, H., Largent-Milnes, T. M., & Bhattacharya, M. R. (2021). Transmembrane protein TMEM184B is necessary for interleukin-31-induced itch. Pain.
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  Nociceptive and pruriceptive neurons in the dorsal root ganglia (DRG) convey sensations of pain and itch to the spinal cord, respectively. One subtype of mature DRG neurons, comprising 6% to 8% of neurons in the ganglia, is responsible for sensing mediators of acute itch and atopic dermatitis, including the cytokine IL-31. How itch-sensitive (pruriceptive) neurons are specified is unclear. Here, we show that transmembrane protein 184B (TMEM184B), a protein with roles in axon degeneration and nerve terminal maintenance, is required for the expression of a large cohort of itch receptors, including those for interleukin 31 (IL-31), leukotriene C4, and histamine. Male and female mice lacking TMEM184B show reduced responses to IL-31 but maintain normal responses to pain and mechanical force, indicating a specific behavioral defect in IL-31-induced pruriception. Calcium imaging experiments indicate that a reduction in IL-31-induced calcium entry is a likely contributor to this phenotype. We identified an early failure of proper Wnt-dependent transcriptional signatures and signaling components in Tmem184b mutant mice that may explain the improper DRG neuronal subtype specification. Accordingly, lentiviral re-expression of TMEM184B in mutant embryonic neurons restores Wnt signatures. Together, these data demonstrate that TMEM184B promotes adult somatosensation through developmental Wnt signaling and promotion of proper pruriceptive gene expression. Our data illuminate a new key regulatory step in the processes controlling the establishment of diversity in the somatosensory system.
• Liktor-Busa, E., Keresztes, A., LaVigne, J., Streicher, J. M., & Largent-Milnes, T. M. (2021). Analgesic Potential of Terpenes Derived from. Pharmacological reviews, 73(4), 98-126.
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  Pain prevalence among adults in the United States has increased 25% over the past two decades, resulting in high health-care costs and impacts to patient quality of life. In the last 30 years, our understanding of pain circuits and (intra)cellular mechanisms has grown exponentially, but this understanding has not yet resulted in improved therapies. Options for pain management are limited. Many analgesics have poor efficacy and are accompanied by severe side effects such as addiction, resulting in a devastating opioid abuse and overdose epidemic. These problems have encouraged scientists to identify novel molecular targets and develop alternative pain therapeutics. Increasing preclinical and clinical evidence suggests that cannabis has several beneficial pharmacological activities, including pain relief. contains more than 500 chemical compounds, with two principle phytocannabinoids, Δ-tetrahydrocannabinol (Δ-THC) and cannabidiol (CBD). Beyond phytocannabinoids, more than 150 terpenes have been identified in different cannabis chemovars. Although the predominant cannabinoids, Δ-THC and CBD, are thought to be the primary medicinal compounds, terpenes including the monoterpenes -myrcene, -pinene, limonene, and linalool, as well as the sesquiterpenes -caryophyllene and -humulene may contribute to many pharmacological properties of cannabis, including anti-inflammatory and antinociceptive effects. The aim of this review is to summarize our current knowledge about terpene compounds in cannabis and to analyze the available scientific evidence for a role of cannabis-derived terpenes in modern pain management. SIGNIFICANCE STATEMENT: Decades of research have improved our knowledge of cannabis polypharmacy and contributing phytochemicals, including terpenes. Reform of the legal status for cannabis possession and increased availability (medicinal and recreational) have resulted in cannabis use to combat the increasing prevalence of pain and may help to address the opioid crisis. Better understanding of the pharmacological effects of cannabis and its active components, including terpenes, may assist in identifying new therapeutic approaches and optimizing the use of cannabis and/or terpenes as analgesic agents.
• Martin, L. F., Moutal, A., Cheng, K., Washington, S. M., Calligaro, H., Goel, V., Kranz, T., Largent-Milnes, T. M., Khanna, R., Patwardhan, A., & Ibrahim, M. M. (2021). Green Light Antinociceptive and Reversal of Thermal and Mechanical Hypersensitivity Effects Rely on Endogenous Opioid System Stimulation. The journal of pain, 22(12), 1646-1656.
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  Benefits of phototherapy were characterized in multiple diseases including depression, circadian rhythm disruptions, and neurodegeneration. Studies on migraine and fibromyalgia patients revealed that green light-emitting diodes (GLED) exposure provides a pragmatic and safe therapy to manage chronic pain. In rodents, GLED reversed hypersensitivity related to neuropathic pain. However, little is known about the underlying mechanisms of GLED efficacy. Here, we sought to understand how green light modulates the endogenous opioid system. We first characterized how exposure to GLED stimulates release of β-endorphin and proenkephalin in the central nervous system of male rats. Moreover, by individually editing each of the receptors, we found that µ- and δ-opioid receptors are required for green light's antinociceptive effect in naïve rats and a model of HIV-induced peripheral neuropathy. We investigated how GLED could increase pain thresholds, and explored its potential in reversing hypersensitivity in a model of HIV-related neuropathy. Through behavioral and gene editing approaches, we identified that green light provides antinociception via modulation of the endogenous opioid system in the spinal cord. This work identifies a previously unknown mechanism by which GLED can improve pain management. Clinical translation of these results will advance the development of an innovative therapy devoid of adverse effects. PERSPECTIVE: Development of new pain management therapies, especially for HIV patients, is crucial as long-term opioid prescription is not recommended due to adverse side effects. Green light addresses this necessity. Characterizing the underlying mechanisms of this potentially groundbreaking and safe antinociceptive therapy will advance its clinical translation.
• Martin, L., Ibrahim, M., Gomez, K., Yu, J., Cai, S., Chew, L. A., Bellampalli, S. S., Moutal, A., Largent-Milnes, T., Porreca, F., Khanna, R., Olivera, B. M., & Patwardhan, A. (2021). Conotoxin contulakin-G engages a neurotensin receptor 2 /R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception. Pain.
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  Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltage-gated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.
• Milnes, T. M., Vanderah, T. W., Langlais, P. R., Aicher, S. A., Balasubramanian, S., Couture, S., Lipinski, A. A., Liktor-Busa, E., & Levine, A. (2021). Sex differences in the expression of the endocannabinoid system within V1M cortex and PAG of Sprague Dawley rats. Biology of sex differences, 12(1), 60.
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  Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented, but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system comprises the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands.
• Milnes, T. M., Vanderah, T. W., Langlais, P. R., Aicher, S., Balasubramanian, S., Couture, S., Lipinski, A., Liktor-Busa, E., & Levine, A. (2021). Inherent Sex Differences in Expression of the Endocannabinoid Systems within Cortex and PAG. Biol Sex Differ..
• Wiese, B. M., Liktor-Busa, E., Couture, S. A., Nikas, S. P., Ji, L., Liu, Y., Makriyannis, A., Spigelman, I., Vanderah, T. W., & Largent-Milnes, T. M. (2021). Brain Penetrant, but not Peripherally Restricted, Synthetic Cannabinoid 1 Receptor Agonists Promote Morphine-Mediated Respiratory Depression. Cannabis and cannabinoid research.
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  Cannabis acceptance and use continues to rise despite the gaps in knowledge regarding the mechanisms of cannabinoids and the endocannabinoid system in many physiological functions, including respiratory influence. With recent evidence of cannabinoid receptor 1 (CB1R) presence in the collection of respiratory neurons in the brainstem, as well as in the peripheral lung tissue, it is vital that the mechanisms involved in central and peripheral CB1R modulation of respiratory function be delineated. In this study we sought to define the roles of central versus peripheral CB1R activation on respiratory depression alone and in combination with morphine using whole body plethysmography. We show that the peripherally restricted CB1 agonist (4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine [PrNMI] 0.3, 0.6, and 1 mg/kg) does not induce respiratory depression, while our previous studies showed that a central penetrating synthetic cannabinoid does induce respiratory depression. Significantly, the combination of morphine with the peripheral CB1 agonist, PrNMI, attenuated morphine-induced respiratory depression. These studies support that a peripherally restricted CB1R agonist may be a unique strategy to attenuate the respiratory depression associated with opioid therapy.
• Wiese, B. M., Liktor-Busa, E., Levine, A., Couture, S. A., Nikas, S. P., Ji, L., Liu, Y., Mackie, K., Makriyannis, A., Largent-Milnes, T. M., & Vanderah, T. W. (2021). Cannabinoid-2 Agonism with AM2301 Mitigates Morphine-Induced Respiratory Depression. Cannabis and cannabinoid research, 6(5), 401-412.
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  An escalating number of fatalities resulting from accidental opioid overdoses typically attributed to respiratory depression continue to define the opioid epidemic. Opioid respiratory depression results from a decrease in reflexive inspiration within the preBötzinger complex in the brainstem. Cannabinoid receptor agonism is reported to enhance opioid analgesia, yet whether cannabinoids enhance or inhibit opioid-induced respiratory depression is unknown. Studies herein sought to define the roles of cannabinoid-1 receptor (CB1R) and cannabinoid-2 receptor (CB2R) on respiratory depression using selective agonists alone and in combination with morphine in male mice. Using whole body plethysmography, the nonselective CB1R and CB2R agonist (Δ-tetrahydrocannabinol) and the CB1R synthetic cannabinoid, AM356, induced respiratory depression, whereas the well-published selective CB2 agonist, JWH 133, and the novel CB2 agonist (AM2301) did not. Moreover, a selective CB2R agonist (AM2301) significantly attenuated morphine sulfate-induced respiratory depression. Notably, findings suggest that attenuation of opioid-induced respiratory depression relies on CB2R activation, supporting selective CB2R agonism as an opioid adjunct therapy.
• Zhang, H., Lipinski, A. A., Liktor-Busa, E., Smith, A. F., Moutal, A., Khanna, R., Langlais, P. R., Largent-Milnes, T. M., & Vanderah, T. W. (2021). The Effects of Repeated Morphine Treatment on the Endogenous Cannabinoid System in the Ventral Tegmental Area. Frontiers in pharmacology, 12, 632757.
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  The therapeutic utility of opioids is diminished by their ability to induce rewarding behaviors that may lead to opioid use disorder. Recently, the endogenous cannabinoid system has emerged as a hot topic in the study of opioid reward but relatively little is known about how repeated opioid exposure may affect the endogenous cannabinoid system in the mesolimbic reward circuitry. In the present study, we investigated how sustained morphine may modulate the endogenous cannabinoid system in the ventral tegmental area (VTA) of Sprague Dawley rats, a critical region in the mesolimbic reward circuitry. Studies here using proteomic analysis and quantitative real-time PCR (qRT-PCR) found that the VTA expresses 32 different proteins or genes related to the endogenous cannabinoid system; three of these proteins or genes (PLCγ2, ABHD6, and CB2R) were significantly affected after repeated morphine exposure (CB2R was only detected by qRT-PCR but not proteomics). We also identified that repeated morphine treatment does not alter either anandamide (AEA) or 2-arachidonoylglycerol (2-AG) levels in the VTA compared to saline treatment; however, there may be diminished levels of anandamide (AEA) production in the VTA 4 h after a single morphine injection in both chronic saline and morphine pretreated cohorts. Treating the animals with an inhibitor of 2-AG degradation significantly decreased repeated opioid rewarding behavior. Taken together, our studies reveal a potential influence of sustained opioids on the endocannabinoid system in the VTA, suggesting that the endogenous cannabinoid system may participate in the opioid-induced reward.
• Doyle, T. M., Janes, K., Chen, Z., Grace, P. M., Esposito, E., Cuzzocrea, S., Largent-Milnes, T. M., Neumann, W. L., Watkins, L. R., Spiegel, S., Vanderah, T. W., & Salvemini, D. (2020). Activation of sphingosine-1-phosphate receptor subtype 1 in the central nervous system contributes to morphine-induced hyperalgesia and antinociceptive tolerance in rodents. Pain.
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  Morphine-induced alterations in sphingolipid metabolism in the spinal cord and increased formation of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) have been implicated in the development of morphine-induced hyperalgesia (OIH; increased pain sensitivity) and antinociceptive tolerance. These adverse effects hamper opioid use for treating chronic pain and contribute to dependence and abuse. S1P produces distinct effects through five G protein-coupled receptors (S1PR1-5) and several intracellular targets. How S1P exerts its effects in response to morphine remains unknown. Here, we report that S1P contributes to the development of OIH and tolerance through S1P1 receptor subtype 1 (S1PR1) signaling in uninjured male and female rodents that can be blocked by targeting S1PR1 with S1PR1 antagonists or RNA silencing. In mouse neuropathic pain models, S1PR1 antagonists blocked the development of tolerance to the anti-allodynic effects of morphine without altering morphine pharmacokinetics and prevented prolonged morphine-induced neuropathic pain. Targeting S1PR1 reduced morphine-induced neuroinflammatory events in the dorsal horn of the spinal cord: increased glial marker expression, mitogen-activated protein kinase p38 and nuclear factor κB activation and increased inflammatory cytokine expression, such as interleukin-1β, a cytokine central in the modulation of opioid-induced neural plasticity. Our results identify S1PR1 as a critical path for S1P signaling in response to sustained morphine and reveal downstream neuroinflammatory pathways impacted by S1PR1 activation. Our data support investigating S1PR1 antagonists as a clinical approach to mitigate opioid-induced adverse effects and repurposing the functional S1PR1 antagonist FTY720, which is FDA-approved for multiple sclerosis, as an opioid adjunct.
• Doyle, T. M., Largent-Milnes, T. M., Chen, Z., Staikopoulos, V., Esposito, E., Dalgarno, R., Fan, C., Tosh, D. K., Cuzzocrea, S., Jacobson, K. A., Trang, T., Hutchinson, M. R., Bennett, G. J., Vanderah, T. W., & Salvemini, D. (2020). Chronic Morphine-Induced Changes in Signaling at the A Adenosine Receptor Contribute to Morphine-Induced Hyperalgesia, Tolerance, and Withdrawal. The Journal of pharmacology and experimental therapeutics, 374(2), 331-341.
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  Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A adenosine receptor (AAR) signaling. Supplementing adenosine signaling with selective AAR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of AAR in the spinal cord with an ADK inhibitor or AAR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%-75%), cleaved caspase 1 (40%-60%), interleukin (IL)-1 (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, AAR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest AAR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that AAR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the AAR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by AAR agonists, suggesting that AAR may be a target for therapeutic intervention with selective AAR agonist as opioid adjuncts.
• Largent-Milnes, T. M., Canals, M., & Streicher, J. M. (2020). Editorial: Novel Molecular Targets for the Treatment of Pain. Frontiers in molecular neuroscience, 13, 625714.
• Levine, A., Liktor-Busa, E., Karlage, K. L., Giancotti, L., Salvemini, D., Vanderah, T. W., & Largent-Milnes, T. M. (2020). DAGLα Inhibition as a Non-invasive and Translational Model of Episodic Headache. Frontiers in pharmacology, 11, 615028.
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  Recent findings suggested that Clinical Endocannabinoid Deficiency underlies the pathophysiology of pain disorders, including migraine and headache. In models of medication overuse headache induced by sustained administration of sumatriptan or morphine, 2-AG levels were selectively depleted in the periaqueductal gray (PAG) and anandamide (AEA) increased in the cortex suggesting distinct regulation of the endocannabinoid system during headache pain. These results led to the hypothesis that blockade of DAGL, to reduce 2-AG levels would induce headache-like behaviors as a new, translationally relevant model of episodic headache. Our study investigated whether non-selective and selective blockade of DAGL, the main biosynthetic enzyme for 2-AG, induced periorbital and hind-paw allodynia, photophobia, anxiety-like behaviors, responsivity to abortive anti-migraine agents, and 2-AG/AEA levels. Injection of non-selective DAGL (DH376, 10 mg/kg, IP) and selective DAGLα (LEI106, 20 mg/kg, IP) inhibitors, but not DAGLβ agents, induced facial sensitivity in 100% and ∼60% of female and male rats, respectively, without induction of peripheral sensitivity. Notably, male rats showed significantly less sensitivity than female rats after DAGLα inhibition, suggesting sexual dimorphism in this mechanism. Importantly, LEI106 induced periorbital allodynia was attenuated by administration of the clinically available abortive antimigraine agents, sumatriptan and olcegepant. Selective DAGLα inhibition induced significant photophobia as measured by the light-dark box, without anxiety like behaviors or changes in voluntary movement. Analysis of AEA and 2-AG levels at the time of peak pain sensitivity revealed reductions in 2-AG in the visual cortex and periaqueductal gray (PAG), without altering anandamide or significantly increasing diacylglycerol levels. These results provide foundational evidence for DAGL-2AG in the induction of headache-like pain and photophobia without extracephalic allodynia, thus modeling the clinical episodic migraine. Mechanistically, behavioral measures of headache sensitivity after DAGL inhibition suggests that reduced 2-AG signaling in the cortex and PAG, but not the trigeminal nucleus caudalis or trigeminal ganglia, drives headache initiation. Therefore, episodic DAGL inhibition, which reduces the time, cost, and invasiveness of currently accepted models of headache, may fill the need for episodic migraine/headache models mirroring clinical presentation. Moreover, use of this approach may provide an avenue to study the transition from episodic to chronic headache.
• Levine, A., Vanderah, T. W., & Largent-Milnes, T. M. (2020). An underrepresented majority: A systematic review utilizing allodynic criteria to examine the present scarcity of discrete animal models for episodic migraine. Cephalalgia : an international journal of headache, 333102420966984.
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  Despite increasing evidence differentiating episodic and chronic migraine, little work has determined how currently utilized animal models of migraine best represent each distinct disease state.
• Liktor-Busa, E., Blawn, K. T., Kellohen, K. L., Wiese, B. M., Verkhovsky, V., Wahl, J., Vivek, A., Palomino, S. M., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2020). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake. PloS one, 15(5), e0227463.
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  Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.
• Lucarini, E., Coppi, E., Micheli, L., Parisio, C., Vona, A., Cherchi, F., Pugliese, A. M., Pedata, F., Failli, P., Palomino, S., Wahl, J., Largent-Milnes, T. M., Vanderah, T. W., Tosh, D. K., Jacobson, K. A., Salvemini, D., Ghelardini, C., & Di Cesare Mannelli, L. (2020). Acute visceral pain relief mediated by A3AR agonists in rats: involvement of N-type voltage-gated calcium channels. Pain.
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  Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently, their activity has been related to the block of N-type voltage-gated Ca channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing postinflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intracolonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 mL 50% EtOH). Visceral hypersensitivity was assessed by measuring the visceromotor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the postinflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in dorsal root ganglia neurons isolated from either control or DNBS-treated rats. The effect on Ca current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.
• Stine, C., Coleman, D. L., Flohrschutz, A. T., Thompson, A. L., Mishra, S., Blagg, B. S., Largent-Milnes, T. M., Lei, W., & Streicher, J. M. (2020). Heat shock protein 90 inhibitors block the antinociceptive effects of opioids in mouse chemotherapy-induced neuropathy and cancer bone pain models. Pain, 161(8), 1798-1807.
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  Heat shock protein 90 (Hsp90) is a ubiquitous signal transduction regulator, and Hsp90 inhibitors are in clinical development as cancer therapeutics. However, there have been very few studies on the impact of Hsp90 inhibitors on pain or analgesia, a serious concern for cancer patients. We previously found that Hsp90 inhibitors injected into the brain block opioid-induced antinociception in tail flick, paw incision, and HIV neuropathy pain. This study extended from that initial work to test the cancer-related clinical impact of Hsp90 inhibitors on opioid antinociception in cancer-induced bone pain in female BALB/c mice and chemotherapy-induced peripheral neuropathy in male and female CD-1 mice. Mice were treated with Hsp90 inhibitors (17-AAG, KU-32) by the intracerebroventricular, intrathecal, or intraperitoneal routes, and after 24 hours, pain behaviors were evaluated after analgesic drug treatment. Heat shock protein 90 inhibition in the brain or systemically completely blocked morphine and oxymorphone antinociception in chemotherapy-induced peripheral neuropathy; this effect was partly mediated by decreased ERK and JNK MAPK activation and by increased protein translation, was not altered by chronic treatment, and Hsp90 inhibition had no effect on gabapentin antinociception. We also found that the Hsp90 isoform Hsp90α and the cochaperone Cdc37 were responsible for the observed changes in opioid antinociception. By contrast, Hsp90 inhibition in the spinal cord or systemically partially reduced opioid antinociception in cancer-induced bone pain. These results demonstrate that Hsp90 inhibitors block opioid antinociception in cancer-related pain, suggesting that Hsp90 inhibitors for cancer therapy could decrease opioid treatment efficacy.
• Thompson, A. L., Grenald, S. A., Ciccone, H. A., BassiriRad, N., Niphakis, M. J., Cravatt, B. F., Largent-Milnes, T. M., & Vanderah, T. W. (2020). The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain. The Journal of pharmacology and experimental therapeutics, 373(2), 230-238.
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  Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid-based therapies is essential, and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.
• Vekariya, R. H., Lei, W., Ray, A., Saini, S. K., Zhang, S., Molnar, G., Barlow, D., Karlage, K. L., Bilsky, E. J., Houseknecht, K. L., Largent-Milnes, T. M., Streicher, J. M., & Ananthan, S. (2020). Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a μ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects. Journal of medicinal chemistry, 63(14), 7663-7694.
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  We previously identified a pyridomorphinan (, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed μ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.
• Chen, Z., Doyle, T. M., Luongo, L., Largent-Milnes, T. M., Giancotti, L. A., Kolar, G., Squillace, S., Boccella, S., Walker, J. K., Pendleton, A., Spiegel, S., Neumann, W. L., Vanderah, T. W., & Salvemini, D. (2019). Sphingosine-1-phosphate receptor 1 activation in astrocytes contributes to neuropathic pain. Proceedings of the National Academy of Sciences of the United States of America, 116(21), 10557-10562.
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  Neuropathic pain afflicts millions of individuals and represents a major health problem for which there is limited effective and safe therapy. Emerging literature links altered sphingolipid metabolism to nociceptive processing. However, the neuropharmacology of sphingolipid signaling in the central nervous system in the context of chronic pain remains largely unexplored and controversial. We now provide evidence that sphingosine-1-phosphate (S1P) generated in the dorsal horn of the spinal cord in response to nerve injury drives neuropathic pain by selectively activating the S1P receptor subtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharmacological inhibition of S1PR1 with multiple antagonists in distinct chemical classes, but not agonists, attenuated and even reversed neuropathic pain in rodents of both sexes and in two models of traumatic nerve injury. These S1PR1 antagonists retained their ability to inhibit neuropathic pain during sustained drug administration, and their effects were independent of endogenous opioid circuits. Moreover, mice with astrocyte-specific knockout of did not develop neuropathic pain following nerve injury, thereby identifying astrocytes as the primary cellular substrate of S1PR1 activity. On a molecular level, the beneficial reductions in neuropathic pain resulting from S1PR1 inhibition were driven by interleukin 10 (IL-10), a potent neuroprotective and anti-inflammatory cytokine. Collectively, our results provide fundamental neurobiological insights that identify the cellular and molecular mechanisms engaged by the S1PR1 axis in neuropathic pain and establish S1PR1 as a target for therapeutic intervention with S1PR1 antagonists as a class of nonnarcotic analgesics.
• Cottier, K. E., Galloway, E. A., Calabrese, E. C., Tome, M. E., Liktor-Busa, E., Kim, J., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2019). Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery. eNeuro, 5(4).
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  Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.
• Hay, M., Polt, R., Heien, M. L., Vanderah, T. W., Largent-Milnes, T. M., Rodgers, K., Falk, T., Bartlett, M. J., Doyle, K. P., & Konhilas, J. P. (2019). A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction. The Journal of pharmacology and experimental therapeutics, 369(1), 9-25.
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  Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor , interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.
• Moutal, A., Luo, S., Largent-Milnes, T. M., Vanderah, T. W., & Khanna, R. (2019). Cdk5-mediated CRMP2 phosphorylation is necessary and sufficient for peripheral neuropathic pain. Neurobiology of pain (Cambridge, Mass.), 5.
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  Neuropathic pain results from nerve injuries that cause ectopic firing and increased nociceptive signal transmission due to activation of key membrane receptors and channels. The dysregulation of trafficking of voltage-gated ion channels is an emerging mechanism in the etiology of neuropathic pain. We identify increased phosphorylation of collapsin response mediator protein 2 (CRMP2), a protein reported to regulate presynaptic voltage-gated calcium and sodium channels. A spared nerve injury (SNI) increased expression of a cyclin dependent kinase 5 (Cdk5)-phosphorylated form of CRMP2 in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) in the ipsilateral (injured) versus the contralateral (non-injured) sites. Biochemical fractionation of spinal cord from SNI rats revealed the increase in Cdk5-mediated CRMP2 phosphorylation to be enriched to pre-synaptic sites. CRMP2 has emerged as a central node in assembling nociceptive signaling complexes. Knockdown of CRMP2 using a small interfering RNA (siRNA) reversed SNI-induced mechanical allodynia implicating CRMP2 expression as necessary for neuropathic pain. Intrathecal expression of a CRMP2 resistant to phosphorylation by Cdk5 normalized SNI-induced mechanical allodynia, whereas mimicking constitutive phosphorylation of CRMP2 resulted in induction of mechanical allodynia in naïve rats. Collectively, these results demonstrate that Cdk5-mediated CRMP2 phosphorylation is both necessary and sufficient for peripheral neuropathic pain.
• Thompson, A. L., Largent-Milnes, T. M., & Vanderah, T. W. (2019). Animal Models for the Study of Bone-Derived Pain. Methods in molecular biology (Clifton, N.J.), 1914, 391-407.
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  Bone pain is a prevalent issue in society today and also is one of the hardest types of pain to control. Pain originating in the bone can be caused by many different entities including metastatic and primary neoplasm, fracture, osteoarthritis as well as numerous other metabolic disorders. In this chapter we describe the methods and protocols that currently are accepted and validated for the study of bone pain in models of metastatic cancer, bicortical fracture and osteoarthritis. These animal models provide invaluable information as to the nature of bone pain and give rise to potential new targets for its treatment and management.
• Wahl, J., Vanderah, T. W., & Largent-Milnes, T. M. (2019). Migraine, a Review of Basic, Clinical, and Translational Approaches to New Treatment. ARCH Women Health Care, 2(6), 1-5.
• Edwards, K. A., Havelin, J. J., Mcintosh, M. I., Ciccone, H. A., Pangilinan, K., Imbert, I., Largent-Milnes, T. M., King, T., Vanderah, T. W., & Streicher, J. M. (2018). A Kappa Opioid Receptor Agonist Blocks Bone Cancer Pain Without Altering Bone Loss, Tumor Size, or Cancer Cell Proliferation in a Mouse Model of Cancer-Induced Bone Pain. The journal of pain : official journal of the American Pain Society, 19(6), 612-625.
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  Breast cancer metastasizes to bone, diminishing quality of life of patients because of pain, fracture, and limited mobility. Cancer-induced bone pain (CIBP) is characterized as moderate to severe ongoing pain, primarily managed by mu opioid agonists such as fentanyl. However, opioids are limited by escalating doses and serious side effects. One alternative may be kappa opioid receptor (KOR) agonists. There are few studies examining KOR efficacy on CIBP, whereas KOR agonists are efficacious in peripheral and inflammatory pain. We thus examined the effects of the KOR agonist U50,488 given twice daily across 7 days to block CIBP, tumor-induced bone loss, and tumor burden. U50,488 dose-dependently blocked tumor-induced spontaneous flinching and impaired limb use, without changing tactile hypersensitivity, and was fully reversed by the KOR antagonist nor-binaltorphimine. U50,488 treatment was higher in efficacy and duration of action at later time points. U50,488 blocked this pain without altering tumor-induced bone loss or tumor growth. Follow-up studies in human cancer cell lines confirmed that KOR agonists do not affect cancer cell proliferation. These studies suggest that KOR agonists could be a new target for cancer pain management that does not induce cancer cell proliferation or alter bone loss.
• Pandit, V., Khan, M., Zakaria, E. R., Largent-Milnes, T. M., Hamidi, M., O'Keeffe, T., Vanderah, T. W., & Joseph, B. (2018). Continuous remote ischemic conditioning attenuates cognitive and motor deficits from moderate traumatic brain injury. The journal of trauma and acute care surgery, 85(1), 48-53.
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  While studies show that single-dose remote ischemic conditioning (RIC) improves outcomes, the effect of continuous (daily) RIC is unknown. Thus, we aimed to investigate the role of continuous RIC on cognitive and motor function following traumatic brain injury (TBI).
• Schaefer, C. P., Arkwright, N. B., Jacobs, L. M., Jarvis, C. K., Hunn, K. C., Largent-Milnes, T. M., Tome, M. E., & Davis, T. P. (2018). Chronic morphine exposure potentiates p-glycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels. PloS one, 13(2), e0192340.
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  The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.
• Zhang, H., Lund, D. M., Ciccone, H. A., Staatz, W. D., Ibrahim, M. M., Largent-Milnes, T. M., Seltzman, H. H., Spigelman, I., & Vanderah, T. W. (2018). Peripherally restricted cannabinoid 1 receptor agonist as a novel analgesic in cancer-induced bone pain. Pain, 159(9), 1814-1823.
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  Many malignant cancers, including breast cancer, have a propensity to invade bones, leading to excruciating bone pain. Opioids are the primary analgesics used to alleviate this cancer-induced bone pain (CIBP) but are associated with numerous severe side effects, including enhanced bone degradation, which significantly impairs patients' quality of life. By contrast, agonists activating only peripheral CB1 receptors (CB1Rs) have been shown to effectively alleviate multiple chronic pain conditions with limited side effects, yet no studies have evaluated their role(s) in CIBP. Here, we demonstrate for the first time that a peripherally selective CB1R agonist can effectively suppress CIBP. Our studies using a syngeneic murine model of CIBP show that both acute and sustained administration of a peripherally restricted CB1R agonist, 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI), significantly alleviated spontaneous pain behaviors in the animals. This analgesic effect by PrNMI can be reversed by a systemic administration but not spinal injection of SR141716, a selective CB1R antagonist. In addition, the cancer-induced bone loss in the animals was not exacerbated by a repeated administration of PrNMI. Furthermore, catalepsy and hypothermia, the common side effects induced by cannabinoids, were measured at the supratherapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety or a decrease in limb movements was detected. Overall, our studies demonstrate that CIBP can be effectively managed by using a peripherally restricted CB1R agonist, PrNMI, without inducing dose-limiting central side effects. Thus, targeting peripheral CB1Rs could be an alternative therapeutic strategy for the treatment of CIBP.
• Grenald, S. A., Doyle, T. M., Zhang, H., Slosky, L. M., Chen, Z., Largent-Milnes, T. M., Spiegel, S., Vanderah, T. W., & Salvemini, D. (2017). Targeting the S1P/S1PR1 axis mitigates cancer-induced bone pain and neuroinflammation. Pain, 158(9), 1733-1742.
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  Metastatic bone pain is the single most common form of cancer pain and persists as a result of peripheral and central inflammatory, as well as neuropathic mechanisms. Here, we provide the first characterization of sphingolipid metabolism alterations in the spinal cord occurring during cancer-induced bone pain (CIBP). Following femoral arthrotomy and syngenic tumor implantation in mice, ceramides decreased with corresponding increases in sphingosine and the bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P). Intriguingly, de novo sphingolipid biosynthesis was increased as shown by the elevations of dihydro-ceramides and dihydro-S1P. We next identified the S1P receptor subtype 1 (S1PR1) as a novel target for therapeutic intervention. Intrathecal or systemic administration of the competitive and functional S1PR1 antagonists, TASP0277308 and FTY720/Fingolimod, respectively, attenuated cancer-induced spontaneous flinching and guarding. Inhibiting CIBP by systemic delivery of FTY720 did not result in antinociceptive tolerance over 7 days. FTY720 administration enhanced IL-10 in the lumbar ipsilateral spinal cord of CIBP animals and intrathecal injection of an IL-10 neutralizing antibody mitigated the ability of systemic FTY720 to reverse CIBP. FTY720 treatment was not associated with alterations in bone metabolism in vivo. Studies here identify a novel mechanism to inhibit bone cancer pain by blocking the actions of the bioactive metabolites S1P and dihydro-S1P in lumbar spinal cord induced by bone cancer and support potential fast-track clinical application of the FDA-approved drug, FTY720, as a therapeutic avenue for CIBP.
• Ibrahim, M. M., Patwardhan, A., Gilbraith, K. B., Moutal, A., Yang, X., Chew, L. A., Largent-Milnes, T., Malan, T. P., Vanderah, T. W., Porreca, F., & Khanna, R. (2017). Long-lasting antinociceptive effects of green light in acute and chronic pain in rats. Pain, 158(2), 347-360.
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  Treatments for chronic pain are inadequate, and new options are needed. Nonpharmaceutical approaches are especially attractive with many potential advantages including safety. Light therapy has been suggested to be beneficial in certain medical conditions such as depression, but this approach remains to be explored for modulation of pain. We investigated the effects of light-emitting diodes (LEDs), in the visible spectrum, on acute sensory thresholds in naive rats as well as in experimental neuropathic pain. Rats receiving green LED light (wavelength 525 nm, 8 h/d) showed significantly increased paw withdrawal latency to a noxious thermal stimulus; this antinociceptive effect persisted for 4 days after termination of last exposure without development of tolerance. No apparent side effects were noted and motor performance was not impaired. Despite LED exposure, opaque contact lenses prevented antinociception. Rats fitted with green contact lenses exposed to room light exhibited antinociception arguing for a role of the visual system. Antinociception was not due to stress/anxiety but likely due to increased enkephalins expression in the spinal cord. Naloxone reversed the antinociception, suggesting involvement of central opioid circuits. Rostral ventromedial medulla inactivation prevented expression of light-induced antinociception suggesting engagement of descending inhibition. Green LED exposure also reversed thermal and mechanical hyperalgesia in rats with spinal nerve ligation. Pharmacological and proteomic profiling of dorsal root ganglion neurons from green LED-exposed rats identified changes in calcium channel activity, including a decrease in the N-type (CaV2.2) channel, a primary analgesic target. Thus, green LED therapy may represent a novel, nonpharmacological approach for managing pain.
• Malekzadeh, P., Hu, J., Sandweiss, A. J., Ameli, N., Bierny, P., Largent-Milnes, T. M., Vanderah, T. W., & Shirazi, F. (2017). Effect of Centruroides antivenom on reversal of methamphetamine-induced hyperkinesis and hyperthermia in rats. Journal of pharmaceutics & pharmacology, 5(1).
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  Methamphetamine (MA) toxicity is a major health concern causing agitation, hyperkinesia, hyperthermia, and even death, affecting 24.7 million people worldwide. It has been observed that MA generates movement disorders in children similar to that of scorpion envenomation. Four cases have been reported where MA intoxication in children were both subjectively and objectively improved as indicated by the reversal of nystagmus and movement disorders following administration of Centruroides antivenom (AV) therapy.
• Moutal, A., Dustrude, E. T., Largent-Milnes, T. M., Vanderah, T. W., Khanna, M., & Khanna, R. (2017). Blocking CRMP2 SUMOylation reverses neuropathic pain. Molecular psychiatry.
• Sandweiss, A. J., Azim, A., Ibraheem, K., Largent-Milnes, T. M., Rhee, P., Vanderah, T. W., & Joseph, B. (2017). Remote ischemic conditioning preserves cognition and motor coordination in a mouse model of traumatic brain injury. The journal of trauma and acute care surgery, 83(6), 1074-1081.
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  Management of traumatic brain injury (TBI) is focused on minimizing or preventing secondary brain injury. Remote ischemic conditioning (RIC) is an established treatment modality that has been shown to improve patient outcomes in different clinical settings by influencing inflammatory insults. In a clinical trial, RIC showed amelioration of SB100 and neuron-specific enolase. The aim of our study was to further elucidate the mechanisms and outcome when applying RIC in a mouse model of traumatic brain injury.
• Sandweiss, A. J., Cottier, K. E., McIntosh, M. I., Dussor, G., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2017). 17-β-Estradiol induces spreading depression and pain behavior in alert female rats. Oncotarget, 8(69), 114109-114122.
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  Test the putative contribution of 17-β-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats.
• Sandweiss, A. J., McIntosh, M. I., Moutal, A., Davidson-Knapp, R., Hu, J., Giri, A. K., Yamamoto, T., Hruby, V. J., Khanna, R., Largent-Milnes, T. M., & Vanderah, T. W. (2017). Genetic and pharmacological antagonism of NK1 receptor prevents opiate abuse potential. Molecular psychiatry.
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  Development of an efficacious, non-addicting analgesic has been challenging. Discovery of novel mechanisms underlying addiction may present a solution. Here we target the neurokinin system, which is involved in both pain and addiction. Morphine exerts its rewarding actions, at least in part, by inhibiting GABAergic input onto substance P (SP) neurons in the ventral tegmental area (VTA), subsequently increasing SP release onto dopaminergic neurons. Genome editing of the neurokinin 1 receptor (NK1R) in the VTA renders morphine non-rewarding. Complementing our genetic approach, we demonstrate utility of a bivalent pharmacophore with dual activity as a μ/δ opioid agonist and NK1R antagonist in inhibiting nociception in an animal model of acute pain while lacking any positive reinforcement. These data indicate that dual targeting of the dopaminergic reward circuitry and pain pathways with a multifunctional opioid agonist-NK1R antagonist may be an efficacious strategy in developing future analgesics that lack abuse potential.Molecular Psychiatry advance online publication, 9 May 2017; doi:10.1038/mp.2017.102.
• Deekonda, S., Cole, J., Sunna, S., Rankin, D., Largent-Milnes, T. M., Davis, P., BassiriRad, N. M., Lai, J., Vanderah, T. W., Porecca, F., & Hruby, V. J. (2016). Enkephalin analogues with N-phenyl-N-(piperidin-2-ylmethyl)propionamide derivatives: Synthesis and biological evaluations. Bioorganic & medicinal chemistry letters, 26(1), 222-7.
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  N-Phenyl-N-(piperidin-2-ylmethyl)propionamide based bivalent ligands are unexplored for the design of opioid based ligands. Two series of hybrid molecules bearing N-phenyl-N-(piperidin-2-ylmethyl)propionamide derived small molecules conjugated with an enkephalin analogues with and without a linker (β-alanine) were designed and synthesized. Both bivalent ligand series exhibited remarkable binding affinities from nanomolar to subnanomolar range at both μ and δ opioid receptors and displayed potent agonist activities as well. The replacement of Tyr with Dmt and introduction of a linker between the small molecule and enkephalin analogue resulted in highly potent ligands. Both series of ligands showed excellent binding affinities at both μ (0.6-0.9nM) and δ (0.2-1.2nM) opioid receptors respectively. Similarly, these bivalent ligands exhibited potent agonist activities in both MVD and GPI assays. Ligand 17 was evaluated for in vivo antinociceptive activity in non-injured rats following spinal administration. Ligand 17 was not significantly effective in alleviating acute pain. The most likely explanations for this low intrinsic efficacy in vivo despite high in vitro binding affinity, moderate in vitro activity are (i) low potency suggesting that higher doses are needed; (ii) differences in experimental design (i.e. non-neuronal, high receptor density for in vitro preparations versus CNS site of action in vitro); (iii) pharmacodynamics (i.e. engaging signalling pathways); (iv) pharmacokinetics (i.e. metabolic stability). In summary, our data suggest that further optimisation of this compound 17 is required to enhance intrinsic antinociceptive efficacy.
• Forte, B. L., Slosky, L. M., Zhang, H., Arnold, M. R., Staatz, W. D., Hay, M., Largent-Milnes, T. M., & Vanderah, T. W. (2016). Angiotensin-(1-7)/Mas receptor as an antinociceptive agent in cancer-induced bone pain. Pain, 157(12), 2709-2721.
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  Many cancerous solid tumors metastasize to the bone and induce pain (cancer-induced bone pain [CIBP]). Cancer-induced bone pain is often severe because of enhanced inflammation, rapid bone degradation, and disease progression. Opioids are prescribed to manage this pain, but they may enhance bone loss and increase tumor proliferation, further compromising patient quality of life. Angiotensin-(1-7) (Ang-(1-7)) binds and activates the Mas receptor (MasR). Angiotensin-(1-7)/MasR activation modulates inflammatory signaling after acute tissue insult, yet no studies have investigated whether Ang-(1-7)/MasR play a role in CIBP. We hypothesized that Ang-(1-7) inhibits CIBP by targeting MasR in a murine model of breast CIBP. 66.1 breast cancer cells were implanted into the femur of BALB/cAnNHsd mice as a model of CIBP. Spontaneous and evoked pain behaviors were assessed before and after acute and chronic administration of Ang-(1-7). Tissues were collected from animals for ex vivo analyses of MasR expression, tumor burden, and bone integrity. Cancer inoculation increased spontaneous pain behaviors by day 7 that were significantly reduced after a single injection of Ang-(1-7) and after sustained administration. Preadministration of A-779 a selective MasR antagonist prevented this reduction, whereas pretreatment with the AT2 antagonist had no effect; an AT1 antagonist enhanced the antinociceptive activity of Ang-(1-7) in CIBP. Repeated Ang-(1-7) administration did not significantly change tumor burden or bone remodeling. Data here suggest that Ang-(1-7)/MasR activation significantly attenuates CIBP, while lacking many side effects seen with opioids. Thus, Ang-(1-7) may be an alternative therapeutic strategy for the nearly 90% of patients with advanced-stage cancer who experience excruciating pain.
• Grenald, S. A., Young, M. A., Wang, Y., Ossipov, M. H., Ibrahim, M. M., Largent-Milnes, T. M., & Vanderah, T. W. (2016). Synergistic attenuation of chronic pain using mu opioid and cannabinoid receptor 2 agonists. Neuropharmacology, 116, 59-70.
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  The misuse of prescription opiates is on the rise with combination therapies (e.g. acetaminophen or NSAIDs) resulting in severe liver and kidney damage. In recent years, cannabinoid receptors have been identified as potential modulators of pain and rewarding behaviors associated with cocaine, nicotine and ethanol in preclinical models. Yet, few studies have identified whether mu opioid agonists and CB2 agonists act synergistically to inhibit chronic pain while reducing unwanted side effects including reward liability. We determined if analgesic synergy exists between the mu-opioid agonist morphine and the selective CB2 agonist, JWH015, in rodent models of acute and chronic inflammatory, post-operative, and neuropathic pain using isobolographic analysis. We also investigated if the MOR-CB2 agonist combination decreased morphine-induced conditioned place preference (CPP) and slowing of gastrointestinal transit. Co-administration of morphine with JWH015 synergistically inhibited preclinical inflammatory, post-operative and neuropathic-pain in a dose- and time-dependent manner; no synergy was observed for nociceptive pain. Opioid-induced side effects of impaired gastrointestinal transit and CPP were significantly reduced in the presence of JWH015. Here we show that MOR + CB2 agonism results in a significant synergistic inhibition of preclinical pain while significantly reducing opioid-induced unwanted side effects. The opioid sparing effect of CB2 receptor agonism strongly supports the advancement of a MOR-CB2 agonist combinatorial pain therapy for clinical trials.
• Ramos-Colon, C. N., Lee, Y. S., Remesic, M., Hall, S. M., LaVigne, J., Davis, P., Sandweiss, A. J., McIntosh, M. I., Hanson, J., Largent-Milnes, T. M., Vanderah, T. W., Streicher, J., Porreca, F., & Hruby, V. J. (2016). Structure-Activity Relationships of [des-Arg(7)]Dynorphin A Analogues at the κ Opioid Receptor. Journal of medicinal chemistry, 59(22), 10291-10298.
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  Dynorphin A (Dyn A) is an endogenous ligand for the opioid receptors with preference for the κ opioid receptor (KOR), and its structure-activity relationship (SAR) has been extensively studied at the KOR to develop selective potent agonists and antagonists. Numerous SAR studies have revealed that the Arg(7) residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg(7)]Dyn A analogues found that Arg(7) is not a key residue and even deletion of the residue does not affect biological activities at the KOR. In addition, it was also found that [des-Arg(7)]Dyn A(1-9)-NH2 is a minimum pharmacophore and its modification at the N-terminus leads to selective KOR antagonists. A lead ligand, 14, with high affinity and antagonist activity showed improved metabolic stability and could block antinociceptive effects of a KOR selective agonist, FE200665, in vivo, indicating high potential to treat KOR mediated disorders such as stress-induced relapse.
• Slosky, L. M., BassiriRad, N. M., Symons, A. M., Thompson, M., Doyle, T., Forte, B. L., Staatz, W. D., Bui, L., Neumann, W. L., Mantyh, P. W., Salvemini, D., Largent-Milnes, T. M., & Vanderah, T. W. (2016). The cystine/glutamate antiporter system xc- drives breast tumor cell glutamate release and cancer-induced bone pain. Pain, 157(11), 2605-2616.
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  Bone is one of the leading sites of metastasis for frequently diagnosed malignancies, including those arising in the breast, prostate and lung. Although these cancers develop unnoticed and are painless in their primary sites, bone metastases result in debilitating pain. Deeper investigation of this pain may reveal etiology and lead to early cancer detection. Cancer-induced bone pain (CIBP) is inadequately managed with current standard-of-care analgesics and dramatically diminishes patient quality of life. While CIBP etiology is multifaceted, elevated levels of glutamate, an excitatory neurotransmitter, in the bone-tumor microenvironment may drive maladaptive nociceptive signaling. Here, we establish a relationship between the reactive nitrogen species peroxynitrite, tumor-derived glutamate, and CIBP. In vitro and in a syngeneic in vivo model of breast CIBP, murine mammary adenocarcinoma cells significantly elevated glutamate via the cystine/glutamate antiporter system xc. The well-known system xc inhibitor sulfasalazine significantly reduced levels of glutamate and attenuated CIBP-associated flinching and guarding behaviors. Peroxynitrite, a highly reactive species produced in tumors, significantly increased system xc functional expression and tumor cell glutamate release. Scavenging peroxynitrite with the iron and mangano-based porphyrins, FeTMPyP and SRI10, significantly diminished tumor cell system xc functional expression, reduced femur glutamate levels and mitigated CIBP. In sum, we demonstrate how breast cancer bone metastases upregulate a cystine/glutamate co-transporter to elevate extracellular glutamate. Pharmacological manipulation of peroxynitrite or system xc attenuates CIBP, supporting a role for tumor-derived glutamate in CIBP and validating the targeting of system xc as a novel therapeutic strategy for the management of metastatic bone pain.
• Deekonda, S., Wugalter, L., Kulkarni, V., Rankin, D., Largent-Milnes, T. M., Davis, P., Bassirirad, N. M., Lai, J., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2015). Discovery of 5-substituted tetrahydronaphthalen-2yl-methyl with N-phenyl-N-(piperidin-4-yl)propionamide derivatives as potent opioid receptor ligands. Bioorganic & medicinal chemistry, 23(18), 6185-94.
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  A new series of novel opioid ligands have been designed and synthesized based on the 4-anilidopiperidine scaffold containing a 5-substituted tetrahydronaphthalen-2yl)methyl group with different N-phenyl-N-(piperidin-4-yl)propionamide derivatives to study the biological effects of these substituents on μ and δ opioid receptor interactions. Recently our group reported novel 4-anilidopiperidine analogues, in which several aromatic ring-contained amino acids were conjugated with N-phenyl-N-(piperidin-4-yl)propionamide and examined their biological activities at the μ and δ opioid receptors. In continuation of our efforts in these novel 4-anilidopiperidine analogues, we took a peptidomimetic approach in the present design, in which we substituted aromatic amino acids with tetrahydronaphthalen-2yl methyl moiety with amino, amide and hydroxyl substitutions at the 5th position. In in vitro assays these ligands, showed very good binding affinity and highly selective toward the μ opioid receptor. Among these, the lead ligand 20 showed excellent binding affinity (2 nM) and 5000 fold selectivity toward the μ opioid receptor, as well as functional selectivity in GPI assays (55.20 ± 4.30 nM) and weak or no agonist activities in MVD assays. Based on the in vitro bioassay results the lead compound 20 was chosen for in vivo assessment for efficacy in naïve rats after intrathecal administration. Compound 20 was not significantly effective in alleviating acute pain. This discrepancy between high in vitro binding affinity, moderate in vitro activity, and low in vivo activity may reflect differences in pharmacodynamics (i.e., engaging signaling pathways) or pharmacokinetics (i.e., metabolic stability). In sum, our data suggest that further optimization of this compound 20 is required to enhance in vivo activity.
• Deekonda, S., Wugalter, L., Rankin, D., Largent-Milnes, T. M., Davis, P., Wang, Y., Bassirirad, N. M., Lai, J., Kulkarni, V., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2015). Design and synthesis of novel bivalent ligands (MOR and DOR) by conjugation of enkephalin analogues with 4-anilidopiperidine derivatives. Bioorganic & medicinal chemistry letters, 25(20), 4683-8.
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  We describe the design and synthesis of novel bivalent ligands based on the conjugation of 4-anilidopiperidine derivatives with enkephalin analogues. The design of non-peptide analogues is explored with 5-amino substituted (tetrahydronaphthalen-2yl) methyl containing 4-anilidopiperidine derivatives, while non-peptide-peptide ligands are explored by conjugating the C-terminus of enkephalin analogues (H-Xxx-DAla-Gly-Phe-OH) to the amino group of 4-anilidopiperidine small molecule derivatives with and without a linker. These novel bivalent ligands are evaluated for biological activities at μ and δ opioid receptors. They exhibit very good affinities at μ and δ opioid receptors, and potent agonist activities in MVD and GPI assays. Among these the lead bivalent ligand 17 showed excellent binding affinities (0.1 nM and 0.5 nM) at μ and δ opioid receptors respectively, and was found to have very potent agonist activities in MVD (56 ± 5.9 nM) and GPI (4.6 ± 1.9 nM) assays. In vivo the lead bivalent ligand 17 exhibited a short duration of action (
• Fawley, J. A., Hofmann, M. E., Largent-Milnes, T. M., & Andresen, M. C. (2015). Temperature differentially facilitates spontaneous but not evoked glutamate release from cranial visceral primary afferents. PloS one, 10(5), e0127764.
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  Temperature is fundamentally important to all biological functions including synaptic glutamate release. Vagal afferents from the solitary tract (ST) synapse on second order neurons in the nucleus of the solitary tract, and glutamate release at this first central synapse controls autonomic reflex function. Expression of the temperature-sensitive Transient Receptor Potential Vanilloid Type 1 receptor separates ST afferents into C-fibers (TRPV1+) and A-fibers (TRPV1-). Action potential-evoked glutamate release is similar between C- and A-fiber afferents, but TRPV1 expression facilitates a second form of synaptic glutamate release in C-fibers by promoting substantially more spontaneous glutamate release. The influence of temperature on different forms of glutamate release is not well understood. Here we tested how temperature impacts the generation of evoked and spontaneous release of glutamate and its relation to TRPV1 expression. In horizontal brainstem slices of rats, activation of ST primary afferents generated synchronous evoked glutamate release (ST-eEPSCs) at constant latency whose amplitude reflects the probability of evoked glutamate release. The frequency of spontaneous EPSCs in these same neurons measured the probability of spontaneous glutamate release. We measured both forms of glutamate from each neuron during ramp changes in bath temperature of 4-5 °C. Spontaneous glutamate release from TRPV1+ closely tracked with these thermal changes indicating changes in the probability of spontaneous glutamate release. In the same neurons, temperature changed axon conduction registered as latency shifts but ST-eEPSC amplitudes were constant and independent of TRPV1 expression. These data indicate that TRPV1-operated glutamate release is independent of action potential-evoked glutamate release in the same neurons. Together, these support the hypothesis that evoked and spontaneous glutamate release originate from two pools of vesicles that are independently modulated and are distinct processes.
• François-Moutal, L., Wang, Y., Moutal, A., Cottier, K. E., Melemedjian, O. K., Yang, X., Wang, Y., Ju, W., Largent-Milnes, T. M., Khanna, M., Vanderah, T. W., & Khanna, R. (2015). A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors. Pain, 156(7), 1247-64.
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  Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.
• Giri, A. K., Apostol, C. R., Wang, Y., Forte, B. L., Largent-Milnes, T. M., Davis, P., Rankin, D., Molnar, G., Olson, K. M., Porreca, F., Vanderah, T. W., & Hruby, V. J. (2015). Discovery of Novel Multifunctional Ligands with μ/δ Opioid Agonist/Neurokinin-1 (NK1) Antagonist Activities for the Treatment of Pain. Journal of medicinal chemistry, 58(21), 8573-83.
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  Multifunctional ligands with agonist bioactivities at μ/δ opioid receptors (MOR/DOR) and antagonist bioactivity at the neurokinin-1 receptor (NK1R) have been designed and synthesized. These peptide-based ligands are anticipated to produce better biological profiles (e.g., higher analgesic effect with significantly less adverse side effects) compared to those of existing drugs and to deliver better synergistic effects than coadministration of a mixture of multiple drugs. A systematic structure-activity relationship (SAR) study has been conducted to find multifunctional ligands with desired activities at three receptors. It has been found that introduction of Dmt (2,6-dimethyl-tyrosine) at the first position and NMePhe at the fourth position (ligand 3: H-Dmt-d-Ala-Gly-NMePhe-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) displays binding as well as functional selectivity for MOR over DOR while maintaining efficacy, potency, and antagonist activity at the NK1R. Dmt at the first position with Phe(4-F) at the fourth position (ligand 5: H-Dmt-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) exhibits balanced binding affinities at MOR and DOR though it has higher agonist activity at DOR over MOR. This study has led to the discovery of several novel ligands including 3 and 5 with excellent in vitro biological activity profiles. Metabolic stability studies in rat plasma with ligands 3, 5, and 7 (H-Tyr-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) showed that their stability depends on modifications at the first and fourth positions (3: T1/2 > 24 h; 5: T1/2 ≈ 6 h; 7: T1/2 > 2 h). Preliminary in vivo studies with these two ligands have shown promising antinociceptive activity.
• Liktor-Busa, E., Blawn, K. T., Kellohen, K. L., Wiese, B. M., Verkhovsky, V., Wahl, J., Vivek, A., Davis, T. P., Vanderah, T. W., & Milnes, T. M. (2019). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake. PLoS 1, preprint available on bioRxiv Dec 20, 2019. doi:https://doi.org/10.1101/2019.12.20.884247
• Slosky, L. M., Largent-Milnes, T. M., & Vanderah, T. W. (2015). Use of Animal Models in Understanding Cancer-induced Bone Pain. Cancer growth and metastasis, 8(Suppl 1), 47-62.
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  Many common cancers have a propensity to metastasize to bone. Although malignancies often go undetected in their native tissues, bone metastases produce excruciating pain that severely compromises patient quality of life. Cancer-induced bone pain (CIBP) is poorly managed with existing medications, and its multifaceted etiology remains to be fully elucidated. Novel analgesic targets arise as more is learned about this complex and distinct pain state. Over the past two decades, multiple animal models have been developed to study CIBP's unique pathology and identify therapeutic targets. Here, we review animal models of CIBP and the mechanistic insights gained as these models evolve. Findings from immunocompromised and immunocompetent host systems are discussed separately to highlight the effect of model choice on outcome. Gaining an understanding of the unique neuromolecular profile of cancer pain through the use of appropriate animal models will aid in the development of more effective therapeutics for CIBP.
• Zhang, H., Largent-Milnes, T. M., & Vanderah, T. W. (2020). Glial neuroimmune signaling in opioid reward. Brain research bulletin, 155, 102-111.
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  The opioid epidemic is a growing public concern affecting millions of people worldwide. Opioid-induced reward is the initial and key process leading to opioid abuse and addiction. Therefore, a better understanding of opioid reward may be helpful in developing a treatment for opioid addiction. Emerging evidence suggests that glial cells, particularly microglia and astrocytes, play an essential role in modulating opioid reward. Indeed, glial cells and their associated immune signaling actively regulate neural activity and plasticity, and directly modulate opioid-induced rewarding behaviors. In this review, we describe the neuroimmune mechanisms of how glial cells affect synaptic transmission and plasticity as well as how opioids can activate glial cells affecting the glial-neuronal interaction. Last, we summarize current attempts of applying glial modulators in treating opioid reward.
• Grenald, S. A., Largent-Milnes, T. M., & Vanderah, T. W. (2014). Animal models for opioid addiction drug discovery. Expert opinion on drug discovery, 9(11), 1345-54.
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  Since ancient times, the opium poppy has been used in a variety of settings, including pain management. Natural and synthetic derivatives of opium are commonly used in medicine today and include drugs, such as morphine, codeine, hydromorphone and oxycodone. Although excellent at inhibiting pain, these narcotics often produce a state of euphoria leading to misuse and abuse by the general population, particularly in young adults. The misuse of prescription opiates has continually increased over the past 10 years despite associated negative outcomes, resulting in opiate psychological dependence, withdrawal and relapse.
• Hegarty, D. M., Hermes, S. M., Largent-Milnes, T. M., & Aicher, S. A. (2014). Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats. Journal of chemical neuroanatomy, 61-62, 1-12.
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  We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings.
• Hofmann, M. E., Largent-Milnes, T. M., Fawley, J. A., & Andresen, M. C. (2014). External QX-314 inhibits evoked cranial primary afferent synaptic transmission independent of TRPV1. Journal of neurophysiology, 112(11), 2697-706.
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  The cell-impermeant lidocaine derivative QX-314 blocks sodium channels via intracellular mechanisms. In somatosensory nociceptive neurons, open transient receptor potential vanilloid type 1 (TRPV1) receptors provide a transmembrane passageway for QX-314 to produce long-lasting analgesia. Many cranial primary afferents express TRPV1 at synapses on neurons in the nucleus of the solitary tract and caudal trigeminal nucleus (Vc). Here, we investigated whether QX-314 interrupts neurotransmission from primary afferents in rat brain-stem slices. Shocks to the solitary tract (ST) activated highly synchronous evoked excitatory postsynaptic currents (ST-EPSCs). Application of 300 μM QX-314 increased the ST-EPSC latency from TRPV1+ ST afferents, but, surprisingly, it had similar actions at TRPV1- ST afferents. Continued exposure to QX-314 blocked evoked ST-EPSCs at both afferent types. Neither the time to onset of latency changes nor the time to ST-EPSC failure differed between responses for TRPV1+ and TRPV1- inputs. Likewise, the TRPV1 antagonist capsazepine failed to prevent the actions of QX-314. Whereas QX-314 blocked ST-evoked release, the frequency and amplitude of spontaneous EPSCs remained unaltered. In neurons exposed to QX-314, intracellular current injection evoked action potentials suggesting a presynaptic site of action. QX-314 acted similarly at Vc neurons to increase latency and block EPSCs evoked from trigeminal tract afferents. Our results demonstrate that QX-314 blocked nerve conduction in cranial primary afferents without interrupting the glutamate release mechanism or generation of postsynaptic action potentials. The TRPV1 independence suggests that QX-314 either acted extracellularly or more likely entered these axons through an undetermined pathway common to all cranial primary afferents.
• Largent-Milnes, T. M., Hegarty, D. M., Aicher, S. A., & Andresen, M. C. (2014). Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons. Journal of neurophysiology, 111(11), 2222-31.
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  Trigeminal sensory afferent fibers terminating in nucleus caudalis (Vc) relay sensory information from craniofacial regions to the brain and are known to express transient receptor potential (TRP) ion channels. TRP channels are activated by H(+), thermal, and chemical stimuli. The present study investigated the relationships among the spontaneous release of glutamate, temperature, and TRPV1 localization at synapses in the Vc. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from Vc neurons (n = 151) in horizontal brain-stem slices obtained from Sprague-Dawley rats. Neurons had basal sEPSC rates that fell into two distinct frequency categories: High (≥10 Hz) or Low (
• Largent-Milnes, T. M., Brookshire, S. W., Skinner, D. P., Hanlon, K. E., Giuvelis, D., Yamamoto, T., Davis, P., Campos, C. R., Nair, P., Deekonda, S., Bilsky, E. J., Porreca, F., Hruby, V. J., & Vanderah, T. W. (2013). Building a better analgesic: multifunctional compounds that address injury-induced pathology to enhance analgesic efficacy while eliminating unwanted side effects. The Journal of pharmacology and experimental therapeutics, 347(1), 7-19.
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  The most highly abused prescription drugs are opioids used for the treatment of pain. Physician-reported drug-seeking behavior has resulted in a significant health concern among doctors trying to adequately treat pain while limiting the misuse or diversion of pain medications. In addition to abuse liability, opioid use is associated with unwanted side effects that complicate pain management, including opioid-induced emesis and constipation. This has resulted in restricting long-term doses of opioids and inadequate treatment of both acute and chronic debilitating pain, demonstrating a compelling need for novel analgesics. Recent reports indicate that adaptations in endogenous substance P/neurokinin-1 receptor (NK1) are induced by chronic pain and sustained opioid exposure, and these changes may contribute to processes responsible for opioid abuse liability, emesis, and analgesic tolerance. Here, we describe a multifunctional mu-/delta-opioid agonist/NK1 antagonist compound [Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(CF3)2 (TY027)] that has a preclinical profile of excellent antinociceptive efficacy, low abuse liability, and no opioid-related emesis or constipation. In rodent models of acute and neuropathic pain, TY027 demonstrates analgesic efficacy following central or systemic administration with a plasma half-life of more than 4 hours and central nervous system penetration. These data demonstrate that an innovative opioid designed to contest the pathology created by chronic pain and sustained opioids results in antinociceptive efficacy in rodent models, with significantly fewer side effects than morphine. Such rationally designed, multitargeted compounds are a promising therapeutic approach in treating patients who suffer from acute and chronic pain.
• Lozano-Ondoua, A. N., Hanlon, K. E., Symons-Liguori, A. M., Largent-Milnes, T. M., Havelin, J. J., Ferland, H. L., Chandramouli, A., Owusu-Ankomah, M., Nikolich-Zugich, T., Bloom, A. P., Jimenez-Andrade, J. M., King, T., Porreca, F., Nelson, M. A., Mantyh, P. W., & Vanderah, T. W. (2013). Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 28(1), 92-107.
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  Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB(2) ) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB(2) agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB(1) /CB(2) agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB(2) agonists reduce breast cancer-induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB(2) agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB(2) -mediated effects in vivo were reversed by concurrent treatment with a CB(2) antagonist/inverse agonist but not with a CB(1) antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB(2) agonists as a novel treatment for breast cancer-induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.
• Nair, P., Yamamoto, T., Largent-Milnes, T. M., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2013). Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 23(17), 4975-8.
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  The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-O-[3',5'-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.
• Marshall, T. M., Herman, D. S., Largent-Milnes, T. M., Badghisi, H., Zuber, K., Holt, S. C., Lai, J., Porreca, F., & Vanderah, T. W. (2012). Activation of descending pain-facilitatory pathways from the rostral ventromedial medulla by cholecystokinin elicits release of prostaglandin-E₂ in the spinal cord. Pain, 153(1), 86-94.
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  Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and "anti-opioid" by actions on pain-modulatory cells within the rostral ventromedial medulla (RVM). One consequence of activation of RVM CCK₂ receptors may be enhanced spinal nociceptive transmission; but how this might occur, especially in states of pathological pain, is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold after ligation of L₅/L₆ spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time-related increase in (prostaglandin-E₂) PGE₂ measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE₂ was approximately 5-fold and was observed at approximately 80 minutes post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-hydroxyindoleacetic acid (5-HIAA), a 5-hydoxytryptophan (5-HT) metabolite, as compared with controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT₃ antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE₂ and 5-HT in the spinal cord.
• Tumati, S., Largent-Milnes, T. M., Keresztes, A. I., Yamamoto, T., Vanderah, T. W., Roeske, W. R., Hruby, V. J., & Varga, E. V. (2012). Tachykinin NK₁ receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation. European journal of pharmacology, 684(1-3), 64-70.
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  Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK₁ (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK₁ receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK₁ receptor antagonist (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK₁ antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK₁ receptor antagonists may provide a novel paradigm for long-term pain management.
• Tumati, S., Largent-Milnes, T. M., Keresztes, A., Ren, J., Roeske, W. R., Vanderah, T. W., & Varga, E. V. (2012). Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist. Journal of neuroimmunology, 244(1-2), 23-31.
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  Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.
• Hanlon, K. E., Herman, D. S., Agnes, R. S., Largent-Milnes, T. M., Kumarasinghe, I. R., Ma, S. W., Guo, W., Lee, Y., Ossipov, M. H., Hruby, V. J., Lai, J., Porreca, F., & Vanderah, T. W. (2011). Novel peptide ligands with dual acting pharmacophores designed for the pathophysiology of neuropathic pain. Brain research, 1395, 1-11.
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  The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted. CCK has been shown to act as an endogenous "anti-analgesic" peptide and neuropathic pain conditions promote endogenous CCK release in CNS regions of pain modulation. Administration of CCK into nuclei of the rostral ventromedial medulla induces pronociceptive behaviors in rats. RSA 504 and RSA 601 are novel bifunctional compounds developed to target neuropathic pain by simultaneously acting as agonists at two distinct opioid receptors and antagonizing CCK receptors in the CNS. RSA 504 and RSA 601 demonstrate agonist activity in vitro and antihypersensitivity to mechanical and thermal stimuli in vivo using the spinal nerve ligation model of neuropathic pain. Intrathecal administration of RSA 504 and RSA 601 did not demonstrate antinociceptive tolerance over 7 days of administration and did not display motor impairment or sedation using a rotarod. These are the first behavioral studies that demonstrate how multi-targeted molecule design can address the pathology of neuropathic pain. These compounds with δ and μ opioid agonist activity and CCK antagonist activity within one molecule offer a novel approach with efficacy for neuropathic pain while lacking the side effects typically caused by conventional opioid therapies.
• Tumati, S., Roeske, W. R., Largent-Milnes, T. M., Vanderah, T. W., & Varga, E. V. (2011). Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats. Journal of neuroscience methods, 199(1), 62-8.
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  Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.
• Yamamoto, T., Nair, P., Largent-Milnes, T. M., Jacobsen, N. E., Davis, P., Ma, S., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2011). Discovery of a potent and efficacious peptide derivative for δ/μ opioid agonist/neurokinin 1 antagonist activity with a 2',6'-dimethyl-L-tyrosine: in vitro, in vivo, and NMR-based structural studies. Journal of medicinal chemistry, 54(7), 2029-38.
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  Multivalent ligands with δ/μ opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and had significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt(1) incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus) and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug.
• Largent-Milnes, T. M., & Vanderah, T. W. (2010). Recently patented and promising ORL-1 ligands: where have we been and where are we going?. Expert opinion on therapeutic patents, 20(3), 291-305.
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  The interactions of nociceptin/orphanin FQ (N/OFQ) and the opioid receptor-like receptor 1 (nociceptin opioid peptide--NOP) have been implicated in a variety of systems including cardiovascular, respiratory, immune, and the central and peripheral nervous systems.
• Largent-Milnes, T. M., Yamamoto, T., Nair, P., Moulton, J. W., Hruby, V. J., Lai, J., Porreca, F., & Vanderah, T. W. (2010). Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance. British journal of pharmacology, 161(5), 986-1001.
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  The use of opioids in treating pain is limited due to significant side effects including somnolence, constipation, analgesic tolerance, addiction and respiratory depression. Pre-clinical studies have shown that neurokinin 1 (NK(1) ) receptor antagonists block opioid-induced antinociceptive tolerance and may inhibit opioid-induced rewarding behaviours. Here, we have characterized a bifunctional peptide with both opioid agonist and NK(1) antagonist pharmacophores in a rodent model of neuropathic pain.
• Lozano-Ondoua, A. N., Wright, C., Vardanyan, A., King, T., Largent-Milnes, T. M., Nelson, M., Jimenez-Andrade, J. M., Mantyh, P. W., & Vanderah, T. W. (2010). A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss. Life sciences, 86(17-18), 646-53.
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  Cannabinoid CB(2) agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB(2) agonist, does not demonstrate central nervous system side effects seen with CB(1) agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB(2) selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB(2) agonist administered over a 7day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation.
• Largent-Milnes, T. M., Guo, W., Wang, H., Burns, L. H., & Vanderah, T. W. (2008). Oxycodone plus ultra-low-dose naltrexone attenuates neuropathic pain and associated mu-opioid receptor-Gs coupling. The journal of pain : official journal of the American Pain Society, 9(8), 700-13.
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  Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in mu-opioid receptor (MOR)-G protein coupling from G(i/o) to G(s) that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L(5)/L(6) spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to G(s) in the damaged (ipsilateral) spinal dorsal horn. This MOR-G(s) coupling occurred without changing G(i/o) coupling levels and without changing the expression of MOR or Galpha proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-G(s) coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-G(s) coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this G(s) coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-G(s) coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain.
• Vanderah, T. W., Largent-Milnes, T., Lai, J., Porreca, F., Houghten, R. A., Menzaghi, F., Wisniewski, K., Stalewski, J., Sueiras-Diaz, J., Galyean, R., Schteingart, C., Junien, J. L., Trojnar, J., & Rivière, P. J. (2008). Novel D-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral kappa-opioid receptors. European journal of pharmacology, 583(1), 62-72.
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  Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.
• Yamamoto, T., Nair, P., Vagner, J., Largent-Milnes, T., Davis, P., Ma, S. W., Navratilova, E., Moye, S., Tumati, S., Lai, J., Yamamura, H. I., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2008). A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of medicinal chemistry, 51(5), 1369-76.
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  A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Poster Presentations

• Riegel, A. C., Vanderah, T. W., Milnes, T. M., Langlais, P. R., Majuta, L., Smith-Flint, A., Franca-Solomon, G., Barber, K. R., & Vizcarra, V. S. (2021, November). Targeted manipulation of PFC 5HT2A receptors and KV7 channels attenuates chronic neuropathic pain in rats.. Society for Neuroscience.