Todd W Vanderah

Interests

Teaching

Nervous System and Neuropharmacology

Research

Chronic pain and opioid addiction

Courses

Scholarly Contributions

Journals/Publications

• Coffee, Z., Speight, C., Reyes-Walsh, L., Foreman, J., Davis-Dick, L., Vanderah, T. W., & Gordon, J. S. (2025). Empowering Primary Care Nurse Practitioners: A Multilevel Approach to Combating the Opioid Crisis. Journal of Advanced Nursing, 81(Issue 6). doi:10.1111/jan.16694
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  Aims: We offer a literature-driven, empirically informed, and highly warranted recommendation for a multilevel approach tailored to nurse practitioners. This approach aimed to drive change at the individual level (nurse practitioner), dyadic level (nurse–patient therapeutic relationship), and systems level (organisational culture, education, and policy) to strengthen nurse practitioners' capacity to deliver optimal opioid use disorder care. Background: The opioid overdose epidemic is a global public health crisis, with the United States facing the most severe impact. Access to evidence-based treatment for opioid use disorder remains a significant barrier. Primary care nurse practitioners can play a crucial role in expanding access and bridging the treatment gap. Addressing factors influencing access to, quality of, and effectiveness of treatment requires urgent and careful consideration. Design: This position paper highlights the multilevel barriers that inhibit nurse practitioners in managing opioid use disorder, negatively impacting treatment access and opioid use disorder-related outcomes. Method: The Advanced Practice Provider conceptual model was adapted to guide this paper. This adapted model illustrates the interconnected layers among nurse practitioners, clinical care, education, professional development, and organisational culture and policy in caring for patients with opioid use disorder. Conclusions: Equipping primary care nurse practitioners with the necessary tools to both understand opioid addiction and empower patients experiencing it can have a profound impact. This impact benefits the provider and patient and extends to addressing the opioid crisis at multiple levels. Future research should explore nurse practitioners' experiences when working with individuals with opioid use disorder, identify barriers that hinder positive interactions with patients seeking treatment, and pragmatically test and implement multilevel interventions designed to holistically benefit providers and patient outcomes. Impact to Nursing: This American-based reflection offers valuable insights to nurse practitioners worldwide as they consider effective strategies for addressing opioid use disorder in primary care settings.
• 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. (2025). Activation of sphingosine-1-phosphate receptor subtype 1 in the central nervous system contributes to morphine-induced hyperalgesia and antinociceptive tolerance in rodents: erratum. Pain, 166(Issue 5). doi:10.1097/j.pain.0000000000003630
• De La Rosa, J. S., Brady, B. R., Ibrahim, M. M., Herder, K. E., Wallace, J. S., Padilla, A. R., & Vanderah, T. W. (2024). Co-occurrence of chronic pain and anxiety/depression symptoms in U.S. adults: Prevalence, functional impacts, and opportunities. Pain, 165(Issue 3). doi:10.1097/j.pain.0000000000003056
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  Co-occurrence of chronic pain and clinically significant symptoms of anxiety and/or depression is regularly noted in the literature. Yet, little is known empirically about population prevalence of co-occurring symptoms, nor whether people with co-occurring symptoms constitute a distinct subpopulation within US adults living with chronic pain or US adults living with anxiety and/or depression symptoms (A/D). To address this gap, this study analyzes data from the 2019 National Health Interview Survey, a representative annual survey of self-reported health status and treatment use in the United States (n = 31,997). Approximately 12 million US adults, or 4.9% of the adult population, have co-occurring chronic pain and A/D symptoms. Unremitted A/D symptoms co-occurred in 23.9% of US adults with chronic pain, compared with an A/D prevalence of 4.9% among those without chronic pain. Conversely, chronic pain co-occurred in the majority (55.6%) of US adults with unremitted A/D symptoms, compared with a chronic pain prevalence of 17.1% among those without A/D symptoms. The likelihood of experiencing functional limitations in daily life was highest among those experiencing co-occurring symptoms, compared with those experiencing chronic pain alone or A/D symptoms alone. Among those with co-occurring symptoms, 69.4% reported that work was limited due to a health problem, 43.7% reported difficulty doing errands alone, and 55.7% reported difficulty participating in social activities. These data point to the need for targeted investment in improving functional outcomes for the nearly 1 in 20 US adults living with co-occurring chronic pain and clinically significant A/D symptoms.
• Barber, K. R., Vizcarra, V. S., Zilch, A., Majuta, L., Diezel, C. C., Culver, O. P., Hughes, B. W., Taniguchi, M., Streicher, J. M., Vanderah, T. W., & Riegel, A. C. (2023). The Role of Ryanodine Receptor 2 in Drug-Associated Learning. bioRxiv : the preprint server for biology.
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  Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.
• Chalasani, P., Ehsani, S., Farr, K., Jiralerspong, S., Junak, M., Mohab, I., Roe, D., Segar, J., & Vanderah, T. (2023). Abstract P4-04-02: Evaluation of Dronabinol to Decrease Opioid Use for Cancer- Induced Bone Pain. Cancer Research, 83(5_Supplement), P4-04-02-P4-04-02. doi:10.1158/1538-7445.sabcs22-p4-04-02
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  Abstract Purpose Bone metastases (BM) from breast cancer cause significant complications including pain, hypercalcemia, spinal cord compression, and pathologic fractures, collectively referred to as skeletal related events (SREs). Of all SREs, bone pain impacts the quality of life (QOL) most significantly. Cancer- induced bone pain (CIBP) is difficult to treat with limited treatment options and requires a multimodal approach. Management of CIBP is primarily by opioids which have notable side effects like sedation, constipation, and concern for addiction. In addition, pre-clinical evidence suggests that opioids accelerate bone loss and increase risk of fractures. There is an unmet need for novel analgesic therapy interventions to optimize QOL for patients with BM from metastatic breast cancer. Preclinical studies have shown that the endogenous cannabinoid (CB) system is involved in pain modulation, bone regulation, immunity, and restraint of cancer pathogenesis. CB2 receptor activation has been shown to inhibit proinflammatory cytokines/chemokines in pre-clinical models. Treatment with selective CB2 agonist in mice with BM led to significant antinociception, decreased cancer-induced bone degradation, and reduced side effects of morphine. In addition to alleviating pain, CB2 agonists were shown to enhance bone growth/strength in these mice. Based on this pre-clinical data, we hypothesized that the addition of a CB2 agonist will improve pain symptoms and decrease opioid requirement in patients with bone metastases from breast cancer. We proceeded to conduct a pilot study by repurposing a clinically approved CB2/CB1 agonist, dronabinol. Methods We conducted a prospective, single site study among patients with BM from breast cancer at our center (NCT03661892). Patients had to have been on opioids for CIBP for at least 4 weeks and not using marijuana or CBD products. Patients were treated with 10mg dronabinol BID for 8 weeks. Our primary objective was to determine the proportion who decrease their opioid use by ≥ 20%. The null hypothesis value was 5% of women would have a 20% decrease. With 14 participants, we could detect an increase from 5% to 29% (n=4) with 80% statistical power using a one-sided alpha level of 0.05. Participants completed Brief Pain Inventory and the European Organization for Research and Treatment of Cancer quality of life questionnaires pre and post treatment. Results Twenty participants consented with 14 patients completing the study and evaluable for primary analysis. No patients received any palliative radiation therapy or other therapies for bone pain within 3 months prior to enrollment to this study. Four patients decreased their opioid use by ≥ 20% meeting the primary objective. Patients reported significant improvement in pain severity, interference scores, quality of life and insomnia based on the questionnaires. There were no grade 4 side effects and only 1 patient had grade 3 adverse event (dizziness) related to study drug. Of the 14 patients who completed the study, 9 desired to continue dronabinol therapy after completion. Conclusion Our pilot study shows that the addition of dronabinol resulted in decreased opioid requirements for CIBP in patients with metastatic breast cancer. Patient-reported outcomes also demonstrated improved pain and QOL with the addition of dronabinol. While we did not see any significant AEs tolerability may be of concern due to CB1 psychoactive effects. Our results are promising and warrant further investigation into evaluating CB2 agonists for improved pain control from CIBP and to decrease opioid use. Citation Format: Jennifer Segar, Kiah Farr, Mary Junak, Denise Roe, Sima Ehsani, Sao Jiralerspong, Ibrahim Mohab, Todd Vanderah, Pavani Chalasani. Evaluation of Dronabinol to Decrease Opioid Use for Cancer- Induced Bone Pain [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-04-02.
• De La Rosa, J. S., Brady, B. R., Ibrahim, M. M., Herder, K. E., Wallace, J. S., Padilla, A. R., & Vanderah, T. W. (2023). Co-occurrence of chronic pain and anxiety/depression symptoms in U.S. adults: prevalence, functional impacts, and opportunities. Pain.
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  Co-occurrence of chronic pain and clinically significant symptoms of anxiety and/or depression is regularly noted in the literature. Yet, little is known empirically about population prevalence of co-occurring symptoms, nor whether people with co-occurring symptoms constitute a distinct subpopulation within US adults living with chronic pain or US adults living with anxiety and/or depression symptoms (A/D). To address this gap, this study analyzes data from the 2019 National Health Interview Survey, a representative annual survey of self-reported health status and treatment use in the United States (n = 31,997). Approximately 12 million US adults, or 4.9% of the adult population, have co-occurring chronic pain and A/D symptoms. Unremitted A/D symptoms co-occurred in 23.9% of US adults with chronic pain, compared with an A/D prevalence of 4.9% among those without chronic pain. Conversely, chronic pain co-occurred in the majority (55.6%) of US adults with unremitted A/D symptoms, compared with a chronic pain prevalence of 17.1% among those without A/D symptoms. The likelihood of experiencing functional limitations in daily life was highest among those experiencing co-occurring symptoms, compared with those experiencing chronic pain alone or A/D symptoms alone. Among those with co-occurring symptoms, 69.4% reported that work was limited due to a health problem, 43.7% reported difficulty doing errands alone, and 55.7% reported difficulty participating in social activities. These data point to the need for targeted investment in improving functional outcomes for the nearly 1 in 20 US adults living with co-occurring chronic pain and clinically significant A/D symptoms.
• 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, 4. doi:10.3389/fpain.2023.1171188
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  Introduction: 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. Methods: Cortical spreading depression was induced in female Sprague Dawley rats via KCl (potassium chloride) administration, followed by measures of endocannabinoid levels, enzyme activity, and neuroinflammatory markers. Efficacy of inhibiting 2-arachidonoylglycerol hydrolysis to mitigate periorbital allodynia was then tested using reversal and prevention paradigms. Results: We discovered reduced 2-arachidonoylglycerol levels in the periaqueductal grey associated with increased hydrolysis following headache induction. Pharmacological inhibition of the 2-arachidonoylglycerol hydrolyzing enzymes, α/β-hydrolase domain-containing 6 and monoacylglycerol lipase reversed and prevented induced periorbital allodynia in a cannabinoid receptor-dependent manner. Discussion: Our study unravels a mechanistic link between 2-arachidonoylglycerol hydrolysis activity in the periaqueductal grey in a preclinical, rat model of migraine. Thus, 2-arachidonoylglycerol hydrolysis inhibitors represent a potential new therapeutic avenue for the treatment of headache.
• 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.
• Maltagliati, A. J., Paree, J. H., McIntosh, K. L., Moynahan, K. F., & Vanderah, T. W. (2023). Development and evaluation of a pre-clerkship spiral curriculum: data from three medical school classes. Medical education online, 28(1), 2167258.
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  Pre-clerkship curricula of most Liaison Committee on Medical Education (LCME)-accredited medical schools are divided into blocks by organ system, leaving a significant amount of information susceptible to loss due to prolonged nonuse. We describe the implementation of a formal Spiral Curriculum that periodically revisits material from previous blocks. Learners were surveyed on receptivity to the curriculum across three graduating classes at a single medical school. Medical school graduate classes of 2020, 2021, and 2022 were surveyed at the end of their pre-clerkship years (2018-2020). The class of 2022 actually received the Spiraled Curriculum intervention, for which the authors created 500 board exam style multiple-choice questions, periodically administered via mandatory in-class sessions ranging from 10 to 20 questions reviewing content from previous blocks with designated expert faculty. Response rates were 36% ( = 46), 45% ( = 52), and 32% ( = 40) for classes of 2020, 2021, and 2022, respectively. On a Likert scale (1 = strongly disagree, 5 = neutral, 10 = strongly agree), the classes of 2020, 2021, and 2022 provided statistically significant differences in their belief that a Spiraled Curriculum would/did help them retain information as 8.2 (SD 1.7), 8.2 (SD 2.2), and 5.0 (SD 3.0) ( < 0.05). All classes endorsed neutral confidence in the existing pre-clerkship curriculum in themselves to prepare for United Stated Medical Licensing Examination (USMLE) Step 1, and in their retention of previous block material with no statistically significant differences between classes. USMLE Step 1 scores did not differ significantly between classes ( = 0.21). Those who did not receive the Spiral Curriculum were highly receptive to it in theory, while those who actually received the intervention gave a neutral rating. Per survey comments, implementation of a Spiraling Curriculum would ideally be administered as either team-based or self-directed activities, and a Spiraling Curriculum may be difficult to implement in accelerated (18 month) pre-clerkship formats. Question: What is the receptivity of medical students to a formal Spiral curriculum that uses time-spaced repetition sessions of board exam style questions to revisit previous block content of their pre-clerkship years?Findings: In this single-center, quasi-experimental study, the two control group medical school classes had very positive theoretical reception to a Spiral curriculum proposal (rated 8 out of 10) while the class who actually received the Spiral curriculum provided a statistically significant lower neutral rating (rated 5 out of 10), citing preference for a team-based or self-directed format.Meaning: Medical students are strongly in favor of structured time-spaced repetition with board exam style questions to revisit previous material but prefer a format that does not interfere with time to personalize their medical school experience.
• 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.
• Urena, E. S., Diezel, C. C., Serna, M., Hala'ufia, G., Majuta, L., Barber, K. R., Vanderah, T. W., & Riegel, A. C. (2023). K 7 Channel Opener Retigabine Reduces Self-Administration of Cocaine but Not Sucrose in Rats. bioRxiv : the preprint server for biology.
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  The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviors that can be modeled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that K 7/KCNQ channels may contribute in the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drug is not known. Here, we tested the ability of retigabine (ezogabine), a K 7 channel opener, to regulate instrumental behavior in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a CPP assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine-pretreatment attenuated the self-administration of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared to sucrose-SA, cocaine-SA was associated with reductions in the expression of the K 7.5 subunit in the nucleus accumbens, without alterations in K 7.2 and K 7.3. Therefore, these studies reveal a reward specific reduction in SA behavior considered relevant for the study of long-term compulsive-like behavior and supports the notion that K 7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.
• 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.
• 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.
• Lee, N. Y., Mythreye, K., Langlais, P. R., Hund, T. J., Mohler, P. J., Milnes, T. M., Vanderah, T. W., Lee, Y. S., Mouneimne, G., Ellis, N., Lochhead, J. L., Ortiz, H. R., Ahmed, T., Cruz-Flores, P., Kumar, S., Ramonett, A., Pan, C. C., & Kwak, E. A. (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.
• 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.
• Ramonett, A., Kwak, E. A., Ahmed, T., Flores, P. C., Ortiz, H. R., Lee, Y. S., Vanderah, T. W., Largent-Milnes, T., Kashatus, D. F., Langlais, P. R., Mythreye, K., & Lee, N. Y. (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.
• 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.
• 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. (2022). Brain Penetrant, but not Peripherally Restricted, Synthetic Cannabinoid 1 Receptor Agonists Promote Morphine-Mediated Respiratory Depression. Cannabis and cannabinoid research, 7(5), 621-627.
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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.
• 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.
• Cai, S., Moutal, A., Yu, J., Chew, L. A., Isensee, J., Chawla, R., Gomez, K., Luo, S., Zhou, Y., Chefdeville, A., Madura, C., Perez-Miller, S., Bellampalli, S. S., Dorame, A., Scott, D. D., François-Moutal, L., Shan, Z., Woodward, T., Gokhale, V., , Hohmann, A. G., et al. (2021). Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents. Science translational medicine, 13(619), eabh1314.
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  The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics—without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.
• Levine, A., Liktor-Busa, E., Karlage, K. L., Giancotti, L., Salvemini, D., Vanderah, T. W., & Largent-Milnes, T. M. (2021). DAGLα Inhibition as a Non-invasive and Translational Model of Episodic Headache. Frontiers in Pharmacology, 11. doi:10.3389/fphar.2020.615028
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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., Liktor-Busa, E., Lipinski, A. A., Couture, S., Balasubramanian, S., Aicher, S. A., Langlais, P. R., Vanderah, T. W., & Largent-Milnes, T. M. (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.
• Levine, A., Vanderah, T. W., & Largent-Milnes, T. M. (2021). 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, 41(3), 404-416.
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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.
• 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.
• 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.
• Couture, S. A., Ji, L., Largent-Milnes, T. M., Levine, A., Liktor-Busa, E., Liu, Y., Mackie, K., Makriyannis, A., Nikas, S. P., Vanderah, T. W., & Wiese, B. M. (2020). Cannabinoid-2 Agonism with AM2301 Mitigates Morphine-Induced Respiratory Depression. Cannabis and Cannabinoid Research, 6(Issue 5). doi:10.1089/can.2020.0076
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  Introduction: 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. Objective: Cannabinoid receptor agonism is reported to enhance opioid analgesia, yet whether cannabinoids enhance or inhibit opioid-induced respiratory depression is unknown. Methods: 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. Results: Using whole body plethysmography, the nonselective CB1R and CB2R agonist (Δ9-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. Conclusion: Notably, findings suggest that attenuation of opioid-induced respiratory depression relies on CB2R activation, supporting selective CB2R agonism as an opioid adjunct therapy.
• 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.
• Falk, T., Sherman, S. J., Polt, R., Vanderah, T. W., Szabo, L., So, L. Y., & Bartlett, M. J. (2020). A highly selective mu-opioid receptor antagonist does not block L-DOPA-induced dyskenesia in a rat model. BMC Research Notes.
• Largent-Milnes, T. M., Levine, A., & Vanderah, T. W. (2020). An underrepresented majority: A systematic review utilizing allodynic criteria to examine the present scarcity of discrete animal models for episodic migraine. Cephalalgia, 41(3), 404-416. doi:10.1177/0333102420966984
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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.In this review, we seek to characterize accepted preclinical models of migraine-like headache by their ability to recapitulate the clinical allodynic features of either episodic or chronic migraine.From a search of the Pu bMed database for "animal models of migraine", "headache models" and "preclinical migraine", we identified approximately 80 recent (within the past 20 years) publications that utilized one of 10 different models for migraine research. Models reviewed fit into one of the following categories: Dural KCl application, direct electrical stimulation, nitroglycerin administration, inflammatory soup injection, CGRP injection, medication overuse, monogenic animals, post-traumatic headache, specific channel activation, and hormone manipulation. Recapitulation of clinical features including cephalic and extracephalic hypersensitivity were evaluated for each and compared.Episodic migraineurs comprise over half of the migraine population, yet the vast majority of current animal models of migraine appear to best represent chronic migraine states. While some of these models can be modified to reflect episodic migraine, there remains a need for non-invasive, validated models of episodic migraine to enhance the clinical translation of migraine research.
• 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.
• 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, 161(9), 2179-2190.
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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 Ca2+ 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 Ca2+ 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.
• Smith, A. F., Vanderah, T. W., & Erickson, R. P. (2020). Haploinsufficiency of tau decreases survival of the mouse model of Niemann-Pick disease type C1 but does not alter tau phosphorylation. Journal of applied genetics, 61(4), 567-570.
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  Niemann-Pick C1 (NPC1) mouse models show neurofibrillary tangles as do human patients. A previous study in NPC1/tau double-null mutant mice showed that tau knockout nulls and heterozygotes unexpectedly had decreased survival when compared with NPC1 single mutants (Pacheco et al., Hum Molec Genetics 18:956-965, 2009). This was done in a null model of NPC1 (Npc1). We have extended these results to a hypomorphic model (Npc1) and additionally studied tau phosphorylation, which has not been previously done in a tau heterozygote. As before, NPC1/tau double-mutant mice had shortened survival when compared with the NPC1 single mutant. Tau dosage was not affected by the Npc1 mutation. The increased phosphorylation of tau-ser396 previously noted in NPC1 mouse models was also present, but unaffected by the tau knockout, indicating that changes in tau phosphorylation are not the cause of decreased survival in NPC1/tau double mutants. Thus, the reason for shortened survival of NPC1 mouse models with concomitant tau haploinsufficiency is uncertain.
• 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.
• Johnston, B., & Vanderah, T. W. (2019). Lost in the Haze: The Physician's Role in Cannabinoid Prescribing and Advising. The American journal of medicine.
• Khanna, R., Patwardhan, A., Yang, X., Li, W., Cai, S., Ji, Y., Chew, L. A., Dorame, A., Bellampalli, S. S., Schmoll, R. W., Gordon, J., Moutal, A., Vanderah, T. W., Porreca, F., & Ibrahim, M. M. (2019). Development and Characterization of An Injury-free Model of Functional Pain in Rats by Exposure to Red Light. The journal of pain : official journal of the American Pain Society, 20(11), 1293-1306.
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  We report the development and characterization of a novel, injury-free rat model in which nociceptive sensitization after red light is observed in multiple body areas reminiscent of widespread pain in functional pain syndromes. Rats were exposed to red light-emitting diodes (RLED) (LEDs, 660 nm) at an intensity of 50 Lux for 8 hours daily for 5 days resulting in time- and dose-dependent thermal hyperalgesia and mechanical allodynia in both male and female rats. Females showed an earlier onset of mechanical allodynia than males. The pronociceptive effects of RLED were mediated through the visual system. RLED-induced thermal hyperalgesia and mechanical allodynia were reversed with medications commonly used for widespread pain, including gabapentin, tricyclic antidepressants, serotonin/norepinephrine reuptake inhibitors, and nonsteroidal anti-inflammatory drugs. Acetaminophen failed to reverse the RLED induced hypersensitivity. The hyperalgesic effects of RLED were blocked when bicuculline, a gamma-aminobutyric acid-A receptor antagonist, was administered into the rostral ventromedial medulla, suggesting a role for increased descending facilitation in the pain pathway. Key experiments were subjected to a replication study with randomization, investigator blinding, inclusion of all data, and high levels of statistical rigor. RLED-induced thermal hyperalgesia and mechanical allodynia without injury offers a novel injury-free rodent model useful for the study of functional pain syndromes with widespread pain. RLED exposure also emphasizes the different biological effects of different colors of light exposure. PERSPECTIVE: This study demonstrates the effect of light exposure on nociceptive thresholds. These biological effects of red LED add evidence to the emerging understanding of the biological effects of light of different colors in animals and humans. Understanding the underlying biology of red light-induced widespread pain may offer insights into functional pain states.
• Khanna, R., Patwardhan, A., Yang, X., Li, W., Cai, S., Ji, Y., Chew, L. A., Dorame, A., Bellampalli, S. S., Schmoll, R. W., Gordon, J., Moutal, A., Vanderah, T. W., Porreca, F., & Ibrahim, M. M. (2019). Erratum to ‛Development and Characterization of An Injury-free Model of Functional Pain in Rats by Exposure to Red Light': The Journal of Pain 20 (2019) 1293-1306. The journal of pain : official journal of the American Pain Society, 20(12), 1509.
• 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.
• Zhang, H., Largent-Milnes, T. M., & Vanderah, T. W. (2019). Glial Neuroimmune Signaling in Opioid Reward. Brain research bulletin.
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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.
• 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.
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  Breast cancer metastasizes to bone, diminishing quality of life of patients due to 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, while KOR agonists are efficacious in peripheral and inflammatory pain. We thus examined the effects of the KOR agonist U50,488 given 2x 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 (norBNI). 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.
• Moutal, A., Dustrude, E. T., Largent-Milnes, T. M., Vanderah, T. W., Khanna, M., & Khanna, R. (2018). Blocking CRMP2 SUMOylation reverses neuropathic pain. Molecular Psychiatry, 23(Issue 11). doi:10.1038/mp.2017.117
• 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).
• 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.
• Doyle, T. M., Zhang, H., Vanderah, T. W., Salvemini, D., Jacobson, K. A., Grenald, S. A., & Doyle, T. (2017). (192) Targeting A3 adenosine receptor in HIV-1 gp120-induced neuropathic pain. The Journal of Pain, 18(4), S24. doi:10.1016/j.jpain.2017.02.099
• 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.
• Grenald, S. A., Young, M. A., Wang, Y., Ossipov, M. H., Ibrahim, M. M., Largent-Milnes, T. M., & Vanderah, T. W. (2017). 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.
• Hay, M., Vanderah, T. W., Samareh-Jahani, F., Constantopoulos, E., Uprety, A. R., Barnes, C. A., & Konhilas, J. (2017). Cognitive impairment in heart failure: A protective role for angiotensin-(1-7). Behavioral neuroscience, 131(1), 99-114.
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  Patients with congestive heart failure (CHF) have increased hospital readmission rates and mortality if they are concomitantly diagnosed with cognitive decline and memory loss. Accordingly, we developed a preclinical model of CHF-induced cognitive impairment with the goal of developing novel protective therapies against CHF related cognitive decline. CHF was induced by ligation of the left coronary artery to instigate a myocardial infarction (MI). By 4- and 8-weeks post-MI, CHF mice had approximately a 50% and 70% decline in ejection fraction as measured by echocardiography. At both 4- and 8-weeks post-MI, spatial memory performance in CHF mice as tested using the Morris water task was significantly impaired as compared with sham. In addition, CHF mice had significantly worse performance on object recognition when compared with shams as measured by discrimination ratios during the novel object recognition NOR task. At 8-weeks post-MI, a subgroup of CHF mice were given Angiotensin (Ang)-(1-7) (50mcg/kg/hr) subcutaneously for 4 weeks. Following 3 weeks treatment with systemic Ang-(1-7), the CHF mice NOR discrimination ratios were similar to shams and significantly better than the performance of CHF mice treated with saline. Ang-(1-7) also improved spatial memory in CHF mice as compared with shams. Ang-(1-7) had no effect on cardiac function. Inflammatory biomarker studies from plasma revealed a pattern of neuroprotection that may underlie the observed improvements in cognition. These results demonstrate a preclinical mouse model of CHF that exhibits both spatial memory and object recognition dysfunction. Furthermore, this CHF-induced cognitive impairment is attenuated by treatment with systemic Ang-(1-7). (PsycINFO Database Record
• 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.
• Largent-milnes, T. M., Vanderah, T. W., Largent-milnes, T. M., Kim, J. S., Galoway, E., Davis, T. P., & Cottier, K. E. (2017). (143) Transient Decreases in Blood-Brain Barrier Integrity Correspond with Facial Allodynia Duration in a Model of Migraine with Aura. The Journal of Pain, 18(4), S12. doi:10.1016/j.jpain.2017.02.049
• 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.
• Moutal, A., Li, W., Wang, Y., Ju, W., Luo, S., Cai, S., François-Moutal, L., Perez-Miller, S., Hu, J., Dustrude, E. T., Vanderah, T. W., Gokhale, V., Khanna, M., & Khanna, R. (2017). Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides. British journal of pharmacology.
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  N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.
• Moutal, A., Wang, Y., Yang, X., Ji, Y., Luo, S., Dorame, A., Bellampalli, S. S., Chew, L. A., Cai, S., Dustrude, E. T., Keener, J. E., Marty, M. T., Vanderah, T. W., & Khanna, R. (2017). Dissecting the role of the CRMP2-neurofibromin complex on pain behaviors. Pain, 158(11), 2203-2221.
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  Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.
• 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.
• Davis, B., & Vanderah, T. W. (2016). A new paradigm for pain?. The Journal of family practice, 65(9), 598-605.
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  A new way of thinking about pain that occurs in the absence of a pathophysiologic process or injury may alter our approach to conditions like fibromyalgia.
• 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 and Medicinal Chemistry Letters, 26(Issue 1). doi:10.1016/j.bmcl.2015.10.081
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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 (0.6-0.9 nM) and δ (0.2-1.2 nM) 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.
• Deekonda, S., Rankin, D., Davis, P., Lai, J., Vanderah, T. W., Porecca, F., & Hruby, V. J. (2016). Design synthesis and structure-activity relationship of 5-substituted (tetrahydronaphthalen-2yl)methyl with N-phenyl-N-(piperidin-2-yl)propionamide derivatives as opioid ligands. Bioorganic & medicinal chemistry, 24(2), 85-91.
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  Here, we report the design, synthesis and structure activity relationship of novel small molecule opioid ligands based on 5-amino substituted (tetrahydronaphthalen-2-yl)methyl moiety with N-phenyl-N-(piperidin-2-yl)propionamide derivatives. We synthesized various molecules including amino, amide and hydroxy substitution on the 5th position of the (tetrahydronaphthalen-2-yl)methyl moiety. In our further designs we replaced the (tetrahydronaphthalen-2-yl)methyl moiety with benzyl and phenethyl moiety. These N-phenyl-N-(piperidin-2-yl)propionamide analogues showed moderate to good binding affinities (850-4 nM) and were selective towards the μ opioid receptor over the δ opioid receptors. From the structure activity relationship studies, we found that a hydroxyl substitution at the 5th position of (tetrahydronapthalen-2yl)methyl group, ligands 19 and 20, showed excellent binding affinities 4 and 5 nM, respectively, and 1000 fold selectivity towards the μ opioid relative to the delta opioid receptor. The ligand 19 showed potent agonist activities 75±21 nM, and 190±42 nM in the GPI and MVD assays. Surprisingly the fluoro analogue 20 showed good agonist activities in MVD assays 170±42 nM, in contrast to its binding affinity results.
• 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.
• Hanlon, K. E., Lozano-Ondoua, A. N., Umaretiya, P. J., Symons-Liguori, A. M., Chandramouli, A., Moy, J. K., Kwass, W. K., Mantyh, P. W., Nelson, M. A., & Vanderah, T. W. (2016). Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent. Breast cancer (Dove Medical Press), 8, 59-71.
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  Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands.
• Moutal, A., Chew, L. A., Yang, X., Wang, Y., Yeon, S. K., Telemi, E., Meroueh, S., Park, K. D., Shrinivasan, R., Gilbraith, K. B., Qu, C., Xie, J. Y., Patwardhan, A., Vanderah, T. W., Khanna, M., Porreca, F., & Khanna, R. (2016). (S)-lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology. Pain, 157(7), 1448-63.
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  Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.
• 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-Arg7]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 Arg7 residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg7]Dyn A analogues found that Arg7 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-Arg7]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.
• Xie, J. Y., Chew, L. A., Yang, X., Wang, Y., Qu, C., Wang, Y., Federici, L. M., Fitz, S. D., Ripsch, M. S., Due, M. R., Moutal, A., Khanna, M., White, F. A., Vanderah, T. W., Johnson, P. L., Porreca, F., & Khanna, R. (2016). Sustained relief of ongoing experimental neuropathic pain by a CRMP2 peptide aptamer with low abuse potential. Pain, 157(9), 2124-40.
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  Uncoupling the protein-protein interaction between collapsin response mediator protein 2 (CRMP2) and N-type voltage-gated calcium channel (CaV2.2) with an allosteric CRMP2-derived peptide (CBD3) is antinociceptive in rodent models of inflammatory and neuropathic pain. We investigated the efficacy, duration of action, abuse potential, and neurobehavioral toxicity of an improved mutant CRMP2 peptide. A homopolyarginine (R9)-conjugated CBD3-A6K (R9-CBD3-A6K) peptide inhibited the CaV2.2-CRMP2 interaction in a concentration-dependent fashion and diminished surface expression of CaV2.2 and depolarization-evoked Ca influx in rat dorsal root ganglia neurons. In vitro studies demonstrated suppression of excitability of small-to-medium diameter dorsal root ganglion and inhibition of subtypes of voltage-gated Ca channels. Sprague-Dawley rats with tibial nerve injury had profound and long-lasting tactile allodynia and ongoing pain. Immediate administration of R9-CBD3-A6K produced enhanced dopamine release from the nucleus accumbens shell selectively in injured animals, consistent with relief of ongoing pain. R9-CBD3-A6K, when administered repeatedly into the central nervous system ventricles of naive rats, did not result in a positive conditioned place preference demonstrating a lack of abusive liability. Continuous subcutaneous infusion of R9-CBD3-A6K over a 24- to 72-hour period reversed tactile allodynia and ongoing pain, demonstrating a lack of tolerance over this time course. Importantly, continuous infusion of R9-CBD3-A6K did not affect motor activity, anxiety, depression, or memory and learning. Collectively, these results validate the potential therapeutic significance of targeting the CaV-CRMP2 axis for treatment of neuropathic pain.
• 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. (2015). Enkephalin analogues with N-phenyl-N-(piperidin-2-ylmethyl)propionamide derivatives: Synthesis and biological evaluations. Bioorganic & medicinal chemistry letters.
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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.
• 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 (0.1nM and 0.5nM) at μ and δ opioid receptors respectively, and was found to have very potent agonist activities in MVD (56±5.9nM) and GPI (4.6±1.9nM) assays. In vivo the lead bivalent ligand 17 exhibited a short duration of action (
• Dzierlenga, A. L., Clarke, J. D., Hargraves, T. L., Ainslie, G. R., Vanderah, T. W., Paine, M. F., & Cherrington, N. J. (2015). Mechanistic basis of altered morphine disposition in nonalcoholic steatohepatitis. The Journal of pharmacology and experimental therapeutics, 352(3), 462-70.
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  Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0-150 or 0-240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0-12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity.
• Ford, A., Castonguay, A., Cottet, M., Little, J. W., Chen, Z., Symons-Liguori, A. M., Doyle, T., Egan, T. M., Vanderah, T. W., De Konnick, Y., Tosh, D. K., Jacobson, K. A., & Salvemini, D. (2015). Engagement of the GABA to KCC2 signaling pathway contributes to the analgesic effects of A3AR agonists in neuropathic pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(15), 6057-67.
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  More than 1.5 billion people worldwide suffer from chronic pain, yet current treatment strategies often lack efficacy or have deleterious side effects in patients. Adenosine is an inhibitory neuromodulator that was previously thought to mediate antinociception through the A1 and A2A receptor subtypes. We have since demonstrated that A3AR agonists have potent analgesic actions in preclinical rodent models of neuropathic pain and that A3AR analgesia is independent of adenosine A1 or A2A unwanted effects. Herein, we explored the contribution of the GABA inhibitory system to A3AR-mediated analgesia using well-characterized mouse and rat models of chronic constriction injury (CCI)-induced neuropathic pain. The deregulation of GABA signaling in pathophysiological pain states is well established: GABA signaling can be hampered by a reduction in extracellular GABA synthesis by GAD65 and enhanced extracellular GABA reuptake via the GABA transporter, GAT-1. In neuropathic pain, GABAAR-mediated signaling can be further disrupted by the loss of the KCC2 chloride anion gradient. Here, we demonstrate that A3AR agonists (IB-MECA and MRS5698) reverse neuropathic pain via a spinal mechanism of action that modulates GABA activity. Spinal administration of the GABAA antagonist, bicuculline, disrupted A3AR-mediated analgesia. Furthermore, A3AR-mediated analgesia was associated with reductions in CCI-related GAD65 and GAT-1 serine dephosphorylation as well as an enhancement of KCC2 serine phosphorylation and activity. Our results suggest that A3AR-mediated reversal of neuropathic pain increases modulation of GABA inhibitory neurotransmission both directly and indirectly through protection of KCC2 function, underscoring the unique utility of A3AR agonists in chronic pain.
• Forte, B., Slosky, L., Staatz, W., Largent‐Milnes, T., & Vanderah, T. (2015). Angiotensin‐(1‐7) as an Antinociceptive Agent in Cancer‐Induced Bone Pain. The FASEB Journal, 29(S1). doi:10.1096/fasebj.29.1_supplement.897.4
• 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.
• Largent-milnes, T. M., Vanderah, T. W., Symons-liguori, A. M., Slosky, L. M., Largent-milnes, T. M., Forte, B. L., Bui, L., & Bassirirad, N. M. (2015). (277) The cystine-glutamate antiporter system xc- drives tumor cell glutamate release and cancer-induced bone pain. The Journal of Pain, 16(4), S45. doi:10.1016/j.jpain.2015.01.195
• Lee, Y. S., Hall, S. M., Ramos-Colon, C., Remesic, M., Rankin, D., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2015). Blockade of non-opioid excitatory effects of spinal dynorphin A at bradykinin receptors. Receptors & clinical investigation, 2(1).
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  Dynorphin A (Dyn A) is an endogenous opioid ligand that possesses neuroinhibitory (antinociceptive) effects via μ, δ, and κ opioid receptors. However, under chronic pain conditions, up-regulated spinal Dyn A can also interact with bradykinin receptors (BRs) to promote hyperalgesia through a neuroexcitatory(pronociceptive) effect. These excitatory effects cannot be blocked by an opioid antagonist, and thus are non-opioid in nature. On the basis of the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin(BK) and kallidin (KD), Dyn A's interaction with BRs could not be predicted, and provided an opportunity to identify a novel potential neuroexcitatory target. Systematic structure-activity relationship (SAR) studies discovered a minimum pharmacophore of Dyn A, [des-Arg(7)]-Dyn A-(4-11) LYS1044 for antagonist activity at the BRs, along with insights into the key structural features for BRs recognition, i.e., amphipathicity. The des-Tyr fragment of dynorphin does not bind to opioid receptors. Intrathecal administration of des-Tyr dynorphin produces hyperalgesia reminiscent of behaviors seen in peripheral n europathic pain models and at higher doses, neurotoxicity. Our lead ligand LYS1044 negatively modulated Dyn A-(2-13)-induced neuroexcitatory effects in naïve animals and blocked mechanical hypersensitivity and thermal hyperalgesia in a dose-dependent manner in animals with experimental neuropathic pain. Based on these results, ligand LYS1044 might prevent abnormal pain states by blocking the neuroexcitatory effects of increased levels of Dyn A that are seen in experimental models of neuropathic pain and that likely promote excitation mediated by BRs in the spinal cord.
• Little, J. W., Ford, A., Symons-Liguori, A. M., Chen, Z., Janes, K., Doyle, T., Xie, J., Luongo, L., Tosh, D. K., Maione, S., Bannister, K., Dickenson, A. H., Vanderah, T. W., Porreca, F., Jacobson, K. A., & Salvemini, D. (2015). Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states. Brain : a journal of neurology, 138(Pt 1), 28-35.
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  Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.
• Nair, P., Yamamoto, T., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2015). Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 25(17), 3716-20.
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  Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.
• Sandweiss, A. J., & Vanderah, T. W. (2015). The pharmacology of neurokinin receptors in addiction: prospects for therapy. Substance abuse and rehabilitation, 6, 93-102.
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  Addiction is a chronic disorder in which consumption of a substance or a habitual behavior becomes compulsive and often recurrent, despite adverse consequences. Substance p (SP) is an undecapeptide and was the first neuropeptide of the neurokinin family to be discovered. The subsequent decades of research after its discovery implicated SP and its neurokinin relatives as neurotransmitters involved in the modulation of the reward pathway. Here, we review the neurokinin literature, giving a brief historical perspective of neurokinin pharmacology, localization in various brain regions involved in addictive behaviors, and the functional aspects of neurokinin pharmacology in relation to reward in preclinical models of addiction that have shaped the rational drug design of neurokinin antagonists that could translate into human research. Finally, we will cover the clinical investigations using neurokinin antagonists and discuss their potential as a therapy for drug abuse.
• Slosky, L. M., & Vanderah, T. W. (2015). Therapeutic potential of peroxynitrite decomposition catalysts: a patent review. Expert opinion on therapeutic patents, 25(4), 443-66.
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  Peroxynitrite is a cytotoxic oxidant species implicated in a host of pathologies, including inflammatory and neurodegenerative diseases, cancer, radiation injury and chronic pain. With the recognition of the role of peroxynitrite in disease, numerous experimental and therapeutic tools have arisen to probe peroxyntirite's pathophysiological contribution and attenuate its oxidative damage. Peroxynitrite decomposition catalysts (PNDCs) are redox-active compounds that detoxify peroxynitrite by catalyzing its isomerization or reduction to nitrate or nitrite.
• 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.
• De Felice, M., Hall, S. M., Hruby, V. J., Hu, J., Lai, J., Lee, Y. S., Muthu, D., Porreca, F., Ramos-Colon, C., Rankin, D., Sandweiss, A. J., Vanderah, T. W., & Xie, J. Y. (2014). Discovery of Amphipathic Dynorphin A Analogues to Inhibit the Neuroexcitatory Effects of Dynorphin A through Bradykinin Receptors in the Spinal Cord. Journal of the American Chemical Society, 136(18), 6608-6616. doi:10.1021/ja501677q
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  We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg(7)]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A's biological activity, is not necessary, and that a type I β-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.
• Petrov, R. R., Lee, Y. S., Vardanyan, R. S., Liu, L., Ma, S. W., Davis, P., Lai, J., Porreca, F., Vanderah, T. W., & Hruby, V. J. (2013). Effect of anchoring 4-anilidopiperidines to opioid peptides. Bioorganic and Medicinal Chemistry Letters, 23(Issue 11). doi:10.1016/j.bmcl.2013.03.065
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  We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides. © 2013 Elsevier Ltd. All rights reserved.
• Slosky, L. M., Thompson, B. J., Sanchez-Covarrubias, L., Zhang, Y., Laracuente, M. L., Vanderah, T. W., Ronaldson, P. T., & Davis, T. P. (2013). Acetaminophen modulates P-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism. Molecular Pharmacology, 84(Issue 5). doi:10.1124/mol.113.086298
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  Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drugdrug interaction that modulates opioid analgesic efficacy. © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Vanderah, T., 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).
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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.
• Vanderah, T., 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).
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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.
• Vanderah, T., Lozano-Ondoua, A. N., Symons-Liguori, A. M., & Vanderah, T. W. (2013). Cancer-induced bone pain: Mechanisms and models. Neuroscience letters.
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  Cancerous cells can originate in a number of different tissues such as prostate, breast and lung, but often go undetected and are non-painful. Many types of cancers have a propensity to metastasize to the bone microenvironment first. Tumor burden within the bone causes excruciating breakthrough pain with properties of ongoing pain that is inadequately managed with current analgesics. Part of this failure is due to the poor understanding of the etiology of cancer pain. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has features distinctive from other chronic pain states. For example, preclinical models of metastatic cancer often result in the positive modulation of neurotrophins, such as NGF and BDNF, that can lead to nociceptive sensitization. Preclinical cancer models also demonstrate nociceptive neuronal expression of acid-sensing receptors, such as ASIC1 and TRPV1, which respond to cancer-induced acidity within the bone. CIBP is correlated with a significant increase in pro-inflammatory mediators acting peripherally and centrally, contributing to neuronal hypersensitive states. Finally, cancer cells generate high levels of oxidative molecules that are thought to increase extracellular glutamate concentrations, thus activating primary afferent neurons. Knowledge of the unique neuro-molecular profile of cancer pain will ultimately lead to the development of novel and superior therapeutics for CIBP.
• Vanderah, T., Podolsky, A. T., Sandweiss, A., Hu, J., Bilsky, E. J., Cain, J. P., Kumirov, V. K., Lee, Y. S., Hruby, V. J., Vardanyan, R. S., & Vanderah, T. W. (2013). Novel fentanyl-based dual μ/δ-opioid agonists for the treatment of acute and chronic pain. Life sciences.
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  Approximately one third of the adult U.S. population suffers from some type of on-going, chronic pain annually, and many more will have some type of acute pain associated with trauma or surgery. First-line therapies for moderate to severe pain include prescriptions for common mu opioid receptor agonists such as morphine and its various derivatives. The epidemic use, misuse and diversion of prescription opioids have highlighted just one of the adverse effects of mu opioid analgesics. Alternative approaches include novel opioids that target delta or kappa opioid receptors, or compounds that interact with two or more of the opioid receptors.
• Vanderah, T., 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).
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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.
• Bilsky, E. J., Booth, R., Dener, J. M., Hanlon, K., Havelin, J., Holsinger, L. J., Ondoua-Lozano, A., Rodriguez, J., & Vanderah, T. W. (2011). Abstract A219: Efficacy of a spectrum-selective cathepsin inhibitor in a mouse model of bone cancer.. Molecular Cancer Therapeutics, 10(11_Supplement), A219-A219. doi:10.1158/1535-7163.targ-11-a219
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  Abstract Introduction: Increased cathepsin levels and activity have been shown to play a role in a variety of tumors, including lung and breast malignancies, and are correlated to poor patient prognosis. Proteolytic activity of cathepsins S, L, B and K may play a role in degradation of the basement membrane and extracellular matrix allowing a loss of cell adhesion and facilitating tumor metastasis. Cathepsin K inhibition has been reported to suppress bone resorption in women with breast cancer by inhibiting osteoclast function. Studies with cathepsin S inhibitors have demonstrated a reduction in nociception in animals, suggesting a role for cathepsin S in tumor-associated neuropathy. We report the efficacy of the spectrum-selective cathepsin inhibitor VBY-825 in a mouse model of metastatic breast cancer. Results and Discussion: VBY-825 is a potent ketoamide-based inhibitor of cathepsins S, B, K, V, and L with pM or single digit nM affinity on this spectrum of cysteine proteases. VBY-825 has been shown to have potent anti-tumor activity in a spontaneous mouse model of pancreatic cancer (Elie et al, 2010, Biochimie). VBY-825 potently inhibited angiogenesis in tumor cell lines, endothelial cell co-cultures and tumor matrix invasion when assayed in an in vitro 3D tumor cell growth assay. Pharmacokinetic behavior of VBY-825 supported once daily dosing resulting in plasma levels sufficient for sustained inhibition of the target cathepsins. Efficacy of VBY-825 was assessed in a murine bone cancer model in which murine 66.1 breast cancer cells are injected and confined to the intramedullary space of the femur of non-immunocompromised mice. Beginning on post surgical day 7 animals received either VBY-825, zoledronic acid, or 5% dextrose vehicle once daily for 14 days. Similarly to the bisphosphonate, VBY-825 in this model significantly reduced bone matrix degradation. VBY-825 also reduced behaviors associated with spontaneous pain including flinching and guarding with significantly greater efficacy than zoledronic acid suggesting that VBY-825 may also induce analgesia against bone cancer pain. Conclusions: These studies demonstrate that the cathepsin inhibitor VBY-825 is effective in a model of bone cancer and associated chronic pain, demonstrating both analgesic activity as well as protection from bone matrix loss and remodeling. These studies support the role of multiple cathepsins as therapeutic targets in metastatic and primary bone cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A219.
• Davis, P., Hruby, V. J., Jacobsen, N. E., Lai, J., Largent-Milnes, T. M., Ma, S., Nair, P., Porreca, F., Vanderah, T. W., Yamamoto, T., & Yamamura, H. I. (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), 2029-2038. doi:10.1021/jm101023r
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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′-(CF3)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 Dmt1 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.
• De Felice, M., Sanoja, R., Wang, R., Vera-Portocarrero, L., Oyarzo, J., King, T., Ossipov, M. H., Vanderah, T. W., Lai, J., Dussor, G. O., Fields, H. L., Price, T. J., & Porreca, F. (2011). Engagement of descending inhibition from the rostral ventromedial medulla protects against chronic neuropathic pain. Pain, 152(Issue 12). doi:10.1016/j.pain.2011.06.008
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  A puzzling observation is why peripheral nerve injury results in chronic pain in some, but not all, patients. We explored potential mechanisms that may prevent the expression of chronic pain. Sprague Dawley (SD) or Holtzman (HZ) rats showed no differences in baseline sensory thresholds or responses to inflammatory stimuli. However, spinal nerve ligation (SNL)-induced tactile allodynia occurred in approximately 85% of SD and 50% of HZ rats, respectively. No apparent differences were observed in a survey of dorsal root ganglion or spinal neuropathic markers after SNL regardless of allodynic phenotype. SNL-induced allodynia was reversed by administration of lidocaine within the rostral ventromedial medulla (RVM), a site that integrates descending pain modulation via pain inhibitory (ie, OFF) and excitatory (ie, ON) cells. However, in SD or HZ rats with SNL but without allodynia, RVM lidocaine precipitated allodynia. Additionally, RVM lidocaine produced conditioned place preference in allodynic SD or HZ rats but conditioned place aversion in nonallodynic HZ rats. Similarly, RVM U69,593 (kappa opioid agonist) or blockade of spinal α 2 adrenergic receptors precipitated allodynia in previously nonallodynic HZ rats with SNL. All rats showed an equivalent first-phase formalin responses. However, HZ rats had reduced second-phase formalin behaviors along with fewer RVM OFF cell pauses and RVM ON cell bursts. Thus, expression of nerve injury-induced pain may ultimately depend on descending modulation. Engagement of descending inhibition protects in the transition from acute to chronic pain. These unexpected findings might provide a mechanistic explanation for medications that engage descending inhibition or mimic its consequences. © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
• Lee, Y. S., Kulkarani, V., Cowell, S. M., Ma, S. W., Davis, P., Hanlon, K. E., Vanderah, T. W., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2011). Development of potent μ and δ opioid agonists with high lipophilicity. Journal of Medicinal Chemistry, 54(Issue 1). doi:10.1021/jm100982d
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  An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-ylpropionamide moiety at the C-terminal was performed and has resulted in highly potent ligands at μ and δ opioid receptors. In general, ligands with the substitution of d-Nle2 and halogenation of the aromatic ring of Phe4 showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay. © 2010 American Chemical Society.
• Vanderah, T., Fioravanti, B., Kasasbeh, A., Edelmayer, R., Skinner, D. P., Hartings, J. A., Burklund, R. D., De Felice, M., French, E. D., Dussor, G. O., Dodick, D. W., Porreca, F., & Vanderah, T. W. (2011). Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats. Cephalalgia : an international journal of headache, 31(10).
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  Cortical spreading depression (CSD) is a wave of depolarization followed by depression of bioelectrical activity that slowly propagates through the cortex. CSD is believed to be the underlying mechanism of aura in migraine; however, whether CSD can elicit pain associated with migraine headache is unclear.
• Vanderah, T., 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.
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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.
• Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Xu, H., Ferng, A. S., Dussor, G., Vanderah, T. W., & Mantyh, P. W. (2010). A phenotypically restricted set of primary afferent nerve fibers innervate the bone versus skin: Therapeutic opportunity for treating skeletal pain. Bone, 46(Issue 2). doi:10.1016/j.bone.2009.09.013
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  Although musculoskeletal pain is one of the most common causes of chronic pain and physical disability in both developing and developed countries, relatively little is known about the nerve fibers and mechanisms that drive skeletal pain. Small diameter sensory nerve fibers, most of which are C-fiber nociceptors, can be separated into two broad populations: the peptide-rich and peptide-poor nerve fibers. Peptide-rich nerve fibers express substance P (SP) and calcitonin gene-related peptide (CGRP). In contrast, the peptide-poor nerve fibers bind to isolectin B4 (IB4) and express the purinergic receptor P2X3 and Mas-related G protein-coupled receptor member d (Mrgprd). In the present report, we used mice in which the Mrgprd+ nerve fibers express genetically encoded axonal tracers to determine the peptide-rich and peptide-poor sensory nerve fibers that innervate the glabrous skin of the hindpaw as compared to the bone marrow, mineralized bone and periosteum of the femur. Whereas the skin is richly innervated by CGRP+, SP+, P2X3+ and Mrgprd+ sensory nerve fibers, the bone marrow, mineralized bone and periosteum receive a significant innervation by SP+ and CGRP+, but not Mrgprd+ and P2X3+ nerve fibers. This lack of redundancy in the populations of C-fibers that innervate the bone may present a unique therapeutic opportunity for targeting skeletal pain as the peptide-rich and peptide-poor sensory nerve fibers generally express a different repertoire of receptors and channels to detect noxious stimuli. Thus, therapies that target the specific types of C-nerve fibers that innervate the bone may be uniquely effective in attenuating skeletal pain as compared to skin pain.
• Vanderah, T., & Vanderah, T. W. (2010). Delta and kappa opioid receptors as suitable drug targets for pain. The Clinical journal of pain, 26 Suppl 10.
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  Similar to mu opioid receptors, kappa and delta opioid receptors reside in the periphery, the dorsal root ganglion, the spinal cord, and in supraspinal regions associated with pain modulation. Both delta and kappa opioid agonists have been shown to activate pain inhibitory pathways in the central nervous system. Yet, currently there are only a few pharmacologic agents that target kappa receptors, and none that target delta receptors. Spurred by the need for an efficacious analgesic without the unwanted side effects associated with the typical clinical profile of mu opioid agonists, new research has provided insight into why the development of effective kappa and delta opioid receptor agonists has remained elusive thus far, and importantly, how these obstacles may be overcome. For example, for delta opioid agonists to be effective, a state of inflammation may be required as this induces delta opioid receptors to migrate to the surface of neuronal cells and thereby become accessible to delta opioid agonists. Studies have shown that delta opioid agonists can provide relief of inflammatory pain and malignant bone pain. Meanwhile, peripherally restricted kappa opioid agonists have been developed to target kappa opioid receptors located on visceral and somatic afferent nerves for relief of inflammatory, visceral, and neuropathic chronic pain. The recently shown efficacy of these analgesics combined with a possible lower abuse potential and side effect burden than mu opioid receptor agonists makes delta and peripherally restricted kappa opioid receptor agonists promising targets for treating pain.
• Vanderah, T., Hanlon, K. E., & Vanderah, T. W. (2010). Constitutive activity at the cannabinoid CB(1) receptor and behavioral responses. Methods in enzymology, 484.
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  The cannabinoid receptor type 1, found mainly on cells of the central and peripheral nervous system, is a major component of the endogenous cannabinoid system. Constitutive and endogenous activity at cannabinoid receptor type 1 regulates a diverse subset of biological processes including appetite, mood, motor function, learning and memory, and pain. The complexity of cannabinoid receptor type 1 activity is not limited to the constitutive activity of the receptor: promiscuity of ligands associated with and the capability of this receptor to instigate G protein sequestration also complicates the activity of cannabinoid receptor type 1. The therapeutic use of cannabinoid receptor type 1 agonists is still a heavily debated topic, making research on the mechanisms underlying the potential benefits and risks of cannabinoid use more vital than ever. Elucidation of these mechanisms and the quest for agonists and antagonists with greater specificity will allow a greater control of the side effects and risks involved in utilizing cannabinoids as therapeutic agents. In this chapter, we review a small subset of techniques used in the pharmacological application of and the behavioral effects of molecules acting at the paradoxical cannabinoid receptor type 1.
• Vanderah, T., 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).
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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.
• Vanderah, T., 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).
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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.
• Vanderah, T., 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).
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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.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Kulkarni, V., Davis, P., Ma, S. W., Navratilova, E., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2010). Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines. Journal of Medicinal Chemistry, 53(Issue 15). doi:10.1021/jm100157m
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  Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with ?-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3?,5?-(CF 3)2-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold ?-selectivity over the ?-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant ?-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug. © 2010 American Chemical Society.
• Lee, Y. S., Petrov, R., Kulkarni, V., Min, B. J., Ma, S. W., Davis, P., Oyarzo, J., Vanderah, T., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2009). Development of mu/delta opioid ligands: enkephalin analogues containing 4-anilidopiperidine moiety.. Advances in experimental medicine and biology, 611. doi:10.1007/978-0-387-73657-0_225
• Davis, P., Hruby, V. J., Lai, J., Largent-Milnes, T., Ma, S., Moye, S., Nair, P., Navratilova, E., Porreca, F., Tumati, S., Vagner, J., Vanderah, T. W., Yamamoto, T., & Yamamura, H. I. (2008). A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists. Journal of Medicinal Chemistry, 51(5), 1369-1376. doi:10.1021/jm070332f
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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 δ and μ receptors (Ki = 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 (IC50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation (Ke = 26 nM) and good affinity for the hNK1 receptor (Ki = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.
• Vanderah, T., Fioravanti, B., & Vanderah, T. W. (2008). The ORL-1 receptor system: are there opportunities for antagonists in pain therapy?. Current topics in medicinal chemistry, 8(16).
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  Following the cloning of the classical opioid receptors (mu, delta and kappa), the opioid receptor like-1 (ORL-1) was identified as a G-protein coupled receptor (GPCR) with 65% structure homology to the other members of the opioid family. Its endogenous ligand nociception/orphanin FQ (N/OFQ) was discovered shortly thereafter, becoming the subject of investigation in numerous studies. Since activation of the ORL-1 receptor by N/OFQ leads to G alpha(i)-coupling and signal transduction similar to that of opioid receptors, N/OFQ was thought to have a role in pain modulation, similar to that of the endogenous opioids. Surprisingly, studies characterizing N/OFQ's effects on pain transmission yielded conflicting results, attributing to N/OFQ both pronociceptive and antinociceptive actions, depending on doses and routes of administration as well as species and sex of the subjects. With the development of selective and potent ORL-1 antagonists, many scientists believed these contradicting actions would be elucidated. Here we review the recent literature reporting the use of novel ORL-1 antagonists, both peptide and non-peptide, in different models of pain and discuss their use as research tools or potential drug candidates.
• Vanderah, T., Fioravanti, B., De Felice, M., Stucky, C. L., Medler, K. A., Luo, M., Gardell, L. R., Ibrahim, M., Malan, T. P., Yamamura, H. I., Ossipov, M. H., King, T., Lai, J., Porreca, F., & Vanderah, T. W. (2008). Constitutive activity at the cannabinoid CB1 receptor is required for behavioral response to noxious chemical stimulation of TRPV1: antinociceptive actions of CB1 inverse agonists. The Journal of neuroscience : the official journal of the Society for Neuroscience, 28(45).
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  The potential modulation of TRPV1 nociceptive activity by the CB(1) receptor was investigated here using CB(1) wild-type (WT) and knock-out (KO) mice as well as selective CB(1) inverse agonists. No significant differences were detected in baseline thermal thresholds of ICR, CB(1)WT or CB(1)KO mice. Intraplantar capsaicin produced dose- and time-related paw flinch responses in ICR and CB(1)WT mice and induced plasma extravasation yet minimal responses were seen in CB(1)KO animals with no apparent differences in TRPV1 channel expression. Capsaicin-evoked CGRP release from spinal cord tissue and capsaicin-evoked action potentials on isolated skin-nerve preparation were significantly decreased in CB(1)KO mice. Pretreatment with intraplantar galanin and bradykinin, compounds known to sensitize TRPV1 receptors, restored capsaicin-induced flinching in CB(1)KO mice. The possibility that constitutive activity at the CB(1) receptor is required to maintain the TRPV1 receptor in a "sensitized" state was tested using CB(1) inverse agonists. The CB(1) inverse agonists elicited concentration-related inhibition of capsaicin-induced calcium influx in F-11 cells and produced dose-related inhibition of capsaicin-induced flinching in ICR mice. These data suggest that constitutive activity at the CB(1) receptor maintains the TRPV1 channel in a sensitized state responsive to noxious chemical stimuli. Treatment with CB(1) inverse agonists may promote desensitization of the channel resulting in antinociceptive actions against chemical stimulus modalities. These studies propose possible therapeutic exploitation of a novel mechanism providing pain relief by CB(1) inverse agonists.
• Vanderah, T., 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).
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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., & Vanderah, T. W. (2007). Pathophysiology of pain. The Medical clinics of North America, 91(1).
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  The processing and interpretation of pain signals is a complex process that entails excitation of peripheral nerves, local interactions within the spinal dorsal horn, and the activation of ascending and descending circuits that comprise a loop from the spinal cord to supraspinal structures and finally exciting nociceptive inputs at the spinal level. Although the "circuits" described here appear to be part of normal pain processing, the system demonstrates a remarkable ability to undergo neuroplastic transformations when nociceptive inputs are extended over time, and such adaptations function as a pronociceptive positive feedback loop. Manipulations directed to disrupt any of the nodes of this pain facilitatory loop may effectively disrupt the maintenance of the sensitized pain state and diminish or abolish neuropathic pain. Understanding the ascending and descending pain facilitatory circuits may provide for the design of rational therapies that do not interfere with normal sensory processing.
• Yamamoto, T., Nair, P., Davis, P., Ma, S. W., Navratilova, E., Moye, S., Tumati, S., Lai, J., Vanderah, T. W., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2007). Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for δ/μ opioid receptor agonists and neurokinin-1 receptor antagonists. Journal of Medicinal Chemistry, 50(Issue 12). doi:10.1021/jm061369n
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  A series of bifunctional peptides that act as agonists for δ and μ opioid receptors with δ selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at δ/μ opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5- Bzl(CF3)2 (7) had excellent agonist activity for both δ opioid and μ opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states. © 2007 American Chemical Society.
• Ibrahim, M. M., Rude, M. L., Stagg, N. J., Mata, H. P., Lai, J., Vanderah, T. W., Porreca, F., Buckley, N. E., Makriyannis, A., & Malan, T. P. (2006). CB2 cannabinoid receptor mediation of antinociception. Pain, 122(Issue 1-2). doi:10.1016/j.pain.2005.12.018
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  Management of acute pain remains a significant clinical problem. In preclinical studies, CB2 cannabinoid receptor-selective agonists inhibit nociception without producing central nervous system side effects. The CB2 receptor-selective agonist AM1241 produces antinociceptive effects that are antagonized by CB2, but not CB1, receptor-selective antagonists, suggesting that activation of CB2 receptors results in antinociception. However, it has not been possible to definitively demonstrate that these effects are mediated by CB2 receptors, because we have lacked the pharmacological tools to confirm the in vivo receptor selectivity of the antagonists used. Further, recent evidence for cannabinoid-like receptors beyond CB1 and CB2 raises the possibility that AM1241 exerts its antinociceptive effects at uncharacterized CB2-like receptors that are also inhibited by AM630. The experiments reported here further test the hypothesis that CB2 receptor activation inhibits nociception. They evaluated the antinociceptive actions of AM1241 and the less-selective CB2 receptor agonist WIN55,212-2 in wild-type ( CB2+ / + ) mice and in mice with genetic disruption of the CB2 receptor ( CB2- / - mice). AM1241 inhibited thermal nociception in CB2+ / + mice, but had no effect in CB2- / - littermates. WIN55,212-2 produced equivalent antinociception in CB1+ / + and CB1- / - mice, while its antinociceptive effects were reduced in CB2- / - compared to CB2+ / + mice. The effects of morphine were not altered in CB2- / - compared to CB2+ / + mice. These data strongly suggest that AM1241 produces antinociception in vivo by activating CB2 cannabinoid receptors. Further, they confirm the potential therapeutic relevance of CB2 cannabinoid receptors for the treatment of acute pain. © 2006 International Association for the Study of Pain.
• King, T., Rao, S., Vanderah, T., Chen, Q., Vardanyan, A., & Porreca, F. (2006). Differential Blockade of Nerve Injury-Induced Shift in Weight Bearing and Thermal and Tactile Hypersensitivity by Milnacipran. Journal of Pain, 7(Issue 7). doi:10.1016/j.jpain.2006.02.001
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  Antidepressants such as tricyclic antidepressants have become used to treat a variety of chronic pain conditions. However, the side effects are dose-limiting in the treatment of chronic pain. Milnacipran is a norepinephrine/serotonin reuptake inhibitor that does not have the severe side effects associated with traditional tricyclic antidepressants. The effects of intrathecal and systemic administration of milnacipran on spinal nerve ligation (SNL)-induced thermal and mechanical hypersensitivity and shift in weight bearing were determined. Intrathecal administration of milnacipran was found to reverse both SNL-induced thermal and tactile (to von Frey filaments) hypersensitivity, as well as shift in weight bearing. Acute systemic administration of milnacipran also reversed nerve injury-induced thermal hypersensitivity for up to 5 hours but failed to reverse tactile hypersensitivity or shift in weight bearing. Of note, both intrathecal and subcutaneous administration of milnacipran induced thermal antinociception in both SNL and sham rats. Chronic (daily) systemic administration of milnacipran alleviated both thermal hypersensitivity and shift in weight bearing, with both acute and chronic effects observed on thermal hypersensitivity. However, chronic systemic milnacipran administration failed to alleviate tactile hypersensitivity to von Frey filaments. These results indicate that different mechanisms underlie shift in weight bearing, thermal hypersensitivity, and tactile hypersensitivity. Perspective: These results indicate that the ability of milnacipran to relieve nerve injury-induced allodynia, hyperalgesia, and shift in weight bearing depends on the route of administration and the duration of treatment, with alleviation of SNL-induced tactile hypersensitivity and shift in weight bearing as a result of activity within the central-rather than the peripheral-nervous system. © 2006 American Pain Society.
• Porreca, F., Vanderah, T. W., Guo, W., Barth, M., Dodey, P., Peyrou, V., Luccarini, J. M., Junien, J. L., & Pruneau, D. (2006). Antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl] methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl)sulfonyl]methylamino]ethoxy] -N-methylacetamide, fumarate (LF22-0542), a novel nonpeptidic bradykinin B 1 receptor antagonist. Journal of Pharmacology and Experimental Therapeutics, 318(Issue 1). doi:10.1124/jpet.105.098368
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  The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl) phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino] ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B 1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.
• Yue, X., Varga, E. V., Stropova, D., Vanderah, T. W., Yamamura, H. I., & Roeske, W. R. (2006). Chronic morphine-mediated adenylyl cyclase superactivation is attenuated by the Raf-1 inhibitor, GW5074. European Journal of Pharmacology, 540(Issue 1-3). doi:10.1016/j.ejphar.2006.04.033
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  The utility of morphine for the treatment of chronic pain is limited by the development of analgesic tolerance. Adenylyl cyclase (AC) superactivation, induced by chronic opioid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present work, we tested the role of Raf-1 in morphine-mediated AC superactivation in CHO cells stably expressing the human μ-opioid receptor. We found that pretreatment of CHO cells stably expressing the human μ-opioid receptor with the selective Raf-1 inhibitor, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one (GW5074, 10 μM, 60 min) completely abolished chronic morphine-mediated AC superactivation (P < 0.01). This finding indicates that Raf-1 may have a crucial role in compensatory feedback regulation of cellular cAMP levels by clinically important opioid analgesics. © 2006 Elsevier B.V. All rights reserved.
• Albrecht, P. J., Davar, G., Ibrahim, M. M., Khodorova, A., Lai, J., Makriyannis, A., Malan, T. P., Mata, H. P., Porreca, F., Rice, F. L., & Vanderah, T. W. (2005). CB ₂ cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proceedings of the National Academy of Sciences, 102(8), 3093-3098. doi:10.1073/pnas.0409888102
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  CB2 cannabinoid receptor-selective agonists are promising candidates for the treatment of pain. CB2 receptor activation inhibits acute, inflammatory, and neuropathic pain responses but does not cause central nervous system (CNS) effects, consistent with the lack of CB2 receptors in the normal CNS. To date, there has been virtually no information regarding the mechanism of CB2 receptor-mediated inhibition of pain responses. Here, we test the hypothesis that CB2 receptor activation stimulates release from keratinocytes of the endogenous opioid β-endorphin, which then acts at opioid receptors on primary afferent neurons to inhibit nociception. The antinociceptive effects of the CB2 receptor-selective agonist AM1241 were prevented in rats when naloxone or antiserum to β-endorphin was injected in the hindpaw where the noxious thermal stimulus was applied, suggesting that β-endorphin is necessary for CB2 receptor-mediated antinociception. Further, AM1241 did not inhibit nociception in μ-opioid receptor-deficient mice. Hindpaw injection of β-endorphin was sufficient to produce antinociception. AM1241 stimulated β-endorphin release from rat skin tissue and from cultured human keratinocytes. This stimulation was prevented by AM630, a CB2 cannabinoid receptor-selective antagonist and was not observed in skin from CB2 cannabinoid receptor-deficient mice. These data suggest that CB2 receptor activation stimulates release from keratinocytes of β-endorphin, which acts at local neuronal μ-opioid receptors to inhibit nociception. Supporting this possibility, CB2 immunolabeling was detected on β-endorphin-containing keratinocytes in stratum granulosum throughout the epidermis of the hindpaw. This mechanism allows for the local release of β-endorphin, where CB2 receptors are present, leading to anatomical specificity of opioid effects. © 2005 by The National Academy of Sciences of the USA.
• Gavva, N. R., Tamir, R., Qu, Y., Klionsky, L., Zhang, T. J., Immke, D., Wang, J., Zhu, D., Vanderah, T. W., Porreca, F., Doherty, E. M., Norman, M. H., Wild, K. D., Bannon, A. W., Louis, J. C., & Treanor, J. J. (2005). AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. Journal of Pharmacology and Experimental Therapeutics, 313(Issue 1). doi:10.1124/jpet.104.079855
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  The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b] [1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 ± 15.7 nM; rat TRPV1, 85.6 ± 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 ± 192 nM; human TRPV1, 92.7 ± 72.8 nM), heat (IC50 value for rat TRPV1, 21 ± 17 nM; human TRPV1, 15.8 ± 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.
• Lai, J., Vera-portocarrero, L. P., Vanderah, T. W., Porreca, F., Lai, J., & King, T. (2005). Ablation of NK-1 expressing cells in the spinal dorsal horn prevents the development of opioid-induced hyperalgesia and antinociceptive tolerance. The Journal of Pain, 6(3), S17. doi:10.1016/j.jpain.2005.01.065
• Vanderah, T., Xie, J. Y., Herman, D. S., Stiller, C., Gardell, L. R., Ossipov, M. H., Lai, J., Porreca, F., & Vanderah, T. W. (2005). Cholecystokinin in the rostral ventromedial medulla mediates opioid-induced hyperalgesia and antinociceptive tolerance. The Journal of neuroscience : the official journal of the Society for Neuroscience, 25(2).
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  Opioid-induced hyperalgesia is characterized by hypersensitivity to innocuous or noxious stimuli during sustained opiate administration. Microinjection of lidocaine into the rostral ventromedial medulla (RVM), or dorsolateral funiculus (DLF) lesion, abolishes opioid-induced hyperalgesia, suggesting the importance of descending pain facilitation mechanisms. Here, we investigate the possibility that cholecystokinin (CCK), a pronociceptive peptide, may drive such descending facilitation from the RVM during continuous opioid administration. In opioid-naive rats, CCK in the RVM produced acute tactile and thermal hypersensitivity that was antagonized by the CCK2 receptor antagonist L365,260 or by DLF lesion. CCK in the RVM also acutely displaced the spinal morphine antinociceptive dose-response curve to the right. Continuous systemic morphine elicited sustained tactile and thermal hypersensitivity within 3 d. Such hypersensitivity was reversed in a time-dependent manner by L365,260 in the RVM, and blockade of CCK2 receptors in the RVM also blocked the rightward displacement of the spinal morphine antinociceptive dose-response curve. Microdialysis studies in rats receiving continuous morphine showed an approximately fivefold increase in the basal levels of CCK in the RVM when compared with controls. These data suggest that activation of CCK2 receptors in the RVM promotes mechanical and thermal hypersensitivity and antinociceptive tolerance to morphine. Enhanced, endogenous CCK activity in the RVM during sustained morphine exposure may diminish spinal morphine antinociceptive potency by activating descending pain facilitatory mechanisms to exacerbate spinal nociceptive sensitivity. Prevention of opioid-dose escalation in chronic pain states by CCK receptor antagonism represents a potentially important strategy to limit unintended enhanced clinical pain and analgesic tolerance
• Varga, E. V., Hosohata, K., Borys, D., Navratilova, E., Nylen, A., Vanderah, T. W., Porreca, F., Roeske, W. R., & Yamamura, H. I. (2005). Antinociception depends on the presence of G protein γ2- subunits in brain. European Journal of Pharmacology, 508(Issue 1-3). doi:10.1016/j.ejphar.2004.11.062
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  We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein γ2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the γ2 subunit (Gγ2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a δ-opioid receptor agonist. In the present study, we examined the involvement of Gγ2 in antinociception mediated by other (μ- or κ-opioid, cannabinoid, α2-adrenoreceptor) analgesic agents in a warm (55°C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein γ2 subunits in supraspinal antinociception. © 2004 Elsevier B.V. All rights reserved.
• Chen, Q., King, T., Vanderah, T. W., Ossipov, M. H., Malan, T. P., Lai, J., & Porreca, F. (2004). Differential blockade of nerve injury-induced thermal and tactile hypersensitivity by systemically administered brain-penetrating and peripherally restricted local anesthetics. Journal of Pain, 5(Issue 5). doi:10.1016/j.jpain.2004.05.002
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  Systemic administration of local anesthetics has been shown to transiently reverse thermal and tactile hypersensitivity induced by peripheral nerve injury, effects that have been taken as suggesting direct actions on the peripheral nerves. The present study sought to determine whether a central site of action could contribute to, or account for, the effects of lidocaine on nerve injury-induced thermal and tactile hypersensitivity. Systemic lidocaine and its peripherally restricted analogues, QX-314 or QX-222, effectively reversed thermal hypersensitivity after spinal nerve ligation injury. Nerve injury-induced tactile hypersensitivity, however, was reversed by systemic lidocaine but not QX-314 or QX-222. Microinjection of either lidocaine or QX-314 into the rostral ventromedial medulla fully reversed spinal nerve ligation-induced thermal and tactile hypersensitivity. The data strongly suggest that nerve injury-induced thermal and tactile hypersensitivity are mediated through different mechanisms. In addition, the present study supports a prominent contribution of the central nervous system in the activity of systemically given lidocaine against nerve injury-induced tactile and thermal hypersensitivity. Thus, lidocaine might reverse tactile hypersensitivity mainly through its actions within the central nervous system, whereas its reversal of thermal hypersensitivity might occur through either central or peripheral sites. Perspective Nerve injury-induced neuropathic pain has proved remarkably difficult to treat. Systemic administration of ion channel blockers such as lidocaine has been explored for the management of chronic pain. This work indicates that systemic rather than local administration of lidocaine would be more effective in treating tactile allodynia. © 2004 by the American Pain Society.
• Wang, R., Guo, W., Ossipov, M. H., Vanderah, T. W., Porreca, F., & Lai, J. (2004). Erratum: Glial cell line-derived neurotrophic factor normalizes neurochemical changes in injured dorsal root ganglion neurons and prevents the expression of experimental neuropathic pain (Neuroscience 121: 3 (815-824) DOI: 10.1016/S0306-4522(03)00491-3). Neuroscience, 126(Issue 1). doi:10.1016/j.neuroscience.2004.03.032
• Cockayne, D. A., Deng, H., Ibrahim, M. M., Kwan, J., Lai, J., Makriyannis, A., Malan, T. P., Mata, H. P., Porreca, F., Vanderah, T. W., & Zvonok, A. (2003). Activation of CB ₂ cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: Pain inhibition by receptors not present in the CNS. Proceedings of the National Academy of Sciences, 100(18), 10529-10533. doi:10.1073/pnas.1834309100
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  We designed AM1241, a selective CB2 cannabinoid receptor agonist, and used it to test the hypothesis that CB2 receptor activation would reverse the sensory hypersensitivity observed in neuropathic pain states. AM1241 exhibits high affinity and selectivity for CB2 receptors. It also exhibits high potency in vivo. AM1241 dose-dependently reversed tactile and thermal hypersensitivity produced by ligation of the L5 and L6 spinal nerves in rats. These effects were selectively antagonized by a CB2 but not by a CB1 receptor antagonist, suggesting that they were produced by actions of AM1241 at CB2 receptors. AM1241 was also active in blocking spinal nerve ligation-induced tactile and thermal hypersensitivity in mice lacking CB1 receptors (CB1-/- mice), confirming that AM1241 reverses sensory hypersensitivity independent of actions at CB1 receptors. These findings demonstrate a mechanism leading to the inhibition of pain, one that targets receptors localized exclusively outside the CNS. Further, they suggest the potential use of CB2 receptor-selective agonists for treatment of human neuropathic pain, a condition currently without consistently effective therapies. CB2 receptor-selective agonist medications are predicted to be without the CNS side effects that limit the effectiveness of currently available medications.
• Gardell, L. R., Vanderah, T. W., Gardell, S. E., Wang, R., Ossipov, M. H., Lai, J., & Porreca, F. (2003). Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation. Journal of Neuroscience, 23(Issue 23). doi:10.1523/jneurosci.23-23-08370.2003
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  Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing μ-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.
• Hosohata, K., Varga, E. V., Alfaro-Lopez, J., Tang, X., Vanderah, T. W., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). (2S,3R)β-methyl-2′,6′-dimethyltyrosine-L- tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] is a potent, selective δ opioid receptor antagonist in mouse brain. Journal of Pharmacology and Experimental Therapeutics, 304(Issue 2). doi:10.1124/jpet.102.042929
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  The constrained opioid peptide (2S,3R)β-methyl-2′,6′-dimethyltyrosine-L- tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the δ-opioid receptors (Liao et al., 1997). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5′-O-(3-[35S]thiotriphosphate) ([35S]GTPγS) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. δ-(SNC80; (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl) -3-methoxy-benzyl]-N,N-diethyl-benzamide) or μ- (DAMGO; [D-Ala2, Me-Phe4, Gly(ol)5]enkephalin) selective opioid full agonists stimulated [35S]GTPγS binding in mouse brain membranes 150 ± 4.5% and 152 ± 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [35S]GTP±S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a Ke value of 3.6 ± 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the μ-selective opioid agonist, DAMGO. Warm water (55°C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGOmediated antinociception (40 ± 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral δ-opioid antagonist in mouse brain.
• Wessells, H., Hruby, V., Hackett, J., Han, G., Balse-Srinivasan, P., & Vanderah, T. (2003). Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 induces penile erection via brain and spinal melanocortin receptors. Neuroscience, 118(3). doi:10.1016/S0306-4522(02)00866-7
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  Penile erection induced by α-melanocyte-stimulating hormone and melanocortin receptors (MC-R) in areas of the spinal cord and periphery has not been demonstrated. To elucidate sites of the proerectile action of melanocortin peptides, in awake male rats we administered the MC-R agonist Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 (MT-II) i.c.v., intrathecal (i.th.) and i.v. and scored penile erection and yawning. Injection of the MC-R antagonist Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2 (SHU-9119) i.c.v. or i.th. in combination with i.th. MT-II differentiated spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracavernous pressure responses to intracavernosal injection of MT-II in the anesthetized rat. I.c.v., i.th., and i.v. MT-II induced penile erections in a dose-dependent fashion. Yawning was observed with i.c.v. and i.v. MT-II, while spinal injection did not produce this behavior. Intrathecal delivery of MT-II to the lumbosacral spinal cord was more efficacious in inducing erections than i.c.v. or i.v. administration; SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracavernous pressure nor augmented neurostimulated erectile responses. We confirmed the central proerectile activity of MT-II and demonstrated that in addition to a site of action in the brain, the distal spinal cord contains melanocortin receptors that can initiate penile erection independent of higher centers. These results provide new insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
• Gardell, L., Ossipov, M., Vanderah, T., Lai, J., & Porreca, F. (2002). Dynorphin-independent spinal cannabinoid antinociception. Pain, 100(3). doi:10.1016/S0304-3959(02)00173-2
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  Spinal antinociception produced by delta 9-tetrahydro-cannabinol (Δ9-THC) and other cannabinoid agonists has been suggested to be mediated by the release of dynorphin acting at the kappa opioid receptor. Alternatively, as cannabinoid receptors are distributed appropriately in the pain transmission pathway, cannabinoid agonists might act directly at the spinal level to inhibit nociception, without requiring dynorphin release. Here, these possibilities were explored using mice with a deletion of the gene encoding prodynorphin. Antinociceptive dose-response curves were constructed for spinal Δ9-THC and WIN 55,212-2 in prodynorphin knock-out mice and in wild-type littermates. WIN 55,212-2 and Δ9-THC were equipotent in the wild-type and prodynorphin knock-out mice. Spinal pretreatment with a kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), did not alter the dose-response curves for either WIN 55,212-2 or Δ9-THC in prodynorphin knock-out and wild-type mice. However, the same dose of nor-BNI used blocked U50,488H-induced antinociception in both wild-type and prodynorphin knock-out mice, confirming kappa opioid receptor activity. Pretreatment with SR141716A, a cannabinoid receptor antagonist blocked the antinociceptive actions of both WIN 55,212-2 and Δ9-THC. These data support the conclusion that antinociception produced by spinal cannabinoids are likely to be mediated directly through activation of cannabinoid receptors without the requirement for dynorphin release or activation of kappa opioid receptors. © 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.
• Lai, J., Park, E. W., Porreca, F., Rasband, M. N., Trimmer, J. S., & Vanderah, T. W. (2001). Distinct potassium channels on pain-sensing neurons. Proceedings of the National Academy of Sciences, 98(23), 13373-13378. doi:10.1073/pnas.231376298
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  Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.
• Malan, T., Ibrahim, M., Deng, H., Liu, Q., Mata, H., Vanderah, T., Porreca, F., & Makriyannis, A. (2001). CB2 cannabinoid receptor-mediated peripheral antinociception. Pain, 93(3). doi:10.1016/S0304-3959(01)00321-9
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  Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence implicates the CB1 receptor in the production of antinociception. However, the capacity of CB2 receptors, which are located outside the central nervous system (CNS), to produce antinociception is not known. Using AM1241, a CB2 receptor-selective agonist, we demonstrate that CB2 receptors produce antinociception to thermal stimuli. Injection of AM1241 in the hindpaw produced antinociception to a stimulus applied to the same paw. Injection of an equivalent dose of AM1241 into the paw contralateral to the side of testing did not. The antinociceptive actions of AM1241 were blocked by the CB2 receptor-selective antagonist AM630, but not by the CB1 receptor-selective antagonist AM251. AM1241 also produced antinociception when injected systemically (intraperitoneally). The antinociceptive actions of systemic AM1241 were blocked by injection of AM630 into the paw where the thermal stimulus was applied, but not the contralateral paw. These findings demonstrate the local, peripheral nature of CB2 cannabinoid antinociception. AM1241 did not produce the CNS cannabinoid effects of hypothermia, catalepsy, inhibition of activity or impaired ambulation, while this tetrad of effects was produced by the mixed CB1/CB2 receptor agonist WIN55,212-2. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB2 receptors. CB2 receptor agonists may have promise clinically for the treatment of pain without CNS cannabinoid side effects. © 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.
• Vanderah, T. W., Gardell, L. R., Burgess, S. E., Ibrahim, M., Dogrul, A., Zhong, C. M., Zhang, E. T., Malan, T. P., Ossipov, M. H., Lai, J., & Porreca, F. (2000). Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance. Journal of Neuroscience, 20(Issue 18). doi:10.1523/jneurosci.20-18-07074.2000
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  The nonopioid actions of spinal dynorphin may promote aspects of abnormal pain after nerve injury. Mechanistic similarities have been suggested between opioid tolerance and neuropathic pain. Here, the hypothesis that spinal dynorphin might mediate effects of sustained spinal opioids was explored. Possible abnormal pain and spinal antinociceptive tolerance were evaluated after intrathecal administration of [D-Ala2, N-Me-Phe4, Gly-ol5]enke phalin (DAMGO), an opioid μ agonist. Rats infused with DAMGO, but not saline, demonstrated tactile allodynia and thermal hyperalgesia of the hindpaws (during the DAMGO infusion) and a decrease in antinociceptive potency and efficacy of spinal opioids (tolerance), signs also characteristic of nerve injury. Spinal DAMGO elicited an increase in lumbar dynorphin content and a decrease in the μ receptor immunoreactivity in the spinal dorsal horn, signs also seen in the postnerve-injury state. Intrathecal administration of dynorphin A(1-17) antiserum blocked tactile allodynia and reversed thermal hyperalgesia to above baseline levels (i.e., antinociception). Spinal dynorphin antiserum, but not control serum, also reestablished the antinociceptive potency and efficacy of spinal morphine. Neither dynorphin antiserum nor control serum administration altered baseline non-noxious or noxious thresholds or affected the intrathecal morphine antinociceptive response in saline-infused rats. These data suggest that spinal dynorphin promotes abnormal pain and acts to reduce the antinociceptive efficacy of spinal opioids (i.e., tolerance). The data also identify a possible mechanism for previously unexplained clinical observations and offer a novel approach for the development of strategies that could improve the long-term use of opioids for pain.
• Wegert, S., Ossipov, M., Nichols, M., Bian, D., Vanderah, T., Malan, T., & Porreca, F. (1997). Differential activities of intrathecal MK-801 or morphine to alter responses to thermal and mechanical stimuli in normal or nerve-injured rats. Pain, 71(1). doi:10.1016/S0304-3959(97)03337-X
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  Nerve ligation injury in rats results in reduced nociceptive and non- nociceptive thresholds, similar to some aspects of clinical conditions of neuropathic pain. Since underlying mechanisms of hyperalgesia and allodynia may differ, the present study investigated the pharmacology of morphine and MK-801 in rats subjected to a tight ligation of the L5 and L6 nerve roots or to a sham-operation procedure. Response to acute nociception was measured by (a) withdrawal of a hindpaw from a radiant heat source, (b) withdrawal of the tail from a radiant heat source or (c) the latency to a rapid flick of the tail following immersion in water at different noxious temperatures. Mechanical thresholds were determined by measuring response threshold to probing the hindpaw with von Frey filaments. Nerve ligation produced a significant, stable and long-lasting decrease in threshold to mechanical stimulation (i.e., tactile allodynia) when compared to sham-operated controls. Standardization of the diameter of the filaments (to that of the largest filament) did not alter the response threshold in nerve injured animals. Nerve ligation produced decreased response latency of the ipsilateral paw (i.e., hyperalgesia) when compared to that of sham-operated rats. Tail-flick latencies to thermal stimuli induced by water at constant temperatures (48°, 52°or 55°C) or by radiant heat were not significantly different between nerve-injured and sham-operated groups. At doses which were not behaviorally toxic, MK-801 had no effect on tactile allodynia. At these doses, MK-801 blocked decreased paw withdrawal latency to radiant heat in nerve-injured rats, but did not significantly elevate the response threshold of sham-operated rats. Systemic (i.p.) or intracerebroventricular (i.c.v.) doses of morphine previously shown to be antiallodynic in nerve-ligated rats did not affect the response to probing with von Frey filaments in sham- operated controls. Intrathecal (i.t.) morphine did not change paw withdrawal thresholds elicited by yon Frey filaments of either nerve-ligated rats (as previously reported) or of sham-operated rats at doses maximally effective against thermal stimuli applied to the tail or foot. Spinal morphine produced dose-dependent antinociception in both nerve-injured and sham-operated groups in the foot-flick test but was less potent in the nerve-injured group. Presuppression of hyperalgesia of the foot with i.t. MK-801 in nerve-injured animals did not alter the potency of i.t. morphine. I.t. morphine was also active in the tail-flick tests with decreased potency in nerve-injured animals and, at some stimulus intensities, with a decreased efficacy as well. These data emphasize the distinction between the inactivity of morphine to suppress mechanical withdrawal thresholds (as elicited by yon Frey filaments) and the activity of this compound to block the response to an acute thermal nociceptive stimulus in sham-operated or nerve-injured rats. It appears that nerve ligation injury produces a thermal allodynia/hyperalgesia which is likely dependent upon opioid-sensitive small-diameter primary afferent fibers and a mechanical allodynia which may be largely independent of small-fiber input.
• Edsall, S. A., Knapp, R. J., Vanderah, T. W., Roeske, W. R., Consroe, P., & Yamamura, H. I. (1996). Antisense oligodeoxynucleotide treatment to the brain cannabinoid receptor inhibits antinociception. NeuroReport, 7(Issue 2). doi:10.1097/00001756-199601310-00052
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  Studies using agonists from at least three major cannabinoid ligand groups suggest the mediation of several distinct effects (e.g. psychotropic, analgesia, and antiemetic) by the recently cloned CB1 cannabinoid receptor. However, other studies suggest the presence of multiple cannabinoid receptors and at least one other receptor (CB2) has been cloned. The present investigation was undertaken to determine whether one of the potential therapeutic actions of cannabinoids (i.e. antinociception) is mediated by the CB1 receptor using the antisense oligodeoxynucleotide 'knock-down' approach. Synthetic oligodeoxynucleotides complementary to the 5' end of the coding region of the mouse CB1 receptor mRNA were administered to mice by the intracerebroventricular (i.c.v.) route twice daily for 3 days. Mismatch oligodeoxynucleotides of similar sequence, but containing six mismatched positions out of the 18 nucleotides within the oligodeoxynucleotide were administered to other mice. Treatment with antisense oligodeoxynucleotides, but not mismatched oligodeoxynucleotides, greatly inhibited the antinociceptive response of the cannabinoid agonist CP-55,940. Untreated mice and those treated with mismatched oligodeoxynucleotides showed similar, full response antinociception after CP-55,940 administration. The data provides strong evidence that the CB1 receptor-ligand interaction is essential for the antinociceptive effect.
• Vanderah, T., Bernstein, R., Yamamura, H., Hruby, V., & Porreca, F. (1996). Enhancement of morphine antinociception by a CCKB antagonist in mice is mediated via opioid delta receptors. Journal of Pharmacology and Experimental Therapeutics, 278(1).
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  This study investigated the possible involvement of opioid δ receptors in the modulation of morphine antinociceptive potency produced by L365,260, a CCKB antagonist, Intracerebroventricular (i.c.v.), intrathecal (i.th.) or subcutaneous (s.c.) L365,260 alone did not produce any antinociceptive actions in the mouse warm-water tail-flick test. Treatment with L.365,260 by any of these routes produced a leftward shift of the corresponding morphine dose-effect curve that was blocked by pretreatment with a receptor-selective dose of s.c. naltrindole, an opioid δ receptor antagonist. Pretreatment with i.c.v. antisera to [Leu5]enkephalin also blocked the leftward displacement of the i.c.v. morphine dose-effect curve resulting from L.365,260 but did not directly alter the i.c.v. morphine doseeffect curve; antisera to [Met5]enkephalin did not alter the effects of morphine or the modulation of morphine antinociception produced by L365,260. Repeated pretreatment with L365,260 resulted in a progressive decrease in the magnitude of the morphine modulatory action (i.e., L365,260 "tolerance"). In these "L365,260-tolerant" mice, the dose-effect curve for i.c.v. [D-Ala2, Glu4]deltorphin (a selective δ agonist) was displaced to the right by approximately 8.2-fold. The i.c.v. administration of [Leu5]enkephalin produced a leftward displacement of the i.c.v. morphine dose-effect curve that diminished after repeated administration (i.e., [Leu5]enkephalin "tolerance"). In "[Leu5]enkephalin-tolerant" mice, L.365,260 failed to produce the leftward shift of the morphine dose-effect curve seen in control animals. That is, two-way antinociceptive cross-tolerance was observed between an opioid δ agonist and a CCKB receptor antagonist. Intracerebroventricular thiorphan, a peptidase inhibitor, did not elicit antinociception directly. Co-administration of thiorphan with L365,260 elicited significant antinociception that was blocked by naltrindole or antisera to [Leu5]enkephalin; antisera to [Met5]enkephalin had no effect. Repeated administration of i.c.v. [D-Ala2, Glu4]deltorphin resulted in a progressively decreasing antinociceptive effect (i.e., [D-Ala2, Glu4]deltorphin "tolerance"). In "[D-Ala2, Glu4]deltorphintolerant" mice, the thiorphan/L365,260 antinociceptive effect was inhibited. Collectively, these data suggest that CCK interacts at the CCKB receptor to inhibit tonically the release and/or availability of an endogenous substance acting at opioid δ receptors. The subsequent enhancement of morphine antinociceptive potency may reflect the well-known modulation of morphine antinociception produced by opioid δ receptor agonists. In this case, the latter may be [Leu5]enkephalin or a [Leu5]enkephalin-like substance.
• Vanderah, T. W., Lai, J., Yamamura, H. I., & Porreca, F. (1994). Antisense oligodeoxynucleotide to the CCKB receptor produces naltrindole- and [Leu5]enkephalin antiserum-sensitive enhancement of morphine antinociception. NeuroReport, 5(Issue 18). doi:10.1097/00001756-199412000-00049
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  Cholecystokinin (CCK) has been shown to attenuate, while CCK antagonists enhance, the antinociceptive activity of morphine, suggesting that this peptide may act as an endogenous modulator of the opioid system. Here, we have investigated the effects of administration of a synthetic oligodeoxynucleotide (oligo) complementary to the 5' coding region of the cloned mouse CCK„ receptor (antisense), or a mismatch oligo, on the antinociceptive effects of morphine. Intracerebroventricular (i.c.v.) treatment of mice with CCK„ antisense, but not mismatch, oligo for 3 days resulted in an enhancement of the antinociceptive potency of i.c.v. morphine, as indicated by an approximately 6-fold leftward shift of the dose-effect curve. The antinociceptive effects of morphine in control and CCK„ antisense-treated animals were investigated in the presence or absence of naltrindole, an opioid 8 receptor antagonist, as well as in the presence or absence of antisera directed against either [Leu5]- or [Mets]enkephalin. The enhanced potency of morphine in mice pretreated with CCK„ antisense oligo was blocked by a-selective dose of naltrindole and antisera to [Leu5]enkephalin, but not [Met5]enkephalin; naltrindole, or antisera towards [Leu5]enkephalin or [Met5]enkephalin did not produce antinociceptive effects when given alone and did not alter the antinociceptive actions of morphine in control mice. These data suggest that CCK may act via CCK„ receptors to tonically inhibit the release of [Leu5]enkephalin, or a [Leu5]enkephalin-like peptide. The enhancement of morphine antinociception seen in the presence of blockade of the CCK„ receptor may be the result of the well-known enhancement of morphine antinociception by opioid 8 agonists. © Rapid Communications of Oxford Ltd.
• Mattia, A., Vanderah, T., Raffa, R. B., Vaught, J. L., Tallarida, R. J., & Porreca, F. (1993). Characterization of the unusual antinociceptive profile of tramadol in mice. Drug Development Research, 28(Issue 2). doi:10.1002/ddr.430280208
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  The present study characterized the antinociceptive effects of tramadol with emphasis on the site of action, the possible development of antinociceptive tolerance, and possible cross‐tolerance to morphine. Both tramadol and morphine produced antinociception as assessed in male, ICR mice using the warm water (55°C) tail‐flick test. Intrathecal (i.t.) tramadol produced an antinociceptive response, but with a shallow dose‐response curve (DRC), but i.c.v. tramadol was ineffective up to toxic doses (> 380 nmol). Simultaneous co‐administration of tramadol by the i.c.v. and i.t. routes in a 1:1 fixed dose‐ratio resulted in a synergistic interaction. Construction of a naloxone dose‐response curve against an A90 dose of agonist yielded an AD50 (and 95% C.L.) of 0.19 (0.14–0.33) and 0.55 (0.38–0.74) μmol/kg against morphine and tramadol, respectively; both agonists were completely antagonized by naloxone. In mice pretreated twice daily (8:00 a.m. and 5:00 p.m.) with s.c. morphine the DRC for morphine was displaced 16‐fold to the right, indicating significant development of antinociceptive tolerance. The same pretreatment resulted in only a 3‐fold right‐ward shift in the tramadol DRC. Chronic twice‐daily (8:00 a.m. and 5:00 p.m.) pretreatment with tramadol resulted in only an approximate 3‐ and 4‐fold rightward displacement in the tramadol and morphine DRCs, respectively. These results suggest that tramadol has an unusual antinociceptive mechanism which may reflect a primary site of action at spinal sites with a synergistic spinal/supraspinal interaction. Further, the lack of complete cross‐tolerance between tramadol and morphine supports the suggestion of a non‐opioid mechanism for this compound, whereas the complete antagonism by naloxone apparently reflects the opioid component of its mechanism in this test. Finally, the minimal development of tolerance to tramadol antinociception reinforces the view of a favorable tolerance‐dependence profile for this compound. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.
• Wild, K., McCormick, J., Bilsky, E., Vanderah, T., McNutt, R., Chang -, K., & Porreca, F. (1993). Antinociceptive actions of BW373U86 in the mouse. Journal of Pharmacology and Experimental Therapeutics, 267(2).
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  This study explored the antinociceptive properties of (±)-4-[(α-F*)- α-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinol]-3-hydroxybenzyl]-N,N- diethyl-benzamide dihydrochloride (BW373U86) a nonpeptidic compound proposed to be a delta opioid agonist, using three models of nociception and four routes of administration in mice. BW373U86 produced dose- and time-dependent, naloxone-sensitive antinociception in both the tail-flick and tail-pinch assays when given intrathecally (i.t.). However, at doses up to 187 nmol/mouse, i.c.v. BW373U86 was inactive in the tail-flick or tail-pinch assays. Additionally, at doses up to 187 μmol/kg, BW373U86 was not active after i.p. or p.o. administration in these endpoints. Further, in the tail- flick test, i.c.v. BW373U86 did not antagonize the antinociceptive effects of i.c.v. [D-Pen2,D-Pen5]enkephalin or [D-Ala2,Glu4]deltorphin, but partially antagonized the effects of i.c.v. morphine. In the acetic-acid abdominal constriction assay. BW373U86 produced dose-dependent antinociceptive effects when given by the i.p., i.c.v. or i.t., but not by the p.o., routes. Intrathecal BW373U86 was 663-fold more potent in the abdominal constriction assay than when given by the same route in the tail- flick test. The effects of an A70 dose of i.p. or i.c.v. BW373U86 in the abdominal constriction assay were partially antagonized by i.c.v. naloxone, but not by i.c.v. N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, where Aib is α- aminoisobutyric acid (ICI-174,864) or naltrindole. In contrast, i.t. naloxone. ICI-174,864 or naltrindole antagonized the antinociceptive effect of i.p. or i.t. BW373U86 in the abdominal constriction assay. These data suggest that BW373U86 can be classified as a partial opioid agonist with actions at delta as well as mu receptors. The actions of lower doses of BW373U86 at spinal levels appears to involve a delta mechanism whereas supraspinal effects appear to be associated with interactions at mu sites.

Presentations

• Coffee, Z., Vanderah, T. W., Gordon, J. S., Taylor-Piliae, R. E., & Badger, T. A. (2024).

   Pilot feasibility of a virtual Tai Chi intervention for opioid use disorder and co-occurring symptoms.

  . Poster presented at the California Society of Addiction Medicine Conference. San Francisco, CA..

Poster Presentations

• Coffee, Z., Vanderah, T. W., Gordon, J. S., Taylor-Piliae, R. E., & Badger, T. A. (2024). Pilot feasibility of a virtual Tai Chi intervention for opioid use disorder and co-occurring symptoms.. California Society of Addiction Medicine Conference.
• Vanderah, T. W., Ibrahim, M. M., Jiralerspong, S., Ehsani Chimeh, S., Roe, D., Junak, M., Farr, K., & Segar, J. (2022, December). Evaluation of Dronabinol to Decrease Opioid Use for Cancer- Induced Bone Pain. San Antonio Breast Cancer Symposium. San Antonio Texas.
• 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.

Creative Productions

• Vanderah, T. W., Rice, A., Ricker, M. A., & Min Simpkins, A. A. (2020. FLEXcast - EPISODE 2: Time Management.