Arjun Muralidharan

A small molecule angiotensin II type 2 receptor (AT2R) antagonist produces analgesia in a rat model of neuropathic pain by inhibition of p38 Mitogen-Activated Protein Kinase (MAPK) and p44/p42 MAPK activation in the dorsal root ganglia

OBJECTIVE There is an unmet clinical need for novel analgesics for neuropathic pain. This study was designed to elucidate the mechanism through which EMA300, a small molecule antagonist of the angiotensin II type 2 receptor (AT₂R) with >1,000-fold selectivity over the angiotensin II type 1 receptor, produces analgesia in a rodent model of neuropathic pain. DESIGN AND METHODS Groups of AT₂R knockout, hemizygotes, and wild-type mice with a chronic constriction injury (CCI) of the sciatic nerve received single intraperitoneal (i.p.) bolus doses of EMA300 (100 or 300 mg/kg), and analgesic efficacy was assessed. Groups of control, sham-operated, and CCI rats were euthanized and perfusion fixed. Lumbar dorsal root ganglia (DRGs) were removed for investigation of the mechanism through which EMA300 alleviates neuropathic pain. RESULTS EMA300 analgesia was abolished in AT₂R knockout CCI mice with intermediate responses in the hemizygotes, affirming the AT₂R as the target mediating EMA300 analgesia. In CCI rats, DRG immunofluorescence (IF) levels for angiotensin II, the main endogenous ligand of the AT₂R, were increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. Mean DRG IF levels for activated p38 (pp38) and activated p44/p42 (pp44/pp42) MAPK were also increased ∼1.5-2.0-fold (P < 0.05) cf. sham-controls. At the time of peak EMA300 analgesia in CCI rats, mean DRG levels of pp38 MAPK and pp44/pp42 MAPK (but not angiotensin II) were reduced to match the respective levels in sham-controls. CONCLUSION Augmented angiotensin II/AT₂R signaling in the DRGs of CCI rats is attenuated by EMA300 to block p38 MAPK and p44/p42 MAPK activation, a mechanism with clinical validity for alleviating neuropathic pain.

Pain, analgesia and genetics

Objectives  In the clinical setting, there is marked intersubject variability in the intensity of pain reported by patients with apparently similar pain states, as well as widely differing analgesic dosing requirements between individuals to produce satisfactory pain relief with tolerable side‐effects. Genetic and environmental factors as well as their interaction are implicated, and these are discussed in this review.

Pathobiology and management of prostate cancer-induced bone pain: recent insights and future treatments

Prostate cancer (PCa) has a high propensity for metastasis to bone. Despite the availability of multiple treatment options for relief of PCa-induced bone pain (PCIBP), satisfactory relief of intractable pain in patients with advanced bony metastases is challenging for the clinicians because currently available analgesic drugs are often limited by poor efficacy and/or dose-limiting side effects. Rodent models developed in the past decade show that the pathobiology of PCIBP comprises elements of inflammatory, neuropathic and ischemic pain arising from ectopic sprouting and sensitization of sensory nerve fibres within PCa-invaded bones. In addition, at the cellular level, PCIBP is underpinned by dynamic cross talk between metastatic PCa cells, cellular components of the bone matrix, factors associated with the bone microenvironment as well as peripheral components of the somatosensory system. These insights are aligned with the clinical management of PCIBP involving use of a multimodal treatment approach comprising analgesic agents (opioids, NSAIDs), radiotherapy, radioisotopes, cancer chemotherapy agents and bisphosphonates. However, a major drawback of most rodent models of PCIBP is their short-term applicability due to ethical concerns. Thus, it has been difficult to gain insight into the mal(adaptive) neuroplastic changes occurring at multiple levels of the somatosensory system that likely contribute to intractable pain at the advanced stages of metastatic disease. Specifically, the functional responsiveness of noxious circuitry as well as the neurochemical signature of a broad array of pro-hyperalgesic mediators in the dorsal root ganglia and spinal cord of rodent models of PCIBP is relatively poorly characterized. Hence, recent work from our laboratory to develop a protocol for an optimized rat model of PCIBP will enable these knowledge gaps to be addressed as well as identification of novel targets for drug discovery programs aimed at producing new analgesics for the improved relief of intractable PCIBP.

Analgesic Efficacy and Mode of Action of a Selective Small Molecule Angiotensin II Type 2 Receptor Antagonist in a Rat Model of Prostate Cancer-Induced Bone Pain

OBJECTIVE The pathobiology of prostate cancer (PCa)-induced bone pain (PCIBP) has both inflammatory and neuropathic components. Previously, we showed that small molecule angiotensin II type 2 receptor (AT2 R) antagonists with >1,000-fold selectivity over the angiotensin II type 1 receptor produced dose-dependent analgesia in a rat model of neuropathic pain. Here, we assessed the analgesic efficacy and mode of action of the AT2 R antagonist, EMA200, in a rat model of PCIBP. METHODS At 14-21 days after unilateral intratibial injection of AT3B PCa cells, rats exhibiting hindpaw hypersensitivity received single intravenous bolus doses of EMA200 (0.3-10 mg/kg) or vehicle, and analgesic efficacy was assessed. The mode of action was investigated using immunohistochemical, Western blot, and/or molecular biological methods in lumbar dorsal root ganglia (DRGs) removed from drug-naïve and EMA200-treated PCIBP rats relative to sham-control rats. RESULTS Intravenous bolus doses of EMA200 produced dose-dependent analgesia in PCIBP rats. Lumbar DRG levels of angiotensin II, nerve growth factor (NGF), tyrosine kinase A (TrkA), phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p44/p42 MAPK, but not the AT2 R, were increased significantly (P < 0.05) in PCIBP rats, c.f. the corresponding levels for sham controls. EMA200 produced analgesia in PCIBP rats by reducing elevated angiotensin II levels in the lumbar DRGs to attenuate augmented angiotensin II/AT2 R signaling. This in turn reduced augmented NGF/TrkA signaling in the lumbar DRGs. The net result was inhibition of p38 MAPK and p44/p42 MAPK activation. CONCLUSION Small molecule AT2 R antagonists are worthy of further investigation as novel analgesics for relief of intractable PCIBP and other pain types where hyperalgesia worsens symptoms.

Pharmacogenetics of pain and analgesia

Pain severity ratings and the analgesic dosing requirements of patients with apparently similar pain conditions may differ considerably between individuals. Contributing factors include those of genetic and environmental origin with epigenetic mechanisms that enable dynamic gene–environment interaction, more recently implicated in pain modulation. Insight into genetic factors underpinning inter‐patient variability in pain sensitivity has come from rodent heritability studies as well as familial aggregation and twin studies in humans. Indeed, more than 350 candidate pain genes have been identified as potentially contributing to heritable differences in pain sensitivity. A large number of genetic association studies conducted in patients with a variety of clinical pain types or in humans exposed to experimentally induced pain stimuli in the laboratory setting, have examined the impact of single‐nucleotide polymorphisms in various target genes on pain sensitivity and/or analgesic dosing requirements. However, the findings of such studies have generally failed to replicate or have been only partially replicated by independent investigators. Deficiencies in study conduct including use of small sample size, inappropriate statistical methods and inadequate attention to the possibility that between‐study differences in environmental factors may alter pain phenotypes through epigenetic mechanisms, have been identified as being significant.

Optimization and characterization of a rat model of prostate cancer-induced bone pain using behavioral, pharmacological, radiological, histological and immunohistochemical methods.

The major limitation of currently utilized rodent models of prostate cancer (PCa)-induced bone pain (PCIBP) involving intra-osseous injection of PCa cells, is their relatively short-term applicability due to progressive deterioration of animal health necessitating euthanasia. Here, we describe establishment of an optimized rat model of PCIBP where good animal health was maintained for at least 90-days following unilateral intra-tibial injection (ITI) of PCa cells. We have characterized this model using behavioral, pharmacological, radiological, histological and immunohistochemical methods. Our findings show that following unilateral ITI of 4×10(4) AT3B PCa cells (APCCs), there was temporal development of bilateral hindpaw hypersensitivity that was fully developed between days 14 and 21 post-ITI. Although there was apparent spontaneous reversal of bilateral hindpaw sensitivity that was maintained until at least day 90 post-ITI, administration of bolus doses of the opioid receptor antagonist, naloxone, rescued the pain phenotype in these animals. Hence, upregulation of endogenous opioid signaling mechanisms appears to underpin apparent spontaneous resolution of hindpaw hypersensitivity. Importantly, the histological and radiological assessments confirmed that tumor formation and development of osteosclerotic metastases was confined to the APCC-injected tibial bones. In our rat model of PCIBP, single bolus doses of morphine, gabapentin, meloxicam and amitriptyline produced dose-dependent relief of mechanical allodynia and thermal hyperalgesia in the bilateral hindpaws. The optimized rat model of PCIBP characterized herein has potential to provide new insights into the pathophysiological mechanisms associated with long-term (mal)adaptive pain due to advanced PCa-induced bony metastases and for screening novel compounds with potential for improved alleviation of this condition.

Targeting angiotensin II type 2 receptor pathways to treat neuropathic pain and inflammatory pain

Introduction: Neuropathic pain and chronic inflammatory pain are large unmet medical needs. Over the past two decades, numerous ’pain targets’ have been identified for analgesic drug discovery. Despite promising results in rodent pain models, many compounds modulating such targets lacked efficacy in clinical trials. An exception is oral EMA401, a small-molecule angiotensin II type 2 receptor (AT2R) antagonist. Areas covered: Herein, angiotensin II/AT2R signaling-induced hyperexcitability and abnormal sprouting of cultured dorsal root ganglion neurons, together with radioligand binding, pharmacokinetics, analgesic efficacy and mode of action of small-molecule AT2R antagonists in rodent models of peripheral neuropathic and chronic inflammatory pain, are reviewed. The findings of a successful Phase IIa clinical trial of EMA401 in patients with neuropathic pain are presented in brief. Expert opinion: The functional importance of angiotensin II/AT2R signaling has remained enigmatic for decades, and there are no clinically available medications that target the AT2R. However, on the basis of preclinical findings and recent clinical trial data showing that the peripherally restricted, small-molecule AT2R antagonist, EMA401, successfully alleviated neuropathic pain in a Phase II clinical trial, the AT2R is receiving considerable attention as a new therapeutic target with human validation for the relief of peripheral neuropathic and chronic inflammatory pain conditions.

Comparison of Burrowing and Stimuli-Evoked Pain Behaviors as End-Points in Rat Models of Inflammatory Pain and Peripheral Neuropathic Pain

Establishment and validation of ethologically-relevant, non-evoked behavioral end-points as surrogate measures of spontaneous pain in rodent pain models has been proposed as a means to improve preclinical to clinical research translation in the pain field. Here, we compared the utility of burrowing behavior with hypersensitivity to applied mechanical stimuli for pain assessment in rat models of chronic inflammatory and peripheral neuropathic pain. Briefly, groups of male Sprague-Dawley rats were habituated to the burrowing environment and trained over a 5-day period. Rats that burrowed ≤ 450 g of gravel on any 2 days of the individual training phase were excluded from the study. The remaining rats received either a unilateral intraplantar injection of Freunds complete adjuvant (FCA) or saline, or underwent unilateral chronic constriction injury (CCI) of the sciatic nerve- or sham-surgery. Baseline burrowing behavior and evoked pain behaviors were assessed prior to model induction, and twice-weekly until study completion on day 14. For FCA- and CCI-rats, but not the corresponding groups of sham-rats, evoked mechanical hypersensitivity developed in a temporal manner in the ipsilateral hindpaws. Although burrowing behavior also decreased in a temporal manner for both FCA-and CCI- rats, there was considerable inter-animal variability. By contrast, mechanical hyperalgesia and mechanical allodynia in the ipsilateral hindpaws of FCA- and CCI-rats respectively, exhibited minimal inter-animal variability. Our data collectively show that burrowing behavior is altered in rodent models of chronic inflammatory pain and peripheral neuropathic pain. However, large group sizes are needed to ensure studies are adequately powered due to considerable inter-animal variability.

Establishment and Characterization of a Novel Rat Model of Mechanical Low Back Pain Using Behavioral, Pharmacologic and Histologic Methods

Chronic low back pain (LBP), the leading cause of disability globally, is notoriously difficult to treat. Most rodent models of LBP mimic lumbar radicular pain rather than mechanical LBP. Here, we describe establishment of a new rat model of mechanical LBP that is devoid of a neuropathic component. Groups of adult male Sprague Dawley rats were anesthetized and their lumbar L4/L5 and L5/L6 intervertebral disks (IVDs) were punctured (0.5 mm outer diameter, 2mm-deep) 5 (LPB-5X), or 10 (LBP-10X) times per disk. Sham-rats underwent similar surgery, but without disk puncture. Baseline noxious pressure hyperalgesia of lumbar axial deep tissues, mechanical allodynia in the hindpaws and gait were assessed prior to surgery and once-weekly until study completion on day 49. The model was also characterized using pharmacologic and histologic methods. Good animal health was maintained for ≥ 49 days post-surgery. For LBP- but not sham-rats, there was temporal development of noxious pressure hyperalgesia in lumbar axial deep tissues at days 14–49 post-surgery. Importantly, there were no between-group differences in von Frey paw withdrawal thresholds or gait parameters until study completion. On day 49, significant histologic changes were observed in the L4/L5 and L5/L6 IVDs for LBP-10X rats, but not sham-rats. In LBP-10X rats, single bolus doses of morphine produced dose-dependent relief of primary and secondary mechanical hyperalgesia in lumbar axial deep tissues at L4/L5 and L1, respectively. In conclusion, our new rat model has considerable potential for providing novel insight on the pathobiology of mechanical LBP and for analgesic efficacy assessment of novel compounds.

Attenuation of the Infiltration of Angiotensin II Expressing CD3+ T-Cells and the Modulation of Nerve Growth Factor in Lumbar Dorsal Root Ganglia – A Possible Mechanism Underpinning Analgesia Produced by EMA300, An Angiotensin II Type 2 (AT2) Receptor Antagonist

Recent preclinical and proof-of-concept clinical studies have shown promising analgesic efficacy of selective small molecule angiotensin II type 2 (AT2) receptor antagonists in the alleviation of peripheral neuropathic pain. However, their cellular and molecular mechanism of action requires further investigation. To address this issue, groups of adult male Sprague–Dawley rats with fully developed unilateral hindpaw hypersensitivity, following chronic constriction injury (CCI) of the sciatic nerve, received a single intraperitoneal bolus dose of the small molecule AT2 receptor antagonist, EMA300 (10 mg kg-1), or vehicle. At the time of peak EMA300-mediated analgesia (∼1 h post-dosing), groups of CCI-rats administered either EMA300 or vehicle were euthanized. A separate group of rats that underwent sham surgery were also included. The lumbar (L4–L6) dorsal root ganglia (DRGs) were obtained from all experimental cohorts and processed for immunohistochemistry and western blot studies. In vehicle treated CCI-rats, there was a significant increase in the expression levels of angiotensin II (Ang II), but not the AT2 receptor, in the ipsilateral lumbar DRGs. The elevated levels of Ang II in the ipsilateral lumbar DRGs of CCI-rats were at least in part contributed by CD3+ T-cells, satellite glial cells (SGCs) and subsets of neurons. Our findings suggest that the analgesic effect of EMA300 in CCI-rats involves multimodal actions that appear to be mediated at least in part by a significant reduction in the otherwise increased expression levels of Ang II as well as the number of Ang II-expressing CD3+ T-cells in the ipsilateral lumbar DRGs of CCI-rats. Additionally, the acute anti-allodynic effects of EMA300 in CCI-rats were accompanied by rescue of the otherwise decreased expression of mature nerve growth factor (NGF) in the ipsilateral lumbar DRGs of CCI-rats. In contrast, the increased expression levels of TrkA and glial fibrillary acidic protein in the ipsilateral lumbar DRGs of vehicle-treated CCI-rats were not attenuated by a single bolus dose of EMA300. Consistent with our previous findings, there was also a significant decrease in the augmented levels of the downstream mediators of Ang II/AT2 receptor signaling, i.e., phosphorylated-p38 mitogen-activated protein kinase (MAPK) and phosphorylated-p44/p42 MAPK, in the ipsilateral lumbar DRGs.