12-(S)-Hydroxyeicosatetraenoic Acid and GPR31 Signaling in Spinal Cord in Neuropathic Pain.

Neuropathic pain is a pressing unmet medical need requiring novel nonopioid-based therapeutic approaches. Using unbiased transcriptomic analysis, we found that the expression of Gpr31, a G protein-coupled receptor, increased in the dorsal horn of the spinal cord in rats with traumatic nerve injury-induced neuropathic pain. Daily intrathecal injections of siGpr31 reversed behavioral hypersensitivities in a time-dependent manner. GPR31, a Gα i protein-coupled receptor, has recently been cloned and is a receptor for 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. The lack of commercially available GPR31 antagonists has hampered the understanding of this receptor in pathophysiological states, including pain. To investigate this, our first approach was to identify novel GPR31 antagonists. Using a multidisciplinary approach, including in silico modeling, we identified the first highly potent and selective small-molecule GPR31 antagonist, SAH2. Here, we characterize the pharmacological activity in well-described models of neuropathic pain in rodents and provide evidence that 12-(S)-HETE/GPR31-dependent behavioral hypersensitivities are mediated through mitogen-activated protein kinase (MAPK) activation in the spinal cord. Our studies provide the pharmacological rationale for investigating contributions of GPR31 along the pain neuroaxis and the development of nonopioid GPR31-targeted strategies. SIGNIFICANCE STATEMENT: We have identified the first highly selective GPR31 antagonist. Using this antagonist, we have demonstrated that GPR31 signaling in the spinal cord is pronociceptive and MAPK pathways provided signaling mechanisms downstream of GPR31 activation in these processes.

Modulation of the nitric oxide/cGMP pathway in cardiac contraction and relaxation: Potential role in heart failure treatment.

Evidence exists that heart failure (HF) has an overall impact of 1-2 % in the global population being often associated with comorbidities that contribute to increased disease prevalence, hospitalization, and mortality. Recent advances in pharmacological approaches have significantly improved clinical outcomes for patients with vascular injury and HF. Nevertheless, there remains an unmet need to clarify the crucial role of nitric oxide/cyclic guanosine 3',5'-monophosphate (NO/cGMP) signalling in cardiac contraction and relaxation, to better identify the key mechanisms involved in the pathophysiology of myocardial dysfunction both with reduced (HFrEF) as well as preserved ejection fraction (HFpEF). Indeed, NO signalling plays a crucial role in cardiovascular homeostasis and its dysregulation induces a significant increase in oxidative and nitrosative stress, producing anatomical and physiological cardiac alterations that can lead to heart failure. The present review aims to examine the molecular mechanisms involved in the bioavailability of NO and its modulation of downstream pathways. In particular, we focus on the main therapeutic targets and emphasize the recent evidence of preclinical and clinical studies, describing the different emerging therapeutic strategies developed to counteract NO impaired signalling and cardiovascular disease (CVD) development.

Editorial: Small molecules and biologics for future purinergic therapeutics.

Receptor agonists and antagonists and other modulators of purinergic signalling have potential as novel therapeutics for a broad range of diseases and conditions. This special issue focuses on compounds or approaches that are either in clinical trials or headed in that direction. It is intended to serve as an up-to-date description of selected efforts to discover and develop new small molecular purinergic drugs.

Spinal A3 adenosine receptor activation acutely restores morphine antinociception in opioid tolerant male rats.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A3 adenosine receptor (A3 AR) in opioid analgesic tolerance. Intrathecal administration of the A3 AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7 days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A3 AR expression was not affected. Collectively, our findings indicate that spinal A3 AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

Activation of GPR183 by 7α,25-Dihydroxycholesterol Induces Behavioral Hypersensitivity through Mitogen-Activated Protein Kinase and Nuclear Factor-κB.

Emerging evidence implicates the G-protein coupled receptor (GPCR) GPR183 in the development of neuropathic pain. Further investigation of the signaling pathways downstream of GPR183 is needed to support the development of GPR183 antagonists as analgesics. In rodents, intrathecal injection of its ligand, 7α,25-dihydroxycholesterol (7α,25-OHC), causes time-dependent development of mechano-and cold- allodynia (behavioral hypersensitivity). These effects are blocked by the selective small molecule GPR183 antagonist, SAE-14. However, the molecular mechanisms engaged downstream of GPR183 in the spinal cord are not known. Here, we show that 7α,25-OHC-induced behavioral hypersensitivity is Gα i dependent, but not ß-arrestin 2-dependent. Non-biased transcriptomic analyses of dorsal-horn spinal cord (DH-SC) tissues harvested at the time of peak hypersensitivity implicate potential contributions of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB). In support, we found that the development of 7α,25-OHC/GPR183-induced mechano-allodynia was associated with significant activation of MAPKs (extracellular signal-regulated kinase [ERK], p38) and redox-sensitive transcription factors (NF-κB) and increased formation of inflammatory and neuroexcitatory cytokines. SAE-14 blocked these effects and behavioral hypersensitivity. Our findings provide novel mechanistic insight into how GPR183 signaling in the spinal cord produces hypersensitivity through MAPK and NF-κB activation. SIGNIFICANCE STATEMENT: Using a multi-disciplinary approach, we have characterized the molecular mechanisms underpinning 7α,25-OHC/GPR183-induced hypersensitivity in mice. Intrathecal injections of the GPR183 agonist 7α,25-OHC induce behavioral hypersensitivity, and these effects are blocked by the selective GPR183 antagonist SAE-14. We found that 7α,25-OHC-induced allodynia is dependent on MAPK and NF-κB signaling pathways and results in an increase in pro-inflammatory cytokine expression. This study provides a first insight into how GPR183 signaling in the spinal cord is pronociceptive.

Role of Adenosine Kinase in Sphingosine-1-Phosphate Receptor 1-Induced Mechano-Hypersensitivities.

Emerging evidence implicates the sphingosine-1-phosphate receptor subtype 1 (S1PR1) in the development of neuropathic pain. Continued investigation of the signaling pathways downstream of S1PR1 are needed to support development of S1PR1 antagonists. In rodents, intrathecal (i.th.) injection of SEW2871, a selective S1PR1 agonist, activates the nod-like receptor family, pyrin domain containing 3 inflammasome, increases interleukin-1ß (IL-1ß) and causes behavioral hypersensitivity. I.th. injection of a IL-1ß receptor antagonist blocks SEW2871-induced hypersensitivity, suggesting that IL-1ß contributes to S1PR1's actions. Interestingly, previous studies have suggested that IL-1ß increases the expression/activity of adenosine kinase (ADK), a key regulator of adenosine signaling at its receptors (ARs). Increased ADK expression reduces adenosine signaling whereas inhibiting ADK restores the action of adenosine. Here, we show that SEW287-induced behavioral hypersensitivity is associated with increased expression of ADK in astrocytes of the dorsal horn of the spinal cord. Moreover, the ADK inhibitor, ABT702, blocks SEW2871-induced hypersensitivity. These findings link ADK activation to S1PR1. If SEW2871-induced pain is mediated by IL-1ß, which in turn activates ADK and leads to mechano-allodynia, then blocking ADK should attenuate IL-1ß effects. In support of this idea, recombinant rat (rrIL-1ß)-induced allodynia was blocked by at least 90% with ABT702, functionally linking ADK to IL-1ß. Moreover, the selective A3AR antagonist, MRS1523, prevents the ability of ABT702 to block SEW2871 and IL-1ß-induced allodynia, implicating A3AR signaling in the beneficial effects exerted by ABT702. Our findings provide novel mechanistic insight into how S1PR1 signaling in the spinal cord produces hypersensitivity through IL1-ß and ADK activation.

Sphingosine-1-phosphate receptor 1 activation in astrocytes contributes to neuropathic pain.

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 S1pr1 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.

Adenosine A3 Receptor (A3AR) Agonist for the Treatment of Bleomycin-Induced Lung Fibrosis in Mice.

Adenosine receptors (ARs) are involved in the suppression and development of inflammatory and fibrotic conditions. Specifically, AR activation promotes differentiation of lung fibroblasts into myofibroblasts, typical of a fibrotic event. Pulmonary fibrosis is a severe disease characterized by inflammation and fibrosis of unknown etiology and lacking an effective treatment. The present investigation explored the action of MRS5980, a new, highly potent and selective A3AR agonist, in an established murine model of lung fibrosis. The effects of either vehicle or MRS5980 were studied in mice following intratracheal bleomycin administration. We evaluated the role of the A3AR agonist on lung stiffness, studying the airway resistance to inflation, oxidative stress (8-OHdG and MDA), inflammation, pro- and anti-inflammatory marker levels (IL-1ß, IL-6, TNF-α, IL-10 and IL-17A) and fibrosis establishment, evaluating transforming growth factor (TGF)-ß expression and α-smooth muscle actin (α-SMA) deposition in lungs. Bleomycin administration increased lung stiffness, TGF-ß levels, α-SMA deposition, and inflammatory and oxidative stress markers. The treatment with MRS5980 attenuated all the analyzed functional, biochemical and histopathological markers in a dose-dependent manner. Our findings support the therapeutic potential of A3AR agonists in lung fibrosis by demonstrating reduced disease progression, as indicated by decreased inflammation, TGF-ß expression and fibrotic remodeling.

Targeting reactive nitroxidative species in preclinical models of migraine.

BACKGROUND: Reactive nitroxidative species, such as nitric oxide but particularly peroxynitrite, have been strongly implicated in pain mechanisms. Targeting peroxynitrite is anti-nociceptive in pain models, but little is known about its role in migraine mechanisms. Given the need to validate novel targets for migraine headache, our objective was to study the potential of reactive nitroxidative species, particularly peroxynitrite, as novel targets for drug discovery and their role in migraine mechanisms. METHODS: We recorded neuronal activity in rats with extracellular electrodes and examined the effects of targeting nitric oxide or peroxynitrite on ongoing and cranial-evoked firing rates of central trigeminocervical neurons. We injected calcitonin gene-related peptide (which produces migraine-like headache in migraineurs) and characterized neuronal responses to cranial stimulation and on behavioral responses to nociceptive periorbital stimulation and determined the effects of targeting reactive nitroxidative species on the mediated changes. RESULTS: L-NAME (nitric oxide synthase inhibitor) and Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride (FeTPPS; peroxynitrite decomposition catalyst) inhibited ongoing and dural-evoked responses of trigeminocervical neurons, without affecting normal facial-cutaneous responses. Calcitonin gene-related peptide caused activation and sensitization of dural-responsive trigeminovascular neurons with hypersensitivity to intracranial and extracranial stimulation, and reduction of periorbital withdrawal thresholds. Only the peroxynitrite decomposition catalyst prevented these neuronal and behavioral nociceptive responses. DISCUSSION: The data support that calcitonin gene-related peptide mediates the underlying neurobiological mechanisms related to the development of migraine-like headache. They also confirm the role of nitric oxide and implicate peroxynitrite production along the trigeminovascular migraine pathway in these mechanisms. The data also support peroxynitrite as a novel and potentially effective target for migraine treatment. The current drug development focus on peroxynitrite decomposition catalysts for chronic pain disorders should therefore extend to migraine.

Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

The Deductive Reasoning Strategy Enables Biomedical Breakthroughs.

G protein-coupled receptors (GPCRs) transmit the signals of a variety of hormones and neurotransmitters and are targets of more than 30% of all FDA-approved drugs. We developed an approach for identifying the endogenous ligands for a family of orphan GPCRs that enables the development of novel therapeutics for the potential treatment of a wide variety of disorders including pain, diabetes, appetitive behaviors, infertility and obesity. With this approach, we have deorphanized five previously orphaned GPCRs.

Novel Non-Opioid Based Therapeutics for Chronic Neuropathic Pain.

Chronic neuropathic pain is currently a major health issue in U.S. complicated by the lack of non-opioid analgesic alternatives. Our investigations led to the discovery of major signaling pathways involved in the transition of acute to chronic neuropathic pain and the identification of several targets for therapeutic intervention. Our translational approach has facilitated the advancement of novel medicines for chronic neuropathic pain that are in advanced clinical development and clinical trials.

Adenosine A3 receptor as a novel therapeutic target to reduce secondary events and improve neurocognitive functions following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common pathological condition that presently lacks a specific pharmacological treatment. Adenosine levels rise following TBI, which is thought to be neuroprotective against secondary brain injury. Evidence from stroke and inflammatory disease models suggests that adenosine signaling through the G protein-coupled A3 adenosine receptor (A3AR) can provide antiinflammatory and neuroprotective effects. However, the role of A3AR in TBI has not been investigated. METHODS: Using the selective A3AR agonist, MRS5980, we evaluated the effects of A3AR activation on the pathological outcomes and cognitive function in CD1 male mouse models of TBI. RESULTS: When measured 24 h after controlled cortical impact (CCI) TBI, male mice treated with intraperitoneal injections of MRS5980 (1 mg/kg) had reduced secondary tissue injury and brain infarction than vehicle-treated mice with TBI. These effects were associated with attenuated neuroinflammation marked by reduced activation of nuclear factor of kappa light polypeptide gene enhancer in B cells (NFκB) and MAPK (p38 and extracellular signal-regulated kinase (ERK)) pathways and downstream NOD-like receptor pyrin domain-containing 3 inflammasome activation. MRS5980 also attenuated TBI-induced CD4+ and CD8+ T cell influx. Moreover, when measured 4-5 weeks after closed head weight-drop TBI, male mice treated with MRS5980 (1 mg/kg) performed significantly better in novel object-placement retention tests (NOPRT) and T maze trials than untreated mice with TBI without altered locomotor activity or increased anxiety. CONCLUSION: Our results provide support for the beneficial effects of small molecule A3AR agonists to mitigate secondary tissue injury and cognitive impairment following TBI.

Sphingosine-1-phosphate receptor subtype 1 activation in the central nervous system contributes to morphine withdrawal in rodents.

Opioid therapies for chronic pain are undermined by many adverse side effects that reduce their efficacy and lead to dependence, abuse, reduced quality of life, and even death. We have recently reported that sphingosine-1-phosphate (S1P) 1 receptor (S1PR1) antagonists block the development of morphine-induced hyperalgesia and analgesic tolerance. However, the impact of S1PR1 antagonists on other undesirable side effects of opioids, such as opioid-induced dependence, remains unknown. Here, we demonstrate that naloxone-precipitated morphine withdrawal in mice altered de novo sphingolipid metabolism in the dorsal horn of the spinal cord and increased S1P that accompanied the manifestation of several withdrawal behaviors. Blocking de novo sphingolipid metabolism with intrathecal administration of myriocin, an inhibitor of serine palmitoyltransferase, blocked naloxone-precipitated withdrawal. Noteworthy, we found that competitive (NIBR-15) and functional (FTY720) S1PR1 antagonists attenuated withdrawal behaviors in mice. Mechanistically, at the level of the spinal cord, naloxone-precipitated withdrawal was associated with increased glial activity and formation of the potent inflammatory/neuroexcitatory cytokine interleukin-1ß (IL-1ß); these events were attenuated by S1PR1 antagonists. These results provide the first molecular insight for the role of the S1P/S1PR1 axis during opioid withdrawal. Our data identify S1PR1 antagonists as potential therapeutics to mitigate opioid-induced dependence and support repurposing the S1PR1 functional antagonist FTY720, which is FDA-approved for multiple sclerosis, as an opioid adjunct.

GPR183-Oxysterol Axis in Spinal Cord Contributes to Neuropathic Pain.

Neuropathic pain is a debilitating public health concern for which novel non-narcotic therapeutic targets are desperately needed. Using unbiased transcriptomic screening of the dorsal horn spinal cord after nerve injury we have identified that Gpr183 (Epstein-Barr virus-induced gene 2) is upregulated after chronic constriction injury (CCI) in rats. GPR183 is a chemotactic receptor known for its role in the maturation of B cells, and the endogenous ligand is the oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC). The role of GPR183 in the central nervous system is not well characterized, and its role in pain is unknown. The profile of commercially available probes for GPR183 limits their use as pharmacological tools to dissect the roles of this receptor in pathophysiological settings. Using in silico modeling, we have screened a library of 5 million compounds to identify several novel small-molecule antagonists of GPR183 with nanomolar potency. These compounds are able to antagonize 7α,25-OHC-induced calcium mobilization in vitro with IC50 values below 50 nM. In vivo intrathecal injections of these antagonists during peak pain after CCI surgery reversed allodynia in male and female mice. Acute intrathecal injection of the GPR183 ligand 7α,25-OHC in naïve mice induced dose-dependent allodynia. Importantly, this effect was blocked using our novel GPR183 antagonists, suggesting spinal GPR183 activation as pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify this receptor as a potential target for therapeutic intervention. SIGNIFICANCE STATEMENT: We have identified several novel GPR183 antagonists with nanomolar potency. Using these antagonists, we have demonstrated that GPR183 signaling in the spinal cord is pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify it as a potential target for therapeutic intervention.

Chronic Morphine-Induced Changes in Signaling at the A3 Adenosine Receptor Contribute to Morphine-Induced Hyperalgesia, Tolerance, and Withdrawal.

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 A3 adenosine receptor (A3AR) signaling. Supplementing adenosine signaling with selective A3AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A3AR in the spinal cord with an ADK inhibitor or A3AR 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)-1ß (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A3AR 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 A3AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A3AR 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 A3AR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1ß neuroinflammation in spinal cord. These effects are attenuated by A3AR agonists, suggesting that A3AR may be a target for therapeutic intervention with selective A3AR agonist as opioid adjuncts.

Antioxidant modulation of sirtuin 3 during acute inflammatory pain: The ROS control.

Oxidative stress induced post-translational protein modifications are associated with the development of inflammatory hypersensitivities. At least 90% of cellular reactive oxygen species (ROS) are produced in the mitochondria, where the mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), is located. MnSOD's ability to reduce ROS is enhanced by the mitochondrial NAD+-dependent deacetylase sirtuin (SIRT3). SIRT3 can reduce ROS levels by deacetylating MnSOD and enhancing its ability to neutralize ROS or by enhancing the transcription of MnSOD and other oxidative stress-responsive genes. SIRT3 can be post-translationally modified through carbonylation which results in loss of activity. The contribution of post-translational SIRT3 modifications in central sensitization is largely unexplored. Our results reveal that SIRT3 carbonylation contributes to spinal MnSOD inactivation during carrageenan-induced thermal hyperalgesia in rats. Moreover, inhibiting ROS with natural and synthetic antioxidants, prevented SIRT3 carbonylation, restored the enzymatic activity of MnSOD, and blocked the development of thermal hyperalgesia. These results suggest that therapeutic strategies aimed at inhibiting post-translational modifications of SIRT3 may provide beneficial outcomes in pain states where ROS have been documented to play an important role in the development of central sensitization.

Design and in vivo activity of A3 adenosine receptor agonist prodrugs.

Prodrugs (MRS7422, MRS7476) of highly selective A3 adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively, were synthesized by succinylation of the 2' and 3' hydroxyl groups, and the parent, active drug was shown to be readily liberated upon incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury model, 3 µmol/kg, p.o.), a known A3AR effect. MRS7476 (5 mg/kg, p.o., twice daily) was found to protect against non-alcoholic steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 production, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2 levels) or inflammation. Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls. Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body A3AR knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables water solubility and in vivo activity of masked A3AR agonists in models of two disease conditions.

Modulation of Nitric Oxide Synthases by Oxidized LDLs: Role in Vascular Inflammation and Atherosclerosis Development.

The maintenance of physiological levels of nitric oxide (NO) produced by eNOS represents a key element for vascular endothelial homeostasis. On the other hand, NO overproduction, due to the activation of iNOS under different stress conditions, leads to endothelial dysfunction and, in the late stages, to the development of atherosclerosis. Oxidized LDLs (oxLDLs) represent the major candidates to trigger biomolecular processes accompanying endothelial dysfunction and vascular inflammation leading to atherosclerosis, though the pathophysiological mechanism still remains to be elucidated. Here, we summarize recent evidence suggesting that oxLDLs produce significant impairment in the modulation of the eNOS/iNOS machinery, downregulating eNOS via the HMGB1-TLR4-Caveolin-1 pathway. On the other hand, increased oxLDLs lead to sustained activation of the scavenger receptor LOX-1 and, subsequently, to NFkB activation, which, in turn, increases iNOS, leading to EC oxidative stress. Finally, these events are associated with reduced protective autophagic response and accelerated apoptotic EC death, which activates atherosclerotic development. Taken together, this information sheds new light on the pathophysiological mechanisms of oxLDL-related impairment of EC functionality and opens new perspectives in atherothrombosis prevention.

Pain and Poppies: The Good, the Bad, and the Ugly of Opioid Analgesics.

Treating pain is one of the most difficult challenges in medicine and a key facet of disease management. The isolation of morphine by Friedrich Sertürner in 1804 added an essential pharmacological tool in the treatment of pain and spawned the discovery of a new class of drugs known collectively as opioid analgesics. Revered for their potent pain-relieving effects, even Morpheus the god of dreams could not have dreamt that his opium tincture would be both a gift and a burden to humankind. To date, morphine and other opioids remain essential analgesics for alleviating pain. However, their use is plagued by major side effects, such as analgesic tolerance (diminished pain-relieving effects), hyperalgesia (increased pain sensitivity), and drug dependence. This review highlights recent advances in understanding the key causes of these adverse effects and explores the effect of chronic pain on opioid reward. SIGNIFICANCE STATEMENT: Chronic pain is pervasive and afflicts >100 million Americans. Treating pain in these individuals is notoriously difficult and often requires opioids, one of the most powerful and effective classes of drugs used for controlling pain. However, their use is plagued by major side effects, such as a loss of pain-relieving effects (analgesic tolerance), paradoxical pain (hyperalgesia), and addiction. Despite the potential side effects, opioids remain the pharmacological cornerstone of modern pain therapy. This review highlights recent breakthroughs in understanding the key causes of these adverse effects and explores the cellular control of opioid systems in reward and aversion. The findings will challenge traditional views of the good, the bad, and the ugly of opioids.