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Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

Presbyterian College School of Pharmacy

1. Non-steroidal anti-inflammatory drugs (NSAIDs) are effective anti-inflammatory and analgesic drugs although they also induce unwanted side effects due to the inhibition of the physiological effects regulated by prostaglandins. This has led to the search for new compounds with fewer side effects, such as the nitro-NSAIDs (NO-NSAIDs). Paracetamol is an analgesic drug devoid of some of the side effect of the NSAIDs but without anti-inflammatory activity. NCX-701 is a nitric oxide releasing version of paracetamol with anti-inflammatory and analgesic properties. 2. We have tested, in the single motor unit technique, the antinociceptive actions of intravenous cumulative doses of NCX-701 vs paracetamol, studying their antinociceptive effects in responses to noxious mechanical and electrical stimulation (wind-up). 3. Paracetamol did not induce any significant effect at the doses tested (maximum of 480 micromol kg(-1), 72.5 mg kg(-1)). NCX-701 however was very effective in reducing responses to noxious mechanical stimulation (32+/-10% of control response) and wind-up (ED(50) of 147+/-1 micromol kg(-1), 41.5+/-0.3 mg kg(-1)). The inhibition was not reversed by 1 mg kg(-1) of the opioid antagonist naloxone. In control experiments performed with either the vehicle or the NO donor NOC-18, no significant changes were observed in the nociceptive responses studied. 4. We conclude that NCX-701 is a very effective non-opioid antinociceptive agent in normal animals and its action is located mainly at central areas. The antinociceptive effect was not due solely to the release of NO.

NCX-701 (nitroparacetamol) is an effective antinociceptive agent in rat withdrawal reflexes and wind-up.

New generations of cyclooxygenase (COX) inhibitors are more potent and efficacious than their traditional parent compounds. They are also safer than the classic non-steroidal anti-inflammatory drugs (NSAIDs) and are starting to be used not only for low to moderate intensity pain, but also for high intensity pain. Three different strategies have been followed to improve the pharmacological profile of COX inhibitors: 1. Development of COX-2 selective inhibitors. This is based on the initial hypothesis that considered COX-2 as the enzyme responsible for the generation of prostaglandins only in inflammation, and, therefore, uniquely responsible for inflammation, pain and fever. Initial expectations gave rise to controversial results, still under discussion. The second generation of these compounds is being developed and should contribute to clarifying both their efficacy and the specific functions of the COX enzymes. 2. Modified non-selective COX inhibitors. Molecules like nitro-NSAIDs or tromethamine salt derivatives have been synthesized considering that both COX-1 and COX-2 are responsible for the synthesis of prostaglandins involved either in homeostatic functions or inflammation. Nitroaspirin, nitroparacetamol or dexketoprofen trometamol are some examples of molecules that are already showing an important clinical efficacy. The modifications performed in their structures seem to lower the unwanted side effects as well as to enhance their analgesic efficacy. 3. Combined therapy of classic NSAIDs with other drugs. This strategy looks for improvements in the incidence of adverse effects or to take advantage of the synergistic enhancement of their therapeutic effects. Some of the molecules resulting from these strategies are very valuable as therapeutic agents and open a wide range of possibilities in the treatment of high intensity pain, including neuropathic pain, and opiate sparing therapy.

Antinociception and the new COX inhibitors: research approaches and clinical perspectives.

Neuropathic pain may be primarily driven by immune responses in peripheral nerves. Peripherally released catecholamines may exacerbate neuropathic pain and also modulate immune responses in a complex and sometimes opposing manner by actions on multiple adrenoceptor subtypes. We showed previously that injection of the alpha2-adrenoceptor agonist clonidine at the site of peripheral nerve injury reduces pain behavior and local tissue pro-inflammatory cytokine content in rats. The current study used a model of acute inflammatory neuritis to test the efficacy and mechanisms of action of alpha2-adrenoceptor stimulation to reduce pain. Zymosan, injected on the sciatic nerve, caused hypersensitivity to mechanical stimuli ipsilateral to injection and contralaterally, so-called mirror image pain. Ipsilateral hypersensitivity was inhibited dose-dependently by perineural injection of clonidine. Zymosan increased leukocyte number at the site of injection 3 d later as well as their content of interleukin 1alpha (IL-1alpha), IL-1beta, and IL-6. Perineural clonidine prevented both the increase in leukocyte number and cytokine expression induced by zymosan. Additionally, clonidine reduced the capacity of leukocytes to express pro-inflammatory cytokines as assessed by treatment of cells ex vivo with lipopolysaccharide, whereas no repression of IL-10 production occurred. Clonidine reduced the number of macrophages and lymphocytes as well as their expression of tumor necrosis factor alpha. All of the effects of clonidine were prevented by coadministration of an alpha2A-adrenoceptor-preferring antagonist. These results suggest that alpha2-adrenoceptor stimulation transforms cytokine gene expression, especially in macrophages and lymphocytes from a pro- to an anti-inflammatory profile in the setting of neuritis, likely relieving neuritis-induced pain by this mechanism.

Alpha2-adrenoceptor stimulation transforms immune responses in neuritis and blocks neuritis-induced pain.

The combination of numerous classic drugs with nitric oxide donors has led to the development of new compounds with promising therapeutic activities in a great variety of situations, including cardiovascular and respiratory systems, ocular pressure, inflammation, and pain. One of the first compounds developed was NCX-701 or nitroparacetamol, resulting from the combination of paracetamol, a classic and popular analgesic used in a great number of over-the-counter medications because of its antipyretic and analgesic properties, and a nitrooxybutyroyl moiety, which releases nitric oxide at a low but steady level. Although paracetamol is devoid of most of the gastrointestinal toxicity associated with aspirin-like drugs, this type of compounds was first designed to take advantage of the cytoprotective properties of nitric oxide when released at low concentrations. However, the combination of these molecules also resulted in an unexpected enhancement of the analgesic activity of paracetamol. In fact, NCX-701 has been shown to be effective in acute nociception as well as in neuropathic pain, situations in which paracetamol and other COX inhibitors are devoid of any effect. In addition, NCX-701 is more potent and, in some circumstances, more effective than its parent compound in different models of inflammatory pain. Furthermore, whereas paracetamol lacks any effective antiinflammatory action, NCX-701 might reduce inflammation. All these results taken together imply that the mechanism of action of NCX-701 is different from that of paracetamol, although it is not yet established for either molecule. NCX-701 appears to be a promising compound in the treatment of different types of pain, with a likely better profile of side effects than its parent molecule, paracetamol. Although recent clinical trials provided data consistent with the preclinical profile of NCX-701, further studies are needed to support its clinical use.

Nitroparacetamol (NCX-701) and pain: first in a series of novel analgesics.

An alarming portion of patients develop persistent or chronic pain following surgical procedures, but the mechanisms underlying the transition from acute to chronic pain states are not fully understood. In general, endocannabinoids (ECBs) inhibit nociceptive processing by stimulating cannabinoid receptors type 1 (CB(1)) and type 2 (CB(2)). We have previously shown that intrathecal administration of a CB(2) receptor agonist reverses both surgical incision-induced behavioral hypersensitivity and associated over-expression of spinal glial markers. We therefore hypothesized that endocannabinoid signaling promotes the resolution of acute postoperative pain by modulating pro-inflammatory signaling in spinal cord glial cells.

Evidence for a role of endocannabinoids, astrocytes and p38 phosphorylation in the resolution of postoperative pain.

Anti-nociceptive tolerance to opioids is a well-described phenomenon, which severely limits the clinical efficacy of opioids for the treatment of chronic pain syndromes. The mechanisms that drive anti-nociceptive tolerance, however, are less well understood. We have previously shown that glia have a central role in the development of morphine tolerance and that administration of a glial modulating agent attenuated tolerance formation. Recently, we have demonstrated that morphine enhances microglial Iba1 expression and P2X4 receptor-mediated microglial migration via direct mu opioid receptor signaling in in vitro microglial cultures. We hypothesize that P2X4 receptors drive morphine tolerance and modulate morphine-induced spinal glial reactivity. Additionally, we hypothesize that perivascular microglia play a role in morphine tolerance and that P2X4 receptor expression regulates perivascular microglia ED2 expression. To test these hypotheses, rats were implanted with osmotic minipumps releasing morphine or saline subcutaneously for seven days. Beginning three days prior to morphine treatment, P2X4 receptor antisense oligonucleotide (asODN) was injected intrathecally daily, to selectively inhibit P2X4 receptor expression. P2X4 receptor asODN treatment inhibited morphine-induced P2X4 receptor expression and blocked anti-nociceptive tolerance to systemically administered morphine. P2X4 receptor asODN treatment also attenuated the morphine-dependent increase of spinal ionized calcium binding protein (Iba1), glial fibrillary acidic protein (GFAP) and mu opioid receptor protein expression. Chronic morphine also decreased perivascular microglial ED2 expression, which was reversed by P2X4 receptor asODN. Together, these data suggest that the modulation of P2X4 receptor expression on microglia and perivascular microglia may prove an attractive target for adjuvant therapy to attenuate opioid-induced anti-nociceptive tolerance.

Inhibition of microglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and mu opioid receptor protein expression while enhancing perivascular microglial ED2.

Depression and pain: does ketamine improve the quality of life of patients in chronic pain by targeting their mood?

Peripheral nerve injury generally results in spinal neuronal and glial plastic changes associated with chronic behavioral hypersensitivity. Spinal mitogen-activated protein kinases (MAPKs), eg, p38 or extracellular signal-regulated kinases (ERKs), are instrumental in the development of chronic allodynia in rodents, and new p38 inhibitors have shown potential in acute and neuropathic pain patients. We have previously shown that the cannabinoid type 2 receptor agonist JWH015 inhibits ERK activity by inducing MAPK phosphatase (MKP)-1 and MKP-3 (the major regulators of MAPKs) in vitro in microglial cells. Therefore, we decided to investigate the role of these phosphatases in the mechanisms of action of JWH015 in vivo using the rat L5 nerve transection model of neuropathic pain. We observed that peripheral nerve injury reduced spinal MKP-1/3 expression and activity and that intrathecal JWH015 reduced established L5 nerve injury-induced allodynia, enhanced spinal MKP-1/3 expression and activity, and reduced the phosphorylated form of p38 and ERK-1/2. Triptolide, a pharmacological blocker of MKP-1 and MKP-3 expression, inhibited JWH015's effects, suggesting that JWH015 exerts its antinociceptive effects by modulating MKP-1 and MKP-3. JWH015-induced antinociception and MKP-1 and MKP-3 expression were inhibited by the cannabinoid type 2 receptor antagonist AM630. Our data suggest that MKP-1 and MKP-3 are potential targets for novel analgesic drugs.

Spinal cannabinoid receptor type 2 agonist reduces mechanical allodynia and induces mitogen-activated protein kinase phosphatases in a rat model of neuropathic pain.

The mechanisms that drive the normal resolution of acute postoperative pain are not completely understood. We hypothesize a pivotal role of a major spinal mitogen-activated protein kinase (MAPKs) regulator, MAPK phosphatase (MKP)-3, in the resolution of postoperative pain. We used wild-type and MKP-3 knock-out (KO) mice, a paw incision model of acute postoperative pain, and behavioral and molecular biology experiments. We observed persistent mechanical allodynia in mice lacking MKP-3 (postoperative day 21), concurrently with persistent phosphorylation of spinal p38 and extracellular signal-regulated kinases (ERK)-1/2 on postoperative day 12, while both MAPK phosphorylation and allodynia resolved on postoperative day 7 in wild-type mice. Spinal p-ERK was expressed mainly in neurons and microglia, while spinal p-p38 was expressed mostly in microglia in MKP-3 KO mice, and their selective pharmacological inhibition reduced the persistent allodynia observed in these mice. Our findings strongly suggest that dysregulation of MKP-3 prevents spontaneous resolution of acute postoperative pain and drives its transition to persistent pain via persistent neuronal and microglial MAPK phosphorylation in the spinal cord.

Spinal mitogen-activated protein kinase phosphatase-3 (MKP-3) is necessary for the normal resolution of mechanical allodynia in a mouse model of acute postoperative pain.

Mitogen-activated protein kinases (MAPKs) play critical roles in the central nervous system immune responses through glial function, which are regulated with relative selectivity (or preference) by MAPK phosphatases (MKP). Phosphorylated extracellular signal-regulated protein kinase (p-ERK) is preferentially dephosphorylated by MKP-3, which display little activity over p-p38 and p-c-Jun NH2-terminal kinases (p-JNK). It has been proposed that these substrate preferences may vary depending on tissue or functional cellular processes. Since astrocytes display a prominent activity of JNK>ERK under stressed or reactive phenotype, we hypothesize that MKP-3 possess a similar or differential substrate preference in astrocytes for JNK and ERK (ERK=JNK or JNK>ERK). We generated transient expression of MKP-3 by transfecting a specific cDNA in primary rat neonatal brain cortex astrocytes. Cells were stimulated with lipopolysaccharide (LPS), and MAPKs and downstream pro-inflammatory products were measured by Western blot and ELISA analyses. MKP-3 expression in primary astrocytes reduced LPS-induced p-ERK and p-p38 by ∼50%, and p-JNK by ∼75%, and moderately reduced nitrite oxide (NO), while completely blocked Interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). We confirmed MKP-3 specific activity by developing a BV-2 microglia cell line stably overexpressing MKP-3 and using a specific siRNA against MKP-3. Our data demonstrate MKP-3 has differential substrate preference in astrocytes compared to other cells types, since it preferentially dephosphorylated p-JNK over p-ERK. Our results indicate also that astrocytic immune functions can be modulated by MKP-3 induction, a strategy that could be beneficial in neurological conditions in which astrocytes play a pathophysiological role, i.e. persistent pain.

Article	Year
NCX-701 (nitroparacetamol) is an effective antinociceptive agent in rat withdrawal reflexes and wind-up. British journal of pharmacology\| PubMed ID: 11906970	2002
Antinociception and the new COX inhibitors: research approaches and clinical perspectives. CNS drug reviews\| PubMed ID: 14530796	2003
Alpha2-adrenoceptor stimulation transforms immune responses in neuritis and blocks neuritis-induced pain. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16192389	2005
Nitroparacetamol (NCX-701) and pain: first in a series of novel analgesics. CNS drug reviews\| PubMed ID: 17894645	2007
Evidence for a role of endocannabinoids, astrocytes and p38 phosphorylation in the resolution of postoperative pain. PloS one\| PubMed ID: 20531936	2010
Inhibition of microglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and mu opioid receptor protein expression while enhancing perivascular microglial ED2. Pain\| PubMed ID: 20573450	2010
Depression and pain: does ketamine improve the quality of life of patients in chronic pain by targeting their mood? Anesthesiology\| PubMed ID: 21841467	2011
Spinal cannabinoid receptor type 2 agonist reduces mechanical allodynia and induces mitogen-activated protein kinase phosphatases in a rat model of neuropathic pain. The journal of pain : official journal of the American Pain Society\| PubMed ID: 22901764	2012
Spinal mitogen-activated protein kinase phosphatase-3 (MKP-3) is necessary for the normal resolution of mechanical allodynia in a mouse model of acute postoperative pain. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24155322	2013
Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) displays a p-JNK-MAPK substrate preference in astrocytes in vitro. Neuroscience letters\| PubMed ID: 24861519	2014

E. Alfonso Romero-Sandoval

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