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2.  Tachykinin NK1 receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation 
European journal of pharmacology  2012;684(1-3):64-70.
Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK1 receptor antagonist (N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in morphine-withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK1 antagonist pharmacophor yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK1 receptor antagonists may provide a novel paradigm for long-term pain management.
PMCID: PMC3565540  PMID: 22724132
opioid-induced hyperalgesia; spinal glia; Tachykinin NK1 receptor; Tachykinin NK1 receptor antagonist
3.  Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist 
Journal of Neuroimmunology  2012;244(1-2):23-31.
Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine–mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.
doi:10.1016/j.jneuroim.2011.12.021
PMCID: PMC3298577  PMID: 22285397
morphine; spinal glia; CB2 agonist; hyperalgesia; allodynia; pain sensitization
4.  EAACI: A European Declaration on Immunotherapy. Designing the future of allergen specific immunotherapy 
Allergy today is a public health concern of pandemic proportions, affecting more than 150 million people in Europe alone. In view of epidemiological trends, the European Academy of Allergy and Clinical Immunology (EAACI) predicts that within the next few decades, more than half of the European population may at some point in their lives experience some type of allergy.
Not only do allergic patients suffer from a debilitating disease, with the potential for major impact on their quality of life, career progression, personal development and lifestyle choices, but they also constitute a significant burden on health economics and macroeconomics due to the days of lost productivity and underperformance. Given that allergy triggers, including urbanization, industrialization, pollution and climate change, are not expected to change in the foreseeable future, it is imperative that steps are taken to develop, strengthen and optimize preventive and treatment strategies.
Allergen specific immunotherapy is the only currently available medical intervention that has the potential to affect the natural course of the disease. Years of basic science research, clinical trials, and systematic reviews and meta-analyses have convincingly shown that allergen specific immunotherapy can achieve substantial results for patients, improving the allergic individuals’ quality of life, reducing the long-term costs and burden of allergies, and changing the course of the disease. Allergen specific immunotherapy not only effectively alleviates allergy symptoms, but it has a long-term effect after conclusion of the treatment and can prevent the progression of allergic diseases.
Unfortunately, allergen specific immunotherapy has not yet received adequate attention from European institutions, including research funding bodies, even though this could be a most rewarding field in terms of return on investments, translational value and European integration and, a field in which Europe is recognized as a worldwide leader. Evaluation and surveillance of the full cost of allergic diseases is still lacking and further progress is being stifled by the variety of health systems across Europe. This means that the general population remains unaware of the potential use of allergen specific immunotherapy and its potential benefits.
We call upon Europe’s policy-makers to coordinate actions and improve individual and public health in allergy by:
Promoting awareness of the effectiveness of allergen specific immunotherapy
Updating national healthcare policies to support allergen specific immunotherapy
Prioritising funding for allergen specific immunotherapy research
Monitoring the macroeconomic and health economic parameters of allergy
Reinforcing allergy teaching in medical disciplines and specialties
The effective implementation of the above policies has the potential for a major positive impact on European health and well-being in the next decade.
doi:10.1186/2045-7022-2-20
PMCID: PMC3514324  PMID: 23110958
Allergy; Asthma; Rhinitis; Immunotherapy; Health economics; Quality of life
5.  Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist 
Journal of medicinal chemistry  2011;54(7):2467-2476.
A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.
doi:10.1021/jm1016285
PMCID: PMC3096782  PMID: 21413804
NK1 receptor antagonists; opioids; multitarget drug design; designed multiple ligands
6.  Sustained morphine treatment augments prostaglandin E2-evoked Calcitonin Gene-Related Peptide release from primary sensory neurons in a PKA- dependent manner 
European journal of pharmacology  2010;648(1-3):95-101.
Tissue damage leads to pain sensitization due to peripheral and central release of excitatory mediators such as prostaglandin E2 (PGE2). PGE2 sensitizes spinal pain neurotransmitter such as calcitonin gene-related peptide (CGRP) release via activation of cyclic AMP (cAMP)/Protein kinase A (PKA) -dependent signaling mechanisms. Our previous data demonstrates that sustained morphine pretreatment sensitizes adenylyl cyclase(s) (AC) toward the direct stimulator, forskolin, in cultured primary sensory neurons (AC superactivation). In the present work we investigated the hypothesis that morphine pretreatment also sensitizes ACs toward Gs protein-coupled excitatory modulators (such as PGE2), leading to augmented PKA-dependent CGRP release from PGE2-stimulated primary sensory dorsal root ganglion (DRG) neurons. Our results show that sustained morphine treatment potentiated PGE2-mediated cAMP formation and augmented PGE2-evoked CGRP release from cultured primary sensory neurons in a PKA-dependent manner. Our data suggests that attenuation of AC-superactivation in primary sensory neurons may prevent the development of opioid-induced hyperalgesia.
doi:10.1016/j.ejphar.2010.08.042
PMCID: PMC2955884  PMID: 20826131
Morphine; PGE2; PKA; CGRP release; DRG neurons; opioid-induced hyperalgesia; Gs protein signaling
7.  Intrathecal Raf-1-selective siRNA attenuates sustained morphinemediated thermal hyperalgesia 
European journal of pharmacology  2008;601(1-3):207-208.
Studies have demonstrated that long-term opioid treatment leads to an increased sensitivity to painful (hyperalgesia) or normally innocuous (allodynia) stimuli. The molecular mechanisms that lead to paradoxical pain sensitization upon chronic opioid treatment are not completely understood. Enhanced excitatory pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release in the dorsal horn of the spinal cord may play a role in sustained morphine-mediated paradoxical pain. Recently we have demonstrated that inhibition of Raf-1 attenuates sustained morphine treatment-mediated augmentation of CGRP release in vitro, in cultured primary sensory neurons. In the present study, we show that knockdown of spinal Raf-1 levels in vivo by intrathecal administration of Raf-1-specific siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats.
doi:10.1016/j.ejphar.2008.10.033
PMCID: PMC2640499  PMID: 18976650
Opioids; Morphine; Raf-1; Hyperalgesia
8.  Quantitative Evaluation of Human δ Opioid Receptor Desensitization Using the Operational Model of Drug Action 
Molecular pharmacology  2007;71(5):1416-1426.
Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human δ opioid receptors (hDOR) by measuring guanosine 5′-O-(3-[35S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of β-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of β-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced β-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.
doi:10.1124/mol.106.030023
PMCID: PMC2694736  PMID: 17322005
9.  Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner 
European journal of pharmacology  2008;584(2-3):272-277.
Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.
doi:10.1016/j.ejphar.2008.02.013
PMCID: PMC2375088  PMID: 18328477
Morphine; CGRP; Raf-1; cAMP; PKA; Dorsal root ganglion neurons
10.  Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling 
European journal of pharmacology  2007;581(1-2):19-29.
Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD = 0.2 ± 0.03 nM and 2 ± 0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB1 receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940- and WIN 55,212 - bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD = 27 ± 8 nM and KD = 10.7 ± 4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.
doi:10.1016/j.ejphar.2007.11.053
PMCID: PMC2279194  PMID: 18162180
trafficking; G proteins; PWR spectroscopy; functional selectivity
11.  Cell Signaling and Trafficking of Human Melanocortin Receptors in Real Time Using Two-photon Fluorescence and Confocal Laser Microscopy: Differentiation of Agonists and Antagonists 
Chemical biology & drug design  2006;68(4):183-193.
Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.
doi:10.1111/j.1747-0285.2006.00432.x
PMCID: PMC2547351  PMID: 17105482
β-arrestin; green fluorescence protein; melanocortin receptors; MTII; rhodamine; SHU-9119; two-photon fluorescence laser scanning microscopy
12.  New Paradigms and Tools in Drug Design for Pain and Addiction 
The AAPS journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
13.  New paradigms and tools in drug design for pain and addiction 
The AAPS Journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
14.  Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL 
Molecular and Cellular Biology  2004;24(21):9317-9326.
A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IκB kinase β (IKK-β), IKK-α, and IKK-γ/N, leading to changes in NF-κB-dependent gene expression. However, it is not clear how the NF-κB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-α)-dependent but not interleukin-1-dependent NF-κB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-κB activity. SIMPL interacts with nuclear p65 in a TNF-α-dependent manner to promote endogenous NF-κB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-α activation of NF-κB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-α-specific induction of gene expression.
doi:10.1128/MCB.24.21.9317-9326.2004
PMCID: PMC522234  PMID: 15485901

Results 1-14 (14)