Opiate Tolerance Research Articles

Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders.

Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.

0171 Parabrachial-CGRP neurons regulate awakenings to pain stimulus

Abstract Introduction The external lateral part of the PB (PBel), receives spinal afferents carrying pain-signals, which are then relayed to other brainstem and forebrain arousal areas. Many PBel neurons that express calcitonin gene-related peptide [CGRP; PBelCGRP] also receive spinal afferents carrying pain signals. Selective activation of PBelCGRP neurons, in CGRP-CreER mice, cause short latency cortical arousal, while inhibition of these neurons prevents waking to hypercapnia. Also, blocking CGRP receptors has a potential to reverse opiate tolerance in rodents, however, mechanism of action or the circuitry remains unknown. Methods To test if PBelCGRP is the critical relay node transmitting pain to induce cortical arousals, we conducted either genetic deletion or acute optogenetic inhibition of this node, in both inflammatory pain and opto-pain models. All mice were implanted for recording sleep.Inflammatory-pain model: CGRP-creER mice were bilaterally injected with AAV-Flex-DTA (n=10) to delete CGRP neurons in PBel or AAV-GFP (n=6; control). Both groups received a foot injection of either 5% formalin or saline (10 µl) and were recorded for the sleep.Opto-pain model: CGRP-ChR2 mice were injected in the PB with AAV-Flex-JAWS (n=5) and implanted with bilateral optical fibers directed to the PB, and miniature probes in the foot pad to facilitate stimulation of the nociceptors. We recorded sleep while stimulating nociceptors every 5 min with or without opto-inhibition of PBelCGRP. Results In mice with intact CGRP neurons, foot stimulation with formalin produced 100% wakefulness for the first three hours, while with deletion of PBCGRP neurons, pain induced sleep loss could be attenuated in both males (by 83%) and females(by 60%). This recovery of NREM sleep significantly correlated with loss of PBelCGRP. In the opto-pain model, opto-stimulation of nociceptors at 10Hz for 5 sec woke up mice in 100% of the trials with a short latency of 4.4 ± 0.78 sec, while opto-inhibition of PBelCGRP that expressed JAWS, by red laser that preceded the nociceptive stimulus by 20s, prevented stimulus-induced awakenings. Conclusion This suggests that PBelCGRP is the main relay node for awakening to pain. We are now investigating the terminal fields of the PBelCGRP neurons to assess which of them contribute to pain induced arousal. Support (If Any) NS112175

Cannabinoid treatment of opiate addiction

Opioid abuse is a growing global problem. Current therapies for opioid abuse target withdrawal symptoms and have several adverse side effects. There are no treatments to address opioid-induced neural adaptations associated with abuse and addiction. Preclinical research demonstrates interactions between the endogenous opioid and cannabinoid systems, suggesting that cannabinoids may be used to treat opioid addiction and dependence. The aim of this review is to assess how cannabinoids affect behavioural and molecular measures of opioid dependence and addiction-like behaviour in animal models. It appears that cannabidiol and cannabinoid receptor 1 (CB1R) antagonists have potential for treating drug-craving and drug-seeking behaviour, based on evidence from preclinical animal models. Ligands which inhibit the action of cannabinoid degradation enzymes also show promise in reducing opioid withdrawal symptoms and opioid self-administration in rodents. Agonists of CB1R could be useful for treating symptoms of opioid withdrawal; however, the clinical utility of these drugs is limited by side effects, the potential for cannabinoid addiction and an increase in opiate tolerance induced by cannabinoid consumption. The mechanisms by which cannabinoids reduce opioid addiction-relevant behaviours include modulation of cannabinoid, serotonin, and dopamine receptors, as well as signalling cascades involving ERK-CREB-BDNF and peroxisome proliferator-activated receptor-α. Identifying the receptors involved and their mechanism of action remains a critical area of future research.

Inhibition of fatty acid amide hydrolase in the CNS prevents and reverses morphine tolerance in male and female mice.

Fatty acid amide hydrolase (FAAH) is an intracellular serine amidase that terminates the signalling of various lipid messengers involved in pain regulation, including anandamide and palmitoylethanolamide. Here, we investigated the effects of pharmacological or genetic FAAH removal on tolerance to the anti-nociceptive effects of morphine. We induced tolerance in male and female mice by administering twice-daily morphine for 7 days while monitoring nociceptive thresholds by the tail immersion test. The globally active FAAH inhibitor URB597 (1 and 3 mg·kg-1 , i.p.) or the peripherally restricted FAAH inhibitor URB937 (3 mg·kg-1 , i.p.) were administered daily 30 min prior to morphine, alone or in combination with the cannabinoid CB1 receptor antagonist AM251 (3 mg·kg-1 , i.p.), the CB2 receptor antagonist AM630 (3 mg·kg-1 , i.p.), or the PPAR-α antagonist GW6471 (4 mg·kg-1 , i.p.). Spinal levels of FAAH-regulated lipids were quantified by LC/MS-MS. Gene transcription was assessed by RT-qPCR. URB597 prevented and reversed morphine tolerance in both male and female mice. This effect was mimicked by genetic FAAH deletion, but not by URB937. Treatment with AM630 suppressed, whereas treatment with AM251 or GW6471, attenuated the effects of URB597. Anandamide mobilization was enhanced in the spinal cord of morphine-tolerant mice. mRNA levels of the anandamide-producing enzyme N-acyl-phosphatidylethanolamine PLD (NAPE-PLD) and the palmitoylethanolamide receptor PPAR-α, but not those for CB2 , CB1 receptors or FAAH, were elevated in spinal cord CONCLUSION AND IMPLICATIONS: FAAH-regulated lipid signalling in the CNS modulated opiate tolerance, suggesting FAAH as a potential target for opiate-sparing medications.

Generating highly potent and efficacious antibodies to the cholecystokinin B (CCK‐B) receptor by ribosome display for the treatment of neuropathic pain

Development of effective non‐opioid therapeutics tailored to specific chronic pain syndromes is sorely needed. Our recent microchip gene expression profile identified upregulated cholecystokinin B (CCK‐B) receptor in trigeminal ganglia (TG) as one suitable target among many others. Thus, the CCK‐B receptor is an ideal candidate to impact both nociceptive and limbic components of chronic pain. More importantly, its inhibition can reverse opiate tolerance. Based on the literature and the gene expression changes over time, the CCK‐B receptor is implicated as one of the persisting functional targets suitable for development of therapeutics to inhibit chronic pain‐ and anxiety‐related behaviors. Here we describe the isolation and characterization of a panel of CCK‐B receptor specific antibodies using ribosome display technology. Furthermore, we demonstrate in vivo efficacy of our antibodies in a relevant model of chronic neuropathic pain. This is the first example, to date, of scFv antibodies that can block the CCK‐B receptor directly with high potency and specificity. This highlights the exciting potential therapeutic opportunity of an antibody for blocking this type of receptor.Support or Funding InformationThis work was supported by Startup dollars from Loyola University Medical Center (RD) and University of New Mexico (KW).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co-administration of Phycocyanobilin and/or Phase 2-Inducer Nutraceuticals for Prevention of Opiate Tolerance.

Chronic use of opiates for control of chronic pain is complicated by the development of tolerance and hyperalgesia, and hence usually entails dose escalation and diminished efficacy. Our evolving understanding of the mechanisms mediating induction of morphine tolerance may enable discovery of adjunct measures which can prevent this tolerance; this essay proposes that certain nutraceuticals may have utility in this regard. Considerable evidence now points to an obligate role for production of peroxynitrite and other oxidants in the dorsal horn in development of morphine tolerance. Various isoforms of NADPH oxidase are the chief source of the superoxide which gives rise to these oxidants. Since heme oxygenase, via its products bilirubin and carbon monoxide, functions as a physiological inhibitor of various isoforms of NADPH oxidase, phase 2-inducing nutraceuticals with blood brain-barrier permeability such as lipoic acid, an effective inducer of heme oxygenase-1, may have potential for prevention of morphine tolerance; indeed, this has been demonstrated in a mouse study. The phycocyanobilin (PhyCB) chromophore of spirulina, a structural analog of biliverdin, shares bilirubin's ability to inhibit NAPDH oxidase complexes; hence, administration of spirulina or of PhyCB-enriched spirulina extracts merits evaluation in rodent models of morphine tolerance. Uric acid quenches peroxynitrite-derived radicals, and its plasma level can be boosting via supplementation with inosine; indeed, administration of inosine has been shown to counteract development of hyperalgesia in rodents. If practical doses of these agents can be shown to prevent morphine tolerance and hyperalgesia in rodents, their use as adjuvants to clinical opiate therapy should be assessed.

Exploring Morphine-Triggered PKC-Targets and Their Interaction with Signaling Pathways Leading to Pain via TrkA.

It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain.

The effect of metformin on morphine analgesic tolerance and dependence in rats

Opiate tolerance and dependence is a worldwide public health problem and gives a significant burden to society. The aim of this study was to evaluate the effects of metformin (MET) on development and expression of morphine tolerance and dependence in rats. For induction of tolerance, morphine sulfate was injected (10 mg/kg, twice a day, s.c.) for 7 days. Animals received metformin (5 and 50 mg/kg, orally, daily) during the examination period for assessing the development of morphine tolerance and dependence. In order to evaluate the expression of morphine tolerance and dependence, single doses of MET (5 and 50 mg/kg, orally) were administered on day 7. Tail flick test was performed to assess the induction of morphine tolerance. For evaluation of morphine dependence, naloxone-induced jumping (5 mg/kg, s.c.) was monitored. Our results showed that 7 days coadministration of 50 mg/kg of MET significantly reduced the development of morphine analgesic tolerance versus morphine + saline treated rats (P < 0.001). Treatment with 50 mg/kg MET reduced the incidence and frequency of jumping in naloxone injected animals (P < 0.01). It is notable that single dose administration of MET, did not prevent the expression of analgesic tolerance and physical dependence to morphine. Based on these results, it can be concluded that MET attenuates the development of morphine analgesic tolerance and dependence in rats.

Inhibition of morphine tolerance by MrgC receptor via modulation of interleukin-1β and matrix metalloproteinase 9 in dorsal root ganglia in rats

Opiate tolerance is a critical issue in pain management. Previous studies show that activation of Mas-related gene (Mrg) C receptor can modulate the development of morphine tolerance. This study was designed to investigate the underlying mechanism(s). Intrathecal (i.t.) administration of morphine (20µg) increased the expression of interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) in small- and medium-sized neurons in dorsal root ganglia (DRG). Co-administration of bovine adrenal medulla 8–22 (BAM8-22), a selective MrgC receptor agonist, via i.t. route inhibited the increase of IL-1β and MMP-9 in the DRG. Exposure of DRG cultures to morphine (3.3μM) for 3 or 5 days, but not for 1 day, induced an increase in MMP-9 mRNA expression. The treatment with BAM8-22 (10nM) for 20, 40 or 60min abolished chronic (5 days) morphine-induced increase of MMP-9 mRNA in the cultured DRG. The treatment with BAM8-22 for 1h inhibited chronic morphine-induced increase of MMP-9 and IL-1β mRNA in DRG but these effects were abolished by MrgC receptor antibody. The treatment with BAM8-22 for 24 and 72h respectively inhibited and enhanced morphine-induced expression of MMP-9 and IL-1β mRNA in the cultured DRG. The BAM8-22-induced inhibition and enhancement were abolished by MrgC receptor antibody. The results suggest that the inhibition of IL-1β and MMP-9 expressions in DRG underlain the modulation of morphine tolerance by the acute activation of MrgC receptors. The chronic activation of MrgC receptors can facilitate morphine-induced increase of MMP-9 and IL-1β expressions in DRG.

Anti-opioid Effects of RFRP-3 on Magnocellular Neuron Activity in Morphine-naïve and Morphine-treated Female Rats.

Neuropeptide FF receptors (NPFFR1 and NPFFR2) have been proposed to possess anti-opioid properties, and be involved in the development of opiate tolerance and dependence. However, there is no evidence to date supporting such opioid effects at the cellular level in vivo. Using in vivo electrophysiological recordings from vasopressin and oxytocin neurons in the supraoptic nucleus, we aimed to determine the effects of NPFFRs on opiate inhibition, tolerance, and dependence at a cellular level. Both vasopressin and oxytocin neurons are acutely inhibited by opioids and develop opiate tolerance. Oxytocin neurons also develop cellular opiate dependence and undergo withdrawal hyperexcitation upon cessation of opiate administration. Here, the classical μ-opioid receptor agonist, morphine robustly inhibited the spontaneous firing rate of vasopressin and oxytocin neurons, and this inhibition was attenuated by pretreatment with the NPFFR1 agonist, RFamide-related peptide-3. In rats infused with morphine for 6 d, vasopressin neurons were unresponsive to morphine, indicating the development of cellular tolerance, but pretreatment with the NPFFR antagonist, GJ14, restored acute morphine inhibition. In morphine-infused rats, RFamide related peptide-3 did not induce withdrawal excitation in oxytocin neurons and GJ14 did not reverse naloxone-precipitated withdrawal excitation. This is the first evidence of anti-opioid effects of the NPFFR system at a cellular level in vivo. Our results suggest that the anti-opioid properties of the NPFFR system reduce morphine sensitivity during tolerance but that it is not involved in dependence.

NPYFa, A Chimeric Peptide of Met-Enkephalin, and NPFF Induces Tolerance-Free Analgesia.

Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies indicated that NPYFa induced an early onset, potent, dose-dependent and prolonged antinociception. Additionally, antagonists (MOR, KOR, and DOR) pretreatment studies determined a KOR-mediated antinociception activity of the ligand. Further, in vitro binding studies using the Eu-GTP-γS binding assay on cell lines expressing opioid and NPFF receptors showed binding to both the opioid and neuropeptide FF receptors suggesting a multiple receptor binding character of NPYFa. Moreover, chronic (6 days) treatment with NPYFa exhibited an absence of tolerance development subsequent to its analgesia. The current study proposes NPYFa as a potent, long-acting antinociceptor lacking tolerance development as well as a probe to study opioid analgesia and the associated complex mechanisms of tolerance development.

Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model

ABSTRACTDuring the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

EphB2 reverse signaling regulates learned opiate tolerance via hippocampal function

Despite significant progress, many uncertainties remain regarding molecular and cellular mechanisms governing opiate tolerance. We report that loss of EphB2 receptor reverse signaling results in a marked acceleration of morphine tolerance in vivo. EphB2 null mice exhibited no significant difference in brain or blood morphine metabolism, mu opiate receptor affinity or binding capacity. Motor and sensory performance for EphB2 null mice was also comparable to controls for both morphine naïve or tolerized states. Regional distributions of mu opioid receptor, CGRP and substance P were also unaltered in EphB2 null mice. However EphB2 null mice, but not animals homozygous for kinase dead version of EphB2, exhibited significant modification of context-dependent anti-nociceptive responses following chronic morphine treatment. To verify the changes seen in EphB2 null mice arise from impairment of hippocampal learning, discreet bilateral lesions of the dorsal hippocampus were produced in wildtype mice demonstrating striking similarities to that seen in EphB2 null mice for opiate-dependent behavior. The results demonstrate that EphB2 reverse signaling plays a unique and requisite role in inhibiting the development of opiate-dependent tolerance in vivo.

Methadone Reverses Analgesic Tolerance Induced by Morphine Pretreatment.

Background:Opiates such as morphine are the most powerful analgesics, but their protracted use is restrained by the development of tolerance to analgesic effects. Recent works suggest that tolerance to morphine might be due to its inability to promote mu opioid receptor endocytosis, and the co-injection of morphine with a mu opioid receptor internalizing agonist like [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin reduces tolerance to morphine. So far, no studies have been conducted to evaluate the ability of methadone to reduce morphine tolerance in morphine-pretreated animals, a treatment sequence that could be encountered in opiate rotation protocol. We investigated the ability of methadone (a mu opioid receptor internalizing agonist used in therapy) to reverse morphine tolerance and the associated cellular mechanisms in the periaqueductal gray matter, a region involved in pain control.Methods:We measured analgesic response following a challenge dose of morphine in the hot plate test and investigated regulation of mu opioid receptor (coupling and endocytosis) and some cellular mechanisms involved in tolerance such as adenylate cyclase superactivation and changes in N-methyl-d-aspartate receptor subunits expression and phosphorylation state.Results:A chronic treatment with morphine promoted tolerance to its analgesic effects and was associated with a lack of mu opioid receptor endocytosis, adenylate cyclase overshoot, NR2A and NR2B downregulation, and phosphorylation of NR1. We reported that a methadone treatment in morphine-treated mice reversed morphine tolerance to analgesia by promoting mu opioid receptor endocytosis and blocking cellular mechanisms of tolerance.Conclusions:Our data might lead to rational strategies to tackle opiate tolerance in the frame of opiate rotation.

Editorial: Monitoring endogenous GPCRs: lessons for drug design.

Editorial: Monitoring endogenous GPCRs: lessons for drug design.

Reversal of morphine tolerance by a compound with NPFF receptor subtype-selective actions

Neuropeptide FF (NPFF) modulates opiate actions. It has pro-nociceptive effects, primarily through the NPFF receptor 1 subtype, and anti-nociceptive effects, primarily through the NPFFR2 subtype. AC-263093 is a small l, organic, systemically active molecule that was previously shown to functionally activate NPFFR2, but not NPFFR1. It was hypothesized that AC-263093 would attenuate morphine tolerance. Rats were tested for radiant heat tail-flick latency before and after 5mg/kg morphine sulfate s.c. They were then rendered morphine-tolerant by continuous subcutaneous infusion of 17.52mg/kg/day morphine sulfate. On the seventh day of infusion, they were retested for analgesia 10 and 20min after 5mg/kg morphine sulfate s.c. Tolerance was indicated by reduction of morphine analgesia from the pre-infusion test. Fifty minutes prior to morphine challenge, rats received either 10mg/kg i.p. AC-263093 or injection vehicle alone. AC-2623093-treated rats had far smaller tolerance scores than control rats. This drug effect was significant, p=0.015. The same dose of AC-263093 had almost no analgesic effect in non-tolerant, saline-infused rats. In vitro experiments revealed that AC-263093 had equal affinity for NPFFR1 and NPFFR2, and functionally inactivated NPFFR1, in addition to its previously shown ability to activate NPFFR2. Thus, altering the balance between activation of NPFF receptor subtypes may provide one approach to reversing opiate tolerance.

The role of P2X receptors in morphine tolerance

Background The analgesic potency of morphine degrades rapidly after repeated administration,which is known as morphine antinociceptive tolerance.Series of progress have been made towards illustrating the mechanisms underlying morphine antinociceptive tolerance,but the details still remains unknown.Most of the researches in the past years prevailingly focused on neuronal mechanisms of adaptation,sensitization,and so on.Recently,much attention has been paid to investigate the critical role of glia and purinergic recepters in morphine antinociceptive tolerance.Objective We summarize the research progress of P2X recepter a subtype of purinergic recepters in morphine antinociceptive tolerance in this review.Content We described the research history as well as distribution of purinergic recepters,and reviewed respectively the role of 7 subtypes of P2X receptors in morphine tolerance.Trend The P2X recepters play a prominent role in morphine and neuropathic pain.Further studies will be helpful to illuminate the complicated mechanism of morphine tolerance and promote its clinical therapy. Key words: P2X; Purinergic recepter; Morphine tolerance; Opiate tolerance; Glia

Part B provider status would guide payers today, in future

“Tom” and “Ann” were not quite old enough for Medicare when they went to see Dan Buffington, PharmD, MBA, for medication therapy management (MTM). The couple’s monthly out-of-pocket prescription drug costs were about $900, and they were struggling to cover them. They went to Buffington for help. “We did an MTM consultation, and between a combination of patient-assistance programs and other strategies, we were able to reduce their costs from close to $1,000 to about $100 a month,” Buffington said. Buffington’s patients at Clinical Pharmacy Services in Tampa, FL, are typically on 10 to 15 medications and seeing about five physicians. “People are very happy to find someone who will be a bridge across all these doctors and monitor their therapy,” he said. They are not unlike many Americans. Nearly one-half of Americans older than 65 years have two or more of the nine most common chronic diseases, which makes treatment and management of those diseases particularly complicated. This may explain in part why one-third of hospital readmissions are medication-related and why a growing number of Americans seek MTM. In addition to managing multiple medications across numerous conditions and specialists, Buffington manages patients on chronic pain medications in order to help them avoid opiate tolerance or dependence. He tracks patient adherence, assesses regimens for possible drug interactions, educates patients, and refines regimens for patients who could take fewer drugs. Arguably, nearly one-half of U.S. seniors, and many younger patients as well, would benefit from this type of care. But Medicare, which is managed by CMS, does not recognize Buffington and other pharmacists as health care providers who are eligible for payment with CMS funds. As a result, neither do most other payers, such as commercial insurance and health plans. ■Dan Buffington relies on various ways to be paid for services for which other providers could simply bill.■Pharmacists are a pivotal part of future solutions in health care models. ■Dan Buffington relies on various ways to be paid for services for which other providers could simply bill.■Pharmacists are a pivotal part of future solutions in health care models. “Pharmacists are a pivotal part of future solutions in health care models, whether it’s controlling costs of readmissions, identification of drug-related problems, or controlling pharmaceutical spending. Pharmacists are integral to all those and just simply not able to be functionally paid,” he said. Medicare Part B covers care delivered by doctors, nurses, physical therapists, speech pathologists, and many other providers, but not pharmacists. So Buffington relies on a number of means, and some technicalities, in order to be paid for services for which other health care providers could simply bill. Some of his patients get care from Buffington by proxy through the physician on staff in his practice. Others get care because their Medicare Part D prescription drug plan requested it. Some carry one of the few health insurance policies that cover pharmacist-delivered MTM. And others pay out of pocket. Profiles in provider statusPharmacists explain how their patients would benefit from provider status in a new series of profiles appearing in Pharmacy Today and on pharmacist.com. Pharmacists explain how their patients would benefit from provider status in a new series of profiles appearing in Pharmacy Today and on pharmacist.com. “What Medicare does, secondary payers will do as well,” Buffington said. “There’s a laundry list of secondary impacts that achieving Medicare Part B- eligible provider status would resolve.” He is careful to point out, however, that achieving provider status isn’t just about ensuring MTM billing codes become included in standard fee schedules. “There are many other health care professionals who live in that elective service model every day and they do fine,” he said. More than fixing fees, Medicare Part B provider status would mean Medicare recognizes pharmacists as full members of the allied health professions, not simply providers of an elective service. “In fact, out of all the different health care professions, pharmacists’ services have some of the strongest and most impactful return on investment data,” Buffington added. While the Affordable Care Act may shift many health care providers out of the fee-for-service payment model and into bundled payment, that will not diminish the importance of provider status for pharmacists, according to Buffington. “Down the road, through health care reform, the new patient-centered medical homes and ACOs [accountable care organizations] could be confused and perceive that MTM is not a covered service under Medicare, simply because we are not included in the list of eligible providers for payment,” noted Buffing- ton. “We need to do something that is correct today and that simultaneously includes pharmacists in a position to be part of the solution in future health care delivery and payment models. And that, simply put, is correcting the Medicare Part B-eligible provider status issue.”

Serine 363 is Required for NOPR Desensitization, Internalization, and Arrestin Signaling

Nociceptin/Orphanin FQ peptide receptor (NOPR) plays a key role in pain modulation, opiate tolerance, and responsivity to stress and anxiety. C‐terminal phosphorylation at serine (S), threonine (T), and tyrosine (Y) residues are required for μ and κ opioid receptor internalization, desensitization, arrestin recruitment, and mitogen‐activated protein kinase phosphorylation (pMAPK), so we generated mutations in the NOPR c‐terminus. We showed that NOPR‐YFP internalized shortly after nociceptin treatment while NOPR‐S363A blocked internalization. We determined time course and concentration‐dependence of NOPR‐YFP‐mediated pMAPK. NOPR‐YFP‐induced pERK peaked at 10min following treatment and pJNK peaked at 30min. NOPR‐S363A showed reduced pJNK levels. We determined that NOPR‐YFP internalization is blocked by arrestin3 and GRK3 shRNAs and that NOPR‐S363A internalization and pJNK can be rescued via a dominant positive arrestin3. These data implicate GRK/arrestin in NOPR MAPK signaling and highlight the potential for the development of functionally selective NOPR‐ligands. This work was partially supported by NIH grant R00DA034929 (to MRB), T32DA007261 (to SDC) and HHMI SURF Program (to NZ).

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.