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Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min., (© 2020 Wiley-VCH GmbH.)

We recently reported (Verzillo, et al. J. Neurochem: 130, 790-796, 2014) that chronic systemic morphine selectively up-regulates mRNA encoding two C-terminal μ-opioid receptor (MOR) splice variants, MOR-1C1 and MOR-1B2 (MOR-1B2/-1C1). Given the known disconnects between changes in levels of mRNA and corresponding protein, it is essential to directly demonstrate that chronic opioid treatment elevates functional MOR-1B2/-1C1 protein prior to inferring relevance of these MOR variants to spinal opioid tolerance mechanisms. Accordingly, we investigated the ability of chronic opioid exposure to up-regulate MOR protein in Chinese hamster ovary cells stably transfected with rat MOR variants MOR-1B2, MOR-1C1, or MOR-1 (considered to be the predominant MOR). Findings revealed that chronic treatment with the clinically relevant opioids morphine, oxycodone and hydrocodone substantially up-regulated membrane MOR-1B2/-1C1 protein. This up-regulation was abolished by the protein synthesis inhibitor cycloheximide, eliminating contributions from receptor redistribution. The increment in MOR-1B2/-1C1 protein was paralleled by a significant increment in opioid agonist-stimulated GTPγS-binding (reflective of increased aggregate MOR G protein coupling) indicating that up-regulated MOR-1B2/-1C1 represented functional receptors. Strikingly, these tolerance-associated adaptations of MOR-1B2/-1C1 differed considerably from those of MOR-1. Antithetical regulation of MOR-1B2/-1C1 and MOR-1 by chronic opioids has significant implications for the design of new therapeutic agents to counteract opioid analgesic tolerance and accompanying addiction. Since chronic opioids induce MOR-1B2/-1C1 up-regulation in spinal cord of males, but not females, elucidating cellular compartments and intracellular pathways mediating MOR-1B2/-1C1 up-regulation and defining their unique signaling attributes would enable a precision medicinal approach to pain management and addiction therapy. In the spinal cord of males, but not females, chronic morphine up-regulates mRNA encoding two mu-opioid receptor (MOR) variants, MOR-1B2 and MOR-1C1 (MOR-1B2/-1C1). We now demonstrate that chronic treatment with the clinically relevant opioids morphine, hydrocodone or oxycodone up-regulates MOR-1B2/-1C1 functional protein, which is dependent on de novo protein synthesis. Findings underscore the importance of unique signaling attributes of MOR variants to sexually dimorphic tolerance mechanisms., (© 2015 International Society for Neurochemistry.)

Unlabelled: Interactions between exogenous and endogenous opioids are not commonly investigated as a basis for sexually dimorphic opioid analgesia. We investigated the influence of spinal endomorphin 2 (EM2), an endogenous mu-opioid receptor (MOR) ligand, on the spinal antinociception produced by intrathecally administered opioids. Activation of spinal MORs facilitated spinal EM2 release. This effect was sexually dimorphic, occurring in males but not in females. Although activational effects of testosterone were required for opioid facilitation of spinal EM2 release in males, the absence of this facilitation in females did not result from either insufficient levels of testosterone or mitigating effects of estrogens. Strikingly, in males, the contribution of spinal EM2 to the analgesia produced by intrathecally applied MOR agonists depended on their analgesic efficacy relative to that of EM2. Spinal EM2 released by the higher efficacy MOR agonist sufentanil diminished sufentanil's analgesic effect, whereas EM2 released by the lower efficacy morphine had the opposite effect on spinal morphine antinociception. Understanding antithetical contributions of endogenous EM2 to intrathecal opioid antinociception not only enlightens the selection of opioid medications for pain management but also helps to explain variable sex dependence of the antinociception produced by different opioids, facilitating the acceptance of sexually dimorphic antinociception as a basic tenet., Perspective: The male-specific MOR-coupled enhancement of spinal EM2 release implies a parallel ability to harness endogenous EM2 antinociception. The inferred diminished ability of females to utilize the spinal EM2 antinociceptive system could contribute to their greater frequency and severity of chronic pain syndromes., (Copyright © 2015 American Pain Society. Published by Elsevier Inc. All rights reserved.)

The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction., (© 2014 International Society for Neurochemistry.)

The present study examined efficacy of a series of opioid agonists and then using chronic in vivo treatment protocols, determined tolerance to opioid agonist stimulated [(35)S]GTPγS (guanosine 5'-O-(3-[(35)S] thio)triphosphate) binding in mouse spinal cord membranes and compared it directly to spinal analgesic tolerance. The [(35)S]GTPγS binding assay was used to estimate efficacy (E(max) and τ; Operational Model of Agonism) of a series of opioid agonists for G-protein activation in mouse spinal cord. The rank order of opioid agonist efficacy determined in the [(35)S]GTPγS assay using the Operational Model and E(max) was similar. These efficacy estimates correlated with historical analgesic efficacy estimates. For tolerance studies, mice were continuously treated s.c. for 7days with morphine, oxycodone, hydromorphone, etorphine or fentanyl and [(35)S]GTPγS studies were conducted in spinal cord membranes. Other mice were tested in i.t. analgesia dose response studies (tailflick). Tolerance to DAMGO ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin) or morphine stimulated [(35)S]GTPγS binding (decrease in E(max)) was observed following etorphine and fentanyl treatment only. These treatment protocols downregulate μ-opioid receptor density whereas morphine, oxycodone and hydromorphone do not. Spinal analgesic tolerance was observed following all treatment protocols examined (morphine, oxycodone and etorphine). Opioid antagonist treatment that specifically upregulates (chronic naltrexone) or downregulates (clocinnamox) μ-opioid receptor density produced a corresponding change in opioid agonist stimulated [(35)S]GTPγS binding. Although receptor downregulation and G-protein uncoupling are among potential mechanisms of opioid tolerance, the present results suggest that uncoupling in mouse spinal cord plays a minor role and that the [(35)S]GTPγS assay is particularly responsive to changes in μ-opioid receptor density., (Copyright © 2011 Elsevier Inc. All rights reserved.)

On the basis of efficacy, opioid antagonists are classified as inverse opioid agonists (e.g. naltrexone) or neutral opioid antagonists (e.g. 6β-naltrexol). This study examined the interaction between naltrexone and 6β-naltrexol in the precipitated opioid withdrawal syndrome in morphine dependent mice. Furthermore, the possible contribution of constitutive opioid receptor activity to precipitated withdrawal was evaluated using increasing levels of morphine dependence. In the first experiment, low doses of 6β-naltrexol antagonized naltrexone precipitated withdrawal while high doses acted additively. All doses of naltrexone increased 6β-naltrexol's potency to precipitate withdrawal. The next experiment examined changes in antagonist potency to precipitate withdrawal with increasing morphine dependence. Mice were exposed to morphine for 1-6 days and then withdrawal was precipitated. Naltrexone was more potent than 6β-naltrexol at all the time points. The ED(50) of both drugs decreased at the same rate suggesting that increased dependence produced no change in constitutive opioid receptor activity. Taken together these results indicate that the functional efficacy of 6β-naltrexol is dose-dependent and that constitutive opioid receptor activity did not change as opioid dependence increased from 1 to 6 days., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Rationale: Analgesic efficacy of opioids and dosing protocol have been shown to influence analgesic tolerance., Objective: This study tested the hypothesis that there is an inverse relationship between analgesic efficacy and tolerance following continuous infusion of opioid analgesics. Furthermore, it was hypothesized that analgesic efficacy plays a minor role in determining the magnitude of tolerance following intermittent or acute administration, and that acute and intermittent administration of opioid agonists produces less tolerance than continuous infusion., Materials and Methods: Analgesic (tailflick) efficacy (tau) of etorphine, methadone, oxycodone, and hydrocodone was determined using the operational model of agonism. To induce tolerance, mice were injected with opioid agonists once (acute), once per day for 7 days (intermittent) or continuously infused for 7 days. Dose-response studies were conducted using morphine following treatment., Results: The order of analgesic efficacy was etorphine > methadone > oxycodone congruent with hydrocodone. Infusion of the higher analgesic efficacy drug etorphine produced significantly less tolerance than the lower analgesic efficacy drugs oxycodone, methadone, and hydrocodone at equi-effective doses. In general, intermittent and acute treatment produced less tolerance compared to continuous infusion even at similar daily doses., Conclusion: Taken together, intermittent and acute opioid agonist administration produces minimal tolerance compared to continuous infusion. Furthermore, there is an inverse relationship between analgesic efficacy and tolerance following continuous infusion. These results suggest that opioid analgesic tolerance may be increased when sustained release dosing formulations or continuous infusions are employed clinically.

Opioid antagonists can be classified as inverse agonists and neutral antagonists. In the opioid-dependent state, neutral antagonists are significantly less potent in precipitating withdrawal than inverse agonists. Consequently, neutral opioid antagonists may offer advantages over inverse agonists in the management of opioid overdose. In this study, the relative potency of three opioid antagonists to block opioid analgesia and toxicity and precipitate withdrawal was examined. First, the potency of two opioid inverse agonists (naltrexone and naloxone) and a neutral antagonist (6beta-naltrexol) to antagonize fentanyl-induced analgesia and lethality was determined. The order of potency to block analgesia was naltrexone > naloxone > 6beta-naltrexol (17, 4, 1), which was similar to that to block lethality (13, 2, 1). Next, the antagonists were compared using withdrawal jumping in fentanyl-dependent mice. The order of potency to precipitate withdrawal jumping was naltrexone > naloxone 6beta-naltrexol (1107, 415, 1). The relative potencies to precipitate withdrawal for the inverse agonists compared with the neutral antagonist were dramatically different from that for antagonism of analgesia and lethality. Finally, the effect of 6beta-naltrexol pretreatment on naloxone-precipitated jumping was determined in morphine and fentanyl-dependent mice. 6beta-Naltrexol pretreatment decreased naloxone precipitated withdrawal, indicating that 6beta-naltrexol is a neutral antagonist. These data demonstrate that inverse agonists and neutral antagonists have generally comparable potencies to block opioid analgesia and lethality, whereas the neutral opioid antagonist is substantially less potent in precipitating opioid withdrawal. These results support suggestions that neutral antagonists may have advantages over inverse agonists in the management of opioid overdose.

Dosing protocol and analgesic efficacy have been proposed to be important determinants of the magnitude of opioid tolerance. The present study examined the effect of acute, intermittent and continuous treatment with the low analgesic efficacy agonist morphine on analgesic tolerance. Mice were implanted s.c. with a 25 mg morphine pellet for 1-7 days. Other mice were implanted s.c. with two 25 mg, or one 75 mg morphine pellet for 7 days. The release of morphine from subcutaneous implanted pellets was quantitated using a spectrophotometric assay. In other studies, mice were injected with morphine once (18.5-185 mg/kg/day; approximately 10-100 times ED(50) for morphine analgesia) or once/day for 7 days. Controls were implanted with a placebo pellet or injected with saline. Analysis of drug release from a 25 mg pellet indicated that release was greatest during the first 24 h, declined and then remained relatively constant. The amount of morphine released over 7 days by a 75 mg pellet (23.9 mg) was more than that of a single 25 mg pellet (15.4 mg) but less than two 25 mg pellets (30.8 mg). Following treatment, morphine cumulative dose-response studies were conducted (tail flick). Continuous treatment with morphine using pellet implantation produced a dose-dependent shift in the morphine ED(50) by 3.3, 5.8 and 8.5 fold for one 25 mg pellet, one 75 mg pellet and two 25 mg pellets, respectively. Acute and intermittent morphine administration produced substantially less analgesic tolerance than continuous release of morphine by implant pellets. The maximum shift in the ED(50) was 1.6 for acute treatment and 2.7 for 7 day intermittent treatment; despite a larger total daily dose. The present results indicate that continuous treatment with morphine results in greater analgesic tolerance than acute or intermittent morphine treatment even at comparable daily doses. These results are consistent with the suggestion that intermittent dosing has reduced risk of producing opioid tolerance.