5-HT1A Serotonergic, α-Adrenergic and Opioidergic Receptors Mediate the Analgesic Efficacy of Vortioxetine in Mice.

Vortioxetine is a multimodal antidepressant drug that affects several brain neurochemicals and has the potential to induce various pharmacological effects on the central nervous system. Therefore, we investigated the centrally mediated analgesic efficacy of this drug and the mechanisms underlying this effect. Analgesic activity of vortioxetine (5, 10 and 20 mg/kg, p.o.) was examined by tail-clip, tail-immersion and hot-plate tests. Motor performance of animals was evaluated using Rota-rod device. Time course measurements (30-180 min) showed that vortioxetine (10 and 20 mg/kg) administrations significantly increased the response latency, percent maximum possible effect and area under the curve values in all of the nociceptive tests. These data pointed out the analgesic effect of vortioxetine on central pathways carrying acute thermal and mechanical nociceptive stimuli. Vortioxetine did not alter the motor coordination of mice indicating that the analgesic activity of this drug was specific. In mechanistic studies, pre-treatments with p-chlorophenylalanine (serotonin-synthesis inhibitor), NAN-190 (serotonin 5-HT1A receptor antagonist), α-methyl-para-tyrosine (catecholamine-synthesis inhibitor), phentolamine (non-selective α-adrenoceptor blocker), and naloxone (non-selective opioid receptor blocker) antagonised the vortioxetine-induced analgesia. Obtained findings indicated that vortioxetine-induced analgesia is mediated by 5-HT1A serotonergic, α-adrenergic and opioidergic receptors, and contributions of central serotonergic and catecholaminergic neurotransmissions are critical for this effect.

Determination of buprenorphine, norbuprenorphine, naloxone, and their glucuronides in urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their glucuronide conjugates BUP-G, NBUP-G, and NAL-G in urine samples was developed. The method, omitting a hydrolysis step, involved non-polar solid-phase extraction, liquid chromatography on a C18 column, electrospray positive ionization, and mass analysis by multiple reaction monitoring. Quantification was based on the corresponding deuterium-labelled internal standards for each of the six analytes. The limit of quantification was 0.5 µg/L for BUP and NAL, 1 µg/L for NAL-G, and 3 µg/L for NBUP, BUP-G, and NBUP-G. Using the developed method, 72 urine samples from buprenorphine-dependent patients were analysed to cover the concentration ranges encountered in a clinical setting. The median (maximum) concentration was 4.2 µg/L (102 µg/L) for BUP, 74.7 µg/L (580 µg/L) for NBUP, 0.9 µg/L (85.5 µg/L) for NAL, 159.5 µg/L (1370 µg/L) for BUP-G, 307.5 µg/L (1970 µg/L) for NBUP-G, and 79.6 µg/L (2310 µg/L) for NAL-G.

Formulation of a recombinant HIV-1 polytope candidate vaccine with naloxone/alum mixture: induction of multi-cytokine responses with a higher regulatory mechanism.

The potency of a vaccine highly depends upon the nature of the adjuvant used. There are a variety of ineffective vaccines, such as HIV-1 vaccine candidates, that need to be optimized with new adjuvant formulations to improve vaccine potency and efficacy. Studies show the potency of naloxone (NLX)/alum mixture in the induction of Th1/Th2 response for vaccine. However, other immunologic patterns inducing by this adjuvant and its immunoregulatory effect is unclear. In this regard, the aim of the present study was to investigate the effect of the NLX/alum mixture, as an adjuvant, on cytokine networks and immunoregulatory activity for an HIV-1 polytope vaccine. BALB/c mice were divided into six groups (n = 6) and immunized subcutaneously with 10 µg of the vaccine formulated with NLX/alum, NLX, alum, and Freund's adjuvants. At the same time, the mice in the control groups received an equal volume of PBS or NLX. The lymphocyte proliferation assay was carried out using the BrdU method. ELISA was used to measure the levels of IFN-γ, IL-2, IL-4, IL-10, IL-12, and IL-17 cytokines, total IgG, as well as IgG1 and IgG2a subtypes in serum samples. Our findings showed that mice receiving the NLX/alum-adjuvanted vaccine exhibited increased antibody levels compared with other groups. In addition, there was a considerable difference in the levels of IgG1, IgG2a, IFN-γ, IL-2, IL-10, IL-12, and IL-17 in mice receiving the NLX/alum-adjuvanted vaccine as compared with other groups. The NLX/alum mixture, as an adjuvant, may have a positive effect on the induction of multi-cytokine responses, as well as the increased level of IL-10, showing its higher immunogenicity with a higher immunoregulatory mechanism.

Ligand based conformational space studies of the µ-opioid receptor.

BACKGROUND: G protein-coupled receptors (GPCRs) comprise a family of membrane proteins that can be activated by a variety of external factors. The µ-opioid receptor (MOR), a class A GPCR, is the main target of morphine. Recently, enhanced sampling molecular dynamics simulations of a constitutively active mutant of MOR in its apo form allowed us to capture the novel intermediate states of activation, as well as the active state. This prompted us to apply the same techniques to wild type MOR in complex with ligands, in order to explore their contributions to the receptor conformational changes in the activation process. METHODS: MOR was modeled in complex with agonists (morphine, BU72), a partial agonist (naloxone benzoylhydrazone) and an antagonist (naloxone). Replica exchange with solute tempering (REST2) molecular dynamics simulations were carried out for all systems. Trajectory frames were clustered, and the activation state of each cluster was assessed by two different methods. RESULTS: Cluster sizes and activation indices show that while agonists stabilized structures in a higher activation state, the antagonist behaved oppositely. Morphine tends to drive the receptor towards increasing R165-T279 distances, while naloxone tends to increase the NPxxYA motif conformational change. CONCLUSIONS: Despite not observing a full transition between inactive and active states, an important conformational change of transmembrane helix 5 was observed and associated with a ligand-driven step of the process. GENERAL SIGNIFICANCE: The activation process of GPCRs is widely studied but still not fully understood. Here we carried out a step forward in the direction of gaining more details of this process.

Physical Compatibility and Chemical Stability of Fentanyl and Naloxone Hydrochloride in 0.9% Sodium Chloride Injection Solution for Patient-Controlled Analgesia Administration.

BACKGROUND AND OBJECTIVE: The combination of naloxone hydrochloride (NH) and fentanyl citrate (FC) in patient-controlled analgesia (PCA) is examined to reduce the risk of opioid-induced nausea and vomiting. However, there are no such commercially available drug mixtures, and there is also no published evidence on the compatibility and stability of NH and FC. Thus, the primary purpose of the current research is to investigate the physical compatibility and chemical stability of NH when mixed with FC over a 72-h period in a 0.9% sodium chloride injection solution for PCA administration under storage at 4°C and 25°C. METHODS: Test solutions of 20 µg/mL FC and 4 µg/mL NH were prepared and stored in polyvinyl chloride (PVC) bags or glass bottles with a 0.9% sodium chloride injection solution as the diluent. During the 72-h storage period at 4°C or 25°C without light protection, the concentrations of the test drugs were assayed via high-performance liquid chromatography (HPLC), and the physical compatibility was determined with the naked eye. Furthermore, pH measurement of each sample was also performed with a pH meter. RESULTS: The percentages of the initial concentrations of FC and NH in the various solutions were maintained at a minimum of 98% over the 72-h study period. All of the mixtures remained clear and colourless throughout the observation period, and no precipitation or turbidity was observed in any of the batches. CONCLUSION: The 20 µg/mL FC test solution was physically compatible and chemically stable with the 4 µg/mL NH test solution when stored at 4°C or 25°C in PVC bags or glass bottles containing the 0.9% sodium chloride injection solution.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors.

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Effect of Extreme Temperature on Naloxone Nasal Spray Dispensing Device Performance.

INTRODUCTION: The opioid epidemic has led to the wide-spread distribution of naloxone to emergency personnel and to the general public. Recommended storage conditions based on prescribing information are between 15°C and 25°C (59°F and 77°F), with excursions permitted between 4°C and 40°C (39°F and 104°F). Actual storage likely varies widely with potential exposures to extreme temperatures outside of these ranges. These potentially prolonged extreme temperatures may alter the volume of naloxone dispensed from the nasal spray device, which could result in suboptimal efficacy. STUDY OBJECTIVE: The aim of this study was to assess the naloxone volume deployed following nasal spray device storage at extreme temperatures over an extended period of time. METHODS: Naloxone nasal spray devices were exposed to storage temperatures of -29°C (-20°F), 20°C (68°F), and 71°C (160°F) to simulate extreme temperatures and a control for 10 hours. First, the density was measured under each temperature condition. Following the density calculation part of the experiment, the mass of naloxone dispensed from each nasal spray device at each temperature was captured and used to calculate volume: calculated volume (microliter, µl) = spray mass (mg converted to g)/mean density (g/mL). Measurements and calculations are reported as means with standard deviation and standard error, and a one-way ANOVA was used to evaluate mean dispensed volume differences at different temperatures. RESULTS: There was no difference in the mean volume deployed at -29°C (-20°F), 20°C (68°F), and 71°C (160°F), and measurements were 101.44µl (SD = 9.56; SE = 5.52), 99.01µl (SD = 6.31; SE = 3.64), and 108.28µl (SD = 2.04; SE = 1.18), respectively; P value = .289, F-statistic value = 1.535. CONCLUSION: The results of this study suggest that naloxone nasal spray devices will dispense the appropriate volume, even when stored at extreme temperatures outside of the manufacturer's recommended range.

Development of Hydrogels for Microneedle-Assisted Transdermal Delivery of Naloxone for Opioid-Induced Pruritus.

Transdermal naloxone delivery could be a potential option for treating opioid-induced pruritus, but naloxone does not permeate skin well because of its hydrophilic nature. Microneedles (MNs) could overcome the skin barrier by painlessly creating microchannels in the skin to permit naloxone absorption to therapeutic levels. This study investigated how ionization correlates with naloxone permeation across MN-treated skin. Hydrogels containing 0.2, 0.5, or 1% naloxone were formulated with 1% cross-linked polyacrylic acid (polymer) and adjusted to pH 5, 6.5, or 7.4. Porcine skin was treated with MNs and naloxone gel, and in vitro permeation studies were performed using an in-line diffusion setup. Gel structural properties were evaluated using rheology. All gels had viscoelastic properties and good spreadability. Naloxone permeation through intact skin was highest from pH 7.4 gels when naloxone is unionized, in contrast with undetectable concentrations permeated from pH 5 gels with 100% ionization. Combining MN treatment with pH 5 gels significantly enhanced permeation and resulted in steady-state flux that would achieve therapeutic delivery. Absorption lag time was affected by MN length and naloxone gel concentration. Polymer concentration did not influence drug permeability. This study demonstrates that transdermal naloxone delivery with MNs is a viable treatment option for opioid-induced pruritus.

Modulation of µ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

BACKGROUND AND PURPOSE: Adverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on µ-opioid receptor binding and signalling induced by the µ-opioid receptor ligands fentanyl, DAMGO, and naloxone. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with µ-opioid receptors were used to study opioid ligand binding, [35 S]-GTPγS binding, and cAMP reduction at physiological and acidic pH. We used µ-opioid receptors mutated at H2976.52 to A (MOR-H2976.52 A) to delineate ligand-specific interactions with H2976.52 . KEY RESULTS: Low pH and the mutant receptor MOR-H2976.52 A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G-protein activation were decreased under these conditions. Fentanyl-induced signalling was not influenced by pH and largely independent of H2976.52 . CONCLUSIONS AND IMPLICATIONS: Our investigations indicate that low pH selectively impairs µ-opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52 . We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52 .

The effects of heat and freeze-thaw cycling on naloxone stability.

PURPOSE: The availability of take home naloxone (THN) was increased for Canadians in 2016, including access to kits via pharmacies. Unlike typical over-the-counter (OTC) and prescription drugs, THN kits may be stored in non-standard conditions, including in vehicles, backpacks, and out of doors. To evaluate whether these non-standard storage conditions affect stability, we investigated the impact of heat and freeze-thaw cycling on naloxone hydrochloride stability. METHODS: To assess the effect of heat, naloxone hydrochloride ampoules were exposed to 80 °C in a temperature-controlled oven for 8 h followed by 16 h at room temperature. To assess the effect of freeze-thaw cycles, naloxone hydrochloride ampoules were exposed to - 20 °C for 16 h followed by 8 h at 4 °C. The impact of these conditions on naloxone hydrochloride stability was evaluated each day for 1 week and after 2 and 4 weeks. The concentration of remaining naloxone hydrochloride was quantified using high-performance liquid chromatography (HPLC). Naloxone hydrochloride ampoules stored at room temperature served as the experimental control. RESULTS: Naloxone hydrochloride ampoules exhibit no changes in drug concentration following exposure to heat or freeze-thaw cycles for up to 28 days compared to ampoules maintained at room temperature (as indicated in the product monograph). CONCLUSIONS: Naloxone hydrochloride remains chemically stable following exposure to heat or freeze-thaw cycles after 28 days. If THN kits are stored in non-standard conditions (for up to 28 days) the active naloxone is likely to remain stable. Despite this, pharmacists should continue to emphasize the importance of appropriate storage of THN kits to ensure optimal efficacy should naloxone administration be required in an emergency situation.

Caged Naloxone: Synthesis, Characterization, and Stability of 3- O-(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX).

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3- O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as "caged naloxone", 3- O-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of α R,5 R,9 R,13 S,14 S and α S,5 R,9 R,13 S,14 S diastereomers. Using long-range and heteronuclear NMR correlations, the 1H NMR and 13C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products.

Synthesis of novel norsufentanil analogs via a four-component Ugi reaction and in vivo, docking, and QSAR studies of their analgesic activity.

Novel substituted amino acid tethered norsufentanil derivatives were synthesized by the four-component Ugi reaction. Norsufentanil was reacted with succinic anhydride to produce the corresponding carboxylic acid. The resulting carboxylic acid has undergone a multicomponent reaction with different aldehydes, amines, and isocyanides to produce a library of the desired compounds. In all cases, amide bond rotation was observed in the NMR spectra. In vivo analgesic activity of the synthesized compounds was evaluated by a tail flick test. Very encouraging results were obtained for a number of the synthesized products. Some of the synthesized compounds such as 5a, 5b, 5h, 5j, and 5r were found to be more potent than sufentanil, sufentanil citrate, and norsufentanil. Binding modes between the compounds and mu and delta-opioid receptors were studied by molecular docking method. The relationship between the molecular structural features and the analgesic activity was investigated by a quantitative structure-activity relationship model. The results of the molecular modeling studies and the in vivo analgesic activity suggested that the majority of the synthesized compounds were more potent than sufentanil and norsufentanil.

A structural insight into the negative effects of opioids in analgesia by modulating the TLR4 signaling: An in silico approach.

Opioids are considered the gold standard therapy for pain. However, TLR-dependent negative effects in analgesia have highlighted the complexities in the pharmacodynamics of opioids. While successive studies have reported that morphine and Morphine-3-glucuronide (M3G) activate the TLR4 pathway, the structural details of this mechanism are lacking. Here, we have utilized various computational tools to reveal the structural dynamics of the opioid-bound TLR4/MD2 complex, and have proposed a potential TLR4 activation mechanism. Our results support previous findings, and include the novel insight that the stable binding of morphine and naloxone, but not M3G, in the MD2 cavity, is TLR4 dependent. Morphine interacts with MD2 near its Phe126 loop to induce the active conformation (MD2C); however, this binding is likely reversible, and the complex gains stability upon interaction with TLR4. M3G also induces the MD2C state, with both the Phe126 loop and the H1 loop being involved in MD2-M3G complex stability. Remarkably, naloxone, which requires TLR4 interaction for complex stability, switches the conformation of the gating loop to the inactive state (MD2°). Cumulatively, our findings suggest that ligand binding and receptor clustering occur successively in opioid-induced TLR4 signaling, and that MD2 plasticity and pocket hydrophobicity are crucial for the recognition and accommodation of ligands.

Comparison of (+)- and (-)-Naloxone on the Acute Psychomotor-Stimulating Effects of Heroin, 6-Acetylmorphine, and Morphine in Mice.

Toll-like receptor 4 (TLR4) signaling is implied in opioid reinforcement, reward, and withdrawal. Here, we explored whether TLR4 signaling is involved in the acute psychomotor-stimulating effects of heroin, 6-acetylmorphine (6-AM), and morphine as well as whether there are differences between the three opioids regarding TLR4 signaling. To address this, we examined how pretreatment with (+)-naloxone, a TLR4 active but opioid receptor (OR) inactive antagonist, affected the acute increase in locomotor activity induced by heroin, 6-AM, or morphine in mice. We also assessed the effect of pretreatment with (-)-naloxone, a TLR4 and OR active antagonist, as well as the pharmacokinetic profiles of (+) and (-)-naloxone in the blood and brain. We found that (-)-naloxone reduced acute opioid-induced locomotor activity in a dose-dependent manner. By contrast, (+)-naloxone, administered in doses assumed to antagonize TLR4 but not ORs, did not affect acute locomotor activity induced by heroin, 6-AM, or morphine. Both naloxone isomers exhibited similar concentration versus time profiles in the blood and brain, but the brain concentrations of (-)-naloxone reached higher levels than those of (+)-naloxone. However, the discrepancies in their pharmacokinetic properties did not explain the marked difference between the two isomers' ability to affect opioid-induced locomotor activity. Our results underpin the importance of OR activation and do not indicate an apparent role of TLR4 signaling in acute opioid-induced psychomotor stimulation in mice. Furthermore, there were no marked differences between heroin, 6-AM, and morphine regarding involvement of OR or TLR4 signaling.

Amorphous Formulation and in Vitro Performance Testing of Instantly Disintegrating Buccal Tablets for the Emergency Delivery of Naloxone.

The aim of this study was to develop a freeze-dried buccal tablet for the rapid delivery of naloxone in opioid overdose. The tablet composition was optimized to produce an amorphous matrix, which was confirmed by the absence of peaks associated with crystallinity observed by differential scanning calorimetry and powder X-ray diffraction. Tablets with high gelatin content lacked adequate porosity. Mannitol was added to the formulation to bridge and intercalate gelatin's tight polymer aggregates, however sodium bicarbonate was also required to prevent crystallization within the tablets. A linear reduction in mannitol's recrystallization enthalpy was observed with increasing sodium bicarbonate concentration (ΔrecryH = -20.3[NaHCO3] + 220.9; r(2) = 0.9, n = 18). The minimum sodium bicarbonate concentration for full inhibition of mannitol crystallization was 10.9% w/w. Freeze-dried tablets with lower amounts of sodium bicarbonate possessed a crystalline fraction that PXRD identified as mannitol hemihydrate from the unique peak at 9.7° 2θ. Mannitol's greater affinity for both ions and residual water rather than its affinity for self-association was the mechanism for the inhibition of crystallization observed here. The optimized tablet (composition mannitol 24% w/w (4.26 mg), gelatin 65% w/w (11.7 mg), sodium bicarbonate 11% w/w (1.98 mg), and naloxone 800 µg) formed predominantly amorphous tablets that disintegrated in less than 10 s. Optimized tablets were chemically and physically stable over 9 months storage at 25 °C. As speed of drug liberation is the critical performance attribute for a solid dosage form designed to deliver drug in an emergency, a novel imaging based in vitro disintegration assay for buccal tablets was developed. The assay was optimized with regard to conditions in the buccal cavity: i.e., temperature 33-37 °C, volume of medium (0.1-0.7 mL), and use of mucin-containing biorelevant medium. The disintegration assay was sensitive to temperature, medium volume, and medium composition; naloxone tablet disintegration was extremely rapid, with full disintegration ranging from 5 to 20 s. In conclusion, rapidly disintegrating tablets have been developed which are suitable for proof-of-concept clinical trial in humans to determine the pharmacokinetics of naloxone delivered via the buccal route.

Simultaneous determination of buprenorphine, norbuprenorphine and naloxone in human plasma by liquid chromatography/tandem mass spectrometry.

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for simultaneous quantification of naloxone, buprenorphine and its metabolite norbuprenorphine in human plasma. Human plasma samples were extracted using a single step liquid-liquid extraction, and then separated on an Imtakt Unison UK-C18 column (2.1×50mm, 3µm) using alkaline mobile phases with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation, precision, accuracy, recoveries and stability were determined. The linear range was 20-10000pg/mL for buprenorphine and norbuprenorphine; and 1-500pg/mL for naloxone. The correlation coefficient (R(2)) values for all three analytes were ≥0.995. The precision and accuracy for intra-day and inter-day were <11.0%. The recoveries were >63% and matrix effects were tracked by the deuterated internal standards (IS) with the IS-normalized matrix factor ranging from 0.96 to 1.33 for all three analytes. The validated method was successfully applied in a clinical pharmacokinetic study with low dose administration of sublingual buprenorphine and naloxone.

Agonist stimulation at human µ opioid receptors in a [(35)S]GTPγS incorporation assay: observation of "bell-shaped" concentration-response relationships under conditions of strong receptor G protein coupling.

CONTEXT: The appearance of "bell"- (or "inverted U"-) shaped agonist concentration-response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. OBJECTIVE: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. METHODS: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [(35)S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10-300 mM) and the GDP concentration (0.3-30 µM). RESULTS: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [(35)S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. CONCLUSION: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.

Species-specific lipophilicity of morphine antagonists.

Complete sets of microscopic acid-base and partition equilibrium constants were experimentally determined for therapeutically important morphine derivatives, including the widely used antagonists naloxone and naltrexone. The acid-base microequilibria were characterized by combining pH-potentiometry and deductive methods using synthesized auxiliary compounds. Microscopic protonation equilibria show that approximately three times as many zwitterionic microspecies than non-charged ones exist in oxymorphone and naltrexone solutions. On the other hand, the non-charged microspecies is the dominant one in the case of naloxone, although its concentration is only 1.34 times higher than that of its zwitterionic protonation isomer. Partition coefficients of the individual microspecies were determined by a combination of experimentally measured distribution constants and deductive methods. The contribution ratio of the non-charged versus zwitterionic species to the overall lipophilicity is quantified and depicted in terms of species-specific lipophilicities over the entire pH range for each compound. Our lipophilicity values allowed the molecular interpretation of the classical pharmacologic observation that naloxone has a faster onset for antagonist activity, and a concomitant shorter duration of action.

Remifentanil Ameliorates Liver Ischemia-Reperfusion Injury Through Inhibition of Interleukin-18 Signaling.

BACKGROUND: Hepatic injury induced by ischemia-reperfusion (I/R) after transplantation or lobectomy is a major clinical problem. The potential benefit of remifentanil in these hepatic surgeries remains unknown. The current study investigated whether remifentanil protects the liver against I/R injury in a rat model and whether the underlying mechanism involves the modulation of interleukin (IL)-18 signaling. METHODS: Male Sprague-Dawley rats were subjected to 45 minutes of partial hepatic ischemia followed by 6 hours of reperfusion. Then, they received an intravenous saline or remifentanil (0.4, 2, or 10 µg/kg per minute) infusion from 30 minutes before ischemia until the end of ischemia with or without previous administration of naloxone, a nonselective opioid receptor antagonist. Serum aminotransferase, hepatic morphology, and hepatic neutrophil infiltration were analyzed. The expression of hepatic IL-18; IL-18-binding protein (BP); and key cytokines downstream of IL-18 signaling were measured. RESULTS: Remifentanil significantly decreased serum aminotransferase levels and profoundly attenuated the liver histologic damages. Liver I/R injury increased the expression of both hepatic IL-18 and IL-18BP. Although remifentanil pretreatment significantly decreased I/R-induced IL-18 expression, it further upregulated IL-18BP levels in liver tissues. The I/R-induced increases of hepatic interferon-γ, tumor necrosis factor-α and IL-1ß expression, and neutrophil infiltration were also significantly reduced by remifentanil. Naloxone inhibited the remifentanil-induced downregulation of IL-18, but not the elevation of IL-18BP, and significantly attenuated its protective effects on liver I/R injury. CONCLUSIONS: Remifentanil protects the liver against I/R injury. Modulating the hepatic IL-18/IL-18BP balance and inhibiting IL-18 signaling mediate, at least in part, the hepatoprotective effects of remifentanil.

Pharmaceutical and pharmacokinetic characterization of a novel sublingual buprenorphine/naloxone tablet formulation in healthy volunteers.

CONTEXT: Bitter taste, as well as dissolve time, presents a significant challenge for the acceptability of formulations for oral transmucosal drug delivery. OBJECTIVE: To characterize a novel sublingual tablet formulation of buprenorphine/naloxone with regards to pharmacokinetics, dissolve time and formulation acceptability. METHODS: Dry mixing techniques were employed to produce a small and fast dissolving buprenorphine/naloxone sublingual tablet formulation, OX219 (Zubsolv®), using sucralose and menthol as sweetener and flavor to mask the bitter taste of the active ingredients. Two cross-over studies were performed in healthy volunteers to evaluate pharmacokinetics, dissolve time and acceptability of OX219 5.7/1.4 mg tablets compared to the commercially available buprenorphine/naloxone formulations Suboxone® tablets and films (8/2 mg). RESULTS: Buprenorphine exposure was equivalent in OX219 and Suboxone tablets. Sublingual dissolve times were significantly shorter for OX219 than for Suboxone tablets and were similar to Suboxone films. The OX219 formulation received significantly higher subjective ratings for taste and overall acceptability than both Suboxone formulations. OX219 was preferred over Suboxone tablet and film formulations by 77.4% and 88.9% of subjects, respectively. CONCLUSIONS: A sublingual tablet formulation with an improved acceptability has been successfully developed.