Effect of naloxone-3-glucuronide and N-methylnaloxone on the motility of the isolated rat colon after morphine

The effect of the opioid antagonists naloxone-3-glucuronide and N-methylnaloxone on rat colon motility after morphine stimulation was measured. The rat model consisted of the isolated, vascularly perfused colon. The antagonists (10(-4) M, intraluminally) and morphine (10(-4) M, intra-arterially) were administered from 20 to 30 and from 10 to 50 min, respectively. Colon motility was determined by the luminal outflow. The antagonist concentrations in the luminal and venous outflow were measured by high-performance liquid chromatography. Naloxone-3-glucuronide and N-methylnaloxone reversed the morphine-induced reduction of the luminal outflow to baseline within 10 and 20 min, respectively. These antagonists were then excreted in the luminal outflow and could not be found in the venous samples. Naloxone, produced by hydrolysis or demethylation, was not detectable. In conclusion, highly polar naloxone derivatives peripherally antagonize the motility-lowering effect of morphine in the perfused isolated rat colon, are stable, and are not able to cross the colon-mucosal blood barrier.

The effect of naloxone-3-glucuronide on colonic transit time in healthy men after acute morphine administration: a placebo-controlled double-blinded crossover preclinical volunteer study

BACKGROUND: Constipation is a significant side effect of opioid therapy. We have previously demonstrated that naloxone-3-glucuronide (NX3G) antagonizes the motility-lowering-effect of morphine in the rat colon. AIM: To find out whether oral NX3G is able to reduce the morphine-induced delay in colonic transit time (CTT) without being absorbed and influencing the analgesic effect. METHODS: Fifteen male volunteers were included. Pharmacokinetics: after oral administration of 0.16 mg/kg NX3G, blood samples were collected over a 6-h period. Pharmacodynamics: NX3G or placebo was then given at the start time and every 4 h thereafter. Morphine (0.05 mg/kg) or placebo was injected s.c. 2 h after starting and thereafter every 6 h for 24 h. CTT was measured over a 48-h period by scintigraphy. Pressure pain threshold tests were performed. RESULTS: Neither NX3G nor naloxone was detected in the venous blood. The slowest transit time was observed during the morphine phase, which was significantly different from morphine with NX3G and placebo. The pain perception was not significantly influenced by NX3G. CONCLUSIONS: Orally administered NX3G is able to reverse the morphine-induced delay of CTT in humans without being detected in peripheral blood samples. Therefore, NX3G may improve symptoms of constipation in-patients using opioid medication without affecting opioid-analgesic effects.

The role of morphine-6-glucuronide (M6G) in pain control

Morphine-6beta-D-glucuronide (M6G) is an analgesically active metabolite of morphine, accounting for approximate to10% of the morphine dose when administered by systemic routes to humans. Although M6G is more hydrophilic than morphine, it crosses the blood-brain barrier, albeit relatively slowly. For this reason, it is generally thought that, after chronic dosing, M6G contributes significantly to the analgesic effects of systemically administered morphine. Owing to its polar nature, M6G is cleared from the systemic circulation primarily via renal elimination. As M6G accumulates in patients with renal impairment, there is an increased risk of M6G-induced respiratory depression in renal failure patients who are being dosed chronically with systemic morphine. Consistent with its analgesic and respiratory depressant properties, M6G binds to the p-opioid receptor in a naloxone-reversible manner. Although the affinity of M6G for the mu-opioid receptor is similar to or slightly less than that of morphine, preclinical studies in rodents show that M6G is one to two orders of magnitude more potent than morphine when administered by central routes. This major discrepancy between the markedly higher intrinsic antinociceptive potency of M6G relative to morphine, despite their similar p-opioid receptor binding affinities, is difficult to reconcile. It has been proposed that M6G mediates its pain-relieving effects through a novel 'M6G opioid receptor', while others have argued that M6G may have higher efficacy than morphine for transduction of intracellular events. When administered by parenteral routes to rodents, M6G's antinociceptive potency is no more than twofold higher than morphine. In humans, the analgesic efficacy and respiratory depressant potency of M6G relative to morphine have been assessed in a number of short-term studies involving the intrathecal or intravenous routes of administration. For example, in hip replacement patients, intrathecal M6G provided excellent postoperative analgesia but the occurrence of late respiratory depression in 10% of these patients raised serious concern about safety. In postoperative patients, intravenous M6G administered by means of patient-controlled analgesia (PCA), or bolus plus PCA, produced no analgesia in one study and limited analgesia in another. Similarly, there was a lack of significant analgesia in healthy volunteers who received intravenous M6G for the alleviation of experimental pain (carbon dioxide applied to the nasal mucosa). In contrast, satisfactory analgesia was produced by bolus doses of intravenous M6G administered to patients with cancer pain, and to healthy volunteers with experimentally-induced ischaemic, electrical or thermal (ice water) pain. Studies to date in healthy volunteers suggest that intravenous M6G may be a less potent respiratory depressant and have a lower propensity for producing nausea and vomiting than morphine. However, it is unclear whether equi-analgesic doses of M6G and morphine were compared. Clearly, more extensive short-term trials, together with studies involving chronic M6G administration, are necessary before the potential clinical utility of M6G as an analgesic drug in its own right can be determined.

Entwicklung eines Screeningverfahrens zum simultanen Nachweis von Opioiden in menschlichen Körperflüssigkeiten, Geweben und Haaren für forensisch-toxikologische und klinische Untersuchungen

In den letzten Jahren stieg in Deutschland der Gebrauch bzw. Missbrauch von Opioid-Analgetika zunehmend an. Das entwickelte Verfahren sollte unter Einbeziehung neuer Substanzen möglichst viele verschiedene Opioide und auch ihre pharmakologisch aktiven Stoffwechselprodukte berücksichtigen.rnVor Analyse wurden Blut-, Serum- oder Urinproben mit Phosphatpuffer versetzt und mittels Festphasenextraktion an C18-Säulenmaterial aufgearbeitet. Post-Mortem-Gewebematerial wurde mit isotonischer Kochsalzlösung versetzt, homogenisiert und anschließend durch eine Festphasenextraktion aufgereinigt. Haarproben wurden nach Zerkleinerung mit Methanol unter Ultrabeschallung extrahiert. Die Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie (Elektrosprayionisation im positiven Modus) erwies sich als geeignetes Verfahren für die simultane Bestimmung der Opioide in biologischem Probenmaterial (Körperflüssigkeiten, Gewebe und Haaren). Der Multi-Analyt Assay erlaubt die quantitative Analyse von 35 verschiedenen Opioiden. Die Analyten wurden durch eine Phenyl-Hexyl Säule und einen Wasser/Acetonitril Gradienten durch eine UPLC 1290 Infinity gekoppelt mit einem 6490 Triple Quadrupol von Agilent Technologies separiert.rnDie LC/MS Methode zur simultanen Bestimmung von 35 Opioiden in Serum und Haaren wurde nach den Richtlinien der Gesellschaft für Toxikologische und Forensische Chemie (GTFCh) validiert. Im Fall der Serumvalidierung lagen die Nachweisgrenzen zwischen 0.02 und 0.6 ng/ml und die Bestimmungsgrenzen im Bereich von 0.1 bis 2.0 ng/ml. Die Kalibrationskurven waren für die Kalibrationslevel 1 bis 6 linear. Wiederfindungsraten lagen für alle Verbindungen zwischen 51 und 88 %, außer für Alfentanil, Bisnortiliidn, Pethidin und Morphin-3-Glucuronid. Der Matrixeffekt lag zwischen 86 % (Ethylmorphin) und 105 % (Desomorphin). Für fast alle Analyten konnten akzeptable Werte bei der Bestimmung der Genauigkeit und Richtigkeit nach den Richtlinien der GTFCh erhalten werden. Im Fall der Validierung der Haarproben lagen die Nachweisgrenzen zwischen 0.004 und 0.6 ng/Probe und die Bestimmungsgrenzen zwischen 0.1 ng/Probe und 2.0 ng/Probe. Für die Kalibrationslevel 1 bis 6 waren alle Kalibrationsgeraden linear. Die Wiederfindungsraten lagen für die Opioide im Bereich von 73.5 % (Morphin-6-Glucuronid) und 114.1 % (Hydrocodon). Die Werte für die Bestimmung der Richtigkeit lagen zwischen - 6.6 % (Methadon) und + 11.7 % (Pholcodin). Präzisionsdaten wurden zwischen 1.0 % für Dextromethorphan und 11.5 % für Methadon ermittelt. Die Kriterien der GTFCh konnten bei Ermittlung des Matrixeffekts für alle Substanzen erfüllt werden, außer für 6-Monoacetylmorphin, Bisnortilidin, Meperidin, Methadon, Morphin-3-glucuronid, Morphin-6-glucuronid, Normeperidin, Nortilidin und Tramadol.rnZum Test des Verfahrens an authentischem Probenmaterial wurden 206 Proben von Körperflüssigkeiten mit Hilfe der simultanen LC/MS Screening Methode untersucht. Über 150 Proben wurden im Rahmen von forensisch-toxikologischen Untersuchungen am Instituts für Rechtsmedizin Mainz analysiert. Dabei konnten 23 der 35 Opioide in den realen Proben nachgewiesen werden. Zur Untersuchung der Pharmakokinetik von Opioiden bei Patienten der anästhesiologischen Intensivstation mit Sepsis wurden über 50 Blutproben untersucht. Den Patienten wurde im Rahmen einer klinischen Studie einmal täglich vier Tage lang Blut abgenommen. In den Serumproben wurde hauptsächlich Sufentanil (0.2 – 0.8 ng/ml in 58 Fällen) und Piritramid (0.4 – 11 ng/ml in 56 Fällen) gefunden. Außerdem wurden die Proben von Körperflüssigkeiten und Gewebe von 13 verschiedenen Autopsiefällen mit Hilfe des Multi-Analyt Assays auf Opioide untersucht.rnIn einem zweiten Schritt wurde die Extraktions- und Messmethode zur Quantifizierung der 35 Opioide am Forensic Medicine Center in Ho Chi Minh City (Vietnam) etabliert. Insgesamt wurden 85 Herzblutproben von Obduktionsfällen mit Verdacht auf Opiatintoxikation näher untersucht. Der überwiegende Teil der untersuchten Fälle konnte auf eine Heroin- bzw. Morphin-Vergiftung zurückgeführt werden. Morphin wurde in 68 Fällen im Konzentrationsbereich 1.7 – 1400 ng/ml und der Heroinmetabolit 6-Monoactetylmorphin in 34 Fällen (0.3 – 160 ng/ml) nachgewiesen werden.rnSchließlich wurden noch 15 Haarproben von Patienten einer psychiatrischen Klinik, die illegale Rauschmittel konsumiert hatten, mit Hilfe der simultanen Opioid-LC/MS Screeningmethode gemessen. Die Ergebnisse der Untersuchung wurden mit früheren Auswertungen von gaschromatographischen Analysen verglichen. Es zeigte sich eine weitgehende Übereinstimmung der Untersuchungsergebnisse für die Opioide 6-Monoacetylmorphin, Morphin, Codein, Dihydrocodein und Methadon. Mit der LC/MS Methode konnten weitere Substanzen, wie zum Beispiel Bisnortilidin, Dextromethorphan und Tramadol in den Haarproben gefunden werden, die bislang nicht entdeckt worden waren.rn

Morphine treatment during pregnancy modulates behavioral selection in lactating rats

Previous studies have demonstrated that treatment of postpartum female rats with morphine inhibits maternal behavior and stimulates foraging. Exposure to drugs of abuse may result in a progressive enhancement of their reinforcing effects. Puerperal treatment with morphine leads to reverse tolerance to this drug. The present study investigated whether repeated morphine treatment during late pregnancy may influence the effects of different morphine dosages on behavioral selection in lactating rats. Females were simultaneously exposed to pups and insects, and the choice between taking care of the pups and hunting insects was observed. Female Wistar rats were treated with morphine (3.5 mg/kg/day, subcutaneous [s.c.]) or saline for 5 days beginning on pregnancy day 17. On day 5 of lactation, animals were acutely challenged with morphine (0.5, 1.0, or 1.5 mg/kg, s.c.; MM0.5, MM1.0, and MM1.5 groups, respectively) or saline (MS group) and tested for predatory hunting and maternal behavior. Control groups were pretreated with saline and challenged with morphine (SM0.5, SM1.0, and SM1.5 groups) or saline (SS group). Animals treated with morphine during late pregnancy and acutely challenged with 1.0 mg/kg morphine (MM1.0 group) exhibited significantly decreased maternal behavior and enhanced hunting. This effect was not evident with the 0.5 mg/kg dose. The 1.5 mg/kg morphine dose decreased maternal behavior and increased hunting in both the MM1.5 group and in animals challenged with morphine after previous saline treatment (SM1.5 group). These results provide evidence of plasticity of the opioidergic role in behavioral selection during lactation. (C) 2010 Elsevier Inc. All rights reserved.

Ventilatory responses of healthy subjects to intravenous combinations of morphine and oxycodone under imposed hypercapnic and hypoxaemic conditions

Aims Previous isobolographic analysis revealed that coadministration of morphine and oxycodone produces synergistic antinociception in laboratory rodents. As both opioids can produce ventilatory depression, this study was designed to determine whether their ventilatory effects were synergistic when coadministered to healthy human subjects. Methods A placebo-controlled, randomized, crossover study was performed in 12 male volunteers. Ventilatory responses to hypoxaemia and hypercapnia were determined from 1-h intravenous infusions of saline ('placebo'), 15 mg morphine sulphate (M), 15 mg oxycodone hydrochloride (O), and their combination in the dose ratios of 1 : 2, 1 : 1, 2 : 1. Drug and metabolite concentrations in serial peripheral venous blood samples were measured by high-performance liquid chromatography-MS/MS. Results 'Placebo' treatment was without significant ventilatory effects. There were no systematic differences between active drug treatments on either the slopes or intercepts of the hypoxaemic and hypercapnia ventilation responses. During drug treatment, the mean minute ventilation at PETCO2 = 55 mmHg (V-E55) decreased to 74% of the subjects' before treatment values (95% confidence interval 62, 87), 68% (57, 80), 69% (59, 79), 68% (63, 73), and 61% (52, 69) for M15, M10/O5, M7.5/O7.5, M5/O10 and O15, respectively. Recovery was more prolonged with increasing oxycodone doses, corresponding to its greater potency and lower clearance compared with morphine. Conclusions Although adverse ventilatory effects of these drugs were found as expected, no unexpected or disproportionate effects of any of the morphine and oxycodone treatments were found that might impede their use in combination for pain management.

Opioids and NMDA receptors: Neuro-excitation and abnormal pain behaviours

In the clinical setting, chronic administration of high doses of systemic morphine may result in neuro-excitatory behaviours such as myoclonus and allodynia in some patients. Additionally, high doses of m-opioid agonists such as morphine administered chronically by the intrathecal route in both rats and humans, as well as DAMGO in rats, have been reported to produce neuro-excitatory behaviours. However, more recently, it has begun to be appreciated that even at normal analgesic doses, opioids such as morphine are capable not only of activating pain inhibitory systems (analgesia/antinociception), but they also activate pain facilitatory systems such that post-opioid allodynia/hyperalgesia may be evident after cessation of opioid treatment. Whilst it is well documented that opioid receptors mediate the inhibitory effects of opioid analgesics, the excitatory and pro-nociceptive effects of opioids appear to involve indirect activation of N-methyl-D-aspartate (NMDA) receptors, such that the extent of pain relief produced may be the net effect of these two opposing actions. Apart from the NMDA-nitric oxide (NO) pro-nociceptive signaling cascade, considerable evidence also implicates dynorphin A as well as the endogenous anti-opioid peptides cholecystokinin (CCK), neuropeptide FF (NPFF) and orphanin FQ/nociceptin, in mediating opioid-induced neuro-excitation and abnormal pain behaviours. Apart from the neuro-excitatory effects that may be produced by the parent opioid, systemic administration of some opioid analgesics such as morphine and hydromorphone in rats and humans results in their rapid conversion to 3-glucuronide metabolites that also contribute significantly to the neuro-excitatory and abnormal pain behaviours produced

A structural basis for the inhibition of collagen-stimulated platelet function by quercetin and structurally related flavonoids

Background and purpose: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A2 receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure–activity relationships with regard to platelet function is also lacking. Experimental approach: Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3′-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCγ2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy. Key results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCγ2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety. Conclusions and implications: The structure–activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling.

Infusão contínua de morfina ou fentanil, associados à licodaína e cetamina, em cães anestesiados com isofluorano

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Infusão contínua de morfina ou fentanil, associados à lidocaína e cetamina, em cães anestesiados com isofluorano

A técnica de analgesia multimodal, por meio da infusão contínua de fármacos, pode ser empregada para diminuir a incidência de sensibilização central durante a anestesia. Avaliaram-se as características cardiorrespiratórias, durante o procedimento de artroscopia de joelho, em cães anestesiados com isofluorano e monitorados por meio do índice biespectral, submetidos à infusão contínua de morfina ou fentanil, associada à lidocaína e cetamina. Utilizaram-se 16 cães adultos, machos ou fêmeas, os quais foram distribuídos aleatoriamente em dois grupos, denominados MLK - que recebeu morfina (3,3μg/kg/min), lidocaína (50μg/kg/min) e cetamina (10μg/kg/min) ou FLK - em que foi substituída a morfina pelo fentanil (0,03μg/kg/min). Os cães foram pré-tratados com levomepromazina (0,5mg/kg IV), induzidos à anestesia com propofol (5mg/kg) e mantidos com isofluorano, ajustando-se a concentração para obterem-se valores de índice biespectral entre 55 e 65. As mensurações da frequência cardíaca (FC), dos parâmetros eletrocardiográficos (ECG), das pressões arteriais sistólica (PAS), diastólica (PAD) e média (PAM), da tensão de dióxido de carbono expirado (EtCO2), da saturação de oxi-hemoglobina (SpO2), da frequência respiratória (FR) e da temperatura esofágica (T) iniciaram-se 30 minutos após a indução (M0) e continuaram após o início da infusão das soluções, em intervalos de 15 minutos (M15 a M75). Diferenças entre os grupos foram registradas para duração do complexo QRS (M60), para FC e T, entre M30 e M75, com MLK apresentando médias maiores que FLK, que registrou médias maiores que MLK para a SpO2 (M60), para os intervalos QT (M30 e M75) e RR (M0, M60 e M75). Concluiu-se que o emprego de morfina ou fentanil, associados à lidocaína e cetamina, promove efeitos semelhantes e não compromete as características avaliadas.

Flupirtine enhances the anti-hyperalgesic effects of morphine in a rat model of prostate bone metastasis

Objective Current treatments for cancer pain are often inadequate, particularly when metastasis to bone is involved. The addition to the treatment regimen of another drug that has a complementary analgesic effect may increase the overall analgesia without the necessity to increase doses, thus avoiding dose-related side effects. This project investigated the synergistic effect of the addition of the potassium channel (KCNQ2–3) modulator flupirtine to morphine treatment in a rat model of prostate cancer-induced bone pain. Design Syngeneic prostate cancer cells were injected into the right tibia of male Wistar rats under anesthesia. This led to expanding tumor within the bone in 2 weeks, together with the concurrent development of hyperalgesia to noxious heat. Paw withdrawal thresholds from noxious heat were measured before and after the maximum non-sedating doses of morphine and flupirtine given alone and in combinations. Dose-response curves for morphine (0.13–5.0 mg/kg ip) and flupirtine (1.25–10.0 mg/kg ip) given alone and in fixed-dose combinations were plotted and subjected to an isobolographic analysis. Results Both morphine (ED50 = 0.74 mg/kg) and flupirtine (ED50 = 3.32 mg/kg) caused dose-related anti-hyperalgesia at doses that did not cause sedation. Isobolographic analysis revealed that there was a synergistic interaction between flupirtine and morphine. Addition of flupirtine to morphine treatment improved morphine anti-hyperalgesia, and resulted in the reversal of cancer-induced heat hyperalgesia. Conclusions These results suggest that flupirtine in combination with morphine may be useful clinically to provide better analgesia at lower morphine doses in the management of pain caused by tumors growing in bone.