Morphine-6-glucuronide Research Articles

Cation‐selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

BackgroundMorphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system.Methodology and Principal FindingsMicroscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies.Conclusions/SignificanceThe presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

Contribution of UDP-Glucuronosyltransferase 1A1 and 1A8 to Morphine-6-Glucuronidation and Its Kinetic Properties

The metabolic conversion of morphine to morphine-6-glucuronide (M6G) seems to play a significant role in mediation of the clinical effect of morphine because of the superior analgesic effect of M6G. Therefore, it would be of great interest to clarify the specificity of morphine-6-glucuronidation by UDP glucuronosyltransferase (UGT) isozymes. We investigated the specificity of morphine-6-glucuronidation catalyzed by recombinant human UGT isozymes in microsomes from baculovirus-infected insect cells. The morphine glucuronidation activity of recombinant human UGT isozymes incubated with morphine and UDP-glucuronic acid was determined by high-performance liquid chromatography with a fluorescence detector. Not only UGT2B7, which is well known to catalyze morphine-6-glucuronidation, but also UGT1A1 and 1A8 effectively catalyzed morphine-6-glucuronidation at relatively low morphine concentrations (<100 muM). The kinetics of both isozymes at the low substrate concentrations showed hyperbolic Michaelis-Menten kinetics. However, as the morphine concentration approached 100 muM, morphine-6-glucuronidation activity gradually decreased, and the kinetics closely resembled substrate inhibition Michaelis-Menten kinetic behavior. The K(m) values were 67.9 and 68.1 muM and the K(si) values were 218.9 and 88.0 muM for UGT1A1 and 1A8, respectively. These kinetics are basically different from that of morphine-6-glucuronidation by UGT2B7, which suggested biphasic Michaelis-Menten kinetic behavior. Furthermore, to estimate the contribution of these UGT isozymes in M6G formation in vivo, the expression levels of UGT1A1 and 1A8 mRNA in human liver and intestine were determined by reverse transcription real-time polymerase chain reaction. The results strongly suggest that UGT1A1 and UGT1A8 are isozymes involved in morphine-6-glucuronidation in vivo, as is UGT2B7 in humans.

UGT2B7 promoter variant −840G&gt;A contributes to the variability in hepatic clearance of morphine in patients with sickle cell disease

The purpose of the study was to determine if UDP-glucuronosyltransferase (UGT) 2B7 allelic variants encoding for UGT2B7, primary enzyme responsible for morphine glucuronidation contribute to the variability in the hepatic clearance of morphine in sickle cell disease (SCD). Twenty-four hour PK study of morphine and UGT2B7 variants genotyping was performed in 20 SCD patients in a steady state of health. Presence of the -840G allele (GG and GA) was associated with lower morphine metabolites/morphine AUC ratio compared with AA genotype (1.8 +/- 0.5 vs. 3.0 +/- 1.8 for M6G/M and 10.1 +/- 2.7 vs. 15.7 +/- 9.4 for M3G/M) (P = 0.03). Presence of UGT2B7 -840G allele is associated with significantly reduced glucuronidation of morphine and thus contributes to the variability in hepatic clearance of morphine in SCD.

Serum and cerebrospinal fluid morphine pharmacokinetics after single doses of intravenous and intramuscular morphine after hip replacement surgery

To compare the time course of morphine and metabolite concentrations in serum and cerebrospinal fluid (CSF) after intravenous and intramuscular administration after surgery. This was a randomized double-blind, double-dummy study in patients who had undergone hip replacement surgery. Morphine (M, 10 mg) was administered intravenously (IV) or intramuscularly (IM). Arterial blood and CSF samples (from a spinal catheter) were drawn simultaneously at 10, 30, 60, and 120 min after administration. Morphine and metabolites [morphine-3-glucuronide (M-3-G), morphine-6-glucuronide (M-6-G), and normorphine (NM)] were determined by a validated liquid chromatography-tandem mass spectrometry method. Thirty-eight patients were included: 13 men and 25 women, 20 in the IV, 18 in the IM group. Serum concentrations of M after 10 min were consistently higher after IM than IV, concentrations of M-3-G and M-6-G after IM surpassed those of IV after 45 min. NM was not found. None of the metabolites was found in CSF. CSF morphine concentrations and CSF/serum concentration ratios were consistently higher after IV compared to IM. The mean AUC(CSF)/AUC(serum) (0-120 min) concentration ratios were 0.18 and 0.09 after IV and IM, respectively. The uptake of morphine to the CSF was consistently higher after IV administration than after IM already after 10 min. The higher CSF concentration may be caused by an initially higher morphine blood/CSF gradient following IV morphine injection. The pharmacokinetic findings are compatible with a more rapid and extensive initial effect of IV morphine compared with IM.

Morphine in Postoperative Patients: Pharmacokinetics and Pharmacodynamics of Metabolites

There is great variability in the need for morphine in the postoperative period. We performed a pharmacokinetic-pharmacodynamic study considering the potential effect of the two main metabolites of morphine. Fifty patients with moderate to severe pain received morphine as an IV titration, followed by IM administration postoperatively. The plasma concentration of morphine, morphine-6-glucuronide (M-6-G), morphine-3-glucuronide (M-3-G), and pain intensity were measured at frequent intervals. Pharmacokinetic and pharmacodynamic fitting was performed with the software NONMEM. The pharmacokinetics were largely predictable. M-6-G and M-3-G clearances were markedly decreased in patients with renal failure. The pharmacodynamics was less predictable, with an important interindividual variability. M-6-G was 7.8 times more potent than morphine, but the average time to peak concentration in the effect compartment after a bolus injection of morphine was 4.25 h for M-6-G, when compared to 0.33 h for morphine. M-3-G showed mild inhibition of the analgesic properties of morphine and of M-6-G. The time to M-3-G peak concentration in the effect compartment after a bolus injection of morphine was 10 h. M-6-G is a potent opioid agonist and M-3-G a mild opioid antagonist. Both are poorly excreted in patients with renal failure. However, the metabolism of morphine was rapid when compared to the transfer of metabolites through the blood-brain barrier, which appears to be the limiting process. Because poor analgesia due to M-3-G's effect may occur in some patients after 1 or 2 days, a switch to other molecules should be considered.

CLINICAL STUDY: Endogenous morphine signaling via nitric oxide regulates the expression of CYP2D6 and COMT: autocrine/paracrine feedback inhibition

We determined changes in mRNA expression in specific enzymes involved in the biosynthesis of morphine in human white blood cells via microarray. Leukocyte exposure to morphine down-regulated catechol-O-methyl transferase (COMT) and CYP2D6 by approximately 50% compared with control values. The treatment did not alter DOPA decarboxylase and dopamine beta-hydroxylase expression, demonstrating the specificity of morphine actions. The verification of the microarray data was accomplished via real-time Taqman reverse transcriptase polymerase chain reaction (RT-PCR) focused on CYP2D6 and COMT expression in different blood samples treated with morphine. The analysis showed similar changes in the expression of CYP2D6 and COMT mRNA. The expression was reduced by 47 +/- 7% for CYP2D6, substantiating the microarray finding of a 54% reduction. Furthermore, exposure of white blood cells to 10(-6) M S-nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, reduced the expression of CYP2D6 and COMT. Prior naloxone (10(-6) M) or N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) addition abrogated morphine's down-regulating activity, demonstrating morphine was initiating its actions via stimulating constitutive NO synthase derived NO release via the mu3 opiate receptor splice variant. In the past we demonstrated that UDP-glucurosyltransferase is involved in metabolizing morphine to morphine 6-glucuronide in adrenal chromaffin cells. In the present study its expression was not found in controls and morphine-treated cells, suggesting that morphine 6-glucuronide may not be synthesized in white blood cells. Taken together, it appears that morphine has the ability to modulate its own synthesis via autocrine and paracrine signaling.

Pharmacokinetics of morphine-6-glucuronide following oral administration in healthy volunteers

After oral administration, morphine-6-glucuronide (M6G) displays an atypical absorption profile with two peak plasma concentrations. A proposed explanation is that M6G is hydrolysed to morphine in the colon, which is then absorbed and subsequently undergoes metabolism in the liver to morphine-3-glucuronide (M3G) and M6G. The aims of this study were to confirm and elucidate the biphasic absorption profile as well as clarify the conversion of M6G to morphine after a single oral administration of M6G in healthy volunteers. The study was conducted accordingly to a nonblinded, randomised, balanced three-way crossover design in eight healthy male subjects. The subjects received 200 mg oral M6G, 50 mg oral M6G and 30 mg oral morphine. Blood samples were collected until 72 h after M6G administration and until 9 h after morphine administration. Paracetamol and sulfasalazine were coadministered with M6G as markers for the gut contents reaching the duodenum and colon, respectively. The plasma concentration peaks of M6G were seen at 4.0 (2.0-6.0) and 18 (12.0-24.0) h after 200 mg M6G and at 3.5 (2.0-6.0) and 21.3 (10.0-23.3) h after 50 mg M6G, which was in agreement with previously published results. The K(M6G_abs)/K(M6G_M6G) ratio was found to be 10. The pharmacokinetic profile of M6G after oral administration was confirmed and with the presence of M3G and morphine in plasma after oral administration of M6G, proof seems to be found of the constant and prolonged absorption of M6G. The K(M6G_abs)/K(M6G_M6G) ratio of 10 indicates that the second absorption peak of M6G consists of approximately 10 times more absorbed M6G than reglucuronidated M6G. However, further studies are required to determine the precise kinetics of the second absorption peak.

Multidrug Resistance Proteins 2 and 3 Provide Alternative Routes for Hepatic Excretion of Morphine-Glucuronides

Glucuronidation is a major hepatic detoxification pathway for endogenous and exogenous compounds, resulting in the intracellular formation of polar metabolites that require specialized transporters for elimination. Multidrug resistance proteins (MRPs) are expressed in the liver and can transport glucuronosyl-conjugates. Using morphine as a model aglycone, we demonstrate that morphine-3-glucuronide (M3G), the predominant metabolite, is transported in vitro by human MRP2 (ABCC2), a protein present in the apical membrane of hepatocytes. Loss of biliary M3G secretion in Mrp2(-/-) mice results in its increased sinusoidal transport that can be attributed to Mrp3. Combined loss of Mrp2 and Mrp3 leads to a substantial accumulation of M3G in the liver, from which it is transported across the sinusoidal membrane at a low rate, resulting in the prolonged presence of M3G in plasma. Our results show that murine Mrp2 and Mrp3 provide alternative routes for the excretion of a glucuronidated substrate from the liver in vivo.

A Randomized, Double-blind, Placebo-controlled Pilot Study of IV Morphine-6-Glucuronide for Postoperative Pain Relief After Knee Replacement Surgery

To determine the dose-response effect of intravenous morphine-6-glucuronide (M6G) on acute postoperative pain. Patients undergoing knee replacement surgery under spinal anesthesia were randomly assigned to 1 of 4 single intravenous M6G doses, 0 (placebo), 10, 20, or 30 mg/70 kg, administered 150 minutes after the spinal anesthetic was given. Analgesic effects were evaluated by determining the cumulative patient controlled analgesia (PCA) morphine dose, consumed over a 12 and 24 hours period, after the initial dose of M6G. For pain assessments, a 10 cm visual analog scale was used. Data from 41 patients were evaluated (n=10, 10, 10, and 11 in the 0, 10, 20, and 30 mg M6G groups). Only at the highest M6G dose (30 mg/70 kg), morphine PCA consumption was significantly less compared with placebo: over the first 12 postoperative hours mean PCA morphine consumption was 3.0+/-2.0 mg/h after placebo and 1.4+/-0.5 mg/h after 30 mg M6G (P=0.03); over the first 24 h mean PCA morphine consumption was 2.5+/-2.1 mg after placebo and 1.0+/-0.4 mg after 30 mg M6G (P=0.04) (mean+/-SD). Visual analog scale values were similar across all groups during these time periods. The analgesic effect of M6G in postoperative pain was demonstrated with 30 mg/70 kg M6G superior to placebo. At this dose, M6G has a long duration of action as determined by a reduction in the use of morphine PCA over 12 and 24 hours.

Morphine‐3‐glucuronide inhibits morphine induced, but enhances morphine‐6‐glucuronide induced locomotor activity in mice

The main metabolite of morphine, morphine‐3‐glucuronide (M3G) has no opioid effects. Some studies have rather indicated that it antagonizes the antinociceptive and respiratory depressive effects of both morphine and the active metabolite morphine‐6‐glucuronide (M6G). We studied the possible influence of M3G on the psychostimulant properties of morphine and M6G measured by locomotor activity. Mice were given two injections, one with either 80, 240 or 500 μmol/kg M3G or saline followed by an injection of 20 or 30 μmol/kg morphine or M6G. M3G influenced the locomotor activity induced by both morphine and M6G, but in opposite directions. M3G reduced the morphine induced locomotor activity during the first hour following morphine injection in a concentration dependent manner. M3G pretreatment did not significantly influence brain concentrations of morphine indicating that the interaction was of a pharmacodynamic type. In contrast M3G pretreatment increased the M6G induced locomotor activity. M3G pretreatment increased serum and brain M6G concentrations to an extent indicating that this interaction was mainly of a pharmacokinetic type. In conclusion our results disclose complicated interactions between morphine and its two metabolites with respect to induction of locomotor activity and possibly also with respect to mechanisms related to drug reward.

Effect of valerian, valerian/hops extracts, and valerenic acid on glucuronidation in vitro

Valerian preparations alone or in combination with hops are popular over-the-counter products used for sleep disturbances or anxiety. Therefore, it is important to characterize the effect of these products on the activity of human drug-metabolizing enzymes. The inhibitory effects of valerian and valerian/hops extracts as well as valerenic acid (a major constituent of valerian) on glucuronidation were evaluated in human liver microsomes and with expressed uridine 5′-diphosphate (UDP)-glucuronosyltransferases (UGT). Methanolic extracts of two herbal preparations caused significant reductions in the rate of formation of acetaminophen, oestradiol, morphine, and testosterone glucuronides. Oestradiol glucuronidation at the 3-hydroxy position was inhibited by nearly 87% in microsomal incubations. In addition, marked reductions in UGT1A1 and UGT2B7 activities were observed in the presence of the herbal extracts using oestradiol and morphine as probe substrates, respectively. Valerenic acid also demonstrated significant inhibitory effects on the glucuronidation of acetaminophen, oestradiol, and morphine with both microsomes and expressed UGTs. The relatively low IC50 values obtained for valerenic acid in microsomal incubations may indicate that this essential oil contributes to the effects observed with herbal extracts in inhibiting glucuronidation in vitro. Overall, these findings suggest that valerian-containing products may interfere with the glucuronidation of endo- and xenobiotics.

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the μ opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH 2 (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.

Influence of Phenobarbital on Morphine Metabolism and Disposition:LC-MS/MS Determination of Morphine (M) and Morphine-3-Glucuronide (M3G) in Wistar-Kyoto Rat Serum, Bile, and Urine

A simple LC-MS/MS method has been developed and validated for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G) in rat serum, bile, and urine. Deuterated D3-M and D3-M3G were used as internal standards (IS) for M and M3G, respectively. Serum samples were processed by acetonitrile precipitation. Bile samples were prepared by solid-phase extraction (SPE) using Oasis MCX cartridges. Urine samples were directly analyzed after dilution with mobile phase. Chromatography was performed using a Luna C18 column (5 microm, 150x2.1 mm I.D.). The mobile phase consisted of acetonitrile (ACN) and 7.5 mM ammonium formate (pH 9.3) delivered from separate pumps with a simple gradient. The method was validated to quantify M in the range of 1-1000 ng/ml in bile and serum, and 0.025-25 microg/ml in urine. M3G was quantified in the range of 1-1000 ng/ml in serum, 0.1-100 microg/ml in bile, and 0.05-25 microg/ml in urine. The method was applied to study the pharmacokinetics and disposition of M and M3G in Wistar-Kyoto (WKY) rats, and the effect of phenobarbital (PB) on M and M3G disposition. M is metabolized to M3G at a lower rate in male than female rats leading to higher M levels and lower M3G levels in serum, urine, and bile of male than female rats. PB administration induces M glucuronidation to M3G in male, but not female WKY rats, and abolishes the gender differences in M and M3G pharmacokinetics.

Effects of generalized and kidney specific Mrp2 (ABCC2) deficiency on renal elimination of PAH and morphine‐6‐glucuronide

To study the role of Mrp2 for renal organic anion elimination without high genetic background variability, Mrp2 deficient congenic Lewis rats (LEW) were bred. RBF, RPF and GFR were similar in Mrp2 deficient and control LEW. With the Mrp2 and Mrp4 substrate PAH infused at 2.8 and 5.6 mg/kg∗h, FE PAH was 6.9 ± 0.3 and 8.4 ± 0.5 in LEW vs. 9.3 ± 0.6 and 11.9 ± 0.5 in Mrp2 deficient LEW (p < 0.001). Western-blot analysis revealed a fourfold higher renal cortical Mrp4 expression in Mrp2 deficient LEW than in controls. Mrp2 deficient LEW showed a hundredfold reduced biliary morphine-6-glucuronide (M6G) elimination indicating that M6G is a Mrp2 substrate. At the plasma concentrations tested, renal M6G elimination was similar in Mrp2 deficient and control LEW with FE M6G close to 1. To investigate kidney specific Mrp2 deficiency, kidneys were cross-transplanted. FE PAH and FE M6G did not differ between animals with kidney specific Mrp2 deficiency and syngeneically transplanted controls. There was no difference in renal Mrp4 expression between Mrp2 deficient and intact kidney grafts. Renal cortical gene expression profiling revealed 9 more than twofold induced and 4 more than twofold reduced genes in Mrp2 negative grafts transplanted into normal LEW compared to syngeneically transplanted controls. We conclude that generalized but not kidney specific Mrp2 deficiency causes increased FE PAH that is at least in part due to induction of renal Mrp4. Kidney specific Mrp2 deficiency does not compromise renal elimination of PAH and M6G.

Morphine in Children with Cancer

The aim of this study was to contrast protein binding of morphine and morphine-6 glucuronide in cord blood and children with adults and examine impact of chemotherapy and other factors. Morphine binding was measured in spiked samples from 18 adults and 18 neonates (cord blood), and compared with six children with cancer receiving morphine. The influence of the following was examined: Human serum albumin (HSA), alpha-1 acid glycoprotein (AAG), non-esterified fatty acids (NEFA); palmitic acid and oleic acid, pH, vincristine, methotrexate, 6-mercaptopurine and M6G. binding correlated with concentrations of albumin and alphal acid glycoprotein. In vitro, binding was not altered by vincristine, 6-mercaptopurine, methotrexate or M6G. Compared with HSA alone, AAG increased binding, palmitic acid reduced it and oleic acid had no effect. Binding was unaffected by pH in samples from patients. Morphine binding was influenced by concentrations of albumin, AAG and morphine itself, but not by age.

Modulation of Ca2+ Channels by Heterologously Expressed Wild-Type and Mutant Human μ-Opioid Receptors (hMORs) Containing the A118G Single-Nucleotide Polymorphism

The most common single-nucleotide polymorphism (SNP) of the human mu-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca(2+) channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca(2+) channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration-response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC(50) values (nM) in cells expressing wild-type hMOR was: DAMGO (31) >> morphine (76) congruent with M-6-G (77) congruent with endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) >> morphine (39) >> endomorphin I (74) congruent with M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca(2+) current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.

80 POSTER Methylnaltrexone potentiates the effects of 5-fluorouracil (5-FU) and Bevacizumab on inhibition of VEGF-induced endothelial cell proliferation and migration

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, agents that inhibit angiogenesis have important therapeutic implications in numerous malignancies. We examined the effects of methylnaltrexone (MNTX), a peripheral mu opioid receptor antagonist, on agonist-induced human EC proliferation and migration, two key components in angiogenesis. Using human dermal microvascular EC, we observed that morphine sulfate (MS), the active metabolite, morphine-6-glucuronide (M6G), DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]enkaphalin) and VEGF induced migration which were inhibited by pretreatment with MNTX at therapeutically relevant concentration (0.1 μM). The biologically inactive metabolite morphine-3-glucuronide (M3G) did not affect EC migration. We next examined the mechanism(s) by which MNTX inhibits opioid and VEGF-induced angiogenesis using human pulmonary microvascular EC. MS and DAMGO induced Src activation which was required for VEGF receptor transactivation and opioid-induced EC proliferation and migration. MNTX inhibited MS, DAMGO and VEGF induced tyrosine phosphorylation (transactivation) of VEGF receptors 1 and 2. Furthermore, MS, DAMGO and VEGF induced RhoA activation which was inhibited by MNTX or VEGF receptor tyrosine kinase inhibition. Finally, MNTX or silencing RhoA expression (siRNA) blocked MS, DAMGO and VEGF-induced EC proliferation and migration. Taken together, these results indicate that MNTX inhibits opioid-induced EC proliferation and migration via inhibition of VEGF receptor phosphorylation/transactivation with subsequent inhibition of RhoA activation. These results suggest that MNTX inhibition of angiogenesis can be a useful therapeutic intervention for cancer treatment.

Blood–brain distribution of morphine‐6‐glucuronide in sheep

At present there are few data regarding the rate and extent of brain-blood partitioning of the opioid active metabolite of morphine, morphine-6-glucuronide (M6G). In this study the cerebral kinetics of M6G were determined, after a short-term intravenous infusion, in chronically instrumented conscious sheep. Five sheep received an intravenous infusion of M6G 2.2 mg kg(-1) over a four-minute period. Non-linear mixed-effects analysis, with hybrid physiologically based kinetic models, was used to estimate cerebral kinetics from the arterio-sagittal sinus concentration gradients and cerebral blood flow measurements. A membrane limited model was selected as the final model. The blood-brain equilibration of M6G was relatively slow (time to reach 50% equilibration of the deep compartment 5.8 min), with low membrane permeability (PS, population mean, 2.5 ml min(-1)) from the initial compartment (V1, 13.7 ml) to a small deep distribution volume (V2) of 18.4 ml. There was some between-animal variability (%CV) in the initial distribution volume (29%), but this was not identified for PS or V2. Pharmacokinetic modelling of M6G showed a delayed equilibration between brain and blood of a nature that is primarily limited by permeability across the blood-brain-barrier, in accordance with its physico-chemical properties.

Heroin-using drivers: importance of morphine and morphine-6-glucuronide on late clinical impairment

To evaluate the relationship between major heroin metabolites (morphine, morphine-6-glucoronide), pattern of drug use, and late impairment of psychomotor functions. From the database of the Norwegian Institute of Public Health, Oslo, blood morphine concentration in samples from heroin users (n=70) containing only morphine were correlated with results of the clinical test for impairment (CTI). For comparison, test results were explored in individuals without any positive analytical finding in blood samples (n=79) selected from the same database. In the "no drug" cases, 86% were judged as not impaired and 14% as impaired. In the morphine only cases, 20% were judged as not impaired, and 80% as impaired. Both daily users and non-daily users had the same proportion of impaired cases. Median blood morphine concentration (M) was 0.09 micromol/l in the "not impaired" group and 0.15 micromol/l in the "impaired" group (P=0.067). For morphine-6-glucuronide (M6G), the median blood concentration was 0.09 micromol/l in the "not impaired" group and 0.14 micromol/l in the "impaired" group (P=0.030). A significant correlation between concentration quartiles and number of cases determined as "impaired" was found for M6G (P=0.018) and for the sum M+M6G (P=0.013). In our population of heroin-drugged drivers, blood concentrations of M6G and the sum M+M6G appeared to have concentration-dependent effects on the CNS that may lead to impairment as judged from a CTI. Variations in pattern of use did not seem to have any bearing on the judgement of impairment.