Opiate receptor binding properties of morphine-, dihydromorphine-, and codeine 6-O-sulfate ester congeners

A series of 3-O-acyl-6-O-sulfate esters of morphine, dihydromorphine, N-methylmorphinium iodide, codeine, and dihydrocodeine were prepared and evaluated for their ability to bind to mu-, delta-, kappa(1)-, kappa(2)-, and kappa(3)-opiate receptors. Several compounds exhibited good affinity for the mu-opiate receptor. Morphine-3-O-propionyl-6-O-sulfate had four times greater affinity than morphine at the mu-opiate receptor and was the most selective compound at this receptor subtype.     

Pivaloylcodeine,a new codeine derivative,for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC₅₀ were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC₅₀ 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of opiates and cocaine in meconium

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C(8) reversed-phase column using a gradient of acetic acid 1%-acetonitrile as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.     

Determination of codeine and metabolites in plasma and urine using ion-pair high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the simultaneous determination of codeine and seven metabolites is described. The samples are purified by reversed-phase solid-phase extraction. Codeine, norcodeine, codeine-6-glucuronide, norcodeine-6-glucuronide and morphine-3-glucoronide are measured with UV detection. Detection limits are 3 nmol/l (morphine-3-glucuronide) to 20 nmol/l (codeine). Morphine, normorphine and morphine-6-glucuronide are measured with electrochemical detection. Detection limits are 0.4 nmol/l (morphine-6-glucuronide) to 1.0 nmol/l (normorphine). Correlation coefficients better than 0.998 are normally obtained for all compounds. The method was applied to the determination of the kinetics of codeine and its metabolites in plasma and urine samples from healthy volunteers.     

High-performance liquid chromatographic-electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to pharmacokinetic studies

A rapid and selective assay of morphine and its 3- and 6-glucuronides in serum, based on high-performance liquid chromatography-electrospray mass spectrometry has been developed. The analytes and the internal standard, codeine or naltrexone, were subjected to solid-phase extraction, using ethyl solid-phase extraction columns, prior to chromatography. A reversed-phase column and a gradient mobile phase consisting of water and methanol were used. The mass spectrometer was operated in the selected-ion monitoring mode. The following ions were used: m/z 286 for morphine, m/z 300 for codeine, m/z 342 for naltrexone, and m/z 462 for morphine 3- and 6-glucuronides. The limit of quantitation observed with this method was 10 ng/ml morphine, 50 ng/ml morphine-6-glucuronide and 100 ng/ml morphine-3-glucuronide. The present method proved useful for the determination of serum levels of the parent drug and its metabolites in pain patients, heroin addicts and in morphine-treated mice.     

Day-to-day variations during clinical drug monitoring of morphine,morphine-3-glucuronide and morphine-6-glucuronide serum concentrations in cancer patients. A prospective observational study

Background  

The feasibility of drug monitoring of serum concentrations of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) during chronic morphine therapy is not established. One important factor relevant to drug monitoring is to what extent morphine, M6G and M3G serum concentrations fluctuate during stable morphine treatment.     

Mu receptor binding of some commonly used opioids and their metabolites.

The binding affinity to the mu receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding (e.g. morphine-6-glucuronide Ki = 0.6 nM; morphine = 1.2 nM). Decreasing the length of the alkyl group at position 3 decreased the Ki values (morphine less than codeine less than ethylmorphine less than pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 (e.g. hydrocodone, Ki = 19.8 nM) had relatively weak receptor binding, whilst their O-demethylated metabolites (e.g. hydromorphone, Ki = 0.6 nM) had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.     

Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine

A hydrophilic interaction liquid chromatography-time-of-flight mass spectrometry (HILIC-TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50-5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.     

Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography

A high-performance liquid chromatography (HPLC) method has been developed for the determination of morphine and its main metabolites, morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G), in plasma or cerebrospinal fluid. Samples were extracted using on-line solid-phase extraction followed by reversed-phase HPLC with fluorescence detection. Recoveries of 20 ng morphine and morphine glucuronides in plasma were over 95%. The limit of detection using 400 μl of a biological matrix was 0.85, 3.4 and 1.0 ng/ml of M-3-G, M-6-G and morphine, respectively. Inter- and intra-day assay precision was better than 10%. The main advantages of the present described method are increased recoveries (>95%) and a high degree of automation allowing a high speed in routine analysis. The time required for the fully automated analysis of one sample was less than 26 min.     

Rapid,highly sensitive method for the determination of morphine and its metabolites in body fluids by liquid chromatography–mass spectrometry

A rapid, highly sensitive method for the determination of morphine and its metabolites morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and normorphine has been developed using high-performance liquid chromatography–electrospray mass spectrometry, with the deuterated analogues as internal standards. The analytes were extracted automatically using end-capped C₂ solid-phase extraction cartridges. Baseline separation of morphine, M3G and M6G was achieved on a LiChrospher 100 RP-18 end-capped analytical column (125×3 mm I.D., 5 μm particle size) with water–acetonitrile–tetrahydrofuran–formic acid (100:1:1:0.1, v/v) as the mobile phase. Morphine and normorphine coeluate and were separated mass spectrometrically. The mass spectrometer was operated in the selected-ion monitoring mode using m/z 272 for normorphine, m/z 286 for morphine, m/z 462 for morphine-6-glucuronide. Due to an interfering peak, M3G was measured by tandem mass spectrometry in the daughter-ion mode. The limits of quantitation achieved with this method were 1.3 pmol/ml for morphine, 1.5 pmol/ml for normorphine, 1.0 pmol/ml for M6G and 5.4 pmol/ml for M3G in serum or cerebrospinal fluid. The limits of quantitation achieved in urine were 10 pmol/ml for morphine, 20 pmol/ml for normorphine and M6G and 50 pmol/ml for M3G using a sample size of 100 μl. The method described was successfully applied to the determination of morphine and its metabolites in human serum, cerebrospinal fluid and urine in pharmacokinetic and drug interaction studies.     

Opiate alkaloids in Ascaris suum

The parasitic worm Ascaris suum contains the opiate alkaloids morphine and morphine-6-glucuronide as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of morphine in muscle tissue of female and male is 252 +/- 32.68, 1168 +/- 278 and 180 +/- 23.47 (ng/g of wet tissue), respectively. The level of M6G in muscle tissue of female and male is 167 +/- 28.37 and 92 +/- 11.45 (ng/g of wet tissue), respectively. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. The anatomic distribution of morphine was examined by indirect immunofluorescent staining and HPLC of various tissues dissected from male and female adult worms. Immunofluorescence revealed morphine in the subcuticle layers, in the animals' nerve chords and in the female reproductive organs. Morphine was found to be most prevalent in the muscle tissue and there is significantly more morphine in females than males, probably due to the large amounts in the female uterus. Morphine (10(-9) M) and morphine-6-glucuronide (10(-9) M) stimulated the release of NO from Ascaris muscle tissue. Naloxone (10(-7) M), and L-NAME (10(-6) M) blocked (P < 0.005) morphine-stimulated NO release from A. suum muscle. CTOP (10(-7) M) did not block morphine's NO release. However, naloxone could not block M6G stimulated NO release by muscle tissue, whereas CTOP (10(-7) M) blocked its release. These findings were in seeming contradiction to our inability to isolate a mu opiate receptor messenger RNA by RT-PCR using a human mu primer. This suggests that a novel mu opiate receptor was present and selective toward M6G.     

Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method has been developed for the detection, separation and measurement of codeine and its metabolites norcodeine, morphine and normorphine, with their glucuronide conjugates. The glucuronidase Escherichia coli type VIIA hydrolyses codeine-6-glucuronide completely and is used for the construction of the calibration curves of codeine-6-glucuronide. Enzymic hydrolysis of codeine-6-glucuronide depends on the specific activity of the glucuronidase applied. Examples are shown of a volunteer who is able to form morphine from codeine and one who is unable to do so.     

Identification of morphine-6-glucuronide in chromaffin cell secretory granules

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.     

A codeine radioimmunoassay exhibiting insignificant cross-reactivity with morphine: (Preliminary communication)

Antisera to codeine have been raised to an $\text{N}$-butyroylnorcodeine-bovine serum albumin conjugate. These antisera were used, at a final dilution of 1:10, 000 in a radioimmunoassay procedure for codeine utilizing tritiated codeine as label. No cross-reactivity was observed with heroin, 6-monoacetyl-morphine, morphine or codeine-6-glucuronide, but, as might be expected, norcodeine cross-reacts to an appreciable extent with this antiserum. This immunoassay system should be of value in quantitating codeine in biological fluids, and in distinguishing codeine from morphine or its major metabolites.     

Morphine-6-glucuronide contributes to rewarding effects of opiates

It was recently confirmed that a metabolite of morphine, morphine-6-glucuronide (M6G), is a long lasting, powerful analgesic in humans and animals and may account for a major component of clinical opiate analgesia. It is reported here that M6G is also a powerful behavioral reinforcer in the conditioned place preference test in rats, indicating that it has rewarding properties, and is therefore likely to have abuse potential. The induction of a place preference by M6G is blocked by naltrexone, indicating that the rewarding effect of M6G is mediated by opioid receptors. Given systemically M6G is approximately equipotent with morphine. When given intracerebroventricularly to bypass the blood-brain barrier, M6G is 146 times more potent than morphine in the place preference test. Thus 6-substituted metabolites of opiates may play a more significant role in the effects of opiates than has been previously assumed.     

Exploration of the metabolism of dihydrocodeine via determination of its metabolites in human urine using micellar electrokinetic capillary chromatography

After single-dose administration of 40 or 60 mg of dihydrocodeine (DHC, in a slow-release tablet) to four healthy individuals known to be extensive metabolizers of debrisoquine, the urinary excretion of DHC and its four major metabolites, dihydrocodeine-6-glucuronide, nordihydrocodeine, dihydromorphine and nordihydromorphine, was assessed using micellar electrokinetic capillary chromatography (MECC). DHC and two of its metabolites (dihydrocodeine-6-glucuronide and nordihydrocodeine) could be analyzed by direct urine injection, whereas the metabolic pattern was obtained by copolymeric bonded-phase extraction of the solutes from both plain and hydrolyzed urine specimens prior to analysis. The total DHC equivalents exceted within 8 and 24 h were determined to be 30.4 ± 7.7% (n = 5) and 63.8 ± 6.1% (n = 2), respectively, and only about 4% of the excreted DHC equivalents were identified as morphinoids. Furthermore, almost no morphinoid metabolites of DHC could be found after administration of quinidine (200 mg of quinidine sulfate) 2 h prior to DHC intake.     

The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 degrees C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5-500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (< or =20% at lower limit of quantification (LLQ) and < or =15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.     

Mass spectrometric detection of nodularin and desmethylnodularin in mussels and flounders

A rapid and simple method for the determination of morphine (M), normorphine (NM), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma by high-performance liquid chromatographic separation with mass spectrometric detection (HPLC-MS) has been developed. Samples (40 microl) were cleaned-up by protein precipitation with two volumes (80 microl) of acetonitrile and reconstituted in formic acid 0.1% in water. Naloxone was used as internal standard. Analytes were separated on a phenyl-hexyl column using a step-gradient (1 ml/min) of acetonitrile and formic acid in water. Acetonitrile was added post-column (0.3 ml/min). Quantification of morphine and its metabolites was achieved with an Agilent 1100 series HPLC-MS system equipped with electrospray interface set to selected ion-monitoring (SIM) mode. Calibration curves covered a wide range of concentrations (2.44-10,000 nM) and were best fitted with a weighed quadratic equation. The limits of quantification achieved with this method were 2.44 nM for M and 4.88 nM for NM, M3G and M6G. The method proved accurate (85-98%), precise (C.V.<10%) and was successfully applied to a wide range of in vitro and in vivo pharmacokinetic studies in rodents.