Supercritical fluid extraction of drugs in drug addict hair

Opiates were extracted from sixteen hair samples of drug addicts using a supercritical fluid extraction method with supercritical carbon dioxide and a modifier solution of methanol-triethylamine-water (2:2:1, v/v). The concentrations, as determined by GC-MS, ranged from 1.22 to 21.73 (mean 7.60 ng/mg), 0.17 to 1.54 (mean 0.69 ng/mg) and 0.15 to 14.09 ng/mg hair (mean 3.78 ng/mg) for codeine, morphine and 6-monoacetylmorphine, respectively. The reproducibility of the total procedure had a relative standard deviation of 13%, 17% and 14% for codeine, morphine and 6-monoacetylmorphine, respectively. By this method, concentrations of 0.3, 0.2 and 0.1 ng/mg hair for codeine, morphine and 6-monoacetylmorphine, respectively, could be detected. Relative extraction recoveries were 61%, 53% and 96% for codeine, morphine and 6-monoacetylmorphine, respectively.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

Morphine formation from codeine in rat brain: a possible mechanism of codeine analgesia

The O-demethylation of codeine to morphine was demonstrated in rat brain homogenate. Maximal formation occurred at 10 minutes, with a Vmax of 5.93 +/- 0.16 nmol/g brain/h and Km of 37.82 +/- 4.99 microM. The formation was significantly (P less than 0.05) greater in the microvessel-rich brain fraction. Intraperitoneal injection of codeine in the rat resulted in brain concentrations of morphine which could not be solely attributed to transfer of morphine from the blood stream across the blood-brain barrier. Morphine formed in the brain after codeine administration may be an important mechanism for codeine-induced analgesia.     

Endogeneous morphine and codeine in mice — effect of muramyl dipeptide

Administration of morphine exerts many effects on the immune system. On the other hand little attention has been paid to the fact, that endogenous morphine and codeine exists in mammals, including man. This raises the question, whether or not endogenous opiate alkaloids play some role in immunoregulation. In addition muramyl-dipeptide (MDP), product of baterial cell wall degradation and a potent immunomdulatory agent exhibits a broad spectrum of effects including effects on CNS functions. The present study investigated whether or not the endogenous levels of morphine and codeine are affected by administration of MDP in mice. Marked variation was found in spleen, brain, small intestine and heart in morphine and codeine concentrations. The intraperitoneal administration of MDP produced a significant increase in tissue morphine levels 30 minutes after injection.     

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.     

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.     

Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively. This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the -hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.     

Electrochemiluminescence determination of codeine or morphine with an organically modified silicate film immobilizing Ru(bpy)3(2+).

An ECL approach was developed for the determination of codeine or morphine based on tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) immobilized in organically modified silicates (ORMOSILs). Tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMe-DiMOS) were selected as co-precursors for ORMOSILs, which were then immobilized on a surface of glassy carbon electrode (GCE) by a dip-coating process. Ru(bpy)(3)(2+) was immobilized in the ORMOSIL film via ion-association with poly(p-styrenesulphonate). The ORMOSIL-modified GCE presented good electrochemical and photochemical activities. In a flow system, the eluted codeine or morphine was oxidized on the modified GCE and reacted with immobilized Ru(bpy)(3)(2+) at a potential of +1.20 V (vs. Ag/AgCl). The modified electrode was used for the ECL determination of codeine or morphine and showed high sensitivity. The calibration curves were linear in the range 2 x 10(-8)-5 x 10(-5) mol/L for codeine and 1 x 10(-7)-3 x 10(-4) mol/L for morphine. The detection limit was 5 x 10(-9) mol/L for codeine and 3 x 10(-8) mol/L for morphine, at signal:noise ratio (S:N)=3. Both codeine and morphine showed reproducibility with RSD values <2.5% at 1.0 x 10(-6) mol/L. Furthermore, the modified electrode immobilized Ru(bpy)(3)(2+) was applied to the ECL determination of codeine or morphine in incitant samples.     

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.     

Narcotic alkaloids of four papaver species from Iran

Four native Papaver species of Iran, i. e. P. glaucum, P. tenuifolium, P. dubium and P. fugax, were collected from their natural habitat and subjected to HPLC analysis for determination of their morphine, codeine and thebaine content. P. dubium and R. glaucum contained all of the three mentioned narcotic alkaloids, while morphine was not found in P. fugax, and P. tenuifolium was free from codeine.     

Codeine and 6-Acetylcodeine Analgesia in Mice

Summary 1. Acetylation of morphine at the 6-position changes its pharmacology. To see if similar changes are seen with codeine, we examined the analgesic actions of codeine and 6-acetylcodeine.2. Like codeine, 6-acetylcodeine is an effective analgesic systemically, supraspinally and spinally, with a potency approximately a third that of codeine.3. The sensitivity of 6-acetylcodeine analgesia to the mu-selective antagonists β-FNA and naloxonazine confirmed its classification as a mu opioid. However, it differed from the other mu analgesics in other paradigms.4. Antisense mapping revealed the sensitivity of 6-acetylcodeine to probes targeting exons 1 and 2 of the mu opioid receptor gene (Oprm), a profile distinct from either codeine or morphine. Although heroin analgesia also is sensitive to antisense targeting exons 1 and 2, heroin analgesia also is sensitive to the antagonist 3-O-methylnaltrexone, while 6-acetylcodeine analgesia is not.5. Thus, 6-acetylcodeine is an effective mu opioid analgesic with a distinct pharmacological profile.     

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.     

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration

Morphine-3- and morphine-6-glucuronide are morphine’s major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.     

A conspicuous down-regulating effect of morphine on essential steroid hydroxylation reactions and certain drug N-demethylations.

This study was conducted to explore the potency of morphine to induce reductions of specific cytochrome P450 isoenzyme functions. Male Sprague-Dawley rats were treated with escalating doses (20-125 mg/kg per day) of morphine for 2 weeks in order to study the effects on the following cytochrome P450 catalyzed reactions: 16 alpha-hydroxylation of dehydroepienderosterone (DHA) and progesterone; 17 alpha- and 21-hydroxylation of progesterone; N-demethylation of ethymorphine, codeine and morphine as well as O-dealkylation of ethylmorphine and codeine. 16 alpha-Hydroxylation of DHA and progesterone and 17 alpha-hydroxylation of progesterone decreased to 18, 12 and 10% of control activities, respectively. The N-demethylation of ethylmorphine and codeine decreased to 34 and 43% of control activities, respectively. Morphine treatment had no effect on the 21-hydroxylation reactions or the O-dealkylation of ethylmorphine or codeine. A monoclonal antibody (Mab) against rat liver cytochrome P450 2 c/RLM 5 exerted a 66-73% inhibition of the N-demethylation of ethylmorphine and codeine, respectively, whereas the O-dealkylation reactions were not affected. This Mab inhibited the 16 alpha- and 17 alpha-hydroxylation of DHA and progesterone, whereas the 21-hydroxylation reactions were unaffected. The steroid hydroxylation reactions in rat adrenals were not altered upon morphine treatment. Our data suggest that a major part of the 16 alpha- and 17 alpha-steroid hydroxylations are catalyzed by the same (or closely related) cytochrome(s) P450 as the opioid N-demethylation reactions.     

Production of monoclonal antibodies and ELISA for thebaine and codeine

The ratio of hapten and bovine serum albumin in antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. Monoclonal antibodies against thebaine and codeine were produced by hybridoma fused with the sprenocytes immunized with thebaine- and codeine-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. No cross-reaction of anti-thebaine antibody against morphine was observed. Very small cross-reaction appeared in codeine (0.004%). The cross-reaction of anti-codeine antibody against morphine and thebaine was 2.97 and 5.98%, respectively. The full measuring range of the assay extends from 60 pg mL to 1 ng mL for thebaine and 1 ng mL to 100 ng mL for codeine.     

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.     

Possible role of endogenous morphine and codeine on growth regulation of lung tissue

The literature indicates that morphine can inhibit the growth of both small cell and non small cell lung cancer cell lines and that nicotine can reverse this inhibition. In this report we present data showing that mammalian lung tissue contain the opiate alkaloids morphine and codeine and that these alkaloids are also to be found in normal lung cell lines. However, analysis of both small cell and non small cell lung cancer cells indicate that they do not contain these opiate alkaloids endogenously. If morphine exerts an inhibitory effect on proliferation of these cells it is interesting that the lung cancer cell lines lack the opiate alkaloids endogenously. Our studies also present data indicating that the circulating levels of morphine and codeine are elevated in smokers as compared to non smokers which we hypothesize to reflect the invocation of a compensatory mechanism.     

Enhanced codeine and morphine production in suspended Papaver somniferum cultures after removal of exogenous hormones

Summary Morphine and codeine accumulation in Papaver somniferum suspension cultures increased markedly after removal of hormones from the medium. Cultures developed hormone self-sufficiency without organogenesis or development of meristemoids; enhanced synthesis of morphinan alkaloids was not dependent on formation of shoots, roots or embryos. Without exogenous hormones, maximum codeine and morphine concentrations were 3.0 mg g^–1 dry weight and 2.5 mg g^–1 dry weight respectively, up to three times higher than in cultures supplied with hormones. Hormone-deprived cells produced a higher ratio of codeine:morphine than cultures supplied with auxin and cytokinin. Improved alkaloid production was correlated with slower overall growth rate.     

Evaluation of the mass spectrometric fragmentation of codeine and morphine after 13C-isotope biosynthetic labeling

All major fragment ions of codeine and morphine were elucidated using LC-electrospray MS/MS and high resolution FT-ICR-MS combined with an IRMPD system. Nanogram quantities of labeled codeine were isolated and purified from Papaver somniferum seedlings, which were grown for up to 9 days in the presence of [ring-13C6]-l-tyrosine, [ring-13C6]-tyramine and [1,2-13C2], [6-O-methyl 13C]-(R,S)-coclaurine. The labeling degree of codeine up to 57% into morphinans was observed.     

Inhibitory effects of opioids on compound action potentials in frog sciatic nerves and their chemical structures

An opioid tramadol more effectively inhibits compound action potentials (CAPs) than its metabolite mono-O-demethyl-tramadol (M1). To address further this issue, we examined the effects of opioids (morphine, codeine, ethylmorphine and dihydrocodeine) and cocaine on CAPs by applying the air-gap method to the frog sciatic nerve. All of the opioids at concentrations less than 10 mM reduced the peak amplitude of the CAP in a reversible and dose-dependent manner. The sequence of the CAP peak amplitude reductions was ethylmorphine>codeine>dihydrocodeine> or = morphine; the effective concentration for half-maximal inhibition (IC(50)) of ethylmorphine was 4.6 mM. All of the CAP inhibitions by opioids were resistant to a non-specific opioid-receptor antagonist naloxone. The CAP peak amplitude reductions produced by morphine, codeine and ethylmorphine were related to their chemical structures in such that this extent enhanced with an increase in the number of -CH(2) in a benzene ring, as seen in the inhibitory actions of tramadol and M1. Cocaine reduced CAP peak amplitudes with an IC(50) value of 0.80 mM. It is concluded that opioids reduce CAP peak amplitudes in a manner being independent of opioid-receptor activation and with an efficacy being much less than that of cocaine. It is suggested that the substituted groups of -OH bound to the benzene ring of morphine, codeine and ethylmorphine as well as of tramadol and M1, the structures of which are quite different from those of the opioids, may play an important role in producing nerve conduction block.