Active Metabolite Morphine Research Articles

Should Codeine Still be Considered a WHO Essential Medicine?

Codeine continues to be widely used as an analgesic, antidiarrhoeal and antitussive agent. Its analgesic effect depends on its biotransformation to morphine, a strong opioid. The highly variable biotransformation of codeine to morphine, catalysed by CYP2D6, underlies the pronounced interindividual variability of its analgesic response. Randomized controlled trials have demonstrated that codeine administered alone has the poorest analgesic effect among all commonly used analgesics in acute postoperative pain. Moreover, it is highly unlikely that the low dose of codeine contributes to the pain-relieving effect of the non-opioid component in combination analgesic products. In addition, there is a lack of reliable clinical evidence to support the use of codeine as an antitussive in acute or chronic cough. Codeine use, through its active metabolite morphine, has the potential to lead to abuse and dependence. The World Health Organization (WHO) removed codeine from the essential medicines list for children in 2011. Based on the available information in the scientific literature on the efficacy and safety of codeine, the WHO should seriously consider removing it also from the list of essential medicines for adults, which would be a strong signal for all health professionals to prescribe and dispense codeine with the utmost caution.

Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood.

Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run. Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards. The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15%, and the accuracy was between 90 and 115%. This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.

Response to letter regarding article ‘Exploring the link between pholcodine exposure and neuromuscular blocking agent anaphylaxis’

Uyttebroek et al. [1] raise some pertinent questions regarding the utility of various testing modalities in the assessment of neuromuscular blocking agent (NMBA) hypersensitivity. In our cases, clinical history was the major assessment tool for identifying pholcodine as the inciting agent of anaphylaxis [2]. In both patients, the temporal association between pholcodine exposure and clinical reaction was in keeping with drug-induced anaphylaxis and no alternative causes were identified. The positive allergen-specific IgE levels to pholcodine (ImmunoCAP Phadia, Uppsala, Sweden) were used to substantiate the high index of clinical suspicion. As mentioned in the article, skin testing to pholcodine has limited utility because it causes wheal and flare reactions in normal control subjects. While drug provocation challenges are sometimes used to confirm allergy in situations where history and skin/allergen-specific IgE testing are ambiguous, in this circumstance this was not felt appropriate, owing to the severity of systemic reaction in both patients' index events. In one of our cases, a tramadol challenge was performed to give additional options if opiate analgesics were required, and the avoidance of codeine and its active metabolite morphine was recommended. Subsequent evaluation in both our patients was targeted toward the potential for NMBA hypersensitivity given previous studies [3–6], presented in the review, suggesting an association between pholcodine exposure and NMBA anaphylaxis. The recommendations regarding NMBA use in both patients were based on the outcome of skin testing and allergen-specific IgE testing. As Uyttebroek et al. [1] allude to, sensitization to a drug does not always equate with clinically meaningful allergy. However, provocation challenges with various NMBAs, as a means of demonstrating patient tolerance or allergy, are not feasible. Thus, the recommendations were made on the available testing at the time of assessment. The development of basophil activation tests as an adjunct to skin testing and specific IgE testing will no doubt help in the assessment of cases of suspected drug allergy. However, these tests are currently not validated or routinely available in most centres, and sensitivity may be lacking even when cases are carefully phenotyped and selected. The cases presented demonstrate the potential for sensitization to NMBA to occur in the context of pholcodine hypersensitivity. These case studies are reflective, at an individual patient level, of the population-based evidence cited previously [3,5,6]. We agree that consideration of whether the patients are able to tolerate opiates following true pholcodine immediate (IgE) hypersensitivity reactions is an important practical concern, and the specific haptenated products mediating such reactions are unknown. Indeed, codeine is metabolized by the genetically polymorphic isoform CYP2D6 to the active metabolite morphine, adding additional potential variability to potential cross-reactivity and patient tolerance. However, the focus of our article [2] was on the potential association of pholcodine exposure and NMBA allergy, and thus, the presentation of the case studies reflects this. Immunoglobulin E-mediated hypersensitivity reactions to pholcodine and morphine are rare, and this does indeed raise important questions regarding the pathogenesis of pholcodine sensitization and NMBA anaphylaxis. At present, the mechanisms underlying why exposure and sensitization to a substituted ammonium ion in pholcodine can prime NMBA anaphylaxis but not result in allergy to all compounds containing similar substituted ammonium ions remain unresolved.

A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography–tandem mass spectrometry

Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3β-glucuronide (M3G) and morphine 6β-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-μm PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25-1000 ng/mL (r(2) > 0.99) for morphine, 1-1,000 ng/mL (r(2) > 0.99) for M3G, and 2.5-1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1-1,000 ng/mL (r(2) > 0.99) for morphine and M3G, and of 2.5-1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20-50 μL.

In Vitro–In Vivo Extrapolation Predicts Drug–Drug Interactions Arising from Inhibition of Codeine Glucuronidation by Dextropropoxyphene, Fluconazole, Ketoconazole, and Methadone in Humans

Because codeine (COD) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro-in vivo extrapolation approaches were used to identify potential drug-drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD-6-glucuronide (C6G) formation by human liver microsomes (HLM) and demonstrated an 88% reduction in the Michaelis constant (K(m)) (0.29 versus 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UDP-glucuronosyltransferase (UGT) enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S(50) values (0.32 and 0.27 mM) generated in the presence of BSA were comparable with the mean K(m) observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone. Inhibitor constant values generated for dextropropoxyphene (3.5 microM), fluconazole (202 microM), ketoconazole (0.66 microM), and methadone (0.32 microM) predicted 1.60- to 3.66-fold increases in the area under the drug plasma concentration-time curve ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4- and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone potentially affect the intensity and duration of COD analgesia.

Pharmacokinetics of acetaminophen, codeine, and the codeine metabolites morphine and codeine-6-glucuronide in healthy Greyhound dogs

The purpose of this study was to determine the pharmacokinetics of codeine and the active metabolites morphine and codeine-6-glucuronide after i.v. codeine administration and the pharmacokinetics of acetaminophen (APAP), codeine, morphine, and codeine-6-glucuronide after oral administration of combination product containing acetaminophen and codeine to dogs. Six healthy Greyhound dogs were administered 0.734 mg/kg codeine i.v. and acetaminophen (10.46 mg/kg mean dose) with codeine (1.43 mg/kg mean dose) orally. Blood samples were collected at predetermined time points for the determination of codeine, morphine, and codeine-6-glucuronide plasma concentrations by LC/MS and acetaminophen by HPLC with UV detection. Codeine was rapidly eliminated after i.v. administration (T(1/2) = 1.22 h; clearance = 29.94 mL/min/kg; volume of distribution = 3.17 L/kg) with negligible amounts of morphine present, but large amounts of codeine-6-glucuronide (C(max) = 735.75 ng/mL) were detected. The oral bioavailability of codeine was 4%, morphine concentrations were negligible, but large amounts of codeine-6-glucuronide (C(max) = 1952.86 ng/mL) were detected suggesting substantial first pass metabolism. Acetaminophen was rapidly absorbed (C(max) = 6.74 microg/mL; T(max) = 0.85 h) and eliminated (T(1/2) = 0.96 h). In conclusion, the pharmacokinetics of codeine was similar to other opioids in dogs with a short half-life, rapid clearance, large volume of distribution, and poor oral bioavailability. High concentrations of codeine-6-glucuronide were detected after i.v. and oral administration.

Pharmacokinetic changes in the elderly. Do they contribute to drug abuse and dependence?

The elderly frequently use psychoactive drugs including alcohol (ethanol), benzodiazepines and opioid analgesics, which have a propensity to cause abuse and dependence. Theoretically, the changes in pharmacokinetics of these agents in the elderly may modify their abuse and dependence potential. In the elderly, blood alcohol concentrations following an oral dose are higher, alcohol withdrawal syndrome follows a more severe and protracted clinical course and requires treatment with higher doses of chlordiazepoxide than needed for younger adults. However, there is no direct evidence that supports an increased direct abuse and dependence potential of alcohol because of its altered kinetics in the elderly. In the case of oxidatively metabolised benzodiazepine, both age-related pharmacokinetics and pharmacodynamic changes may increase their clinical effects in the elderly. The hypothesis that benzodiazepines have an increased abuse and dependence potential in the elderly has not been tested. Many of the benzodiazepines (e.g. alprazolam, triazolam and midazolam) are metabolised by the cytochrome P450 (CYP)3A subfamily. The pharmacokinetics of these agents may be modified by inhibition of CYP3A due to concurrently administered medications such as selective serotonin reuptake inhibitors. Unfortunately, data on the direct measures of abuse and dependence potential of benzodiazepines are not available in the elderly. Thus, a conclusive statement on the contribution of age-related pharmacokinetic changes to benzodiazepine abuse and dependence cannot be made at the present time. The clinical effects of codeine do not appear to change with age. Codeine is O-demethylated to its active metabolite morphine by the genetically polymorphic CYP2D6 isozyme. The activity of this isozyme is unaltered by age, gender or smoking habits; however, it is subject to potent inhibition by some of the frequently used medications in the elderly, such as the antidepressants paroxetine and fluoxetine. This may result in an impairment in O-demethylation of codeine to morphine and may lead to a decrease in the abuse and dependence potential of codeine. Conversely, those with a very rapid CYP2D6 catalytic activity may have an increased potential for codeine abuse and dependence. The clinical significance of age-related pharmacokinetic changes should be evaluated within the context of clinical practice. Most physicians are inclined to prescribe lower doses to the elderly, which may offset the potential impact of altered pharmacokinetics on the abuse and dependence potential of psychoactive agents. In summary, the available data are not sufficient for a definitive conclusion on whether the pharmacokinetic changes in the elderly translate to an increase in the abuse and dependence potential of alcohol, benzodiazepines or opioids. In particular, the data on age-associated changes in direct measures of abuse potential of these agents are missing. Future comparative systemic pharmacokinetic-pharmacodynamic studies assessing pertinent outcome measures on abuse and dependence potential of commonly used psychoactive drugs are required to resolve the ongoing controversy on risk factors for drug abuse and dependence in the elderly.

Biotransformation and pharmacokinetics of ethylmorphine after a single oral dose.

1. The pharmacokinetics of ethylmorphine after administration of a single dose of the cough mixture Cosylan were investigated in 10 healthy subjects. 2. The median urinary recovery of ethylmorphine and measured metabolites was 77% over 48 h. The median tmax of unchanged ethylmorphine was 45 min, and the terminal elimination t1/2 was 2 h. Ethylmorphine-6-glucuronide was found to be the major metabolite. 3. Two subjects had significantly lower urinary recovery (0.48 h) of morphine and morphine-glucuronides than the remainder. Furthermore, these two had urinary metabolic ratios (MRO) and partial metabolic clearances (CLmO) for O-deethylation of ethylmorphine tentatively classifying them phenotypically as poor metabolisers of the debrisoquine/sparteine type. 4. Genotyping for cytochrome P450 (CYP) 2D6 alleles revealed five homozygote (wt/wt) and five heterozygote subjects. Two subjects phenotypically classified as poor metabolisers were genotypically CYP2D6A/wt and CYP2D6D/wt, respectively. 5. Serum and urine samples taken more than 8 and 24 h after administration of ethyl-morphine respectively, contained morphine and morphine-glucuronides, but no ethylmorphine, ethylmorphine-6-glucuronide or (serum only) norethylmorphine. Norethylmorphine could be detected after hydrolysis of urine samples in all subjects. The urinary recovery of the active metabolites morphine and morphine-6-glucuronide after administration of ethylmorphine varied by a factor of 9 between individuals. 6. The wide variation in recovery of morphine and morphine-glucuronides after oral administration of ethylmorphine could not be explained simply by a difference in CYP2D6 genotype. Constitutional variation in other enzymatic pathways involved in ethylmorphine metabolism is probably crucial. Ratios of morphine to parent drug cannot be used to distinguish the source of morphine after administration of ethylmorphine. Norethylmorphine should be included in urine assays for opiates in forensic toxicology, and no firm conclusions about the source of morphine are possible based on serum samples obtained more than 24 h after drug administration.

Murine macrophage cell lines contain μ3-opiate receptors

Opiate alkaloid-selective, opioid peptide-insensitive μ 3 receptors are present in three murine macrophage cell lines (J774.2; RAW 264.7; BAC1.2F5). The receptor binds morphine, its active metabolite morphine 6-glucuronide and certain other alkaloids, but not morphine 3-glucuronide or any of the opioid peptides tested. The cell lines thus provide valuable model systems for investigation of μ 3-opiate receptors, previously demonstrated to mediate inhibitory effects of morphine on activation of human peripheral blood macrophages (monocytes).

Long term effects of morphine on mesangial cell proliferation and matrix synthesis

Since focal glomerulosclerosis is the predominant glomerular lesion in heroin nephropathy and since mesangial expansion is considered to be a precursor of glomerulosclerosis, we have evaluated the effect of opiates on mesangial cell (MC) proliferation and matrix synthesis. We showed, using a fluorometric assay, that MC are not capable of metabolizing heroin to its active metabolite morphine. Cells exposed to morphine (10(-5) M or 10(-4) M) in prolonged cultures either continuously (Group A) or intermittently (Group B) showed enhanced incorporation of [3H]thymidine when compared to control cells (control, 88600 +/- 26303 cpm/well vs. morphine 10(-4) M-Group A, 321203 +/- 52867, P less than 0.001; control vs. morphine 10(-4) M-Group B, 223126 +/- 46866 cpm/well, P less than 0.01; control, 107593 +/- 42284 cpm/well vs. morphine 10(-5) M - Group A, 267108 +/- 41866 cpm/well, P less than 0.001; control vs. morphine 10(-5) M - Group B, 202317 +/- 24325 cpm/well, P less than 0.05). However, MC incubated with a lower concentration of morphine (10(-6) M) enhanced DNA synthesis when exposed intermittently only (control, 107593 +/- 42284 cpm/well vs. Group B, 219164 +/- 15552 cpm/well, P less than 0.05). This growth stimulating effect of morphine (10(-6) M and 10(-5) M) was also observed at earlier time points, that is, one- and one-and-a-half-week old cultures. However, in one-week-old cultures. morphine in a higher concentration (10(-4) M) showed a suppressive effect (P less than 0.05) on MC proliferation (morphine, 3620 +/- 220 cpm/well vs. control, 4668 +/- 410 cpm/well). This effect not only subsided by one and a half weeks but morphine (10(-4) M) treated cells enhanced MC proliferation. An opioid antagonist, naloxone attenuated the effect of morphine in one and half week old cultures. Morphine at 10(-6) M to 10(-4) M concentrations enhanced incorporation of [3H]proline in the extracellular proline pool (a component of mesangial matrix) when compared to control (control, 309661 +/- 3992 vs. morphine 10(-4) M, 363104 +/- 10539 cpm/well, P less than 0.05 or morphine 10(-5) M, 397954 +/- 31008 cpm/well, P less than 0.001 or morphine 10(-6) M, 384630 +/- 26369 cpm/well, P less than 0.01). In addition, MC incubated with morphine (10(-6) M and 10(-4) M) also enhanced (P less than 0.001) synthesis of laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

Additions and corrections

Long term effects of morphine on mesangial cell proliferation and matrix synthesis. Since focal glomerulosclerosis is the predominant glomerular lesion in heroin nephropathy and since mesangial expansion is considered to be a precursor of glomerulosclerosis, we have evaluated the effect of opiates on mesangial cell (MC) proliferation and matrix synthesis. We showed, using a fluorometric assay, that MC are not capable of metabolizing heroin to its active metabolite morphine. Cells exposed to morphine (10-5 M or 10-4 M) in prolonged cultures either continuously (Group A) or intermittently (Group B) showed enhanced incorporation of [3H]thymidine when compared to control cells (control, 88600 ± 26303 cpm/well vs. morphine 10-4 M -Group A, 321203 ± 52867, P < 0.001; control vs. morphine 10-4 M-Group B, 223126 ± 46866 cpm/well, P < 0.01; control, 107593 ± 42284 cpm/well vs. morphine 10-5 M - Group A, 267108 ± 41866 cpm/well, P < 0.001; control vs. morphine 10-5 M - Group B, 202317 ± 24325 cpm/well, P < 0.05). However, MC incubated with a lower concentration of morphine (10-6 M) enhanced DNA synthesis when exposed intermittently only (control, 107593 ± 42284 cpm/well vs. Group B, 219164 ± 15552 cpm/well, P < 0.05). This growth stimulating effect of morphine (10-6 M and 10-5 M) was also observed at earlier time points, that is, one- and one-and-a-half-week old cultures. However, in one-week-old cultures, morphine in a higher concentration (10-4 M) showed a suppressive effect (P < 0.05) on MC proliferation (morphine, 3620 ± 220 cpm/well vs. control, 4668 ±410 cpm/well). This effect not only subsided by one and a half weeks but morphine (10-4 M) treated cells enhanced MC proliferation. An opioid antagonist, naloxone attenuated the effect of morphine in one and half week old cultures. Morphine at 10-6 M to 10-4 M concentrations enhanced incorporation of [3H]proline in the extracellular proline pool (a component of mesangial matrix) when compared to control (control, 309661 ± 3992 vs. morphine 10-4 M, 363104 ± 10539 cpm/well, P < 0.05 or morphine 10-5 M, 397954 ± 31008 cpm/well, P < 0.001 or morphine 10-6 M, 384630 ± 26369 cpm/well, P < 0.01). In addition, MC incubated with morphine (10-6 M and 10-4 M) also enhanced (P < 0.001) synthesis of laminin. MC incubated with morphine (10-5 M or 10-4, 12 weeks) had a greater number of hillocks (foci of cell proliferation and aggregated matrix) when compared with untreated cells. Morphine treated MC synthesized a decreased amount of PGE2 (129 ± 48 pg/mg protein, P < 0.02) when compared to PGE2 production by untreated cells (493 ±123 pg/mg protein). Morphine pretreated MC when stimulated with arginine vasopressin (AVP, 10-6 M) also produced a lesser amount of PGE2 (214 ± 51 pg/mg protein, P < 0.05) when compared to PGE2 production by control cells under stimulation with AVP (1393 ± 451 pg/mg protein). Our results suggest that morphine may be playing a direct role in mesangial expansion in opiate addicts.