Metatox - Web application for generation of metabolic pathways and toxicity estimation.

Xenobiotics biotransformation in humans is a process of the chemical modifications, which may lead to the formation of toxic metabolites. The prediction of such metabolites is very important for drug development and ecotoxicology studies. We created the web-application MetaTox ( http://way2drug.com/mg ) for the generation of xenobiotics metabolic pathways in the human organism. For each generated metabolite, the estimations of the acute toxicity (based on GUSAR software prediction), organ-specific carcinogenicity and adverse effects (based on PASS software prediction) are performed. Generation of metabolites by MetaTox is based on the fragments datasets, which describe transformations of substrates structures to a metabolites structure. We added three new classes of biotransformation reactions: Dehydrogenation, Glutathionation, and Hydrolysis, and now metabolite generation for 15 most frequent classes of xenobiotic's biotransformation reactions are available. MetaTox calculates the probability of formation of generated metabolite - it is the integrated assessment of the biotransformation reactions probabilities and their sites using the algorithm of PASS ( http://way2drug.com/passonline ). The prediction accuracy estimated by the leave-one-out cross-validation (LOO-CV) procedure calculated separately for the probabilities of biotransformation reactions and their sites is about 0.9 on the average for all reactions.

Postmortem tissue distribution of morphine and its metabolites in a series of heroin-related deaths.

The abuse of heroin (diamorphine) and heroin-related deaths are increasing around the world. The interpretation of the toxicological results from suspected heroin-related deaths is notoriously difficult, especially in cases where there may be limited samples. To help forensic practitioners with heroin interpretation, we determined the concentration of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) in blood (femoral and cardiac), brain (thalamus), liver (deep right lobe), bone marrow (sternum), skeletal muscle (psoas), and vitreous humor in 44 heroin-related deaths. The presence of 6-monoacetylmorphine (6-MAM) in any of the postmortem samples was used as confirmation of heroin use. Quantitation was carried out using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with solid-phase extraction. We also determined the presence of papaverine, noscapine and codeine in the samples, substances often found in illicit heroin and that may help determine illicit heroin use. The results of this study show that vitreous is the best sample to detect 6-MAM (100% of cases), and thus heroin use. The results of the M, M3G, and M6G quantitation in this study allow a degree of interpretation when samples are limited. However in some cases it may not be possible to determine heroin/morphine use as in four cases in muscle (three cases in bone marrow) no morphine, M3G, or M6G were detected, even though they were detected in other case samples. As always, postmortem cases of suspected morphine/heroin intoxication should be interpreted with care and with as much case knowledge as possible.

The Pharmacokinetics of Morphine and Codeine in Human Plasma and Urine after Oral Administration of Qiangli Pipa Syrup.

Papaveris pericarpium, a natural source of morphine and codeine, is the principal active component in many antitussive traditional Chinese medicines. We herein report the first PK study of papaveris pericarpium in human plasma and urine following oral administration of single (15, 30, 60 mL) and multiple dose (15 mL) of Qiangli Pipa Syrup (MOR 0.1 mg/mL, COD 0.028 mg/mL) by monitoring morphine and codeine using a HPLC-MS/MS method. Their Tmax and t1/2 values are independent of dosages, while the AUC0-t linearly increased with higher dosages, indicating linear PK characteristics. AUC0-t increased obviously after multiple doses, indicating possible risk of accumulative toxicity. Urine studies suggested risks of positive opiate drug tests with a cutoff of 300 ng/mL, which lasted 6-14 h at different doses. These results provide important information for clinical safety, efficacy and rational drug use of Qiangli Pipa Syrup and also guide the related judicial expertise of its administration.

CYP2D6 drug-gene and drug-drug-gene interactions among patients prescribed pharmacogenetically actionable opioids.

PURPOSE: When codeine and tramadol are used for pain management, it is imperative that nurses are able to assess for potential drug-gene and drug-drug-gene interactions that could adversely impact drug metabolism and ultimately pain relief. Both drugs are metabolized through the CYP2D6 metabolic pathway which can be affected by medications as well the patient's own pharmacogenotype. The purpose of this brief report is to identify drug-gene and drug-drug-gene interactions in 30 adult patients prescribed codeine or tramadol for pain. METHODS: We used three data sources: (1) six months of electronic health record data on the number and types of medications prescribed to each patient; (2) each patient's CYP2D6 pharmacogenotype, and (3) published data on known CYP2D6 gene-drug and drug-drug-gene interactions. RESULTS: Ten patients (33%) had possible drug-gene or drug-drug-gene interactions. Five patients had CYP2D6 drug-gene interactions indicating they were not good candidates for codeine or tramadol. In addition, five patients had potential CYP2D6 drug-drug-gene interactions with either codeine or tramadol. CONCLUSION: Our findings from this exploratory study underscores the importance of assessing and accounting for drug-gene and drug-drug-gene interactions in patients prescribed codeine or tramadol.

Inducing rat brain CYP2D with nicotine increases the rate of codeine tolerance; predicting the rate of tolerance from acute analgesic response.

Repeated opioid administration produces analgesic tolerance, which may lead to dose escalation. Brain CYP2D metabolizes codeine to morphine, a bioactivation step required for codeine analgesia. Higher brain, but not liver, CYP2D is found in smokers and nicotine induces rat brain, but not liver, CYP2D expression and activity. Nicotine induction of rat brain CYP2D increases acute codeine conversion to morphine, and analgesia, however the role of brain CYP2D on the effects of repeated codeine exposure and tolerance is unknown. Rats were pretreated with nicotine (brain CYP2D inducer; 1mg/kg subcutaneously) or vehicle (saline; 1ml/kg subcutaneously). Codeine (40-60mg/kg oral-gavage) or morphine (20-30mg/kg oral-gavage) was administered daily and analgesia was assessed daily using the tail-flick reflex assay. Nicotine (versus saline) pretreatment increased acute codeine analgesia (1.32-fold change in AUC0-60min; p<0.05) and the rate of loss of peak analgesia (11.42%/day versus 4.20%; p<0.006) across the first four days of codeine administration (time to negligible analgesia). Inducing brain CYP2D with nicotine did not alter acute morphine analgesia (1.03-fold; p>0.8), or the rate of morphine tolerance (8.1%/day versus 7.6%; p>0.9). The rate of both codeine and morphine tolerance (loss in peak analgesia from day 1 to day 4) correlated with initial analgesic response on day 1 (R=0.97, p<001). Increasing brain CYP2D altered initial analgesia and subsequent rate of tolerance. Variation in an individual's initial response to analgesic (e.g. high initial dose, smoking) may affect the rate of tolerance, and thereby the risk for dose escalation and/or opioid dependence.

A Compartmental Analysis for Morphine and Its Metabolites in Young Children After a Single Oral Dose.

BACKGROUND AND OBJECTIVES: Currently, the majority of the surgical procedures performed in paediatric hospitals are done on a day care basis, with post-operative pain being managed by caregivers at home. Pain after discharge of these post-operative children has historically been managed with oral codeine in combination with paracetamol (acetaminophen). Codeine is an opioid, which elicits its analgesic effects via metabolism to morphine and codeine-6-glucuronide. Oral morphine is a feasible alternative for outpatient analgesia; however, the pharmacokinetics of morphine after oral administration have been previously described only sparsely, and there is little information in healthy children. METHODS: The clinical trial included 40 children from 2 to 6 years of age, with an American Society of Anaesthesiologists physical status classification of 1 or 2, who were undergoing surgical procedures requiring opioid analgesia. Morphine was orally administered prior to surgery in one of three doses: 0.1 mg/kg, 0.2 mg/kg and 0.3 mg/kg. Blood samples were collected for plasma morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) concentrations at 30, 60, 90, 120, 180 and 240 min after administration. All analyses were performed with the non-linear mixed-effect modelling software NONMEM version 7.2, using the first-order conditional estimation (FOCE) method. RESULTS: A pharmacokinetic model was developed to simultaneously describe the plasma profiles of morphine and its metabolites M3G and M6G after a single dose of oral morphine in young children (2-6 years of age). The disposition of morphine, M3G and M6G in plasma was best described by a one-compartment model. M3G and M6G metabolite formation was best described by a delay transit compartment, indicating a delay in the appearance of these two major metabolites. CONCLUSION: This model provides a foundation on which to further evaluate the use of oral morphine and its safety in young children. Longer follow-up time for morphine oral doses and incorporation of other important covariates, such as phenotype, will add value and will help overcome the limitations of the presented population pharmacokinetic analysis.

Hair analysis to demonstrate administration of amitriptyline, temazepam, tramadol and dihydrocodeine to a child in a case of kidnap and false imprisonment.

Amitriptyline, temazepam, tramadol and dihydrocodeine are prescription-only-medications that are rarely prescribed to children. Each of these drugs has a sedative effect on the central nervous system; their combined use could cause an exacerbation of the sedative effects. Amitriptyline (a tricyclic antidepressant) can be prescribed to treat nocturnal enuresis; temazepam (a hypnotic) can be used as a premedicant in inpatient and day-case surgery; tramadol (a synthetic opioid analgesic) is used to treat moderate or severe pain, though it is not recommended for children under the age of 12 years and dihydrocodeine (opioid analgesic), which is available in combination with acetaminophen (Co-dydramol), is not recommended for children under the age of 4 years; in children over 4 years, a reduced dose is necessary. The North West Forensic Science Service Laboratory, Euxton, Lancashire, was asked by a British police force to analyze three separate hair samples, which had been collected from a young child following their discovery as a result of a large scale kidnap and false imprisonment investigation. After decontamination and segmentation (20 x 1-cm section), two of the three hair specimens were analyzed by liquid chromatography coupled with tandem mass spectrometry after alkaline (pH 9.5) extraction using methylene chloride/isopropanol/n-heptane (25:10:65, v/v/v). The entire length of each hair specimen tested positive for amitriptyline and nortriptyline (7-314 pg/mg amitriptyline; 7-318 pg/mg nortriptyline), temazepam (2-29 pg/mg), tramadol (60-2000 pg/mg) and dihydrocodeine (10-90 pg/mg) demonstrating that the child had ingested these drugs on more than one occasion prior to the kidnap. In this case, the child's mother and the mothers' partner were found guilty of kidnap, false imprisonment and perverting the course of justice. There are very few studies citing the concentrations of these drugs in children - especially children's hair samples. This case demonstrates the added value of hair testing and emphasizes the importance of using hair samples to complement conventional analysis.

Influence of Cytochrome P450, Family 2, Subfamily D, Polypeptide 6 (CYP2D6) polymorphisms on pain sensitivity and clinical response to weak opioid analgesics.

  CYP2D6 polymorphisms show large geographical and interethnic differences. Variations in CYP2D6 activity may impact upon a patient's pain level and may contribute to interindividual variations in the response to opioids. This paper reviews the evidence on how CYP2D6 polymorphisms might influence pain sensitivity and clinical response to codeine and tramadol. For example, it is shown that (1) CYP2D6 poor metabolizers (PMs) may be less efficient at synthesizing endogenous morphine compared with other metabolizers. In contrast, ultra-rapid metabolizers (UMs) may be more efficient than other metabolizers at synthesizing endogenous morphine, thus strengthening endogenous pain modulation. Additionally, for codeine and tramadol that are bioactivated by CYP2D6, PMs may undergo no metabolite formation, leading to inadequate analgesia. Conversely, UMs may experience quicker analgesic effects but be prone to higher mu-opioid-related toxicity. The literature suggested the potential usefulness of the determination of CYP2D6 polymorphisms in elucidating serious adverse events and in preventing subsequent inappropriate selection or doses of codeine and tramadol. Notably, even though many studies investigated a possible role of the CYP2D6 polymorphisms on pain sensitivity, pharmacokinetics and pharmacodynamics of these drugs, the results of analgesia and adverse effects are conflicting. More studies are required to demonstrate genetically determined unresponsiveness and risk of developing serious adverse events for patients with pain and these should involve larger numbers of patients in different population types.

Lack of impairment due to confirmed codeine use prior to a motor vehicle accident: role of pharmacogenomics.

BACKGROUND: We examined forensic serum toxicology and pharmacogenomics data from a woman on codeine shortly before she caused a motor vehicle accident. METHODS: A woman driving erratically collided with a parked car of a highway seriously injuring 2 men working to repair the parked vehicle. The woman tested positive for codeine, acetaminophen and barbital. She had been taking these medications for 20 years due to migraine headache. Serum toxicology and genotype analysis for cytochrome P450, UDP glucuronosyltransferase, and other metabolizing enzymes were measured. RESULTS: The woman was tried and convicted of driving under the influence resulting in bodily harm and was sentenced to 6 years. Toxicology results on peripheral blood showed a total and free codeine of 840 and 348 µg/L, respectively, and total morphine of 20 µg/L (17, 3, and 0 µg/L for morphine-3-glucuronide, morphine-6-glucuronide, and free morphine, respectively). She was heterozygous for CYP 2D6 *2/*4 (extensive/poor metabolism) and heterozygous for UGT 2B7 *1/*2 (extensive/ultra-rapid metabolism). The woman was also taking fluoxetine and bupropion which are strong inhibitors of CYP 2D6. CONCLUSIONS: Based on her genotype and phenotype and reports by the arresting officer, we suggest that the subject in question was not intoxicated by opiates at the time of her motor vehicle accident and may have been falsely incarcerated.

'Lingering' opiate deaths? Concentration of opiates in medulla and femoral blood.

'Lingering death' cases occur when the circumstances of death indicate an opiate overdose, but measured opiate blood levels are only in the therapeutic range; death results from cardiac and respiratory depression. This study examined the relative concentration of opiates in femoral blood and in the medulla oblongata (sites for cardiac and respiratory control) from 41 cases to determine whether a difference in opiate concentration might explain lingering deaths. Opiates from blood and medulla were analyzed using GC-EI-MS in selective ion monitoring mode. Results were correlated with gross and microscopic findings of the lungs and with cause and manner of death. Opiate concentrations for morphine, codeine and 6-acetylmorphine (6-AM) were higher in the medulla than in blood. The brain: blood ratio for the analytes demonstrated an increasing ratio from morphine, to codeine, to 6-AM (1.42, 2.48 and 4.86), which corresponds to the relative lipophilicity of these analytes. The average right and left lung weights were 762 and 668 g, respectively. Histologic examination showed edema, and/or polarizable microemboli, acute bronchopneumonia and acute bronchitis. The preferential distribution of opiates to medulla suggests that lingering opiate deaths may be explained, at least in part, because of higher relative concentrations of drug in brain, compared with femoral blood.

Codeine-associated pediatric deaths despite using recommended dosing guidelines: three case reports.

This report describes the deaths of three children ages 4-10 years due to codeine toxicity at home. All three children were overweight or obese; however, the codeine doses were within recommended dose ranges for adjusted lean weight. Codeine's analgesic effect depends on its metabolic conversion to morphine in the liver via the drug-metabolizing enzyme CYP2D6. Genetic variation may result in poor analgesia, opioid toxicity, or oversedation. Caregivers must be warned about risks associated with comorbidities including obesity and polypharmacy. Codeine should no longer be prescribed to children due to its poor analgesic effect and risk of opioid toxicity and oversedation.

Distinguishing heroin abuse from codeine administration in the urine of Chinese people by UPLC-MS-MS.

Heroin is a highly addictive drug, and heroin abuse is considered to be a serious criminal act. The major metabolite of heroin, morphine, can usually be detected as evidence of heroin abuse. However, it is difficult to determine heroin use when morphine and codeine are both detected, because codeine use will also result in the presence of morphine in urine. Therefore, it is important to distinguish heroin abuse from codeine administration. In this study, urine samples from 21 volunteers with various ingestion patterns of a compound codeine phosphate oral solution were used as negative controls, and urine samples from 89 alleged heroin users were used as positive controls. Urine from single and multiple doses of codeine administration were collected at different time points for a systematic comparison. After protein precipitation, the urine samples were analyzed for the presence of free morphine, free codeine and their metabolites by ultra-performance liquid chromatography-tandem mass spectrometry. The method of percentiles, with median and standard interquartile ranges, was used to describe and analyze the data based on the normality of the distribution. The ratios of concentration of morphine and morphine to codeine were found to be the possible criteria to distinguish heroin users from codeine users in Chinese people.

Disappearance of 6-acetylmorphine, morphine and codeine from human scalp hair after discontinuation of opiate abuse.

Opiates continue to be used at high rates in East and Southeast Asia. Hair analysis for drugs of abuse has been developed into a powerful and widely used tool in forensic and clinical toxicology. Specifically, testing the proximal segment of scalp hair to confirm morphine (MOR) positive urine samples could solve the poppy seed problem. Human scalp hair grows approximately 1cm per month and can therefore reflect a retrospective timeline of drug exposure. This study is the first to investigate the disappearance of 6-acetylmorphine (6-AM), MOR and codeine (COD) from human scalp hair after the discontinuation of drug use. Thirty-two healthy women (ages 21-51 years) with a known history of heroin abuse, who went to a rehabilitation centre and ceased consuming heroin (for 4-5 months), were recruited into the study. A pharmacokinetic analysis in seven individual hair segments was performed using a first-order kinetic. Assuming a rate of hair growth of 1cm/month, the mean hair elimination half-lives of 6-AM, MOR and COD were 0.88 months (95% CI, 0.74-1.03), 0.73 months (95% CI, 0.64-0.81), and 0.61 months (95% CI, 0.54-0.69), respectively. Our results suggest that to evaluate the discontinuation of opiate abuse after a 6-month period of abstinence, the results from a 3-cm proximal hair segment should be free of 6-AM at the proposed 0.2 ng/mg cutoff level. This finding should become the basis for the interpretation of results from segmental hair analyses in the evaluation of drug abstinence.

More codeine fatalities after tonsillectomy in North American children.

In 2009 we reported the fatal case of a toddler who had received codeine after adenotonsillectomy for obstructive sleep apnea syndrome. The child was an ultra-rapid metabolizer of cytochrome P4502D6 (CYP2D6). We now report 3 additional fatal or life-threatening cases from North America. In the 2 fatal cases, functional gene duplications encoding for CYP2D6 caused a significantly greater production of potent morphine from its parent drug, codeine. A severe case of respiratory depression in an extensive metabolizer is also noted. These cases demonstrate that analgesia with codeine or other opioids that use the CYP2D6 pathway after adenotonsillectomy may not be safe in young children with obstructive sleep apnea syndrome.

Genotypic screening of the main opiate-related polymorphisms in a cohort of 139 sickle cell disease patients.

Because no frequency data are available for the main opiate-related polymorphisms in sickle-cell disease (SCD) populations, we decided to perform such a genotyping in a cohort of 139 individuals. For pharmacodynamics,the OPRM1 A118G and the COMT G322A single nucleotide polymorphisms (SNPs) were chosen for their negative effects on the m receptors [1,2]. For pharmacokinetics [3], important SNPs for the CYP2D6 gene (codeine to morphine conversion) and for three genes involved in morphine elimination (namely CYP3A, UGT2B7, and ABCB1) were genotyped. The allelic frequencies of the OPRM1 and COMT SNPs appeared very low (0.01 to 0.05-no double mutant homozygous),as well as the proportion of CYP2D6 poor metabolizers (1.4%)and CYP3A wild-type (17.9%) which are associated with a low morphine exposure. On the contrary, up to 35% of SCD patients may have unfavorable ABCB1 and UGT2B7 genotypes for a good morphine exposure.Obviously, pharmacokinetic studies with precise phenotype/genotype correlations are required to draw definitive conclusions.

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain.

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.

Role of codeine in palliative care.

Codeine is designated as one of the essential medicines of palliative care for symptoms such as pain and diarrhea. Essential drugs for palliative care are drugs that are effective for the treatment of common symptoms in palliative medicine, easily available, and are affordable. Codeine is recommended for the management of mild to moderate pain and is available as a combination product or as a stand-alone opioid. It is a prodrug and exhibits an affinity to micro-opioid receptors 200 times lower than morphine. Codeine is metabolized in the liver to inactive metabolites, which account for 90 percent of the transformed product, and morphine, which accounts for 10 percent of the transformed product and provides the main analgesic effect. The production of morphine is dependent on cytochrome oxidase 2D6 enzyme activity, which may not be fully active in some populations. The purpose of this review is to examine the efficacy of codeine for common symptoms encountered in palliative medicine, which has led to its designation as an essential medicine for palliative care.