Longitudinal Opioid Surveillance Project Involving Toxicologic Analysis of Postmortem Specimens from 9 Counties in Michigan Suggests the Discovery of New High-Intensity Drug Trafficking Areas.

ABSTRACT: Acetyl fentanyl (AF) is a Schedule I fentanyl analog that has been increasingly seen in heroin and fentanyl polydrug toxicity overdoses in Michigan (MI). Drug users are often unaware of the presence of AF in their drugs because it is often sold mixed into or disguised as heroin. High levels of AF in heroin drug products can cause increased incidence of overdose. This article describes data from a longitudinal opioid surveillance program and details 102 decedents in MI who were found to have evidence of heroin in their postmortem blood. A large portion of these decedents were also found to have evidence of fentanyl and AF. Our data further show significant overlap in incidence rates of AF and heroin-related overdose deaths in several MI counties, suggesting that AF is becoming enmeshed in heroin trafficking. Furthermore, we report unprecedented high incidence rates of AF and heroin-related overdose deaths in Calhoun county, and we propose that it is a high-intensity drug trafficking area. Highways US-131 and US-31 are likely used to transport these drugs. More study is needed into the drug trafficking trends in MI to ascertain drug sources and monitor the ever developing and dangerous polydrug heroin combinations.

Necrotizing enterocolitis and its association with the neonatal abstinence syndrome.

OBJECTIVE: The purpose of this study was to describe an identified association between necrotizing enterocolitis (NEC) and prenatal opioid exposure with neonatal abstinence syndrome (NAS) in late preterm and full-term neonates. STUDY DESIGN: In this single-center retrospective cohort study, we analyzed inborn neonates with the diagnosis of NEC discharged from 2012 through 2017. We compared infants with NEC > 35 weeks' gestation to those with NEC<35 weeks' gestation. We compared gestational age, birth weight, age of onset of symptoms, and incidence of prenatal drug exposure between groups. Significance was determined using Mann-Whitney and Fisher's exact tests. RESULTS: Over the study period, 23 infants were identified with NEC, 9 (39%) were babies > 35 weeks at birth and 14 (61%) < 35 weeks. Those > 35 weeks had a higher birth weight, earlier onset of symptoms, and a higher percentage of prenatal exposure to opioids compared to those < 35 weeks' gestation. We further described seven infants with late gestational age onset NEC associated with prenatal opioid exposure. CONCLUSIONS: In this cohort of infants with NEC discharged over a 6 year period we found a higher than expected percentage of infants born at a later gestational age. We speculate that prenatal opioid exposure might be a risk factor for NEC in neonates born at > 35 weeks.

The role of 6-acetylmorphine in heroin-induced reward and locomotor sensitization in mice.

We have previously demonstrated that heroin's first metabolite, 6-acetylmorphine (6-AM), is an important mediator of heroin's acute effects. However, the significance of 6-AM to the rewarding properties of heroin still remains unknown. The present study therefore aimed to examine the contribution of 6-AM to heroin-induced reward and locomotor sensitization. Mice were tested for conditioned place preference (CPP) induced by equimolar doses of heroin or 6-AM (1.25-5 µmol/kg). Psychomotor activity was recorded during the CPP conditioning sessions for assessment of drug-induced locomotor sensitization. The contribution of 6-AM to heroin reward and locomotor sensitization was further examined by pretreating mice with a 6-AM specific antibody (anti-6-AM mAb) 24 hours prior to the CPP procedure. Both heroin and 6-AM induced CPP in mice, but heroin generated twice as high CPP scores compared with 6-AM. Locomotor sensitization was expressed after repeated exposure to 2.5 and 5 µmol/kg heroin or 6-AM, but not after 1.25 µmol/kg, and we found no correlation between the expression of CPP and the magnitude of locomotor sensitization for either opioid. Pretreatment with anti-6-AM mAb suppressed both heroin-induced and 6-AM-induced CPP and locomotor sensitization. These findings provide evidence that 6-AM is essential for the rewarding and sensitizing properties of heroin; however, heroin caused stronger reward compared with 6-AM. This may be explained by the higher lipophilicity of heroin, providing more efficient drug transfer to the brain, ensuring rapid increase in the brain 6-AM concentration.

Acetyl Fentanyl: Trends and Concentrations in Metro Detroit.

Acetyl fentanyl (N-[1-phenethylpiperidin-4-yl]-N-phenylacetamide) is a potent opioid analgesic with no medicinal uses. We report deaths between 2016 and 2017 at the Medical Examiner's Office in Detroit, MI where acetyl fentanyl was found in the decedent's blood and compare them to previously published deaths between 2015 and 2016. The recent cases (cohort B) had a mean acetyl fentanyl concentration of 0.9 ng/mL (range: 0.1-5.3 ng/mL) and an associated higher concentration of fentanyl along with multiple other drugs present. The older cases (cohort A) had higher concentrations of acetyl fentanyl (mean: 8.9 ng/mL; range: 0.28-37 ng/mL) with lower, yet still toxic, concentrations of fentanyl. We conclude that the cause of death in these recent cases was likely multiple drug toxicity with fentanyl and that the consistently observed lower peripheral blood concentrations of acetyl fentanyl are most likely an artifact in the manufacture of the consumed illicit fentanyl.

Metabolomics analysis of serum in a rat heroin self-administration model undergoing reinforcement based on 1H-nuclear magnetic resonance spectra.

BACKGROUND: Understanding the process of relapse to abused drugs and ultimately developing treatments that can reduce the incidence of relapse remains the primary goal for the study of substance dependence. Therefore, exploring the metabolite characteristics during the relapse stage is valuable. METHODS: A heroin self-administered rat model was employed, and analysis of the 1H-nuclear magnetic resonance-based metabolomics was performed to investigate the characteristic metabolite profile upon reintroduction to the drug after abstinence. RESULTS: Sixteen metabolites in the serum of rats, including phospholipids, intermediates in TCA (Tricarboxylic Acid Cycle) cycle, keto bodies, and precursors for neurotransmitters, underwent a significant change in the reinstatement stage compared with those in the control group. In particular, energy production was greatly disturbed as evidenced by different aspects such as an increase in glucose and decrease in intermediates of glycolysis and the TCA cycle. The finding that the level of 3-hydroxybutyrate and acetoacetate increased significantly suggested that energy production was activated from fatty acids. The concentration of phenylalanine, glutamine, and choline, the precursors of major neurotransmitters, increased during the reinstatement stage which indicated that an alteration in neurotransmitters in the brain might occur along with the disturbance in substrate supply in the circulatory system. CONCLUSIONS: Heroin reinforcement resulted in impaired energy production via different pathways, including glycolysis, the TCA cycle, keto body metabolism, etc. A disturbance in the substrate supply in the circulatory system may partly explain heroin toxicity in the central nervous system. These findings provide new insight into the mechanism underlying the relapse to heroin use.

Metabolites of Heroin in Several Different Post-mortem Matrices.

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

A Cluster of Fentanyl-Laced Heroin Deaths in 2015 in Melbourne, Australia.

The prevalence of opioid use in therapeutic and recreational settings has steadily increased throughout the western world. The addition of fentanyl into heroin products can produce potentially dangerous consequences, even to opioid tolerant individuals who may be unaware of such additions. Following an observed spike of heroin-fentanyl related deaths in Melbourne, Australia, a study was undertaken to determine the prevalence of these cases. All reportable deaths occurring in Victoria during 2015 and submitted to the toxicology laboratory were analysed using LC-MS-MS to confirm the combination of the heroin marker 6-acetylmorphine and/or morphine, and fentanyl. Over 4,000 coronial cases in 2015 underwent toxicological analysis for these drugs, there were nine cases identified that involved fentanyl-laced heroin. There was no specific mention of fentanyl use in any of these cases. All occurred within 2 months and in two distinct locations. The first four deaths occurred within 3 days of each other, in neighboring suburbs. The ages ranged from 25 to 57 years with an average of 40 and median of 37 years, and consisted of eight males and one female. The average and median femoral blood concentration of fentanyl was 18 and 20 ng/mL (range: <1-45 ng/mL), and morphine 140 and 80 ng/mL (range: 20-400 ng/mL), respectively. All nine cases had 6-acetylmorphine detectable in blood. Urine analysis was also performed where available. A syringe, powder and spoon found at the scene of one case were also analysed and found to be positive for both heroin and fentanyl, which supported the likelihood of fentanyl-laced heroin. This is the first reported case series of fatalities involving heroin and fentanyl outside of North America in published literature. These findings may help inform public health and prevention strategies serving to decrease the potential for such fatalities in the future.

Illicit Fentanyl-Related Fatalities in Florida: Toxicological Findings.

Fentanyl induces pharmacological effects and abuse liability comparable to other prescription opioids and heroin. A surge in fentanyl-related fatalities has been periodically reported throughout the USA. The University of Florida Forensic Toxicology Laboratory observed a significant increase in fentanyl-related deaths starting in mid-2014. The present report evaluated toxicological findings, demographics of the decedents and circumstances of death in the postmortem cases that were submitted to the laboratory for toxicological analysis from July 2014 to January 2015 and that were tested for fentanyl in biological specimens. The cases originated from 6 of the 24 Florida Medical Examiner Districts, with the majority from District 12 (Desoto, Manatee and Sarasota counties). The specimens were analyzed for fentanyl by gas chromatography-mass spectrometry; the limit of detection (LOD) was 0.62 ng/mL and the limit of quantification (LOQ) was 2.5 ng/mL. During the 7-month period, the laboratory tested 143 postmortem cases for fentanyl and 50% had quantifiable fentanyl in postmortem blood. Fentanyl concentrations ranged from 2.5 to 68 ng/mL (n = 66; median: 9.8 ng/mL); six cases were positive for fentanyl >LOD but

Antipsychotic-related fatal poisoning, England and Wales, 1993-2013: impact of the withdrawal of thioridazine.

CONTEXT: Use of second generation antipsychotics in England and Wales has increased in recent years whilst prescription of first generation antipsychotics has decreased. METHODS: To evaluate the impact of this change and of the withdrawal of thioridazine in 2000 on antipsychotic-related fatal poisoning, we reviewed all such deaths in England and Wales 1993-2013 recorded on the Office for National Statistics drug poisoning deaths database. We also reviewed antipsychotic prescribing in the community, England and Wales, 2001-2013. Use of routine mortality data: When an antipsychotic was recorded with other drug(s), the death certificate does not normally say if the antipsychotic caused the death rather than the other substance(s). A second consideration concerns intent. A record of "undetermined intent" is likely to have been intentional self-poisoning, the evidence being insufficient to be certain that the individual intended to kill. A record of drug abuse/dependence, on the other hand, is likely to have been associated with an unintentional death. Accuracy of the diagnosis of poisoning: When investigating a death in someone prescribed antipsychotics, toxicological analysis of biological samples collected post-mortem is usually performed. However, prolonged attempts at resuscitation, or diffusion from tissues into blood as autolysis proceeds, may serve to alter the composition of blood sampled after death from that circulating at death. With chlorpromazine and with olanzapine a further factor is that these compounds are notoriously unstable in post-mortem blood. Deaths from antipsychotics: There were 1544 antipsychotic-related poisoning deaths. Deaths in males (N = 948) were almost twice those in females. For most antipsychotics, the proportion of deaths in which a specific antipsychotic featured either alone, or only with alcohol was 30-40%, but for clozapine (193 deaths) such mentions totalled 66%. For clozapine, the proportion of deaths attributed to either intentional self-harm, or undetermined intent was 44%, but for all other drugs except haloperidol (20 deaths) the proportion was 56% or more. The annual number of antipsychotic-related deaths increased from some 55 per year (1.0 per million population) between 1993 and 1998 to 74 (1.5 per million population) in 2000, and then after falling slightly in 2002 increased steadily to reach 109 (1.9 per million population) in 2013. Intent: The annual number of intentional and unascertained intent poisoning deaths remained relatively constant throughout the study period (1993: 35 deaths, 2013: 38 deaths) hence the increase in antipsychotic-related deaths since 2002 was almost entirely in unintentional poisoning involving second generation antipsychotics. Clozapine, olanzapine, and quetiapine were the second generation antipsychotics mentioned most frequently in unintentional poisonings (99, 136, and 99 deaths, respectively). Mentions of diamorphine/morphine and methadone (67 and 99 deaths, respectively) together with an antipsychotic were mainly (84 and 90%, respectively) in either unintentional or drug abuse-related deaths. Deaths and community prescriptions: Deaths involving antipsychotics (10 or more deaths) were in the range 11.3-17.1 deaths per million community prescriptions in England and Wales, 2001-2013. Almost all (96%) such deaths now involve second generation antipsychotics. This is keeping with the increase in annual numbers of prescriptions of these drugs overall (<1 million in 2000, 7 million in 2013), largely driven by increases in prescriptions for olanzapine and quetiapine. In contrast, deaths involving thioridazine declined markedly (from 40 in 2000 to 10 in 2003-2013) in line with the fall in prescriptions for thioridazine from 2001. CONCLUSIONS: The removal of thioridazine has had no apparent effect on the incidence of antipsychotic-related fatal poisoning in England and Wales. That such deaths have increased steadily since 2001 is in large part attributable to an increase in unintentional deaths related to (i) clozapine, and (ii) co-exposure to opioids, principally diamorphine and methadone.

Abnormal functional integration of thalamic low frequency oscillation in the BOLD signal after acute heroin treatment.

Heroin addiction is a severe relapsing brain disorder associated with impaired cognitive control, including deficits in attention allocation. The thalamus has a high density of opiate receptors and is critically involved in orchestrating cortical activity during cognitive control. However, there have been no studies on how acute heroin treatment modulates thalamic activity. In a cross-over, double-blind, vehicle-controlled study, 29 heroin-maintained outpatients were studied after heroin and placebo administration, while 20 healthy controls were included for the placebo condition only. Resting-state functional magnetic resonance imaging was used to analyze functional integration of the thalamus by three different resting state analysis techniques. Thalamocortical functional connectivity (FC) was analyzed by seed-based correlation, while intrinsic thalamic oscillation was assessed by analysis of regional homogeneity (ReHo) and the fractional amplitude of low frequency fluctuations (fALFF). Relative to the placebo treatment and healthy controls, acute heroin administration reduced thalamocortical FC to cortical regions, including the frontal cortex, while the reductions in FC to the mediofrontal cortex, orbitofrontal cortex, and frontal pole were positively correlated with the plasma level of morphine, the main psychoactive metabolite of heroin. Furthermore, heroin treatment was associated with increased thalamic ReHo and fALFF values, whereas fALFF following heroin exposure correlated negatively with scores of attentional control. The heroin-associated increase in fALFF was mainly dominated by slow-4 (0.027-0.073 Hz) oscillations. Our findings show that there are acute effects of heroin within the thalamocortical system and may shed new light on the role of the thalamus in cognitive control in heroin addiction. Future research is needed to determine the underlying physiological mechanisms and their role in heroin addiction.

Clinical and Forensic Diagnosis of Very Recent Heroin Intake by 6-acetylmorphine Immunoassay Test and LC-MS/MS Analysis in Urine and Blood.

AIM: The study evaluates the suitability of a specific immunoassay screening test for 6-acetylmorphine (6-AM) in the setting of suspected very recent heroin consumption for forensic and clinical purposes. MATERIAL AND METHOD: The EMIT® II Plus 6-AM immunoassay was applied in 65 cases that had already tested positive for morphine in urine or blood. Biological samples (n.65 urine and n.53 blood) were obtained from workplace drug tests (WDT n. 5), tests for driving under the influence of drugs (DUID n. 30), vehicle accidents (n. 10), overdoses (n. 12) and heroin-related deaths (n. 8) cases. The 6-AM screening assay results were confirmed with the LC-MS/MS analysis in relation to the cut-off set at 10 ng/mL for both urine and blood. RESULTS: Among the 65 urine samples (all morphine-positive), 38 samples were 6-AM-positive and 27 were 6-AM-negative with 100% agreement between the positive/negative results of the two assays. Among the 53 blood samples (34 positive and 19 negative for the morphine), 16 were 6-AM positive and 37 were negative. Only one of the blood samples, positive for 6-AM by LC-MS/MS at 10.3 ng/mL, was negative by the immunoassay test. Based on the concordance between the results of the 6-AM immunoassay versus the LC-MS/MS, the sensitivity of the 6-AM assay was calculated as 100% and 95% for urine and blood respectively, with a specificity and accuracy of 100% for both biological samples. In addition, the study demonstrated that the 6-AM assay test, originally developed for urine, is also sufficiently sensitive to identify 6-AM in blood. Therefore, it could be applied in cases of vehicle accidents or overdose to distinguish rapidly between very recent heroin use and the intake of other opiates for therapeutic purposes.

Development and validation of a bioanalytical method for the simultaneous determination of heroin, its main metabolites, naloxone and naltrexone by LC-MS/MS in human plasma samples: Application to a clinical trial of oral administration of a heroin/naloxone formulation.

A bioanalytical method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification of heroin, its main metabolites and naloxone. In addition, naltrexone was detected qualitatively. This method was used to analyse human plasma samples from a clinical trial after oral administration of a heroin/naloxone formulation in healthy volunteers. O-methylcodeine was used as an internal standard. Samples were kept in an ice-bath during their processing to minimize the degradation of heroin. A short methodology based on protein precipitation with methanol was used for sample preparation. After protein precipitation, only the addition of a formic acid solution was needed to elute heroin, 6-monoacetylmorphine, morphine, naloxone and naltrexone. Morphine metabolites were evaporated to dryness and reconstituted in a formic acid solution. Chromatographic separation was achieved at 35 °C on an X-Bridge Phenyl column (150 × 4.6 mm, 5 µm) using a gradient elution with a mobile phase of ammonium formate buffer at pH 3.0 and formic acid in acetonitrile. The run time was 8 min. The analytes were monitored using a triple quadrupole mass spectrometer with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The method was found to be linear in a concentration range of 10-2000 ng/mL for M3G and 10-1000 ng/mL for the rest of compounds. Quality controls showed accurate values between -3.6% and 4.0% and intra- and inter-day precisions were below 11.5% for all analytes. The overall recoveries were approximately 100% for all analytes including the internal standard. A rapid, specific, precise and simple method was developed for the determination of heroin, its metabolites, naloxone and naltrexone in human plasma. This method was successfully applied to a clinical trial in 12 healthy volunteers.

Increased functional connectivity in the resting-state basal ganglia network after acute heroin substitution.

Reinforcement signals in the striatum are known to be crucial for mediating the subjective rewarding effects of acute drug intake. It is proposed that these effects may be more involved in early phases of drug addiction, whereas negative reinforcement effects may occur more in later stages of the illness. This study used resting-state functional magnetic resonance imaging to explore whether acute heroin substitution also induced positive reinforcement effects in striatal brain regions of protracted heroin-maintained patients. Using independent component analysis and a dual regression approach, we compared resting-state functional connectivity (rsFC) strengths within the basal ganglia/limbic network across a group of heroin-dependent patients receiving both an acute infusion of heroin and placebo and 20 healthy subjects who received placebo only. Subsequent correlation analyses were performed to test whether the rsFC strength under heroin exposure correlated with the subjective rewarding effect and with plasma concentrations of heroin and its main metabolites morphine. Relative to the placebo treatment in patients, heroin significantly increased rsFC of the left putamen within the basal ganglia/limbic network, the extent of which correlated positively with patients' feelings of rush and with the plasma level of morphine. Furthermore, healthy controls revealed increased rsFC of the posterior cingulate cortex/precuneus in this network relative to the placebo treatment in patients. Our results indicate that acute heroin substitution induces a subjective rewarding effect via increased striatal connectivity in heroin-dependent patients, suggesting that positive reinforcement effects in the striatum still occur after protracted maintenance therapy.

Estimation of the Time Interval between the Administration of Heroin and the Sampling of Blood in Chronic Inhalers.

To develop a model for estimating the time delay between last heroin consumption and blood sampling in chronic drug users. Eleven patients, all heroin inhalers undergoing detoxification, were included in the study. Several plasma samples were collected during the detoxification procedure and analyzed for the heroin metabolites 6-acetylmorphine (6AM), morphine (MOR), morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G), according to a UHPLC/MSMS method. The general linear mixed model was applied to time-related concentrations and a pragmatic four-step delay estimation approach was proposed based on the simultaneous presence of metabolites in plasma. Validation of the model was carried out using the jackknife technique on the 11 patients, and on a group of 7 test patients. Quadratic equations were derived for all metabolites except 6AM. The interval delay estimation was 2-4 days when only M3G present in plasma, 1-2 days when M6G and M3G were both present, 0-1 day when MOR, M6G and M3G were present and <2 h for all metabolites present. The 'jackknife' correlation between declared and actual estimated delays was 0.90. The overall precision of the delay estimates was 8-9 h. The delay between last heroin consumption and blood sampling in chronic drug users can be satisfactorily predicted from plasma heroin metabolites.

Pharmacokinetic correlates of the effects of a heroin vaccine on heroin self-administration in rats.

The purpose of this study was to evaluate the effects of a morphine-conjugate vaccine (M-KLH) on the acquisition, maintenance, and reinstatement of heroin self-administration (HSA) in rats, and on heroin and metabolite distribution during heroin administration that approximated the self-administered dosing rate. Vaccination with M-KLH blocked heroin-primed reinstatement of heroin responding. Vaccination also decreased HSA at low heroin unit doses but produced a compensatory increase in heroin self-administration at high unit doses. Vaccination shifted the heroin dose-response curve to the right, indicating reduced heroin potency, and behavioral economic demand curve analysis further confirmed this effect. In a separate experiment heroin was administered at rates simulating heroin exposure during HSA. Heroin and its active metabolites, 6-acetylmorphine (6-AM) and morphine, were retained in plasma and metabolite concentrations were reduced in brain in vaccinated rats compared to controls. Reductions in 6-AM concentrations in brain after vaccination were consistent with the changes in HSA rates accompanying vaccination. These data provide evidence that 6-AM is the principal mediator of heroin reinforcement, and the principal target of the M-KLH vaccine, in this model. While heroin vaccines may have potential as therapies for heroin addiction, high antibody to drug ratios appear to be important for obtaining maximal efficacy.

A monoclonal antibody specific for 6-monoacetylmorphine reduces acute heroin effects in mice.

Immunotherapy against drugs of abuse is being studied as an alternative treatment option in addiction medicine and is based on antibodies sequestering the drug in the bloodstream and blocking its entry into the brain. Producing an efficient vaccine against heroin has been considered particularly challenging because of the rapid metabolism of heroin to multiple psychoactive molecules. We have previously reported that heroin's first metabolite, 6-monoacetylmorphine (6-MAM), is the predominant mediator for heroin's acute behavioral effects and that heroin is metabolized to 6-MAM primarily prior to brain entry. On this basis, we hypothesized that antibody sequestration of 6-MAM is sufficient to impair heroin-induced effects and therefore examined the effects of a monoclonal antibody (mAb) specific for 6-MAM. In vitro experiments in human and rat blood revealed that the antibody was able to bind 6-MAM and block the metabolism to morphine almost completely, whereas the conversion of heroin to 6-MAM remained unaffected. Mice pretreated with the mAb toward 6-MAM displayed a reduction in heroin-induced locomotor activity that corresponded closely to the reduction in brain 6-MAM levels. Intraperitoneal and intravenous administration of the anti-6-MAM mAb gave equivalent protection against heroin effects, and the mAb was estimated to have a functional half-life of 8 to 9 days in mice. Our study implies that an antibody against 6-MAM is effective in counteracting heroin effects.

The interference of ethanol with heroin-stimulated psychomotor activation in mice is not related to changed brain concentrations of the active metabolites 6MAM or morphine.

It has been suggested that the potentiating effect observed in human beings when combining alcohol and heroin may be due to an interference of ethanol with the pharmacokinetics of heroin, leading to accumulation of the biologically active metabolites, 6-monoacetylmorphine (6MAM) and morphine. However, experimental evidence for this hypothesis is lacking. In this study, we used mice and examined the effect of ethanol on the metabolism of heroin by combining a locomotor activity test, which is a behaviour model representative of psychomotor stimulation, with pharmacokinetic studies in blood and brain tissue. Pre-treatment with ethanol (1 and 2.5 g/kg, po) affected heroin-stimulated (2.5 and 15 µmol/kg, sc) locomotor activation significantly, resulting in a dose-dependent reduction in run distance. However, the change in the activity profiles did not indicate any increase in the concentration of active metabolites. Pharmacokinetic studies in blood and brain supported the behavioural findings, showing no change in the time-versus-concentration curves of either 6MAM or morphine after administration of heroin (15 µmol/kg, sc) to mice pre-treated with ethanol (2.5 g/kg, po). The concentration of heroin itself was elevated, but is probably of minor importance because heroin has low biological activity by itself. The in vivo pharmacokinetic findings were supported by experiments in vitro. In conclusion, studies in mice do not support the hypothesis from epidemiological studies of a pharmacokinetic interaction between alcohol and heroin.

Concentrations of cocaine and benzoylecgonine in femoral blood from cocaine-related deaths compared with venous blood from impaired drivers.

The concentrations of cocaine and its major metabolite benzoylecgonine (BZE) were determined in femoral blood from 132 cocaine-related deaths and compared with venous blood from 988 apprehended drivers. Cocaine and BZE were determined by solid-phase extraction and isotope dilution gas chromatography-mass spectrometry with limits of quantitation of 0.02 mg/L for both substances. Significantly more men (95-98%) than women (2-5%) abused cocaine, although their mean age was about the same (29-30 years). Mean age (±SD) of cocaine-related deaths was 29 ± 7 years, which was not significantly different from 30 ± 8 years in traffic cases (P > 0.05). The median concentration of cocaine in blood in 61 fatalities was 0.10 mg/L compared with 0.06 mg/L in traffic cases (P < 0.001). In drug intoxication deaths, the median concentration of cocaine was 0.13 mg/L (N = 25), which was not significantly different from 0.09 mg/L (N = 36) in other causes of death. Cocaine-related deaths mostly involved mixed drug intoxications including co-ingestion of heroin, cannabis, amphetamines as well as legal drugs, such as benzodiazepines and/or ethanol. The concentrations of cocaine in blood from living and deceased persons overlapped, which makes it infeasible to predict toxicity from the analytical toxicology results alone.

Levels of heroin and its metabolites in blood and brain extracellular fluid after i.v. heroin administration to freely moving rats.

BACKGROUND AND PURPOSE: Heroin, with low affinity for µ-opioid receptors, has been considered to act as a prodrug. In order to study the pharmacokinetics of heroin and its active metabolites after i.v. administration, we gave a bolus injection of heroin to rats and measured the concentration of heroin and its metabolites in blood and brain extracellular fluid (ECF). EXPERIMENTAL APPROACH: After an i.v. bolus injection of heroin to freely moving Sprague-Dawley rats, the concentrations of heroin and metabolites in blood samples from the vena jugularis and in microdialysis samples from striatal brain ECF were measured by ultraperformance LC-MS/MS. KEY RESULTS: Heroin levels decreased very fast, both in blood and brain ECF, and could not be detected after 18 and 10 min respectively. 6-Monoacetylmorphine (6-MAM) increased very rapidly, reaching its maximal concentrations after 2.0 and 4.3 min, respectively, and falling thereafter. Morphine increased very slowly, reaching its maximal levels, which were six times lower than the highest 6-MAM concentrations, after 12.6 and 21.3 min, with a very slow decline during the rest of the experiment and only surpassing 6-MAM levels at least 30 min after injection. CONCLUSIONS AND IMPLICATIONS: After an i.v. heroin injection, 6-MAM was the predominant opioid present shortly after injection and during the first 30 min, not only in the blood but also in rat brain ECF. 6-MAM might therefore mediate most of the effects observed shortly after heroin intake, and this finding questions the general assumption that morphine is the main and most important metabolite of heroin.

Dynamic vaccine blocks relapse to compulsive intake of heroin.

Heroin addiction, a chronic relapsing disorder characterized by excessive drug taking and seeking, requires constant psychotherapeutic and pharmacotherapeutic interventions to minimize the potential for further abuse. Vaccine strategies against many drugs of abuse are being developed that generate antibodies that bind drug in the bloodstream, preventing entry into the brain and nullifying psychoactivity. However, this strategy is complicated by heroin's rapid metabolism to 6-acetylmorphine and morphine. We recently developed a "dynamic" vaccine that creates antibodies against heroin and its psychoactive metabolites by presenting multihaptenic structures to the immune system that match heroin's metabolism. The current study presents evidence of effective and continuous sequestration of brain-permeable constituents of heroin in the bloodstream following vaccination. The result is efficient blockade of heroin activity in treated rats, preventing various features of drugs of abuse: heroin reward, drug-induced reinstatement of drug seeking, and reescalation of compulsive heroin self-administration following abstinence in dependent rats. The dynamic vaccine shows the capability to significantly devalue the reinforcing and motivating properties of heroin, even in subjects with a history of dependence. In addition, targeting a less brain-permeable downstream metabolite, morphine, is insufficient to prevent heroin-induced activity in these models, suggesting that heroin and 6-acetylmorphine are critical players in heroin's psychoactivity. Because the heroin vaccine does not target opioid receptors or common opioid pharmacotherapeutics, it can be used in conjunction with available treatment options. Thus, our vaccine represents a promising adjunct therapy for heroin addiction, providing continuous heroin antagonism, requiring minimal medical monitoring and patient compliance.