Role of aquaporin-4 polarization in extracellular solute clearance.

Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4-/-) or lacking the endfoot pool of AQP4 (Snta1-/-). We confirm that Aqp4-/- mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1-/- mice have reduced clearance only for the 500 kDa [3H]dextran, but not 0.18 kDa [3H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [3H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.

MAIN POINTS: Solute clearance is reduced in mice lacking AQP4 Polarization of AQP4 to the endfeet may have a greater impact on clearance of large versus small molecules Clearance of large but not small solutes is correlated with age within adult age.

Metabolite markers for three synthetic tryptamines N-ethyl-N-propyltryptamine, 4-hydroxy-N-ethyl-N-propyltryptamine, and 5-methoxy-N-ethyl-N-propyltryptamine.

N-Ethyl-N-propyltryptamine (EPT), 4-hydroxy-N-ethyl-N-propyltryptamine (4-OH-EPT), and 5-methoxy-N-ethyl-N-propyltryptamine (5-MeO-EPT) are new psychoactive substances classified as tryptamines, sold online. Many tryptamines metabolize rapidly, and identifying the appropriate metabolites to reveal intake is essential. While the metabolism of 4-OH-EPT and 5-MeO-EPT are not previously described, EPT is known to form metabolites by indole ring hydroxylation among others. Based on general knowledge of metabolic patterns, 5-MeO-EPT is also expected to form ring hydroxylated EPT (5-OH-EPT). In the present study, the aim was to characterize the major metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT, to provide markers for substance identification in forensic casework. The tryptamines were incubated with pooled human liver microsomes at 37°C for up to 4 h. The generated metabolites were separated and detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The major in vitro EPT metabolites were formed by hydroxylation, N-dealkylation, and carbonylation. In comparison, 4-OH-EPT metabolism was dominated by double bond formation, N-dealkylation, hydroxylation, and carbonylation in vitro and hydroxylation or carbonylation combined with double bond loss, carbonylation, N-dealkylation, and hydroxylation in vivo. 5-MeO-EPT was metabolized by O-demethylation, hydroxylation, and N-dealkylation in vitro. The usefulness of the characterized metabolites in forensic casework was demonstrated by identification of unique metabolites for 4-OH-EPT in a human postmortem blood sample with suspected EPT or 4-OH-EPT intoxication.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Heroin metabolism in human blood and its impact for the design of an immunotherapeutic approach against heroin effects.

Immunotherapeutic interventions that block drug effects by binding drug molecules to specific antibodies in the bloodstream have shown promising effects in animal studies. For heroin, which effects are mainly mediated by the metabolites 6-acetylmorphine (6-AM; also known as 6-monoacetylmorphine or 6-MAM) and morphine, the optimal antibody specificity has been discussed. In rodents, 6-AM specific antibodies have been recommended based on the rapid metabolism of heroin to 6-AM in the bloodstream. Since the metabolic rate of heroin in blood is unsettled in humans, we examined heroin metabolism with state-of-the-art analytical methodology (UHPLC-MS/MS) in freshly drawn human whole blood incubated with a wide range of heroin concentrations (1-500 µM). The half-life of heroin was highly concentration dependent, ranging from 1.2-1.7 min for concentrations at or above 25 µM, and gradually increasing to approximately 20 min for 1 µM heroin. At concentrations that can be attained in the bloodstream shortly after an i.v. injection, approximately 70% was transformed into 6-AM within 3 min, similar to previous observations in vivo. Our results indicate that blood enzymes play a more important role for the rapid metabolism of heroin in humans than previously assumed. This points to 6-AM as an important target for an efficient immunotherapeutic approach to block heroin effects in humans.

"Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry.

Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

Comparative Neuropharmacology and Pharmacokinetics of Methamphetamine and Its Thiophene Analog Methiopropamine in Rodents.

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (Emax 12.5 mg/kg; i.p.) compared to methamphetamine (Emax 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

Pharmacokinetics and pharmacodynamics of cyclopropylfentanyl in male rats.

BACKGROUND: Illicitly manufactured fentanyl and its analogs are a major driving force behind the ongoing opioid crisis. Cyclopropylfentanyl is a fentanyl analog associated with many overdose deaths, but limited knowledge is available about its pharmacology. In the present study, we developed a bioanalytical method for the determination of cyclopropylfentanyl and its main metabolite cyclopropylnorfentanyl and evaluated pharmacokinetic-pharmacodynamic relationships in rats. METHOD: An ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of cyclopropylfentanyl and cyclopropylnorfentanyl in rat plasma. Male Sprague-Dawley rats fitted with jugular catheters and temperature transponders received cyclopropylfentanyl (30, 100, and 300 µg/kg) or saline subcutaneously. Blood specimens were withdrawn over an 8-h time period, along with measurements of pharmacodynamic endpoints. RESULTS: The analytical method was validated, and both analytes exhibited a low limit of quantification (15 pg/mL). Cyclopropylfentanyl caused dose-related increases in hot plate latency (ED50 = 48 µg/kg) and catalepsy (ED50 = 87 µg/kg) and produced long-lasting hypothermia at the highest dose. Plasma cyclopropylfentanyl rose rapidly in a dose-related fashion, reaching maximal concentration (Cmax) after 15-28 min, whereas metabolite Cmax occurred later at 45-90 min. Cyclopropylfentanyl Cmax values were similar to concentrations measured in non-fatal intoxications in humans; however, differences in parent drug: metabolite ratio indicated possible interspecies variance in metabolism. CONCLUSION: Our study shows that cyclopropylfentanyl produces typical opioid-like effects in male rats. Cyclopropylfentanyl displays much greater analgesic potency when compared to morphine, suggesting that cyclopropylfentanyl poses increased overdose risk for unsuspecting users.

Does the preparation for intravenous administration affect the composition of heroin injections? A controlled laboratory study.

AIMS: To study whether the preparation procedure, and its acidic and heating conditions, used by heroin users to prepare heroin for intravenous administration affects the final composition of the fluid to be injected. METHODS: Samples from different seizures of illegal heroin provided by the Norwegian police were prepared by adding water and ascorbic acid before heating under controlled conditions in the laboratory. Further, three seizures were prepared with different amounts of ascorbic or citric acid relative to their diacetylmorphine content. Pure diacetylmorphine base or salt was also submitted to the procedure applying two different heating intensities. The seizures and the final product after preparation were analysed for diacetylmorphine, 6-acetylmorphine and morphine using liquid chromatography with tandem mass spectrometry (LC-MS-MS). RESULTS: After preparation, a decrease of 19.8% (25th and 75th percentiles = -29.2 and -15.3) in the initial diacetylmorphine content was observed. Both the 6-acetylmorphine and morphine content increased but, due to their low content in the initial product, diacetylmorphine still represented 83.9% (25th and 75th percentiles = 77.3 and 88.0) of the sum of these three opioids in the final solution. The loss of water during preparation caused an increase in the concentration of diacetylmorphine, 6-acetylmorphine and morphine, depending on the heating intensity applied. The content of these opioids was affected by the quantity and type of acid added in relation to the heroin purity and the level of diacetylmorphine dissolved being proportional to the amount of ascorbic acid, but not citric acid, in the sample with high heroin purity. CONCLUSIONS: Preparation of heroin for intravenous injection appears to change the amount or concentration of diacetylmorphine and its active metabolites, 6-acetylmorphine and morphine in the final product, depending on heroin purity, amount and type of acid used or heating conditions. These circumstances can contribute to unintentional variations in the potency of the final injected solution, and therefore affect the outcome after injection.

Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies.

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

Selectivity and sensitivity of urine fentanyl test strips to detect fentanyl analogues in illicit drugs.

BACKGROUND: Urine fentanyl test strips have been employed to check street drugs for fentanyl and fentanyl analogue contamination, but there is limited evidence for the applicability of fentanyl strips for this purpose. We examined the ability of four commercially-available fentanyl test strips to detect fentanyl and a range of fentanyl analogues currently on the recreational drug market. METHODS: Four brands of fentanyl test strips (Rapid Response, One Step, Nal van Minden, and Rapid Self Test) were examined using single-component drug solutions containing fentanyl, 28 fentanyl analogues, four non-fentanyl synthetic opioids, or eight traditional drugs of abuse. The effect of co-presence of heroin or ascorbic acid on test results was also examined. RESULTS: All test strips detected fentanyl as well as 21-24 of the 28 fentanyl analogues tested. One of the test strip brands gave false positive results in the presence of ascorbic acid. CONCLUSIONS: Fentanyl test strips successfully detected the majority of fentanyl analogues tested. Drug solutions for testing should not be overly dilute, since the test results are highly concentration dependent. Fentanyl test strips have utility as a harm reduction tool, but they are no panacea for overdose since certain fentanyl analogues are not detected.

Prenatal exposure to methadone or buprenorphine alters µ-opioid receptor binding and downstream signaling in the rat brain.

There is a growing concern related to the use of opioid maintenance treatment during pregnancy. Studies in both humans and animals have reported reduced cognitive functioning in offspring prenatally exposed to methadone or buprenorphine; however, little is known about the neurobiological mechanisms underlying these impairments. To reveal possible neurobiological effects of such in utero exposure, we examined brain tissue from methadone- and buprenorphine-exposed rat offspring previously shown to display impaired learning and memory. We studied µ-opioid receptor (MOR) and N-methyl-D-aspartate receptor (NMDAR) binding in the rat offspring cerebrum during development and in the hippocampus at young adulthood. Moreover, we examined activation of the Ca2+ /calmodulin-dependent protein kinase II (CaMKII) and the extracellular signal-regulated kinase (ERK), which are central in the downstream signaling of these receptors. The methadone- and buprenorphine-exposed rat pups displayed reduced MOR binding up to two weeks after birth, whereas the NMDAR binding was unaffected. Prenatal exposure to methadone or buprenorphine also resulted in decreased activation of CaMKII and/or ERK during development, while young adult offspring displayed increased hippocampal ERK activation. In conclusion, our findings suggest that prenatal exposure to exogenous opioids, such as methadone or buprenorphine, may disturb the endogenous opioid system during development, with long-term effects on proteins important for cognitive functioning.

Prenatal exposure to methadone or buprenorphine impairs cognitive performance in young adult rats.

BACKGROUND: Concerns have been raised about the use of opioid maintenance treatment (OMT) during pregnancy and negative effects for the offspring. While neonatal outcomes and short-term effects are relatively well described, studies examining long-term effects in adolescents and adults are absent. The aim of the present study was to examine effects on learning and memory in young adult rats prenatally exposed to methadone or buprenorphine. METHODS: Female rats were implanted with a 28-day osmotic minipump delivering methadone (10 mg/kg/day), buprenorphine (1 mg/kg/day) or vehicle 5 days prior to mating. To examine possible effects on cognitive functioning, young adult offspring were included in three different behavioral tests that examine recognition memory, nonspatial, and spatial learning and memory. In addition, offspring growth and maternal behavior after birh were investigated. RESULTS: Prenatal exposure to methadone or buprenorphine caused impaired recognition memory and nonspatial reference learning and memory in young adult rats compared with the vehicle-treated group. Methadone-exposed offspring, but not the buprenorphine-exposed, also showed reduced long-term spatial memory. We did not observe any changes in maternal behavior or offspring growth after prenatal exposure to methadone or buprenorphine, suggesting that the impaired cognitive functioning is due to the opioid exposure rather than reduced maternal caregiving. CONCLUSION: The present findings of long-term cognitive impairments in methadone- and buprenorphine-exposed offspring points to a negative impact of OMT on neurobiological development.

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.

High Accumulation of Methadone Compared with Buprenorphine in Fetal Rat Brain after Maternal Exposure.

Experimental animal studies are valuable in revealing a causal relationship between prenatal exposure to opioid maintenance treatment (OMT) and subsequent effects; however, previous animal studies of OMT during pregnancy have been criticized for their lack of clinical relevance because of their use of high drug doses and the absence of pharmacokinetic data. Hence, the aim of this study was to determine blood and brain concentrations in rat dams, fetuses, and offspring after continuous maternal exposure to methadone or buprenorphine during gestation and to examine the offspring for neonatal outcomes and withdrawal symptoms. Female rats were implanted with a 28-day osmotic minipump delivering methadone (10 mg/kg per day), buprenorphine (1 mg/kg per day) or vehicle 5 days before mating. Continuous exposure to methadone or buprenorphine induced stable blood concentrations in the dams of 0.25 ± 0.02 µM and 5.65 ± 0.16 nM, respectively. The fetal brain concentration of methadone (1.89 ± 0.35 nmol/g) was twice as high as that in the maternal brain, whereas the fetal brain concentration of buprenorphine (20.02 ± 4.97 pmol/g) was one-third the maternal brain concentration. The opioids remained in the offspring brain several days after the exposure ceased. Offspring prenatally exposed to methadone, but not buprenorphine, displayed reduced body weight and length and increased corticosterone levels. No significant changes in ultrasonic vocalizations were revealed. Our data in rat fetuses and neonates indicate that OMT with buprenorphine may be a better choice than methadone during pregnancy. SIGNIFICANCE STATEMENT: Concern has been raised about the use of opioid maintenance treatment during pregnancy because of the important role of the endogenous opioid system in brain development. Here, we show that the methadone concentration in the fetal rat brain was twice as high as that in the maternal brain, whereas the buprenorphine concentration was one-third the maternal concentration. Furthermore, buprenorphine allowed more favorable birth outcomes, suggesting that buprenorphine may be a better choice during pregnancy.

Evidence for nonlinear accumulation of the ultrapotent fentanyl analog, carfentanil, after systemic administration to male rats.

The current opioid overdose crisis is being exacerbated by illicitly manufactured fentanyl and its analogs. Carfentanil is a fentanyl analog that is 10,000-times more potent than morphine, but limited information is available about its pharmacology. The present study had two aims: 1) to validate a method for quantifying carfentanil and its metabolite norcarfentanil in small-volume samples, and 2) to use the method for examining pharmacodynamic-pharmacokinetic relationships in rats. The analytical method involved liquid-liquid extraction of plasma samples followed by quantitation of carfentanil and norcarfentanil using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was validated following SWGTOX guidelines, and both analytes displayed limits of detection and quantification at 7.5 and 15 pg/mL, respectively. Male Sprague-Dawley rats fitted with jugular catheters and temperature transponders received subcutaneous carfentanil (1, 3 and 10 µg/kg) or saline. Repeated blood specimens were obtained over 8 h, along with pharmacodynamic measures including core temperature and catalepsy scores. Carfentanil produced dose-related hypothermia and catalepsy that lasted up to 8 h. Carfentanil Cmax occurred at 15 min whereas metabolite Cmax was at 1-2 h. Concentrations of both analytes increased in a dose-related fashion, but area-under-the-curve values were much greater than predicted after 10 µg/kg. Plasma half-life for carfentanil increased at higher doses. Our findings reveal that carfentanil produces marked hypothermia and catalepsy, which is accompanied by nonlinear accumulation of the drug at high doses. We hypothesize that impaired clearance of carfentanil in humans could contribute to life-threatening effects of this ultrapotent opioid agonist. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Distinguishing Between Cyclopropylfentanyl and Crotonylfentanyl by Methods Commonly Available in the Forensic Laboratory.

BACKGROUND: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity, and legal status, and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. METHODS: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by ultra-high-performance liquid chromatography was investigated using 3 different stationary phases (high strength silica T3, ethylsiloxane/silica hybrid C18, and Kinetex biphenyl) using gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by tandem mass spectrometry. In addition, the major metabolites of the 2 compounds formed on incubation with human liver microsomes were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. RESULTS: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the ethylsiloxane/silica hybrid C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the 2 analogues formed by human liver microsomes differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the 2 approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in 2 authentic postmortem blood samples. CONCLUSIONS: In this study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.

The active heroin metabolite 6-acetylmorphine has robust reinforcing effects as assessed by self-administration in the rat.

Previous studies have suggested that at least some of the behavioral effects of heroin might be mediated by its active metabolite 6-acetylmorphine (6-AM). The aim of the present study was to investigate the reinforcing effects of 6-AM and its role in mediating those of heroin. We used an intravenous self-administration procedure in male Sprague-Dawley rats including four phases: acquisition, extinction, reinstatement of drug-seeking, and re-acquisition. Independent groups of rats readily learned to self-administer equimolar doses (0.135 µmol/kg) of either 6-AM (44.3 µg/kg) or heroin (50 µg/kg). Under a fixed ratio 1 (FR1) schedule of reinforcement, the rate of responding was the same for 6-AM and heroin, but it was significantly higher for 6-AM than for heroin under a FR2 schedule. A non-contingent infusion ('priming') of 0.068 µmol/kg of either 6-AM or heroin reinstated non-reinforced drug-seeking (relapse). The rats readily re-acquired self-administration behaviour when given access to one of two doses (0.068 and 0.135 µmol/kg) of 6-AM or heroin. Pretreatment with a specific monoclonal antibody (mAb) against 6-AM blocked the priming effect of 6-AM, and modified the rate of lever-pressing on re-acquisition of 6-AM self-administration in a manner compatible with a shift to the right of the dose-effect curve. The mAb did not affect heroin responding. The present results show that 6-AM possesses reinforcing effects similar to those of heroin. The lack of effect of 6-AM mAb on heroin priming and heroin self-administration calls for further studies to clarify the role of heroin and its metabolites in heroin reward. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'.

A Monoclonal Antibody against 6-Acetylmorphine Protects Female Mice Offspring from Adverse Behavioral Effects Induced by Prenatal Heroin Exposure.

Escalating opioid use among fertile women has increased the number of children being exposed to opioids during fetal life. Furthermore, accumulating evidence links prenatal opioid exposure, including opioid maintenance treatment, to long-term negative effects on cognition and behavior, and presses the need to explore novel treatment strategies for pregnant opioid users. The present study examined the potential of a monoclonal antibody (mAb) targeting heroin's first metabolite, 6-acetylmorphine (6-AM), in providing fetal protection against harmful effects of prenatal heroin exposure in mice. First, we examined anti-6-AM mAb's ability to block materno-fetal transfer of active metabolites after maternal heroin administration. Next, we studied whether maternal mAb pretreatment could prevent adverse effects in neonatal and adolescent offspring exposed to intrauterine heroin (3 × 1.05 mg/kg). Anti-6-AM mAb pretreatment of pregnant dams profoundly reduced the distribution of active heroin metabolites to the fetal brain. Furthermore, maternal mAb administration prevented hyperactivity and drug sensitization in adolescent female offspring prenatally exposed to heroin. Our findings demonstrate that passive immunization with a 6-AM-specific antibody during pregnancy provides fetal neuroprotection against heroin metabolites, and thereby prevents persistent adverse behavioral effects in the offspring. An immunotherapeutic approach to protect the fetus against long-term effects of prenatal drug exposure has not been reported previously, and should be further explored as prophylactic treatment of pregnant heroin users susceptible to relapse.

Addressing the Fentanyl Analogue Epidemic by Multiplex UHPLC-MS/MS Analysis of Whole Blood.

BACKGROUND: Fentanyl and fentanyl analogues (fentanyls) are very potent opioids posing a serious threat to the public health. Thousands of overdose deaths across the world are caused by fentanyls, and the numbers are increasing. Rapid mapping of current trends in opioid abuse is necessary to accelerate preventive measures. To ensure this, there is a need for sensitive targeted multiplex MS/MS methods to pinpoint drugs of abuse. We present a fully validated UHPLC-MS/MS method for the determination of 26 fentanyls, including several structural isomers, and the opioid antagonist naloxone in human whole blood. METHODS: Blood samples were prepared by liquid-liquid extraction with ethyl acetate and heptane. The fentanyls were separated with UHPLC, using a Kinetex biphenyl column (2.1 × 100 mm, 1.7 µm; Phenomenex, Verløse, Denmark) with an acidic mobile phase. Quantification was performed by MS/MS. The method was validated according to SWGTOX guidelines. RESULTS: The developed method could successfully separate all 27 analytes, including 7 isomers, and was validated according to SWGTOX guidelines with very low limits of quantification (4-20 pg/mL). The applicability of the method was demonstrated by determination of fentanyls in postmortem blood samples from 2 cases. CONCLUSIONS: A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.

Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC-MS/MS.

A novel ion pair reversed phase ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the stress hormones adrenaline, noradrenaline and corticosterone in rodent blood was developed and fully validated. Separations were performed on an Acquity HSS T3 column (2.1mm i.d.×100mm, 1.8µm) with gradient elution and a runtime of 5.5min. The retention of adrenaline and noradrenaline was substantially increased by employing the ion pair reagent heptafluorobutyric acid (HFBA). Ion pair reagents are usually added to the mobile phase only, but we demonstrate for the first time that including HFBA to the sample reconstitution solvent as well, has a major impact on the chromatography of these compounds. The stability of adrenaline and corticosterone in rodent blood was investigated using the surrogate analytes adrenaline-d3 and corticosterone-d8. The applicability of the described method was demonstrated by measuring the concentration of stress hormones in rodent blood samples.