Madeleine J. Swortwood

In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant

BACKGROUND Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. MATERIALS & METHODS A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive™ Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites. RESULTS/CONCLUSION Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.

Cannabinoid disposition in oral fluid after controlled smoked, vaporized, and oral cannabis administration

Oral fluid (OF) is an important matrix for monitoring drugs. Smoking cannabis is common, but vaporization and edible consumption also are popular. OF pharmacokinetics are available for controlled smoked cannabis, but few data exist for vaporized and oral routes. Frequent and occasional cannabis smokers were recruited as participants for four dosing sessions including one active (6.9% Δ9 -tetrahydrocannabinol, THC) or placebo cannabis-containing brownie, followed by one active or placebo cigarette, or one active or placebo vaporized cannabis dose. Only one active dose was administered per session. OF was collected before and up to 54 (occasional) or 72 (frequent) h after dosing from cannabis smokers. THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) were quantified by liquid chromatography-tandem mass spectrometry. OF cannabinoid Cmax occurred during or immediately after cannabis consumption due to oral mucosa contamination. Significantly greater THC Cmax and significantly later THCV, CBD, and CBG tlast were observed after smoked and vaporized cannabis compared to oral cannabis in frequent smokers only. No significant differences in THC, 11-OH-THC, THCV, CBD, or CBG tmax between routes were observed for either group. For occasional smokers, more 11-OH-THC and THCCOOH-positive specimens were observed after oral dosing than after inhaled routes, increasing % positive cannabinoid results and widening metabolite detection windows after oral cannabis consumption. Utilizing 0.3 µg/L THCV and CBG cut-offs resulted in detection windows indicative of recent cannabis intake. OF pharmacokinetics after high potency CBD cannabis are not yet available precluding its use currently as a marker of recent use. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Maternal Buprenorphine Maintenance and Lactation

Background: In addition to the well-known benefits of human milk and breastfeeding for the mother and infant, breastfeeding may mitigate neonatal abstinence syndrome severity in prenatally opioid-exposed infants. However, lack of conclusive data regarding the extent of the presence of buprenorphine and active metabolites in human milk makes the recommendation of breastfeeding for buprenorphine-maintained women difficult for many providers. Objective: This study seeks to determine the concentrations of buprenorphine and its active metabolites (norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide) in human milk, maternal plasma, and infant plasma of buprenorphine-maintained women and their infants. Methods: Up to 10 buprenorphine-maintained women provided paired breast milk and plasma samples at 2, 3, 4, 14, and 30 days postdelivery, and 9 infants provided plasma samples on day 14 of life. All samples were analyzed via liquid chromatography tandem mass spectrometry to determine concentrations of buprenorphine, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide by a fully validated method. Results: Concentrations of buprenorphine and metabolites are low in human milk and maternal plasma. Breastfed infant plasma concentrations of buprenorphine were low or undetectable and metabolite concentrations undetectable at 14 days of infant age. There were significant correlations between maternal buprenorphine dose and maternal plasma and human milk buprenorphine concentrations. Conclusion: These data find low concentrations of buprenorphine and metabolites in human milk and lend support to the recommendation for lactation among stable buprenorphine-maintained women. However, the correlation between maternal dose and maternal plasma and human milk buprenorphine concentrations bears further study.

Cannabis Edibles: Blood and Oral Fluid Cannabinoid Pharmacokinetics and Evaluation of Oral Fluid Screening Devices for Predicting Δ9-Tetrahydrocannabinol in Blood and Oral Fluid following Cannabis Brownie Administration

BACKGROUND Roadside oral fluid (OF) Δ9-tetrahydrocannabinol (THC) detection indicates recent cannabis intake. OF and blood THC pharmacokinetic data are limited and there are no on-site OF screening performance evaluations after controlled edible cannabis. CONTENT We reviewed OF and blood cannabinoid pharmacokinetics and performance evaluations of the Draeger DrugTest®5000 (DT5000) and Alere™ DDS®2 (DDS2) on-site OF screening devices. We also present data from a controlled oral cannabis administration session. SUMMARY OF THC maximum concentrations (Cmax) were similar in frequent as compared to occasional smokers, while blood THC Cmax were higher in frequent [mean (range) 17.7 (8.0-36.1) μg/L] smokers compared to occasional [8.2 (3.2-14.3) μg/L] smokers. Minor cannabinoids Δ9-tetrahydrocannabivarin and cannabigerol were never detected in blood, and not in OF by 5 or 8 h, respectively, with 0.3 μg/L cutoffs. Recommended performance (analytical sensitivity, specificity, and efficiency) criteria for screening devices of ≥80% are difficult to meet when maximizing true positive (TP) results with confirmation cutoffs below the screening cutoff. TPs were greatest with OF confirmation cutoffs of THC ≥1 and ≥2 μg/L, but analytical sensitivities were <80% due to false negative tests arising from confirmation cutoffs below the DT5000 and DDS2 screening cutoffs; all criteria were >80% with an OF THC ≥5 μg/L cutoff. Performance criteria also were >80% with a blood THC ≥5 μg/L confirmation cutoff; however, positive OF screening results might not confirm due to the time required to collect blood after a crash or police stop. OF confirmation is recommended for roadside OF screening.ClinicalTrials.gov identification number: NCT02177513.

Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells

The liver is a dynamic organ which is both multifunctional and highly regenerative. A major role of the liver is to process both endo and xenobiotics. Cigarettes are an example of a legal and widely used drug which can cause major health problems for adults and constitute a particular risk to the foetus, if the mother smokes during pregnancy. Cigarette smoke contains a complex mixture of thousands of different xenobiotics, including nicotine and polycyclic aromatic hydrocarbons. These affect foetal development in a sex-specific manner, inducing sex-dependant molecular responses in different organs. To date, the effect of maternal smoking on the foetal liver has been studied in vitro using cell lines, primary tissue and animal models. While these models have proven to be useful, poor cell phenotype, tissue scarcity, batch-to-batch variation and species differences have led to difficulties in data extrapolation toward human development. Therefore, in this study we have employed hepatoblasts, derived from pluripotent stem cells, to model the effects of xenobiotics from cigarette smoke on human hepatocyte development. Highly pure hepatocyte populations (>90%) were produced in vitro and exposed to factors present in cigarette smoke. Analysis of ATP levels revealed that, independent of the sex, the majority of smoking derivatives tested individually did not deplete ATP levels below 50%. However, following exposure to a cocktail of smoking derivatives, ATP production fell below 50% in a sex-dependent manner. This was paralleled by a loss metabolic activity and secretory ability in both female and male hepatocytes. Interestingly, cell depletion was less pronounced in female hepatocytes, whereas caspase activation was ~twofold greater, indicating sex differences in cell death upon exposure to the smoking derivatives tested.

Long-term stability of cannabinoids in oral fluid after controlled cannabis administration

Cannabinoid stability in oral fluid (OF) is important for assuring accurate results since OF has become a valid alternative matrix of choice for drug testing. We previously published OF cannabinoid stability studies using Quantisal™, Oral-Eze®, and StatSure™ devices stored at room temperature for 1 week, 4 °C for up to 4 weeks, and at -20 °C up to 24 weeks. Extending refrigerated stability up to 3 months would be helpful for clinical and forensic testing, for re-analysis of OF samples and for batching research analyses. Individual authentic OF pools were prepared after controlled smoking of a 6.9% ∆9 -tetrahydracannabinol cannabis cigarette; the Quantisal™ device was utilized for OF collection. Fifteen healthy volunteers participated in the Institutional Review Board-approved study. Stability for THC, 11-nor-9-carboxy-THC (THCCOOH), ∆9 -tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) were determined after storage at 4 °C for 1, 2, and 3 months. Results within ±20% of baseline concentrations were considered stable. All analytes were stable for up to 2 months at 4 °C for all participants with positive baseline concentrations. Baseline concentrations were highly variable. In total, THC, THCCOOH, THCV, CBD, and CBG were stable for 3 months at 4 °C for pooled positive specimens from 14 of 15, 8 of 9, 7 of 8, 8 of 9, and 9 of 10 participants, respectively. In conclusion, Quantisal™-collected OF specimens should be stored at 4 °C for no more than two months to assure accurate THC, THCCOOH, THCV, CBD, and CBG quantitative results; only one participants OF was unstable at three months. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Simultaneous quantification of buprenorphine, naloxone and phase I and II metabolites in plasma and breastmilk by liquid chromatography–tandem mass spectrometry

Opioid abuse during pregnancy is associated with fetal growth restriction, placental abruption, preterm labor, fetal death, and Neonatal Abstinence Syndrome. Current guidelines for medication-assisted opioid addiction treatment during pregnancy are methadone or buprenorphine monotherapy. Buprenorphine/naloxone combination therapy (Suboxone(®)) has not been thoroughly evaluated during pregnancy and insufficient naloxone safety data exist. While methadone- and buprenorphine-treated mothers are encouraged to breastfeed, no studies to date investigated naloxone concentrations during breastfeeding following Suboxone administration. For this reason, we developed and fully validated a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine, buprenorphine-glucuronide, norbuprenorphine, norbuprenorphine-glucuronide, naloxone, naloxone-glucuronide and naloxone-N-oxide in 100μL human plasma and breastmilk in a single injection following protein precipitation and solid-phase extraction. Lowest limits of quantification were 0.1-2μg/L with 20-100μg/L upper limits of linearity. Bias and imprecision were <±16%. Matrix effects ranged from -57.9 to 11.2 and -84.6 to 29.3% in plasma and breastmilk, respectively. All analytes were stable (within ±20% change from baseline) under all tested conditions (24h room temperature, 72h at 4°C, 3 freeze/thaw cycles at -20°C, and in the autosampler for 72h at 4°C). For proof of concept, buprenorphine and its metabolites were successfully quantified in authentic positive maternal and infant plasma and paired breastmilk specimens. This comprehensive, highly sensitive and specific method detects multiple buprenorphine markers in a small specimen volume.

Naloxone and Metabolites Quantification in Cord Blood of Prenatally Exposed Newborns and Correlations with Maternal Concentrations

Objective To quantify naloxone and metabolite concentrations in newborns prenatally exposed to sublingual buprenorphine/naloxone and to correlate neonatal and maternal metabolite concentrations. Methods This is a prospective observational cohort study. Eleven pregnant women treated for opioid use disorder with sublingual buprenorphine/naloxone were enrolled. Maternal and newborn blood was collected and analyzed for naloxone, buprenorphine, and metabolites via liquid chromatography tandem mass spectrometry. Descriptive statistics and correlation coefficients were utilized to analyze data. Results Maternal daily naloxone and buprenorphine doses were 1 to 5 mg and 4 to 20 mg, respectively; the mean (standard deviation) time from medication until delivery was 9.9 (4.3) hours. Naloxone was below the limits of quantification (LOQ) in five infants and six mothers with a range of less than LOQ to 0.3 μg/L. There was a strong positive correlation between maternal and newborn naloxone concentrations: Spearmans ρ = 0.89 (p < 0.01). There were strong positive correlations between maternal and neonatal assays for the buprenorphine analyte concentrations: buprenorphine ρ = 0.88 (p < 0.01), norbuprenorphine ρ = 0.71 (p = 0.01), and norbuprenorphine-glucuronide ρ = 0.98 (p < 0.01), but not for buprenorphine-glucuronide, ρ = 0.53 (p = 0.10). Conclusion Naloxone and buprenorphine are transferred to the fetus during prenatal exposure to maternal sublingual buprenorphine/naloxone. The quantity of naloxone transferred from maternal circulation is minimal and highly correlated with maternal concentrations.

Quantitative Analysis of Novel Synthetic Opioids, Morphine and Buprenorphine in Oral Fluid by LC–MS-MS

The opioid epidemic has become a national health emergency in the USA. While heroin and prescription opioid abuse is not uncommon, synthetic opioid use has risen dramatically, creating a public safety concern. Like traditional opioids, novel synthetic opioids are abused due to their analgesic and euphoric effects. Some adverse side effects include respiratory distress, nausea and decreased consciousness. Synthetic opioids have emerged into the illicit and online drug market, including AH-7921, MT-45, U-series and W-series. Though originally developed by pharmaceutical companies, these substances are not well studied in humans and comprehensive analytical methods for detecting and quantifying these opioids are limited. Oral fluid is a useful biological matrix for determining recent drug use, does not require a trained medical professional, and can be collected under direct observation, deterring adulteration. The purpose of this research was to develop and validate a comprehensive analytical method for the detection and quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18 and W-15 in oral fluid collected via Quantisal. This was achieved by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The limits of detection and quantitation were 5 ng/mL and 10 ng/mL, respectively. Linearity was observed between 10 and 500 ng/mL (R2 ≥ 0.9959). Bias and imprecision were <±11.1%. Matrix effects ranged from -21.1 to 13.7%. No carryover was detected following injection of the highest calibrator. All analytes were stable (within ±15% change from baseline) under all tested conditions (24 h at room temperature, 72 h at 4°C, and in the autosampler for 60 h at 4°C).

Erratum to: Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells

During manuscript proofing, the following sentence was not deleted in the section “Results” at the end of the paragraph: “Both male and female hepatocytes responded in a similar fashion to cotinine, whereas male hepatocyte function was more sensitive to chrysene, fluorene and naphthalene than female hepatocytes”.