Hands-Free Analytical Urine Testing Technology Validated for Drug-Facilitated Crime Investigations.

Forensic laboratories need quick and simple technology to improve turnaround times, while delivering reliable results. The goal of this study is first to create a simplified workflow to meet new Academy Standards Board requirements for urine testing in drug-facilitated crime investigations and, second, to create "ready-to-go", "hands-free" testing technology to further streamline analytical procedures. A first of its kind, the ToxBox forensic test kit is used to validate a single analytical procedure for opioids, benzodiazepines, cannabinoids, antidepressants, and several other drug classes. Method performance indicators follow accreditation requirements and include accuracy, precision, measurement uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement, and analyte stability. "Hands-free" testing platforms require the use of new suspended-state technology to stabilize NIST-traceable standards premanufactured at precise concentrations in the presence of sample preparation reagents. By suspending all reaction components in the solid state, with air gaps between the phases, reference standards and process controls are built in a "ready-to-go" format and stabilized for long-term storage in the presence of a sample matrix, ß-d-glucuronidase, and enzymatic buffers. "Hands-free" test kits are removed from storage, incubated at either ambient temperature or 60 °C, and assayed using validated methods. This is the first example of how complex forensic testing workflows can be streamlined with new "hands-free" testing strategies to meet analytical challenges associated with quantitative and confirmatory analyses.

Deuterated buprenorphine retains pharmacodynamic properties of buprenorphine and resists metabolism to the active metabolite norbuprenorphine in rats.

Introduction: An active metabolite of buprenorphine (BUP), called norbuprenorphine (NorBUP), is implicated in neonatal opioid withdrawal syndrome when BUP is taken during pregnancy. Therefore, reducing or eliminating metabolism of BUP to NorBUP is a novel strategy that will likely lower total fetal exposure to opioids and thus improve offspring outcomes. Precision deuteration alters pharmacokinetics of drugs without altering pharmacodynamics. Here, we report the synthesis and testing of deuterated buprenorphine (BUP-D2). Methods: We determined opioid receptor affinities of BUP-D2 relative to BUP with radioligand competition receptor binding assays, and the potency and efficacy of BUP-D2 relative to BUP to activate G-proteins via opioid receptors with [35S]GTPγS binding assays in homogenates containing the human mu, delta, or kappa opioid receptors. The antinociceptive effects of BUP-D2 and BUP were compared using the warm-water tail withdrawal assay in rats. Blood concentration versus time profiles of BUP, BUP-D2, and NorBUP were measured in rats following intravenous BUP-D2 or BUP injection. Results: The synthesis provided a 48% yield and the product was ≥99% deuterated. Like BUP, BUP-D2 had sub-nanomolar affinity for opioid receptors. BUP-D2 also activated opioid receptors and induced antinociception with equal potency and efficacy as BUP. The maximum concentration and the area under the curve of NorBUP in the blood of rats that received BUP-D2 were over 19- and 10-fold lower, respectively, than in rats that received BUP. Discussion: These results indicate that BUP-D2 retains key pharmacodynamic properties of BUP and resists metabolism to NorBUP and therefore holds promise as an alternative to BUP.

Pharmacokinetics of bleomycin sclerotherapy in patients with vascular malformations.

Bleomycin, a chemotherapy agent that inhibits synthesis of DNA, has been increasingly utilized in sclerotherapy for patients with vascular malformations. A serious long-term risk of intravenous bleomycin is dose-dependent interstitial pneumonitis. Little is known about absorption and circulating levels of bleomycin when used in sclerotherapy for patients with vascular malformations. This is an Institutional Review Board (IRB)-approved prospective study on patients receiving bleomycin sclerotherapy in the management of vascular malformations. Depending on the type of vascular malformation, bleomycin was administered either in the lumen or interstitial space of the involved lesion. A bleomycin assay measured serum bleomycin plasma concentrations versus time at seven intervals following treatment. Pharmacokinetic parameters were obtained for each participant and included peak plasma concentration (Cmax ), time to reach peak plasma concentration (Tmax ), volume of distribution (Vd ), elimination half-life (t1/2 ), the volume of plasma cleared of the drug per unit time (CL), and total systemic exposure area under the curve (AUC). Fifteen patients were enrolled (5: lymphatic, 4: venous, 6: arteriovenous malformations). Bleomycin was administered interstitially (IS) in 11 patients and intraluminal (IL) in four; median age of 13 years (range: 2-67). Pharmacokinetic analysis revealed terminal elimination half-life (t1/2λz ) of 88.51 (±23.09) and 111.61 (±37.75) minutes for the IS and IL groups, respectively. Vd was 4.86 L (±6.74) and 1.55 L (±0.54) for the IS and IL groups, respectively. AUC was 53.9 (±23.45) and 129.17 (±93.57) mg min/L for the IS and IL groups, respectively. There were no statistically significant differences in t1/2λz , Vd , or AUC parameters between groups. Bleomycin is absorbed systemically when used as a sclerosant for vascular malformations when injected either IS or IL.

Short-Term Safety of Repeated Acetaminophen Use in Patients With Compensated Cirrhosis.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP-protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well-defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2-week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in-person at study initiation (day 1) and on days 3 and 5. APAP-protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full-length keratin 18 [K18], and total high-mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP-protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP-protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short-term administration of low-dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis.

The Spice of Death: Sudden Cardiac Arrest After Novel Psychoactive Substance Exposure.

ABSTRACT: Novel psychoactive substances (NPSs), commonly referred to as "K2" or "spice," are a relatively new toxicology challenge for pediatricians. Adolescents often incorrectly believe that these drugs are safe and can be used without major adverse effects. Although recent legislation attempts to ensure that these drugs are not commercially available, many are able to be purchased online as "not fit for human consumption" or under various misnomers such "incense." In addition, there is a wide chemical variation among these substances, making regulation challenging. Standard urine drug screens test for tetrahydrocannabinol, which may not cross-react with synthetic substances, making NPS poisonings difficult to diagnose. We report a case of fatal cardiac arrest in a 16-year-old adolescent boy temporally associated with use of the NPS, 5F-ADB. The case illustrates the dangerous consequences that these unregulated substances pose to users, as well as the need for the consideration of comprehensive toxicological testing in patients with a history of substance use and sudden cardiac arrest, despite a negative drug screen.

Modifying laboratory testing via home brew during the COVID-19 pandemic.

Rapid development and deployment of diagnostic testing for COVID-19 have been a key component of the public health response to the pandemic. Out of necessity, academic and other clinical laboratories developed laboratory testing innovations for COVID-19 to meet clinical testing demands. In addition to constraints on local testing supplies and equipment, a rapidly changing regulatory framework created challenges for translational scientists. Illustrative examples of approaches used to develop laboratory tests during the early stages of the COVID-19 pandemic demonstrate effective team science approaches to this challenging clinical care and public health emergency. These experiences and the associated lessons learned are relevant to the development of public health response plans for future pandemics.

In Utero Exposure to Norbuprenorphine, a Major Metabolite of Buprenorphine, Induces Fetal Opioid Dependence and Leads to Neonatal Opioid Withdrawal Syndrome.

Buprenorphine is the preferred treatment of opioid use disorder during pregnancy but can cause fetal opioid dependence and neonatal opioid withdrawal syndrome (NOWS). Notably, withdrawal severity is independent of maternal buprenorphine dose, suggesting that interindividual variance in pharmacokinetics may influence risk and severity of NOWS. Using a rat model of NOWS, we tested the hypothesis that clinically relevant doses of the active metabolite norbuprenorphine (NorBUP) can induce in utero opioid dependence, manifested as naltrexone-precipitated withdrawal signs in the neonate. Pregnant Long-Evans rats were implanted with 14-day osmotic minipumps containing vehicle, morphine (positive control), or NorBUP (0.3-10 mg/kg per day) on gestation day 9. By 12 hours post-delivery, an intraperitoneal injection of the opioid antagonist naltrexone (1 or 10 mg/kg) or saline was administered to pups. Precipitated withdrawal signs were graded by raters blinded to treatment conditions. In a separate group, NorBUP concentrations in maternal and fetal blood and brain on gestation day 20 were determined by liquid chromatography-tandem mass spectrometry. Steady-state maternal blood concentrations of NorBUP in dams infused with 1 or 3 mg/kg per day were comparable to values reported in pregnant humans treated with buprenorphine (1.0 and 9.6 ng/ml, respectively), suggesting a clinically relevant dosing regimen. At these doses, NorBUP increased withdrawal severity in the neonate as shown by an evaluation of 10 withdrawal indicators. These findings support the possibility that NorBUP contributes to fetal opioid dependence and NOWS following maternal buprenorphine treatment during pregnancy.

Atypical Pharmacodynamic Properties and Metabolic Profile of the Abused Synthetic Cannabinoid AB-PINACA: Potential Contribution to Pronounced Adverse Effects Relative to Δ9-THC.

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ9-THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ9-THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo, producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ9-THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana.

Multi-laboratory validation of a Δ9-tetrahydrocannabinol LC-MS/MS test kit designed for quantifying THC and marijuana metabolites in blood.

Marijuana legalization has increased the demand for testing of Δ9-tetrahydrocannabinol (THC) and THC metabolites. The THC ToxBox® test kit (THC ToxBox®) is commercially available and supports high-throughput LC-MS/MS analytical methods designed to quantify low levels of THC and THC metabolites in blood. The purpose of this study is to determine if this new test kit meets the rigors of laboratory accreditation and produces equivalent results across six states- and locally-funded laboratories. Each laboratory followed internal method validation procedures established for their clinical (CLIA) or international (ISO17025) accreditation program. Test performance indicators included accuracy, precision, measurement of uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement and analyte stability. Analytes and interferents were resolved within the 6-min analytical runtime, and the 48-well plate pre-manufactured with calibrators, second source quality control material, and internal standards at precise concentrations allowed for simple and consistent sample preparation in less than one hour. Every laboratory successfully validated test kit procedures for forensic use. Differences in sensitivity were generally associated with the use of older equipment. Statistical analysis of results spanning reportable ranges show that laboratories with different instrument platforms produce equivalent results at levels sufficiently low enough to support per se limit testing of THC and THC metabolites (1-5 ng/mL). THC ToxBox® represents a viable option for state- and locally-funded laboratories charged with investigating impaired driving cases involving marijuana use.

Altered metabolism of synthetic cannabinoid JWH-018 by human cytochrome P450 2C9 and variants.

Synthetic cannabinoids (SCBs), synonymous with 'K2', 'Spice' or 'synthetic marijuana', are psychoactive drugs of abuse that frequently result in clinical effects and toxicity more severe than those classically associated with Δ9-tetrahydrocannabinol such as extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. JWH-018 is one of the earliest compounds identified in various SCB products, and our laboratory previously demonstrated that JWH-018 undergoes extensive metabolism by cytochromes P450 (P450), binds to, and activates cannabinoid receptors (CBRs). The major enzyme involved in the metabolism of JWH-018 is CYP2C9, a highly polymorphic enzyme found largely in the intestines and liver, with *1 being designated as the wild type, and *2 and *3 as the two most common variants. Three different major products have been identified in human urine and plasma: JWH-018 (ω)-OH, JWH-018 (ω-1)-OH(R), and JWH-018 (ω-1)-OH(S). The (ω-1)-OH metabolite of JWH-018 is a chiral molecule, and is thus designated as either (ω-1)-OH(R) or (ω-1)-OH(S). Here, in vitro enzyme kinetic assays performed with human recombinant CYP2C9 variants (*1, *2, and *3) revealed that oxidative metabolism by CYP2C9*3 resulted in significantly less formation of (ω)-OH and (ω-1)-OH metabolites. Surprisingly, CYP2C9*2 was roughly 3.6-fold more efficient as the CYP2C9*1 enzyme based on Vmax/Km, increasing the rate of JWH-018 metabolism and allowed for a much more rapid elimination. These results suggest that genetic polymorphisms of P450 enzymes result in the production of varying levels of biologically active JWH-018 metabolites in some individuals, offering a mechanistic explanation for the diverse clinical toxicity often observed following JWH-018 abuse.

A High Throughput Method for Measuring Polycyclic Aromatic Hydrocarbons in Seafood Using QuEChERS Extraction and SBSE.

National Oceanic and Atmospheric Administration (NOAA) Method NMFS-NWFSC-59 2004 is currently used to quantitatively analyze seafood for polycyclic aromatic hydrocarbon (PAH) contamination, especially following events such as the Deepwater Horizon oil rig explosion that released millions of barrels of crude oil into the Gulf of Mexico. This method has limited throughput capacity; hence, alternative methods are necessary to meet analytical demands after such events. Stir bar sorptive extraction (SBSE) is an effective technique to extract trace PAHs in water and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction strategy effectively extracts PAHs from complex food matrices. This study uses SBSE to concentrate PAHs and eliminate matrix interference from QuEChERS extracts of seafood, specifically oysters, fish, and shrimp. This method provides acceptable recovery (65-138%) linear calibrations and is sensitive (LOD = 0.02 ppb, LOQ = 0.06 ppb) while providing higher throughput and maintaining equivalency between NOAA 2004 as determined by analysis of NIST SRM 1974b mussel tissue.

Distinct pharmacology and metabolism of K2 synthetic cannabinoids compared to Δ(9)-THC: mechanism underlying greater toxicity?

K2 or Spice products are emerging drugs of abuse that contain synthetic cannabinoids (SCBs). Although assumed by many teens and first time drug users to be a "safe" and "legal" alternative to marijuana, many recent reports indicate that SCBs present in K2 produce toxicity not associated with the primary psychoactive component of marijuana, ∆(9)-tetrahydrocannabinol (Δ(9)-THC). This mini-review will summarize recent evidence that use of K2 products poses greater health risks relative to marijuana, and suggest that distinct pharmacological properties and metabolism of SCBs relative to Δ(9)-THC may contribute to the observed toxicity. Studies reviewed will indicate that in contrast to partial agonist properties of Δ(9)-THC typically observed in vitro, SCBs in K2 products act as full cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) agonists in both cellular assays and animal studies. Furthermore, unlike Δ(9)-THC metabolism, several SCB metabolites retain high affinity for, and exhibit a range of intrinsic activities at, CB1 and CB2Rs. Finally, several reports indicate that although quasi-legal SCBs initially evaded detection and legal consequences, these presumed "advantages" have been limited by new legislation and development of product and human testing capabilities. Collectively, evidence reported in this mini-review suggests that K2 products are neither safe nor legal alternatives to marijuana. Instead, enhanced toxicity of K2 products relative to marijuana, perhaps resulting from the combined actions of a complex mixture of different SCBs present and their active metabolites that retain high affinity for CB1 and CB2Rs, highlights the inherent danger that may accompany use of these substances.

Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS.

Opioid abuse involving emerging opioid compounds is a growing public health problem, which was highlighted recently by cases of human morbidity and mortality linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the toxicology and metabolism of acetyl fentanyl precludes its detection in human samples. The following study was conducted to test a new analytical procedure for the simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl norfentanyl, in human urine. Metabolic reference standards and deuterium-labeled internal standards were synthesized for use in an assay that coupled solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accuracy (% Relative Error <5%) and inter- and intrarun precision (%CV <20%) of this new method resulted in low levels of quantification (∼1 ng/mL). Similar results were obtained using liquid chromatography columns manufactured with phenyl-hexyl and biphenyl stationary phases (r(2) > 0.98). Preliminary human liver microsomal and in vivo rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450s to acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further toxicokinetic studies are required to fully elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and excretion.

Sulfaphenazole and α-naphthoflavone attenuate the metabolism of the synthetic cannabinoids JWH-018 and AM2201 found in K2/spice.

"K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 µM) and ANF (0.1 - 5.0 µM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

Forensic investigation of K2, Spice, and "bath salt" commercial preparations: a three-year study of new designer drug products containing synthetic cannabinoid, stimulant, and hallucinogenic compounds.

New designer drugs such as K2, Spice, and "bath salts" present a formidable challenge for law enforcement and public health officials. The following report summarizes a three-year study of 1320 law enforcement cases involving over 3000 products described as vegetable material, powders, capsules, tablets, blotter paper, or drug paraphernalia. All items were seized in Arkansas from January 2010 through December 2012 and submitted to the Arkansas State Crime Laboratory for analysis. The geographical distribution of these seizures co-localized in areas with higher population, colleges, and universities. Validated forensic testing procedures confirmed the presence of 26 synthetic cannabinoids, 12 designer stimulants, and 5 hallucinogenic-like drugs regulated by the Synthetic Drug Prevention Act of 2012 and other state statutes. Analysis of paraphernalia suggests that these drugs are commonly used concomitantly with other drugs of abuse including marijuana, MDMA, and methamphetamine. Exact designer drug compositions were unpredictable and often formulated with multiple agents, but overall, the synthetic cannabinoids were significantly more prevalent than all the other designer drugs detected. The synthetic cannabinoids JWH-018, AM2201, JWH-122, JWH-210, and XLR11 were most commonly detected in green vegetable material and powder products. The designer stimulants methylenedioxypyrovalerone (MDPV), 3,4-methylenedioxy-N-methylcathinone (methylone), and α-methylamino-valerophenone (pentedrone) were commonly detected in tablets, capsules, and powders. Hallucinogenic drugs were rarely detected, but generally found on blotter paper products. Emerging designer drug products remain a significant problem and continued surveillance is needed to protect public health.

Targeted metabolomic approach for assessing human synthetic cannabinoid exposure and pharmacology.

Designer synthetic cannabinoids like JWH-018 and AM2201 have unique clinical toxicity. Cytochrome-P450-mediated metabolism of each leads to the generation of pharmacologically active (ω)- and (ω-1)-monohydroxyl metabolites that retain high affinity for cannabinoid type-1 receptors, exhibit Δ(9)-THC-like effects in rodents, and are conjugated with glucuronic acid prior to excretion in human urine. Previous studies have not measured the contribution of the specific (ω-1)-monohydroxyl enantiomers in human metabolism and toxicity. This study uses a chiral liquid chromatography-tandem mass spectroscopy approach (LC-MS/MS) to quantify each specific enantiomer and other nonchiral, human metabolites of JWH-018 and AM2201 in human urine. The accuracy (average % RE = 18.6) and reproducibility (average CV = 15.8%) of the method resulted in low-level quantification (average LLQ = 0.99 ng/mL) of each metabolite. Comparisons with a previously validated nonchiral method showed strong correlation between the two approaches (average r(2) = 0.89). Pilot data from human urine samples demonstrate enantiospecific excretion patterns. The (S)-isomer of the JWH-018-(ω-1)-monohydroxyl metabolite was predominantly excreted (>87%) in human urine as the glucuronic acid conjugate, whereas the relative abundance of the corresponding AM2201-(ω-1)-metabolite was low (<5%) and did not demonstrate enantiospecificity (approximate 50:50 ratio of each enantiomer). The new chiral method provides a comprehensive, targeted metabolomic approach for studying the human metabolism of JWH-018 and AM2201. Preliminary evaluations of specific enantiomeric contributions support the use of this approach in future studies designed to understand the pharmacokinetic properties of JWH-018 and/or AM2201.

K2 toxicity: fatal case of psychiatric complications following AM2201 exposure.

Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23-year-old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over-the-counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH-073 found in smoke residue, no other substances were detected. Psychiatric complications including self-induced, lethal trauma can occur after the use of SC products.

Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors.

K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB1Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB2Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB2Rs (hCB2Rs). The affinity of cannabinoids for hCB2Rs was determined by competition binding studies employing CHO-hCB2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB2Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB2Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ(9)-tetrahydrocannabinol (Δ(9)-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB2R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB2Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB2Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB1 and CB2Rs.

Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB(1)) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (K(m) = 0.81-7.3 µM) and low to high reaction velocities (V(max) = 0.0053-2.7 nmol of product · min(-1) · nmol protein(-1)). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of "K2" parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.