Screening of Environmental Chemicals to Characterize Exposures in Participants With Systemic Lupus Erythematosus.

OBJECTIVE: There is a need to characterize exposures associated with the pathogenesis of systemic lupus erythematosus (SLE). In this pilot study, we explore a hypothesis-free approach that can measure thousands of exogenous chemicals in blood ("exposome") in patients with SLE and unaffected controls. METHODS: This cross-sectional study analyzed a cohort of patients with prevalent SLE (n = 285) and controls (n = 106). Plasma was analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). Mass spectrometry features present in at least 25% of all samples were selected for association analysis (n = 2,737). Features were matched to potential chemicals using available databases. Association analysis of abundances of features with SLE status was performed, adjusting for age and sex. We also explored features associated with SLE phenotypes, sociodemographic factors, and current medication use. RESULTS: We found 30 features significantly associated with SLE status (Bonferroni P < 0.05). Of these, seven matched chemical names based on databases. These seven features included phthalate metabolites, a formetanate metabolite, and eugenol. The abundance of acid pesticides differed between patients with SLE and controls (Bonferroni P < 0.05). Two unmatched features were associated with a history of lupus nephritis, and one with anti-double-stranded DNA antibody production (Bonferroni P < 0.05). Seventeen features varied by self-reported race and ethnicity, including a polyfluoroalkyl substance (analysis of variance P < 1.69 × 10-5 ). Eleven features correlated with antimalarials, 6 with mycophenolate mofetil, and 29 with prednisone use. CONCLUSION: This proof-of-concept study demonstrates that LC-QTOF/MS is a powerful tool that agnostically detects circulating exogenous compounds. These analyses can generate hypotheses of disease-related exposures for future prospective, longitudinal studies.

Mass spectrometry imaging of hair identifies daily maraviroc adherence in HPTN 069/ACTG A5305.

Objective measures of adherence for antiretrovirals used as pre-exposure prophylaxis (PrEP) are critical for improving preventative efficacy in both clinical trials and real-world application. Current objective adherence measures either reflect only recent behavior (eg days for plasma or urine) or cumulative behavior (eg months for dried blood spots). Here, we measured the accumulation of the antiretroviral drug maraviroc (MVC) in hair strands by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) to evaluate adherence behavior longitudinally at high temporal resolution. An MSI threshold for classifying daily adherence was established using clinical samples from healthy volunteers following directly observed dosing of 1 to 7 doses MVC/week. We then used the benchmarked MSI assay to classify adherence to MVC-based PrEP regimens in hair samples collected throughout the 48-week HPTN069/ACTGA5305 study. We found that only ~32% of investigated hair samples collected during the study's active dosing period showed consistent daily PrEP adherence throughout a retrospective period of 30 days, and also found that profiles of daily individual adherence from MSI hair analysis could identify when patients were and were not taking study drug. The assessment of adherence from MSI hair strand analysis was 62% lower than adherence classified using paired plasma samples, the latter of which may be influenced by white-coat adherence. These findings demonstrate the ability of MSI hair analysis to examine daily variability of adherence behavior over a longer-term measurement and offer the potential for longitudinal comparison with risk behavior to target patient-specific adherence interventions and improve outcomes.

Alternative Methods for Therapeutic Drug Monitoring and Dose Adjustment of Tuberculosis Treatment in Clinical Settings: A Systematic Review.

BACKGROUND AND OBJECTIVE: Quantifying exposure to drugs for personalized dose adjustment is of critical importance in patients with tuberculosis who may be at risk of treatment failure or toxicity due to individual variability in pharmacokinetics. Traditionally, serum or plasma samples have been used for drug monitoring, which only poses collection and logistical challenges in high-tuberculosis burden/low-resourced areas. Less invasive and lower cost tests using alternative biomatrices other than serum or plasma may improve the feasibility of therapeutic drug monitoring. METHODS: A systematic review was conducted to include studies reporting anti-tuberculosis drug concentration measurements in dried blood spots, urine, saliva, and hair. Reports were screened to include study design, population, analytical methods, relevant pharmacokinetic parameters, and risk of bias. RESULTS: A total of 75 reports encompassing all four biomatrices were included. Dried blood spots reduced the sample volume requirement and cut shipping costs whereas simpler laboratory methods to test the presence of drug in urine can allow point-of-care testing in high-burden settings. Minimal pre-processing requirements with saliva samples may further increase acceptability for laboratory staff. Multi-analyte panels have been tested in hair with the capacity to test a wide range of drugs and some of their metabolites. CONCLUSIONS: Reported data were mostly from small-scale studies and alternative biomatrices need to be qualified in large and diverse populations for the demonstration of feasibility in operational settings. High-quality interventional studies will improve the uptake of alternative biomatrices in guidelines and accelerate implementation in programmatic tuberculosis treatment.

Development and validation of an LC-MS/MS assay for the quantitative analysis of alprazolam, α-hydroxyalprazolam and hydrocodone in dried blood spots.

Dried blood spot (DBS) has been used as an alternative matrix in drug testing. In forensic testing it offers enhanced stability of analytes and ease of storage that requires minimal space. This is compatible with long term archiving of large numbers of samples for future investigation. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify alprazolam, α-hydroxyalprazolam, and hydrocodone in a DBS sample that has been stored for 17 years. We achieved linear dynamic ranges (0.1-50 ng/mL) that capture wide ranges of concentration of the analytes below and above their reported reference ranges, and limits of detection (0.05 ng/mL) of 40-100X lower than the lower limit of the analyte's reference ranges. The method was validated according to FDA and CLSI guidelines and successfully confirmed and quantified alprazolam and α-hydroxyalprazolam in a forensic DBS sample.

Profound Hyperthermia Associated With Fentanyl and Cocaine Use With Suspected Synephrine Adulteration.

BACKGROUND: Although hyperthermia is described after cocaine intoxication, the two hyperthermic cases discussed were unusual in severity and duration for cocaine alone. Synephrine was found in biological samples of these patients in high concentrations and was suspected to be an adulterant in illicitly obtained drugs. CASE REPORT: Two patients presented to a tertiary care university hospital within 2 days of each other after recreational drug use with delayed and protracted hyperthermia. Synephrine was later found in high concentrations in biological samples as an unexpected drug adulterant. The first patient's presentation came with delayed recognition of hyperthermia and implementation of aggressive cooling measures; he entered multisystem organ failure with prolonged intensive care unit stay and significant morbidity. The second patient's hyperthermia was recognized promptly, and she received early, aggressive cooling, including deep sedation and ice water submersion. She left against medical advice from the hospital at her baseline 3 days after presentation. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Synephrine is a suspected adulterant that may be associated with profound hyperthermia. Early recognition of drug overdose and working knowledge of common adulterants can facilitate early targeted management, such as aggressive cooling measures, which may prevent morbidity and mortality.

Glyphosate exposure and preterm birth: A nested case-control pilot study.

Preterm birth (PTB) is associated with a high risk of infant mortality and long-term adverse health effects. Glyphosate is a broad-spectrum herbicide applied in agricultural and non-agricultural settings. Studies suggested an association between maternal exposure to glyphosate and PTB among mostly racially homogenous populations, though results were inconsistent. The objective of this pilot study was to inform the design of a larger and more definitive study of glyphosate exposure and adverse birth outcomes in a racially-diverse population. Urine was obtained from 26 women with a PTB as cases and 26 women with a term birth as controls, from participants enrolled in a birth cohort in Charleston, South Carolina. We used binomial logistic regression to estimate associations between urinary glyphosate and the odds of PTB, and multinomial regression to estimate associations between maternal racial identity and urinary glyphosate among controls. Glyphosate was unrelated to PTB (odds ratio (OR) = 1.06, 95% CI: 0.61, 1.86). Women who identified as Black had greater odds (OR = 3.83, 95% CI: 0.13, 111.33) of having categorical "high" glyphosate (> 0.28 ng/mL) and lesser odds (OR = 0.79, 95% CI: 0.05, 12.21) of "low" glyphosate (< 0.03 ng/mL) relative to women who identified as white, suggesting a potential racial disparity, although the effect estimates were imprecise and included the null. Given concerns of potential reproductive toxicity of glyphosate, the results merit confirmation in a larger investigation to determine specific sources of glyphosate exposure, incorporating longitudinal urinary glyphosate measures during pregnancy and a comprehensive measure of diet.

Delta-8-Tetrahydrocannabinol Exposure and Confirmation in Four Pediatric Patients.

INTRODUCTION: Delta-8-tetrahydrocannabinol (THC) is a known isomer of delta-9-THC, both found naturally in the Cannabis sativa plant and thought to have similar potency. Delta-8-THC products are widely accessible in retail shops which may lead to a rise in pediatric exposures with substantial clinical effects. CASE REPORT: This is a case series of four pediatric patients that were seen between June and September 2021. The patients presented with varied clinical symptoms including confusion, somnolence, seizure-like activity, hypotension, and tachycardia after exposure to delta-8-THC products obtained in retail shops. Basic urine drug screen immunoassays revealed positive results for cannabinoids in all patients. Subsequent confirmatory drug analysis of residual biological samples of blood and/or urine was sent to the University of California San Francisco Clinical Toxicology and Environment Biomonitoring Laboratory with the assistance of the Drug Enforcement Administration's Toxicology Testing Program (DEA TOX). Confirmatory testing revealed 11-nor-9-carboxy-delta-8-THC, the metabolite of delta-8-THC. Delta-9-THC and its metabolites were not detected on confirmatory testing in any of the cases. DISCUSSION: Clinical effects of delta-8-THC in children include but are not limited to altered mental status, seizure-like activity, and vital sign abnormalities. Delta-8-THC exposure may lead to a positive urine drug screen for cannabinoids, but confirmatory testing is needed to differentiate from delta-9-THC.

Reduction of daily-use parabens and phthalates reverses accumulation of cancer-associated phenotypes within disease-free breast tissue of study subjects.

Estrogenic overstimulation is carcinogenic to the human breast. Personal care products (PCPs) commonly contain xenoestrogens (XE), such as parabens and phthalates. Here, we identified the adverse effects of persistent exposure to such PCPs directly within human estrogen responsive breast tissue of subjects enrolled in a regimen of reduced XE use (REDUXE). Pre- and post-intervention fine needle aspirates (FNAs) of the breast were collected from healthy volunteers who discontinued the use of paraben and phthalate containing PCPs over a 28 d period. Based on high-dimensional gene expression data of matched FNA pairs of study subjects, we demonstrate a striking reversal of cancer-associated phenotypes, including the PI3K-AKT/mTOR pathway, autophagy, and apoptotic signaling networks within breast cells of REDUXE compliant subjects. These, and other altered phenotypes were detected together with a significant reduction in urinary parabens and phthalate metabolites. Moreover, in vitro treatment of paired FNAs with 17ß-estradiol (E2), displayed a 'normalizing' impact of REDUXE on gene expression within known E2-modulated pathways, and on functional endpoints, including estrogen receptor alpha: beta ratio, and S-phase fraction of the cell cycle. In a paradigm shifting approach facilitated by community-based participatory research, REDUXE reveals unfavorable consequences from exposure to XEs from daily-use PCPs. Our findings illustrate the potential for REDUXE to suppress pro-carcinogenic phenotypes at the cellular level towards the goal of breast cancer prevention.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (ßarr2d366) ß-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gßγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB1 activation approximating an n-pentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

Drug testing in the era of new psychoactive substances.

Traditional clinical toxicology involves the analysis of patient urine samples by immunoassays designed to detect opiates/opioids, amphetamine/methamphetamine, benzodiazepines, barbiturates, cocaine metabolite and tetrahydrocannabinol. Expanded drug screens may also include assays for oxycodone, buprenorphine, methadone, 6-monoacetylmorphine, phencyclidine and fentanyl. Patient samples that are positive are commonly reflexed to be run on a liquid chromatography-tandem mass spectrometry confirmatory assay, as are samples that are negative for drugs that are prescribed to the patient. These mass spectrometry assays are targeted and so only detect the drugs or drug metabolites that they were designed to detect. With the explosion of new psychoactive substances in the past decade, it has become necessary for clinical laboratories to reevaluate traditional targeted drug screening approaches. The utility of high-resolution mass spectrometry in this arena has been recognized and this review will discuss the traditional approach to, and the recent advances in clinical toxicology including data collection and interrogation strategies for new psychoactive substances using high-resolution mass spectrometry (HRMS). Various modes of data processing techniques including targeted analysis, suspect screening and non-targeted analysis will also be described using HRMS. Several published methods will be described to demonstrate the utility of various data acquisition and processing techniques using HRMS in NPS analysis specifically.

Agreement Between Measures of Adherence to Isoniazid Preventive Therapy Among People With HIV in Uganda.

Background: Isoniazid (INH) preventative therapy is recommended for people with HIV (PWH) in resource-constrained settings. Valid measures are needed to assess adherence. We aimed to examine agreement between measures overall and by level of social desirability. Methods: PWH with latent tuberculosis (TB) were recruited in Mbarara, Uganda. Past 30-day adherence was measured by the number of days with pill bottle openings using a medication event monitoring system (MEMS) and self-reported number of days pills taken. INH concentration (INH plus acetyl INH and their ratio) in hair samples was measured. We used Bland-Altman plots to examine agreement between adherence measures and calculated the area under the receiver operating characteristics curve (AUROC) to determine if INH hair concentration predicted optimal MEMS-measured adherence (≥90%). Results: A total of 301 participants enrolled; 92% were virologically suppressed, and adherence was high. The median (interquartile range [IQR]) number of pill bottle openings in 30 days was 28 (24-30) compared with 30 (28-30) via self-report. The median INH concentration (IQR) was 36.2 (17.2-62.4), and the INH:acetyl ratio was 2.43 (0.99-3.92). Agreement between self-reported and MEMS adherence was greater at more optimal adherence levels. INH:acetyl INH ratio was not predictive of optimal adherence according to MEMS (AUROC, 0.62; 95% CI, 0.52-0.72) in a subset (n = 161). Conclusions: Lower MEMS adherence levels compared with self-report suggest the need for objective adherence measures. Biologic measures have potential, although in this study INH concentration was not predictive of MEMS measured adherence. More data are needed to assess the accuracy of biologic measures.

Glyphosate exposure in early pregnancy and reduced fetal growth: a prospective observational study of high-risk pregnancies.

BACKGROUND: Prenatal glyphosate (GLY) exposure is associated with adverse reproductive outcomes in animal studies. Little is known about the effects of GLY exposure during pregnancy in the human population. This study aims to establish baseline urine GLY levels in a high-risk and racially diverse pregnancy cohort and to assess the relationship between prenatal GLY exposure and fetal development and birth outcomes. METHODS: Random first trimester urine specimens were collected from high risk pregnant women between 2013 and 2016 as part of the Indiana Pregnancy Environmental Exposures Study (PEES). Demographic and clinical data were abstracted from mother and infant medical records. Urine glyphosate levels were measured as a proxy for GLY exposure and quantified using liquid chromatography-tandem mass spectrometry. Primary outcome variables included gestation-adjusted birth weight percentile (BWT%ile) and neonatal intensive care unit (NICU) admission. Relationships between primary outcome variables and GLY exposure were assessed using univariate and multivariate linear and logistic regression models. RESULTS: Urine GLY levels above the limit of detection (0.1 ng/mL) were found in 186 of 187 (99%) pregnant women. Further analyses were limited to 155 pregnant women with singleton live births. The mean age of participants was 29 years, and the majority were non-Hispanic white (70%) or non-Hispanic Black (21%). The mean (± SD) urine GLY level was 3.33 ± 1.67 ng/mL. Newborn BWT%iles were negatively related to GLY (adjusted slope ± SE = -0.032 + 0.014, p = 0.023). Infants born to women living outside of Indiana's large central metropolitan area were more likely to have a lower BWT%ile associated with mother's first trimester GLY levels (slope ± SE = -0.064 ± 0.024, p = 0.007). The adjusted odds ratio for NICU admission and maternal GLY levels was 1.16 (95% CI: 0.90, 1.67, p = 0.233). CONCLUSION: GLY was found in 99% of pregnant women in this Midwestern cohort. Higher maternal GLY levels in the first trimester were associated with lower BWT%iles and higher NICU admission risk. The results warrant further investigation on the effects of GLY exposure in human pregnancies in larger population studies.

Synthesis and pharmacological evaluation of newly detected synthetic cannabinoid receptor agonists AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA, MDMB-4F-BUTINACA and their analogs.

Synthetic cannabinoid receptor agonists (SCRAs) continue to make up a significant portion new psychoactive substances (NPS) detected and seized worldwide. Due to their often potent activation of central cannabinoid receptors in vivo, use of SCRAs can result in severe intoxication, in addition to other adverse health effects. Recent detections of AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA and MDMB-4F-BUTINACA mark a continuation in the appearance of SCRAs bearing novel tail substituents. The proactive characterization campaign described here has facilitated the detection of several new SCRAs in toxicological case work. Here we detail the synthesis, characterization, and pharmacological evaluation of recently detected SCRAs, as well as a systematic library of 32 compounds bearing head, tail, and core group combinations likely to appear in future. In vitro radioligand binding assays revealed most compounds showed moderate to high affinity at both CB1 (pK i = < 5 to 8.89 ± 0.09 M) and CB2 (pK i = 5.49 ± 0.03 to 9.92 ± 0.09 M) receptors. In vitro functional evaluation using a fluorescence-based membrane potential assay showed that most compounds were sub-micromolar to sub-nanomolar agonists at CB1 (pEC50 = < 5 to 9.48 ± 0.14 M) and CB2 (pEC50 = 5.92 ± 0.16 to 8.64 ± 0.15 M) receptors. An in silico receptor-ligand docking approach was utilized to rationalize binding trends for CB2 with respect to the tail substituent, and indicated that rigidity in this region (i.e., 4-cyanobutyl) was detrimental to affinity.

Host and gut bacteria share metabolic pathways for anti-cancer drug metabolism.

Pharmaceuticals have extensive reciprocal interactions with the microbiome, but whether bacterial drug sensitivity and metabolism is driven by pathways conserved in host cells remains unclear. Here we show that anti-cancer fluoropyrimidine drugs inhibit the growth of gut bacterial strains from 6 phyla. In both Escherichia coli and mammalian cells, fluoropyrimidines disrupt pyrimidine metabolism. Proteobacteria and Firmicutes metabolized 5-fluorouracil to its inactive metabolite dihydrofluorouracil, mimicking the major host mechanism for drug clearance. The preTA operon was necessary and sufficient for 5-fluorouracil inactivation by E. coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice and was prevalent in the gut microbiomes of colorectal cancer patients. The conservation of both the targets and enzymes for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug efficacy and side-effect profiles.

Obstetric risk in pregnancy interacts with hair cortisone levels to reduce gestational length.

Background: Maternal psychological stress has been linked to preterm birth. However, the differential contribution of psychological stress versus stress hormones is not clear. Studies focus primarily on perceived stress and cortisol, with few assessing its inter-convertible hormone cortisone. Furthermore, little is known about the potential moderating roles of obstetric risk and fetal sex in the relationship between maternal stress and gestational length. This gap in knowledge is particularly evident for rural women who typically experience chronic multiple stressors during pregnancy. We explored the relationship of hormonal and psychological stress to gestational length and the effects of obstetric risks and fetal sex on this relationship among Kenyan pregnant women. Methods: The sample included 130 women recruited between 22 to 28 weeks gestation. They completed a clinical and sociodemographic questionnaire together with the Perceived Stress Scale and provided a hair sample for cortisol and cortisone assay. Women underwent an ultrasound to assess weeks of gestation. At delivery, their pregnancy-related health problems were identified using information extracted from medical records to compile each woman's number of pregnancy risks on the Obstetric Medical Risk Index (OMRI). Results: Perceived stress and hair cortisol were not significant predictors of gestational length. However, a greater number of obstetric risks on the OMRI was associated with shorter gestational length. This effect was further explained by the interaction between obstetric risk and hair cortisone (B = 0.709, p = 0.02). Hair cortisone levels of mothers who had a shorter gestation were significantly higher in mothers with 2 or more risks on the OMRI but not among mothers with only one or no risks (t = 2.39, p = 0.02). Fetal sex had no relationship to gestational length and also had no moderating effect on the relationship between any stress-related metric and gestational length. Conclusion: Cortisone levels may increase in anticipation of shorter gestation as a compensatory response to increased obstetric risk. Elevated cortisone may be a more sensitive marker of risk for early delivery than cortisol or psychological stress, with salience for both the male and female fetus.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

Defining Steric Requirements at CB1 and CB2 Cannabinoid Receptors Using Synthetic Cannabinoid Receptor Agonists 5F-AB-PINACA, 5F-ADB-PINACA, PX-1, PX-2, NNL-1, and Their Analogues.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N-alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB1 and CB2, respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds (Ki = 0.32 to >10 000 nM, EC50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain (tert-butyl > iso-propyl > iso-butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB1-mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Structure-activity relationships of valine, tert-leucine, and phenylalanine amino acid-derived synthetic cannabinoid receptor agonists related to ADB-BUTINACA, APP-BUTINACA, and ADB-P7AICA.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB1 and CB2), and in vivo cannabimimetic activity. All compounds showed high affinity for CB1 (K i 0.299-538 nM) and most at CB2 (K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB1 and CB2 in a fluorescence-based membrane potential assay and a ßarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB1/CB2 binding and CB1/CB2 functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg-1 and 10 mg kg-1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg-1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.