Cannabinoid receptor 1 positive allosteric modulator ZCZ011 shows differential effects on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

The cannabinoid receptor type 1 (CB1R) is a promising therapeutic target for various neurodegenerative diseases, including HIV-1-associated neurocognitive disorder (HAND). However, the therapeutic potential of CB1R by direct activation is limited due to its psychoactive side effects. Therefore, research has focused on indirectly activating the CB1R by utilizing positive allosteric modulators (PAMs). Studies have shown that CB1R PAMs (ZCZ011 and GAT211) are effective in mouse models of Huntington's disease and neuropathic pain, and hence, we assess the therapeutic potential of ZCZ011 in a well-established mouse model of neuroHIV. The current study investigates the effect of chronic ZCZ011 treatment (14 days) on various behavioral paradigms and the endocannabinoid system in HIV-1 Tat transgenic female and male mice. Chronic ZCZ011 treatment (10 mg/kg) did not alter body mass, locomotor activity, or anxiety-like behavior regardless of sex or genotype. However, differential effects were noted in hot plate latency, motor coordination, and recognition memory in female mice only, with ZCZ011 treatment increasing hot plate latency and improving motor coordination and recognition memory. Only minor effects or no alterations were seen in the endocannabinoid system and related lipids except in the cerebellum, where the effect of ZCZ011 was more pronounced in female mice. Moreover, AEA and PEA levels in the cerebellum were positively correlated with improved motor coordination in female mice. In summary, these findings indicate that chronic ZCZ011 treatment has differential effects on antinociception, motor coordination, and memory, based on sex and HIV-1 Tat expression, making CB1R PAMs potential treatment options for HAND without the psychoactive side effects.

Systematic Structure-Activity Relationship Study of Nalfurafine Analogues toward Development of Potentially Nonaddictive Pain Management Treatments.

Despite the availability of numerous pain medications, the current array of Food and Drug Administration-approved options falls short in adequately addressing pain states for numerous patients and consequently worsens the opioid crisis. Thus, it is imperative for basic research to develop novel and nonaddictive pain medications. Toward addressing this clinical goal, nalfurafine (NLF) was chosen as a lead and its structure-activity relationship (SAR) systematically studied through design, syntheses, and in vivo characterization of 24 analogues. Two analogues, 21 and 23, showed longer durations of action than NLF in a warm-water tail immersion assay, produced in vivo effects primarily mediated by KOR and DOR, penetrated the blood-brain barrier, and did not function as reinforcers. Additionally, 21 produced fewer sedative effects than NLF. Taken together, these results aid the understanding of NLF SAR and provide insights for future endeavors in developing novel nonaddictive therapeutics to treat pain.

Ethyl Acetate in E-liquids: Implications for Breath Testing.

Electronic cigarette liquids (e-liquids) can contain a variety of chemicals to impart flavors, smells, and pharmacological effects. Surveillance studies have identified hundreds of chemicals used in e-liquids which have known health and safety implications. Ethyl acetate has been identified as a common constituent of e-liquids. Ethyl acetate is rapidly hydrolyzed to ethanol in vivo. Animal studies have demonstrated that inhaling >2000 mg/L ethyl acetate can lead to accumulation of ethanol in the blood at concentrations greater than 1000 mg/L, or 0.10%. A "Heisenberg" e-liquid was submitted to the Laboratory for Forensic Toxicology Research for analysis after a random workplace drug test resulted in a breath test result of 0.019% for a safety-sensitive position employee. Analysis of this sample resulted in the detection of 1488 ± 6 mg/L ethyl acetate. The evaluation of several "Heisenberg" e-liquids determined that these products contain ethyl acetate. The identification of ethyl acetate in e-liquids demonstrates poor regulatory oversight and enforcement that potentially has consequences to preliminary breath ethanol testing and interpretations. The accumulation of ethanol in the breath from the ingestion/inhalation of ethyl acetate from an e-liquid used prior to a breath test may contribute to the detection of ethanol.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

N-oleoyl alanine attenuates nicotine reward and spontaneous nicotine withdrawal in mice.

BACKGROUND: As nicotine dependence represents a longstanding major public health issue, new nicotine cessation pharmacotherapies are needed. Administration of N-oleoyl glycine (OlGly), an endogenous lipid signaling molecule, prevents nicotine-induced conditioned place preference (CPP) through a peroxisome proliferator-activated receptor-alpha (PPARα) dependent mechanism, and also ameliorated withdrawal signs in nicotine-dependent mice. Pharmacological evidence suggests that the methylated analog of OlGly, N-oleoyl alanine (OlAla), has an increased duration of action and may offer translational benefit. Accordingly, OlAla was assessed in nicotine CPP and dependence assays as well as its pharmacokinetics compared to OlGly. METHODS: ICR female and male mice were tested in nicotine-induced CPP with and without the PPARα antagonist GW6471. OlAla was also assessed in nicotine-dependent mice following removal of nicotine minipumps: somatic withdrawal signs, thermal hyper-nociception and altered affective behavior (i.e., light/dark box). Finally, plasma and brain were collected after administration of OlGly or OlAla and analyzed by high-performance liquid chromatography tandem mass spectrometry. RESULTS: OlAla prevented nicotine-induced CPP, but this effect was not blocked by GW6471. OlAla attenuated somatic and affective nicotine withdrawal signs, but not thermal hyper-nociception in nicotine-dependent mice undergoing withdrawal. OlAla and OlGly showed similar time-courses in plasma and brain. CONCLUSIONS: The observation that both molecules showed similar pharmacokinetics argues against the notion that OlAla offers increased metabolic stability. Moreover, while these structurally similar lipids show efficacy in mouse models of reward and dependence, they reduce nicotine reward through distinct mechanisms.

Effects of acute cannabidiol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Background: Some evidence suggests that cannabidiol (CBD) has potential to help alleviate HIV symptoms due to its antioxidant and anti-inflammatory properties. Here we examined acute CBD effects on various behaviors and the endocannabinoid system in HIV Tat transgenic mice. Methods: Tat transgenic mice (female/male) were injected with CBD (3, 10, 30 mg/kg) and assessed for antinociception, activity, coordination, anxiety-like behavior, and recognition memory. Brains were taken to quantify endocannabinoids, cannabinoid receptors, and cannabinoid catabolic enzymes. Additionally, CBD and metabolite 7-hydroxy-CBD were quantified in the plasma and cortex. Results: Tat decreased supraspinal-related nociception and locomotion. CBD and sex had little to no effects on any of the behavioral measures. For the endocannabinoid system male sex was associated with elevated concentration of the proinflammatory metabolite arachidonic acid in various CNS regions, including the cerebellum that also showed higher FAAH expression levels for Tat(+) males. GPR55 expression levels in the striatum and cerebellum were higher for females compared to males. CBD metabolism was altered by sex and Tat expression. Conclusion: Findings indicate that acute CBD effects are not altered by HIV Tat, and acute CBD has no to minimal effects on behavior and the endocannabinoid system.

NCP, a dual kappa and mu opioid receptor agonist, is a potent analgesic against inflammatory pain without reinforcing or aversive properties.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Acute Effects of Monoacylglycerol Lipase Inhibitor ABX1431 on Neuronal Hyperexcitability, Nociception, Locomotion, and the Endocannabinoid System in HIV-1 Tat Male Mice.

Background: Evidence suggests that monoacylglycerol lipase (MAGL) inhibitors can potentially treat HIV symptoms by increasing the concentration of 2-arachidonoylglycerol (2-AG). We examined a selective MAGL inhibitor ABX1431 in the context of neuroHIV. Methods: To assess the effects of ABX1431, we conducted in vitro and in vivo studies. In vitro calcium imaging on frontal cortex neuronal cultures was performed to evaluate the role of ABX1431 (10, 30, 100 nM) on transactivator of transcription (Tat)-induced neuronal hyperexcitability. Following in vitro experiments, in vivo experiments were performed using Tat transgenic male mice. Mice were treated with 4 mg/kg ABX1431 and assessed for antinociception using tail-flick and hot plate assays followed by locomotor activity. After the behavioral experiments, their brains were harvested to quantify endocannabinoids (eCB) and related lipids through mass spectrometry, and cannabinoid type-1 and -2 receptors (CB1R and CB2R) were quantified through western blot. Results: In vitro studies revealed that adding Tat directly to the neuronal cultures significantly increased intracellular calcium concentration, which ABX1431 completely reversed at all concentrations. Preincubating the cultures with CB1R and CB2R antagonists showed that ABX1431 exhibited its effects partially through CB1R. In vivo studies demonstrated that acute ABX1431 increased overall total distance traveled and speed of mice regardless of their genotype. Mass spectrometry and western blot analyses revealed differential effects on the eCB system based on Tat expression. The 2-AG levels were significantly upregulated following ABX1431 treatment in the striatum and spinal cord. Arachidonic acid (AA) was also upregulated in the striatum of vehicle-treated Tat(+) mice. No changes were noted in CB1R expression levels; however, CB2R levels were increased in ABX1431-treated Tat(-) mice only. Conclusion: Findings indicate that ABX1431 has potential neuroprotective effects in vitro partially mediated through CB1R. Acute treatment of ABX1431 in vivo shows antinociceptive effects, and seems to alter locomotor activity, with upregulating 2-AG levels in the striatum and spinal cord.

Investigation of Cannabidiol in the Mouse Drug Discrimination Paradigm.

Introduction: Cannabidiol (CBD) has gained considerable public and scientific attention because of its known and potential medicinal properties, as well as its commercial success in a wide range of products. Although CBD lacks cannabimimetic intoxicating side effects in humans and fails to substitute for cannabinoid type-1 receptor (CB1R) agonists in laboratory animal models of drug discrimination paradigm, anecdotal reports describe it as producing a "pleasant" subjective effect in humans. Thus, we speculated that this phytocannabinoid may elicit distinct subjective effects. Accordingly, we investigated whether mice would learn to discriminate CBD from vehicle. Additionally, we examined whether CBD may act as a CB1R allosteric and whether it would elevate brain endocannabinoid concentrations. Materials and Methods: C57BL/6J mice underwent discrimination training of either CBD or the high-efficacy CB1R agonist CP55,940 from vehicle. Additionally, we examined whether CBD or the CB1R-positive allosteric modulator ZCZ011 would alter the CP55,940 discriminative cue. Finally, we tested whether an acute CBD injection would elevate endocannabinoid levels in brain, and also quantified blood and brain levels of CBD. Results: Mice failed to discriminate high doses of CBD from vehicle following 124 training days, though the same subjects subsequently acquired CP55,940 discrimination. In a second group of mice trained to discriminate CP55,940, CBD neither elicited substitution nor altered response rates. A single injection of 100 or 200 mg/kg CBD did not affect brain levels of endogenous cannabinoids and related lipids and resulted in high drug concentrations in blood and whole brain at 0.5 h and continued to increase at 3 h. Discussion: CBD did not engender an interoceptive stimulus, did not disrupt performance in a food-motivated operant task, and lacked apparent effectiveness in altering brain endocannabinoid levels or modulating the pharmacological effects of a CB1R agonist. These findings support the assertions that CBD lacks abuse liability and its acute administration does not appear to play a functional role in modulating key components of the endocannabinoid system in whole animals.

Polycaprolactone microparticles for the subcutaneous administration of cannabidiol: in vitro and in vivo release.

Cannabidiol (CBD) has become a highly attractive entity in therapeutics. However, its low aqueous solubility, instability and handling problems limit the development of effective CBD formulations. Subcutaneously administered CBD-loaded polycaprolactone microparticles (MP) represent an interesting strategy to overcome these challenges. This work focuses on evaluating the pharmacokinetics of CBD formulated in polymer microparticles for subcutaneous administration and characterising its release. The mean release time (MRLT) parameter is used to compare the release of CBD from two microparticle formulations in vitro and in a mouse model. After the administration of CBD in solution, a bicompartmental distribution is observed due to the extensive diffusion to the brain, being the brain/blood AUC ratio 1.29. The blood and brain mean residence time (MRT) are 0.507 ± 0.04 and 0.257 ± 0.0004 days, respectively. MP prepared with two drug/polymer ratios (15/150-MP and 30/150-MP) are designed, showing similar in vitro dissolution profiles (similarity factor (f2) is 63.21), without statistically significant differences between MRLTin vitro values (4.68 ± 0.63 and 4.32 ± 0.05 days). However, considerable differences in blood and brain profiles between both formulations are detected. The blood and brain MRT values of 15/150-MP are 6.44 ± 0.3 days and 6.15 ± 0.25 days, respectively, whereas significantly lower values 3.91 ± 0.29 days and 2.24 ± 0.64 days are obtained with 30/150-MP. The extended release of CBD during 10 days after a single subcutaneous administration is achieved.

Effects of acute Δ9-tetrahydrocannabinol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Cannabis use is highly prevalent especially among people living with HIV (PLWH). Activation of the anti-inflammatory and neuroprotective endocannabinoid system by phytocannabinoids, i.e. Δ9-tetrahydrocannabinol (THC), has been proposed to reduce HIV symptoms. However, THC's effects on HIV-related memory deficits are unclear. Using HIV-1 Tat transgenic mice, the current study investigates acute THC effects on various behavioral outcomes and the endocannabinoid system. For the rodent tetrad model, THC doses (1, 3, 10 mg/kg) induced known antinociceptive effects, with Tat induction increasing antinociceptive THC effects at 3 and 10 mg/kg doses. Only minor or no effects were noted for acute THC on body temperature, locomotor activity, and coordination. Increased anxiety-like behavior was found for females compared to males, but acute THC had no effect on anxiety. Object recognition memory was diminished by acute THC in Tat(-) females but not Tat(+) females, without affecting males. The endocannabinoid system and related lipids were not affected by acute THC, except for THC-induced decreases in CB1R protein expression levels in the spinal cord of Tat(-) mice. Female sex and Tat induction was associated with elevated 2-AG, AEA, AA, CB1R, CB2R, FAAH and/or MAGL expression in various brain regions. Further, AEA levels in the prefrontal cortex of Tat(+) females were negatively associated with object recognition memory. Overall, findings indicate that acute THC exerts differential effects on antinociception and memory, dependent on sex and HIV Tat expression, potentially in relation to an altered endocannabinoid system, which may be of relevance in view of potential cannabis-based treatment options for PLWH.

High-performance liquid chromatography-tandem mass spectrometry method for the analysis of N-oleoyl glycine and N-oleoyl alanine in brain and plasma.

Interest has increased in the role of N-acyl amino acids in a variety of disease states and as potential pharmacotherapies. Recently, N-oleoyl glycine and N-oleoyl alanine have shown promise in reducing the rewarding effects of drugs of abuse and alleviating withdrawal signs in rodent models. Previously published methods for the quantitation of these analytes by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in tissue were part of extensive lipidomic panels which may result in limited sensitivity and selectivity and also reported low recovery. Presented is a method for the extraction and HPLC-MS/MS analysis of N-oleoyl glycine and N-oleoyl alanine. The bias and precision of the assay were determined to be within ± 20%. The method was shown to be reliable and robust, with over 90% recovery for the low-level analytes. Increasing concentrations of N-oleoyl glycine and N-oleoyl alanine were quantitated in mouse brain and plasma following exogenous administration. This method was developed to serve to support studies investigating the pharmacokinetics and involvement of N-oleoyl glycine and N-oleoyl alanine in drug dependence and other diseases.

The preanalytical stability of emerging cannabinoid analogs (delta-8 THC and its metabolites, delta-10 THC and carboxy-HHC) in urine.

There has been a surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This increase is attributed not only to the use of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol, the most abundant phytocannabinoid components of cannabis and hemp, respectively, but also to the use of many other emerging THC analogs. Structurally, these analogs are similar to ∆9-THC. Urine specimens for drug analysis are often collected offsite and transported to a laboratory for analysis. Screening assays are usually the first step in urine drug testing. These assays are usually qualitative and automated, which for negative specimens, reduce cost and reporting time. The stability of ∆9-THC and its metabolites has been known for some time; however, the stability of emerging analogs has not been elucidated, and therefore, assuming equivalent storage stability can be erroneous. Previous work assessed the cross-reactivity of ∆8-THC and its major metabolites, the ∆10-THC chiral analogs and the chiral 11-COOH-hexahydrocannabinol analogs. Stability was assessed for each analyte at a concentration two times greater than the analytes' determined decision point. Samples were prepared in drug-free urine at three different pHs (4.5, 7 and 9) and stored at three different temperatures (4°C, 20°C and 45°C) in triplicate. Samples were analyzed utilizing the Lin-Zhi International Cannabinoids Enzyme Immunoassay cannabinoid screening kit calibrated at the 25 ng/mL cut-off. Overall, the cannabinoid analogs produced diminishing instrument responses depending on pH and temperature. The parent analogs were not detected after a single day at 45°C regardless of pH. In general, carboxylic acid analogs at the acidic pH (4.5) produced diminished instrument responses when compared to their counterparts stored at neutral (7) and basic (9) pH. The time, storage temperature and pH of urine specimens may affect the screening results of specimens collected for cannabinoid drug screening.

The cross-reactivity of cannabinoid analogs (delta-8-THC, delta-10-THC and CBD), their metabolites and chiral carboxy HHC metabolites in urine of six commercially available homogeneous immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to delta-9-tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids-Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay-Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC-Thermo Fisher Scientific, ONLINE DAT Cannabinoid II-Roche Diagnostics and Syva EMIT®II Plus-Siemens Healthineers) were evaluated at two different cutoff concentrations: 50 ng/mL and 20 or 25 ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross-reactivity was evaluated by preparing each analyte at 20, 50, 100 and 1,000 ng/mL in urine. Analytes that did not cross-react at 1,000 ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: delta-8-THC, 11-OH-delta-8-THC, 11-COOH-delta-8-THC, 6-OH-CBD, 7-OH-CBD, all delta-10-THC and HHC carboxylic acid chiral analogs and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross-react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A and olivetolic acid.

Survey of U.S. Residents and Their Usage of Electronic Cigarettes with Drugs Other Than Nicotine.

Electronic cigarettes (e-cigs), originally intended to be used as cigarette substitutes, have evolved into discreet devices for consuming drugs other than nicotine (DOTNs). Presented are the results of an exploratory survey in which information regarding demographics, e-cig device type, DOTN use, frequency and context of use was collected. The average reported age of respondents was 27.4 years of age (SD = 12.0), and respondents predominantly identified as male (73%). Vape pens (disposable or refillable) were the most reported device across all DOTN classes. Cannabinoids were the most reported class of DOTN used, for both lifetime and past 30-day use. Other DOTNs reported included herbal supplements, amphetamines, caffeine, kratom, vitamins, opiates, DMT, fentanyl, and ketamine. Combinations of DOTNs used in e-cigs and trends in poly-substance use were reported. The most commonly reported context was vaping alone, followed by with friends, at home, and at social events; less commonly reported contexts included when driving, at work, and at school. Results from this study are useful for developing future national surveys to consider a comprehensive substance use-focused strategy that includes vaping, building awareness of DOTN e-cig use, and highlighting public safety issues in driving impairment, crime scene investigations, and death investigations.

The Role of Morphine-Induced Impairment of Intestinal Epithelial Antibacterial Activity in Dysbiosis and its Impact on the Microbiota-Gut-Brain Axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupt the intestinal epithelial layer and cause intestinal dysbiosis. Inhibiting opioid-induced dysbiosis can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. However, the mechanism underlying opioid-induced dysbiosis remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine exposure reduces expression of the antimicrobial peptide, Regenerating islet-derived 3 gamma (Reg3γ), in the ileum resulting in reduced intestinal antimicrobial activity against Gram-positive bacteria, L. reuteri. Fecal samples from morphine-treated mice had reduced levels of the phylum, Firmicutes, concomitant with reduced levels of short-chain fatty acid, butyrate. Fecal microbial transplant (FMT) from morphine-naïve mice restored the antimicrobial activity, the expression of Reg3γ, and prevented the increase in intestinal permeability and the development of antinociceptive tolerance in morphine-dependent mice. Similarly, oral gavage with sodium butyrate dose-dependently reduced the development of antinociceptive tolerance, and prevented the downregulation of Reg3γ and the reduction in antimicrobial activity. The alpha diversity of the microbiome was also restored by oral butyrate in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which morphine disrupts the microbiota-gut-brain axis.

The impact of vaping ethanol-containing electronic cigarette liquids on roadside impairment investigations.

Legal professionals and others have suggested that vaping electronic cigarettes (e-cigs) prior to or during ethanol breath testing may produce false positives. Preliminary breath tests (PBTs) and evidentiary breath tests (EBTs) measure ethanol in exhaled breath and standardized field sobriety tests (SFSTs) are used to assess impairment. Ethanol has been identified in e-cig liquids (e-liquids). Presented are a series of experiments designed to determine the mechanics of vaping ethanol using an e-cig and the effects of vaping ethanol on the SFSTs and breath tests used by law enforcement officers (LEO). Twelve participants (five females, age: 21-32 and seven males, age: 21-55), vaped either one or ten puffs of an e-liquid (0% or 20% ethanol). LEOs assessed impairment using SFSTs (12 and 42 min), PBTs (<1, 27, 32, 37 and 57 min) and EBTs (2, 29, 34, 39 and 59 min) post-vaping. A self-assessment test was administered post-vaping (22 and 52 min). Baseline responses for all measures were collected prior to vaping. Results demonstrated that ethanol in the e-liquids was aerosolized by e-cigs and produced particles that could reach the deep lung tissue based on mean-mass diameter. Ethanol was detected by PBT <3 min after participants vaped one (0.007-0.030 g/210 L) or ten puffs (013-0.074 g/210 L) of a 20% ethanol e-liquid. Ethanol was not detected by PBT at any subsequent time point. Ethanol was not detected by the EBT under any condition. Impairment was not indicated by the SFST. Some subjective effects were reported, but few statistically significant differences between conditions were indicated. A wait period prior to ethanol breath testing is not always mandated, depending on jurisdiction, or observed in all applications, such as workplace testing. The results demonstrate that a wait period must be employed to prevent vaping-related false-positive breath ethanol results.

Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates Cisplatin-Induced Nephropathy in Mice.

Cisplatin is a potent first-line therapy for many solid malignancies, such as breast, ovarian, lung, testicular, and head and neck cancer. However, acute kidney injury (AKI) is a major dose-limiting toxicity in cisplatin therapy, which often hampers the continuation of cisplatin treatment. The endocannabinoid system, consisting of anandamide (AEA) and 2-arachidonoylglycerol and cannabinoid receptors, participates in different kidney diseases. Inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme for the degradation of AEA and AEA-related N-acylethanolamines, elicits anti-inflammatory effects; however, little is known about its role in cisplatin nephrotoxicity. The current study tested the hypothesis that genetic deletion of Faah mitigates cisplatin-induced AKI. Male wild-type C57BL6 (WT) and Faah-/- mice were administered a single dose of intraperitoneal injection of cisplatin (30 mg/kg) and euthanatized 72 hours later. Faah-/- mice showed a reduction of cisplatin-induced blood urea nitrogen, plasma creatinine levels, kidney injury markers, and tubular damage in comparison with WT mice. The renal protection from Faah deletion was associated with enhanced tone of AEA-related N-acylethanolamines (palmitoylethanolamide and oleoylethanolamide), attenuated nuclear factor-κB/p65 activity, DNA damage markers p53 and p21, and decreased expression of the inflammatory cytokine interleukin-1ß, as well as infiltration of macrophages and leukocytes in the kidneys. Notably, a selective FAAH inhibitor (PF-04457845) did not interfere with or perturb the antitumor effects of cisplatin in two head and neck squamous cell carcinoma cell lines, HN30 and HN12. Our work highlights that FAAH inactivation prevents cisplatin-induced nephrotoxicity in mice and that targeting FAAH could provide a novel strategy to mitigate cisplatin-induced nephrotoxicity. SIGNIFICANCE STATEMENT: Mice lacking the Faah gene are protected from cisplatin-induced inflammation, DNA damage response, tubular damage, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to mitigate cisplatin-induced nephrotoxicity.