Assay of Endocannabinoid Uptake.

Endocannabinoids at nanomolar physiological concentrations cross cellular membranes by facilitated diffusion, a process that can be studied by measuring transport kinetics and endocannabinoid trafficking employing radioligands and mass spectrometry. Here, we describe radiosubstrate-based assays using arachidonoyl[1-3H]ethanolamine and 2-arachidonoyl[1,2,3-3H]glycerol to measure cellular endocannabinoid uptake in a three-phase assay with human U937 cells. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS)-based lipidomics was used to interrogate the roles of serum and albumin for endocannabinoid trafficking in U937 cells.

Echinacea as a Potential Force against Coronavirus Infections? A Mini-Review of Randomized Controlled Trials in Adults and Children.

Echinacea purpurea has been shown to broadly inhibit coronaviruses and SARS-CoV-2 in vitro. This review discusses the available clinical evidence from randomized, blinded and controlled human studies. Two RCTs capturing incidence of viral respiratory tract infections during Echinacea preventative treatment were identified including coronavirus infections. Incidence and/or viral loads were measured by RT-PCR and symptom severity was recorded. In a first study, Jawad et al. collected nasopharyngeal swabs from adults (N = 755) over 4 months of continuous prevention. Overall, 24 and 47 enveloped virus infections occurred, including 21 and 33 coronavirus detections (229E; HKU1; OC43) with Echinaforce® extract (2400 mg daily) and placebo, respectively (p = 0.0114). In a separate study, Ogal et al. administered the same extract (1200 mg) or control for 4 months to children (4-12 years) (N = 203). Echinacea reduced the incidence of enveloped virus infections from 47 to 29 (p = 0.0038) whereas 11 and 13 coronavirus detections (229E, OC43, NL63) were counted (p > 0.05). Respiratory symptoms during coronavirus infections were significantly lower with area-under-curve AUC = 75.8 (+/-50.24) versus 27.1 (+/-21.27) score points (p = 0.0036). Importantly, viral loads in nasal secretions were significantly reduced by 98.5% in the Echinacea group, with Ct-values 31.1 [95% CI 26.3; 35.9] versus 25.0 [95% CI 20.5; 29.5] in the control group (p = 0.0479). Results from clinical studies confirm the antiviral activity found for Echinacea in vitro, embracing enveloped respiratory pathogens and therefore coronaviruses as well. Substantiating results from a new, completed study seem to extrapolate these effects to the prevention of SARS-CoV-2 infections. As hypothesized, the established broad antiviral activity of Echinacea extract appears to be inclusive for SARS-CoV-2.

Synthesis and Biological Evaluation of Endocannabinoid Uptake Inhibitors Derived from WOBE437.

WOBE437 ((2E,4E)-N-(3,4-dimethoxyphenethyl)dodeca-2,4-dienamide, 1) is a natural product-derived, highly potent inhibitor of endocannabinoid reuptake. In this study, we synthesized almost 80 analogues of 1 with different types of modifications in the dodecadienoyl domain as well as the dimethoxyphenylethyl head group, and we investigated their effects on anandamide uptake into U937 cells. Intriguingly, none of these analogues was a more potent inhibitor of anandamide uptake than WOBE437 (1). At the same time, a number of WOBE437 variants exhibited potencies in the sub-100 nM range, with high selectivity over inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase; two compounds were virtually equipotent with 1. Interestingly, profound activity differences were observed between analogues in which either of the two methoxy substituents in the head group had been replaced by the same bulkier alkoxy group. Some of the compounds described here could be interesting departure points for the development of potent endocannabinoid uptake inhibitors with more drug-like properties.

Effect of the fixed combination of valerian, lemon balm, passionflower, and butterbur extracts (Ze 185) on the prescription pattern of benzodiazepines in hospitalized psychiatric patients-A retrospective case-control investigation.

Stress is an increasing problem that can result in various psychiatric and somatoform symptoms. Among others, benzodiazepines and valerian preparations are used to treat stress symptoms. The aim of this study was to investigate whether the prescription of a fixed herbal extract combination of valerian, lemon balm, passionflower, and butterbur (Ze 185) changes the prescription pattern of benzodiazepines in hospitalized psychiatric patients. In a retrospective case-control study, anonymized medical record data from 3,252 psychiatric in-house patients were analysed over a 3.5-year period. Cases (n = 1,548) with a prescription of Ze 185 and controls (n = 1,704) were matched by age, gender, hospitalization interval, and main International Classification of Diseases, Version 10 F-diagnoses. The primary objective was to investigate the effect of Ze 185 on the prescription pattern of benzodiazepines. Secondary objectives investigated the prescriptions of concomitant drugs and effectiveness of the hospital stay. Distribution of drug classes was analysed using the WHO's anatomic-therapeutic-chemical code. Data showed that both treatment modalities had a comparable clinical effectiveness but with significantly less prescriptions of benzodiazepines in the Ze 185 group (p = .006). This is of clinical importance because suitable alternatives to benzodiazepines are desirable. To obtain more support for this hypothesis, a dedicated randomized, controlled clinical trial monitoring drug safety is required.

Clinical relevance of St. John's wort drug interactions revisited.

The first clinically relevant reports of preparations of St. John's wort (SJW), a herbal medicine with anti-depressant effects, interacting with other drugs, altering their bioavailability and efficacy, were published about 20 years ago. In 2000, a pharmacokinetic interaction between SJW and cyclosporine caused acute rejection in two heart transplant patients. Since then, subsequent research has shown that SJW altered the pharmacokinetics of drugs such as digoxin, tacrolimus, indinavir, warfarin, alprazolam, simvastatin, or oral contraceptives. These interactions were caused by pregnane-X-receptor (PXR) activation. Preparations of SJW are potent activators of PXR and hence inducers of cytochrome P450 enzymes (most importantly CYP3A4) and P-glycoprotein. The degree of CYP3A4 induction correlates significantly with the hyperforin content in the preparation. Twenty years after the first occurrence of clinically relevant pharmacokinetic drug interactions with SJW, this review revisits the current knowledge of the mechanisms of action and on how pharmacokinetic drug interactions with SJW could be avoided. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

Total Synthesis of the Endocannabinoid Uptake Inhibitor Guineensine and SAR Studies.

Guineensine ((2E,4E,12E)-13-(benzo[d][1,3]dioxol-5-yl)-N-isobutyltrideca-2,4,12-trienamide) is a plant-derived natural product that inhibits reuptake of the endocannabinoid anandamide with sub-micromolar potency. We have established a highly efficient total synthesis of guineensine, which provided the natural product in only five steps from commercially available 3-nonyn-1-ol in 17 % overall yield, relying on the attachment of the benzodioxolyl moiety to the unsaturated fatty acid chain by means of a Suzuki coupling as the key step. Subsequent SAR studies revealed that replacement of the N-isobutyl group in the natural product by various alkyl, arylalkyl, or aryl groups is generally well tolerated, and derivatives could be identified that are slightly more potent anandamide reuptake inhibitors than guineensine itself. In contrast, modifications of the benzodioxolyl moiety led to decreased activity. Intriguingly, a change in the configuration of the C4=C5 double bond from E to Z was found to be very well tolerated, in spite of the associated change in the overall geometry of the molecule.

No Clinically Relevant Interactions of St. John's Wort Extract Ze 117 Low in Hyperforin With Cytochrome P450 Enzymes and P-glycoprotein.

Hypericum perforatum L. (St. John's wort) is used to treat mild-to-moderate depression. Its potential safety risks are pharmacokinetic drug interactions via cytochrome P450 (CYP) enzymes and P-glycoprotein, presumably caused by hyperforin. In a phase I, open-label, nonrandomized, single-sequence study, the low-hyperforin Hypericum extract Ze 117 was investigated using a drug cocktail in 20 healthy volunteers. No pharmacokinetic interactions of Ze 117 were observed for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP3A4, and P-glycoprotein. Area under the curve (AUC) and peak plasma concentration (Cmax ) of the used probe drugs showed 90% confidence intervals (CIs) of the geometric mean ratios of the drugs taken together with Ze 117 vs. probe drug alone, well within the predefined bioequivalence range of 80-125%. Though Ze 117 did not induce dextromethorphan metabolism by CYP2D6, it weakly increased dextromethorphan AUC ratio (mean 147.99, 95% CI 126.32-173.39) but not the corresponding metabolic ratio. Ze 117 does not show clinically relevant pharmacokinetic interactions with important CYPs and P-glycoprotein.

Endocannabinoid Tone Regulates Human Sebocyte Biology.

We have previously shown that endocannabinoids (eCBs) (e.g., anandamide) are involved in the maintenance of homeostatic sebaceous lipid production in human sebaceous glands and that eCB treatment dramatically increases sebaceous lipid production. Here, we aimed to investigate the expression of the major eCB synthesizing and degrading enzymes and to study the effects of eCB uptake inhibitors on human SZ95 sebocytes, thus exploring the role of the putative eCB membrane transporter, which has been hypothesized to facilitate the cellular uptake and subsequent degradation of eCBs. We found that the major eCB synthesizing (N-acyl phosphatidylethanolamine-specific phospholipase D, and diacylglycerol lipase-α and -ß) and degrading (fatty acid amide hydrolase, monoacylglycerol lipase) enzymes are expressed in SZ95 sebocytes and also in sebaceous glands (except for diacylglycerol lipase-α, the staining of which was dubious in histological preparations). eCB uptake-inhibition with VDM11 induced a moderate increase in sebaceous lipid production and also elevated the levels of various eCBs and related acylethanolamides. Finally, we found that VDM11 was able to interfere with the proinflammatory action of the TLR4 activator lipopolysaccharide. Collectively, our data suggest that inhibition of eCB uptake exerts anti-inflammatory actions and elevates both sebaceous lipid production and eCB levels; thus, these inhibitors might be beneficial in cutaneous inflammatory conditions accompanied by dry skin.

Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake.

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived N-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC50 = 10 nM) inhibitor N-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

Assay of Endocannabinoid Uptake.

Endocannabinoids at physiological concentrations are crossing cellular membranes by facilitated diffusion, a process that can be studied by measuring transport kinetics. Here, we describe a radiosubstrate-based assay using arachidonoyl[1-(3)H]ethanolamine or arachidonoyl[1,2,3-(3)H]glycerol to measure the cellular endocannabinoid uptake in a three-phase assay with human U937 cells.

Endocannabinoid transport revisited.

Endocannabinoids are arachidonic acid-derived endogenous lipids that activate the endocannabinoid system which plays a major role in health and disease. The primary endocannabinoids are anandamide (AEA, N-arachidonoylethanolamine) and 2-arachidonoyl glycerol. While their biosynthesis and metabolism have been studied in detail, it remains unclear how endocannabinoids are transported across the cell membrane. In this review, we critically discuss the different models of endocannabinoid trafficking, focusing on AEA cellular uptake which is best studied. The evolution of the current knowledge obtained with different AEA transport inhibitors is reviewed and the confusions caused by the lack of their specificity discussed. A comparative summary of the most important AEA uptake inhibitors and the studies involving their use is provided. Based on a comprehensive literature analysis, we propose a model of facilitated AEA membrane transport followed by intracellular shuttling and sequestration. We conclude that novel and more specific probes will be essential to identify the missing targets involved in endocannabinoid membrane transport.

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 µM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 µM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 µM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice.

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50=290nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure-activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Evidence for bidirectional endocannabinoid transport across cell membranes.

Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as 2-arachidonoylglycerol (2-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and 2-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nm AEA and 1 µm 2-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and 2-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 µm) and OMDM-2 (5 µm), a functional synergism on cellular AEA and 2-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and 2-AG. We show, for the first time, that UCM707 and OMDM-2 inhibit the bidirectional movement of AEA and 2-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and 2-AG trafficking and metabolism.

Cannabinoid receptor trafficking in peripheral cells is dynamically regulated by a binary biochemical switch.

The cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB1 and CB2 receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB2 receptors form oligomers and heterodimers with CB1 receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB2 receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression.