An evaluation of the genotoxicity and 90-day repeated-dose toxicity of a CBD-rich hemp oil.

Currently, there is much interest in the sales and study of consumable Cannabis sativa L. products that contain relatively high levels of cannabidiol (CBD) and low levels of Δ-9-tetrahydrocannabinol. While there are published safety evaluations for extracts containing low concentrations of CBD, toxicological assessments for those with higher concentrations are still scant in the public domain. In this paper, genotoxicity tests and a 90-day repeated-dose toxicity study of an ethanolic extract of C. sativa containing ~85% CBD were performed following relevant OECD guidelines. No increased gene mutations were observed in a bacterial reverse mutation assay compared to controls up to the maximum recommended concentration of the guideline. An in vitro chromosomal aberration assay showed no positive findings in the short-term (3 h) treatment assays. Long-term treatment (20 h) showed an increased number of cells containing aberrations at the highest dose of 2 µg/mL, which was outside of historical control levels, but not statistically significantly different from the controls. An in vivo micronucleus study showed no genotoxic potential of the test item in mice. A 90-day repeated-dose gavage study using 0, 75, 125, and 175 mg/kg bw/day showed several slight findings that were considered likely to be related to an adaptive response to consumption of the extract by the animals but were not considered toxicologically relevant. These included increases in liver and adrenal weights compared to controls. The NOAEL was determined as 175 mg/kg bw/day, the highest dose tested (equivalent to approximately 150 mg/kg bw/day of CBD).

Processing Stabilization of Polyethylene with Grape Peel Extract: Effect of Extraction Technology and Composition.

Dry grape peel powder was extracted by three different techniques, stirred tank reactor, Soxhlet and ultrasound extraction. The composition, physical and chemical structure and inherent stability of the extracts were characterized by various methods. The extracts and reference compounds were added to polyethylene and their stabilization efficiency was determined in multiple extrusion experiments. The composition of the extracts was quite similar. Ten main compounds were identified in the extracts, which contained a considerable number of polyphenols, but only small amounts of quercetin and trans-resveratrol. The extracts proved to be more efficient processing stabilizers than trans-resveratrol and the commercial stabilizer, Irganox 1010, irrespective of the extraction technology used. In spite of their good processing stabilization effect, polymers containing the extracts had poor residual stability. The differences in processing and long-term stabilization must be related to the different structures of the polyphenols contained by the extracts and the reference compounds. The results clearly prove that the IC50 value determined by the DPPH assay is not suitable for the estimation of the efficiency of a compound as a stabilizer for polymers.

Comparative bioavailability study following a single dose intravenous and buccal administration of remdesivir in rabbits.

As remdesivir, the first FDA-approved drug for SARS-CoV-2 infection, can be used only for hospitalized patients due to intravenous administration, there is an urgent need of effective oral antiviral formulations to be used at early stage of infection in an outpatient setting. The present paper reports on the comparative pharmacokinetics of the electrospun nanofiber remdesivir/sulfobutyl ether beta-cyclodextrin formulation after intravenous and buccal administration. It was postulated that oral transmucosal administration avoids remdesivir from metabolic transformation and intact remdesivir can be detected in plasma, but only the active metabolite GS-441524 could be experimentally detected at a significantly lower plasma level, than that provided by the intravenous route. In buccally treated animals, the metabolite GS-441524 appeared only at 1 h after treatment, while in intravenously treated animals, GS-441524 was possible to quantify even at the first time-point of blood collection. Further optimization of formulation is required to improve pharmacokinetics of remdesivir-sulfobutyl ether beta-cyclodextrin formulation upon buccal administration.

Development and Validation of an LC-MS-MS Method for the Quantification of Cyanate in Rat Plasma and Its Application to Toxicokinetic Bioanalysis.

Cyanate has been recognized as a uremic toxin that can adversely affect the clinical status of patients with chronic kidney disease. Besides, its toxicity has been under investigation in mammalian toxicology. If such studies are supplemented with toxicokinetic sampling and bioanalysis, additional information can be acquired about the systemic exposure. In order to serve this need, a liquid chromatography with tandem mass spectrometry (LC-MS-MS) method was elaborated and validated for the quantification of cyanate in rat plasma using its isotope-labeled analog for internal standard. Cyanate was converted to a product compatible with reverse-phase LC-MS-MS via a two-step derivatization reaction with the reagent-anthranilic acid. It was observed that this reagent solution contains the reaction products even if prepared freshly in ultrapure water. The phenomenon was interpreted as the presence of urea and its reactivity with anthranilic acid. Contrary to previous research results where fresh anthranilic acid solution was recommended to use, we have found that the aging of the reagent solution is a crucial factor to eliminate the interference. Thereafter, the optimal pH was selected for the plasma sample and processing conditions. Bioanalytical validation and incurred sample reanalysis confirmed the reliability of the method when the intermediate reaction product was used for detection. Only one freeze-thaw cycle stability could be proven, which highlighted the need to collect two sample aliquots whenever possible. Real samples were analyzed in a toxicity study to evaluate systemic exposure of potassium cyanate at three dose levels. Further on, this method might be adapted to provide additional information about the pathophysiological concentration of cyanate in patients with chronic kidney disease for therapeutic support.

Preparation of Molecularly Imprinted Microspheres by Precipitation Polymerization.

Molecularly imprinted polymers (MIPs) gained an expansively growing interest in the past few decades. After an initial, explorative period of preparing MIPs exclusively with bulk polymerization, new polymer synthesis routes have been adapted to overcome the drawbacks of the traditional method. Among these the most appealing is precipitation polymerization that results in nano- and microspheres with narrow size distribution and makes the production of MIPs more straightforward. Here, we describe a precipitation polymerization protocol for a common small molecule template, propranolol that is carried out in the conventional way, in dilute monomer solution. Moreover, a modified precipitation polymerization protocol from concentrated monomer solution is presented for a diclofenac imprinted polymer which makes the synthesis even more versatile and circumvents the disadvantages of the dilute solution conditions.

Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release.

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity. SIGNIFICANCE STATEMENT: Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

Identification of lipophilic bioproduct portfolio from bioreactor samples of extreme halophilic archaea with HPLC-MS/MS.

Extreme halophilic archaea are a yet unexploited source of natural carotenoids. At elevated salinities, however, material corrosivity issues occur and the performance of analytical methods is strongly affected. The goal of this study was to develop a method for identification and downstream processing of potentially valuable bioproducts produced by archaea. To circumvent extreme salinities during analysis, a direct sample preparation method was established to selectively extract both the polar and the nonpolar lipid contents of extreme halophiles with hexane, acetone and the mixture of MeOH/MTBE/water, respectively. Halogenated solvents, as used in conventional extraction methods, were omitted because of environmental considerations and potential process scale-up. The HPLC-MS/MS method using atmospheric pressure chemical ionization was developed and tuned with three commercially available C40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C50 carotenoid bacterioruberin, and vitamin MK-8 were the most valuable products found in bioreactor samples. In contrast to literature on shake flask cultivations, no anhydrous analogues of bacterioruberin, as by-products of the carotenoid biosynthesis, were detected in bioreactor samples. This study demonstrates the importance of sample preparation and the applicability of HPLC-MS/MS methods on real samples from extreme halophilic strains. Furthermore, from a biotechnological point-of-view, this study would like to reveal the relevance of using controlled and defined bioreactor cultivations instead of shake flask cultures in the early stage of potential bioproduct profiling.

Molecularly imprinted polymer microspheres containing photoswitchable spiropyran-based binding sites.

A versatile approach for the preparation of photoswitchable molecularly imprinted polymers (MIPs) is proposed where the selective recognition and the photoresponsive function are assumed by two different monomers. As a proof of concept, MIP microspheres were synthesized by precipitation polymerization for recognizing terbutylazine, a triazine-type herbicide. Formation of the selective binding sites was based upon H-bonding interactions between the template and the functional monomer methacrylic acid, whereas a polymerizable spiropyran unit was incorporated into the polymer matrix to provide light-controllable characteristics. A trifunctional monomer, trimethylolpropane trimethacrylate, was used as a cross-linker. The imprinted particles exhibited considerable morphological differences compared to their nonimprinted counterparts as observed by scanning electron microscopy. The imprinting effect was confirmed by equilibrium rebinding studies. The photoresponsiveness of the polymer particles was visualized by fluorescence microscopy and further characterized by spectroscopy. The template binding behavior could be regulated by alternating UV and visible light illumination when analyte release and uptake was observed, respectively. Binding isotherms fitted by the Freundlich model revealed the photomodulation of the number of binding sites and their average affinity. This facile synthetic approach may give an attractive starting point to endow currently existing highly selective MIPs with photoswitchable properties, thereby extending the scope of spiropyran-based photoresponsive smart materials.

In situ synthesis of molecularly imprinted nanoparticles in porous support membranes using high-viscosity polymerization solvents.

There is a growing need in membrane separations for novel membrane materials providing selective retention. Molecularly imprinted polymers (MIPs) are promising candidates for membrane functionalization. In this work, a novel approach is described to prepare composite membrane adsorbers incorporating molecularly imprinted microparticles or nanoparticles into commercially available macroporous filtration membranes. The polymerization is carried out in highly viscous polymerization solvents, and the particles are formed in situ in the pores of the support membrane. MIP particle composite membranes selective for terbutylazine were prepared and characterized by scanning electron microscopy and N2 porosimetry. By varying the polymerization solvent microparticles or nanoparticles with diameters ranging from several hundred nanometers to 1 µm could be embedded into the support. The permeability of the membranes was in the range of 1000 to 20,000 Lm⁻² hr⁻¹ bar⁻¹. The imprinted composite membranes showed high MIP/NIP (nonimprinted polymer) selectivity for the template in organic media both in equilibrium-rebinding measurements and in filtration experiments. The solid phase extraction of a mixture of the template, its analogs, and a nonrelated compound demonstrated MIP/NIP selectivity and substance selectivity of the new molecularly imprinted membrane. The synthesis technique offers a potential for the cost-effective production of selective membrane adsorbers with high capacity and high throughput.

Selective solid phase extraction of propranolol on multiwell membrane filter plates modified with molecularly imprinted polymer.

Molecularly imprinted polymers (MIPs) were synthesized in 24-well glass fiber membrane filter plates to obtain a novel type of solid phase extraction device for the cleanup of propranolol. Sample processing parameters like residence time during sample loading, sample volume, pH, sample solvent, type and amount of washing and elution solvents were investigated and optimized. Important differences from the traditional molecularly imprinted solid phase extraction (MISPE) cartridges have been identified. The MIP modified composite membrane suits well for the sample preparation of low volume biological samples. A protocol has been elaborated for the quantitation of propranolol from urine and plasma samples in the clinically relevant concentration ranges. Preliminary validation results indicate that the composite MIP membrane filter plates offer a viable alternative to existing MISPE cartridges and at the same time have advantages like much easier and faster synthesis method and high-throughput analysis.