Self-assembling cannabinomimetics: supramolecular structures of N-alkyl amides

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

Cannabinoid receptor ligands as potential anticancer agents--high hopes for new therapies?

OBJECTIVES: The endocannabinoid system is an endogenous lipid signalling network comprising arachidonic-acid-derived ligands, cannabinoid (CB) receptors, transporters and endocannabinoid degrading enzymes. The CB(1) receptor is predominantly expressed in neurons but is also co-expressed with the CB(2) receptor in peripheral tissues. In recent years, CB receptor ligands, including Delta(9)-tetrahydrocannabinol, have been proposed as potential anticancer agents. KEY FINDINGS: This review critically discusses the pharmacology of CB receptor activation as a novel therapeutic anticancer strategy in terms of ligand selectivity, tissue specificity and potency. Intriguingly, antitumour effects mediated by cannabinoids are not confined to inhibition of cancer cell proliferation; cannabinoids also reduce angiogenesis, cell migration and metastasis, inhibit carcinogenesis and attenuate inflammatory processes. In the last decade several new selective CB(1) and CB(2) receptor agents have been described, but most studies in the area of cancer research have used non-selective CB ligands. Moreover, many of these ligands exert prominent CB receptor-independent pharmacological effects, such as activation of the G-protein-coupled receptor GPR55, peroxisome proliferator-activated receptor gamma and the transient receptor potential vanilloid channels. SUMMARY: The role of the endocannabinoid system in tumourigenesis is still poorly understood and the molecular mechanisms of cannabinoid anticancer action need to be elucidated. The development of CB(2)-selective anticancer agents could be advantageous in light of the unwanted central effects exerted by CB(1) receptor ligands. Probably the most interesting question is whether cannabinoids could be useful in chemoprevention or in combination with established chemotherapeutic agents.

Reactions of CF3-enones with arenes under superelectrophilic activation: a pathway to trans-1,3-diaryl-1-CF3-indanes, new cannabinoid receptor ligands

4-Aryl-1,1,1-trifluorobut-3-en-2-ones ArCH[double bond, length as m-dash]CHCOCF3 (CF3-enones) react with arenes in excess of Brønsted superacids (TfOH, FSO3H) to give, stereoselectively, trans-1,3-diaryl-1-trifluoromethyl indanes in 35-85% yields. The reaction intermediates, the O-protonated ArCH[double bond, length as m-dash]CHC(OH(+))CF3 and the O,C-diprotonated ArHC(+)CH2C(OH(+))CF3 species, have been studied by means of (1)H, (13)C, (19)F NMR, and DFT calculations. Both types of the cations may participate in the reaction, depending on their electrophilicity and electron-donating properties of the arenes. The formation of CF3-indanes is a result of cascade reaction of protonated CF3-enones to form chemo-, regio- and stereoselectively three new C-C bonds. The obtained trans-1,3-diaryl-1-trifluoromethyl indanes were investigated as potential ligands for cannabinoid receptors CB1 and CB2 types. The most potent compound showed sub-micromolar affinity for both receptor subtypes with a 6-fold selectivity toward the CB2 receptor with no appreciable cytotoxicity toward SHSY5Y cells.

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids—arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series—and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki = 21.2 ± 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 μM).

A critical review of both the synthesis approach and the receptor profile of the 8-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide and analogue derivatives

We thank European Commission (project “PET BRAIN: Mapping the brain with PET radiolabeled cannabinoid CB1 ligands”; FP7-People-2009-IAPP; Grant Agreement N.25142).

Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.

Chromatographic analysis and survey studies to evaluate the emerging drugs of synthetic cannabinoids in Scotland and Saudi Arabia

Synthetic cannabinoid receptor agonists or more commonly known as synthetic cannabinoids (SCs) were originally created to obtain the medicinal value of THC but they are an emerging social problem. SCs are mostly produced coated on herbal materials or in powder form and marketed under a variety of brand names, e.g. “Spice”, “K2”. Despite many SCs becoming controlled under drug legislation, many of them remain legal in some countries around the world. In Scotland, SCs are controlled under the Misuse of Drugs Act 1971 and Psychoactive Substances Act 2016 that only cover a few early SCs. In Saudi Arabia, even fewer are controlled. The picture of the SCs-problem in Scotland is vague due to insufficient prevalence data, particularly that using biological samples. Whilst there is evidence of increasing use of SCs throughout the world, in Saudi Arabia, there is currently no data regarding the use of products containing SCs among Saudi people. Several studies indicate that SCs may cause serious toxicity and impairment to health therefore it is important to understand the scale of use within society. A simple and sensitive method was developed for the simultaneous analysis of 10 parent SCs (JWH-018, JWH-073, JWH-250, JWH-200, AM-1248, UR-144, A-796260, AB-FUBINACA, 5F-AKB-48 and 5F-PB-22) in whole blood and 8 corresponding metabolites (JWH-018 4-OH pentyl, JWH-073 3-OH butyl, JWH-250 4-OH pentyl, AM-2201 4-OH pentyl, JWH-122 5-OH pentyl, JWH-210 5-OH pentyl, 5F-AKB-48 (N-4 OH pentyl), 5F-PB-22 3-carboxyindole)in urine using LLE and LC-MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013). All analytes gave acceptable precision, linearity and recovery for analysing blood and urine samples. The method was applied to 1,496 biological samples, a mixture of whole blood and urine. Blood and/or urine samples were analysed from 114 patients presenting at Accident and Emergency in Glasgow Royal Infirmary, in spring 2014 and JuneDecember 2015. 5F-AKB-48, 5F-PB-22 and MDMB-CHMICA were detected in 9, 7 and 9 cases respectively. 904 urine samples from individuals admitted to/liberated from Scottish prisons over November 2013 were tested for the presence of SCs. 5F-AKB-48 (N-4 OH pentyl) was detected in 10 cases and 5F-PB-22 3-carboxyindole in 3 cases. Blood and urine samples from two post-mortem cases in Scotland with suspected ingestion of SCs were analysed. Both cases were confirmed positive for 5F-AKB-48. A total of 463 urine samples were collected from personnel who presented to the Security Forces Hospital in Ryiadh for workplace drug testing as a requirement for their job during July 2014. The results of the analysis found 2 samples to be positive for 5F-PB-22 3carboxyindole. A further study in Saudi Arabia using a questionnaire was carried out among 3 subpopulations: medical professionals, members of the public in and around smoking cafes and known drug users. With regards to general awareness of Spice products, 16%, 11% and 22% of those participants of medical professionals, members of the public in and around smoking cafes and known drug users, respectively, were aware of the existence of SCs or Spice products. The respondents had an overall average of 4.5% who had a friend who used these Spice products. It is clear from the results obtained in both blood and urine testing and surveys that SCs are being used in both Scotland and Saudi Arabia. The extent of their use is not clear and the data presented here is an initial look into their prevalence. Blood and urine findings suggest changing trends in SC use, moving away from JWH and AM SCs to the newer 5F-AKB-48, 5-F-PB-22 and MDMBCHMICA compounds worldwide. In both countries 5F-PB-22 was detected. These findings clarify how the SCs phenomenon is a worldwide problem and how the information of every country regarding what SCs are seized can help and is not specific for that country. The analytes included in the method were selected due to their apparent availability in both countries, however it is possible that some newer analytes have been used and these would not have been detected. For this reason it is important that methods for testing SCs are updated regularly and evolve with the ever-changing availability of these drugs worldwide. In addition, there is little published literature regarding the concentrations of these drugs found in blood and urine samples and this work goes some way towards understanding these.

Evidencia sobre la eficacia de cannabinoides en el dolor neuropático oncológico

Contexto: La eficacia de los cannabinoides en el dolor neuropático es desconocida. El control del dolor es determinante en los pacientes ya que genera un impacto negativo en la calidad de vida de los pacientes. Objetivo: El presente trabajo pretende demostrar la evidencia sobre la eficacia de los medicamentos cannabinoides en el control del dolor neuropático oncológico, mediante la evaluación de la literatura disponible. Metodología: Se realizó una revisión sistemática de literatura incluyendo estudios experimentales, observacionales y revisiones sistemáticas en un periodo de 15 años. Se incluyeron todos los estudios desde el años 2000 con evidencia IB según la escala de evidencia de Oxford. Resultados: Cuatro estudios cumplieron criterios para su inclusión, sin embargo la evidencia es baja y no permite recomendar o descartar los cannabinoides como terapia coadyuvante en control del dolor neuropático oncológico. La combinación de THC/CDB (Sativex®) parece ser un medicamento seguro pues no se reportaron muertes asociadas a su uso, sin embargo la presentación de eventos adversos a nivel gastrointestinal y neurológico podría aumentar el riesgo de interacciones medicamentosas y tener un impacto negativo en la calidad de vida de los pacientes oncológicos. Conclusiones: No hay suficiente literatura y la evidencia no es suficiente para recomendar o descartar el uso de los cannabinoides en dolor neuropático oncológico. Futuros estudios deben realizarse para analizar el beneficio de estos medicamentos. Aunque ética y socialmente hay resistencia para el uso de los cannabinoides, actualmente hay una gran discusión política en el mundo y en Colombia para su aceptación como terapia en el control del dolor.

A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

P2 purinergic receptor mRNA in rat and human sinoatrial node and other heart regions.

It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.

Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.