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.

Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML)

Tyrpsine kinase inhibitors (TKIs) effectively target progenitors and mature leukaemic cells but prove less effective at eliminating leukaemic stem cells (LSCs) in patients with chronic myeloid leukaemia (CML). Several reports indicate that the TGFβ superfamily pathway is important for LSC survival and quiescence. We conducted extensive microarray analyses to compare expression patterns in normal haemopoietic stem cells (HSC) and progenitors with CML LSC and progenitor populations in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML. The BMP/SMAD pathway and downstream signalling molecules were identified as significantly deregulated in all three phases of CML. The changes observed could potentiate altered autocrine signalling, as BMP2, BMP4 (p<0.05), and ACTIVIN A (p<0.001) were all down regulated, whereas BMP7, BMP10 and TGFβ (p<0.05) were up regulated in CP. This was accompanied by up regulation of BMPRI (p<0.05) and downstream SMADs (p<0.005). Interestingly, as CML progressed, the profile altered, with BC patients showing significant over-expression of ACTIVIN A and its receptor ACVR1C. To further characterise the BMP pathway and identify potential candidate biomarkers within a larger cohort, expression analysis of 42 genes in 60 newly diagnosed CP CML patient samples, enrolled on a phase III clinical trial (www.spirit-cml.org) with greater than 12 months follow-up data on their response to TKI was performed. Analysis revealed that the pathway was highly deregulated, with no clear distinction when patients were stratified into good, intermediate and poor response to treatment. One of the major issues in developing new treatments to target LSCs is the ability to test small molecule inhibitors effectively as it is difficult to obtain sufficient LSCs from primary patient material. Using reprogramming technologies, we generated induced pluripotent stem cells (iPSCs) from CP CML patients and normal donors. CML- and normal-derived iPSCs were differentiated along the mesodermal axis to generate haemopoietic and endothelial precursors (haemangioblasts). IPSC-derived haemangioblasts exhibited sensitivity to TKI treatment with increased apoptosis and reduction in the phosphorylation of downstream target proteins. 4 Dual inhibition studies were performed using BMP pathway inhibitors in combination with TKI on CML cell lines, primary cells and patient derived iPSCs. Results indicate that they act synergistically to target CML cells both in the presence and absence of BMP4 ligand. Inhibition resulted in decreased proliferation, irreversible cell cycle arrest, increased apoptosis, reduced haemopoietic colony formation, altered gene expression pattern, reduction in self-renewal and a significant reduction in the phosphorylation of downstream target proteins. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to prevent LSC self-renewal and improve outcome for patients. By successfully developing and validating iPSCs for CML drug screening we hope to substantially reduce the reliance on animal models for early preclinical drug screening in leukaemia.