This approach can help to overcome the main sources of bias from classical observational approaches, by providing a more reliable estimate of the likely underlying causal relationship. There are limitations to this study that should be considered. First, the ALSPAC cohort suffers from attrition, which is higher among the socially disadvantaged . Furthermore, polygenic scores for tobacco smoking initiation were associated with drop out in the ALSPAC . We attempted to minimize the effect of drop-out by using multiple imputation, FIML, and inverse probability weighting which assume MAR missing patterns. Although it is not possible to test the MAR assumption, it was made more plausible as a number of SES variables were found to predict whether participants attended the clinic or not . Second, tobacco and cannabis use were self-reported. However, there is evidence to suggest that self-reported assessments are reliable and valid methods , and the assessment of tobacco and cannabis use yearly over 6 years in a latent variable framework helps to account for measurement error . Third, while the longitudinal approach for each substance used in this study has a number of advantages over using measures at a single timepoint, it was not possible to examine cannabis use without tobacco use as most cannabis users use cannabis in combination with tobacco . We therefore cannot rule out the possibility that observed associations between cannabis use and cognitive functioning are exacerbated by the combined use of cannabis and tobacco. Fourth, different measures of tobacco and cannabis use for the observational and MR analyses were used. Along with deriving latent classes of tobacco and cannabis use, we used the largest GWAS consortia which has identified 341 genetic instruments for ‘smoking initiation’, and the GWAS conducted by Pasman and colleagues which identified 8 genetic instruments for lifetime cannabis use which are continuous measures. To our knowledge it is not currently possible to use a nominal exposure and consequently the effect sizes are not directly comparable. Fifth, it is likely that both the one- and two-sample MR analyses areunderpowered. However, findings using weak instruments tend to bias findings towards the null in the two-sample setting and toward the outcome-risk association in the one-sample setting . Sixth, the main limitation of one- and two-sample MR is that the quality of the pooled results in the GWAS consortia is dependent on the individual studies.
Another limitation is that the same sample may contribute to both GWAS which was the case in the current study as ALSPAC was in both the exposure and outcome. This will bias the MR estimate towards the observed estimate. However, as the MR found no clear evidence for an effect, this suggests it was not biased by overlapping samples. See Lawlor and colleagues for a more comprehensive description of limitations associated with MR studies. Finally, it is possible that the direction of the association could work in both ways, that is, impairments in cognitive functioning may precede tobacco and cannabis square pot use.We were able to include a number of measures to maximize the robustness of our findings: ascertaining the temporal order of exposures and outcomes; controlling for premorbid working memory and brain insults prior to measures of tobacco/cannabis use helped to reduce the possibility of cognitive impairments, or lower cognitive abilities in childhood, influencing tobacco/cannabis use; and it is possible that a common risk factor is influencing both tobacco/cannabis use and lower cognitive function, however MR methods helps to protect against this possibility by minimizing bias from reverse causation and residual confounding.Overall, there was observational evidence that adolescent tobacco and cannabis use were associated with subsequent cognitive functioning, highlighting impairments in a range of cognitive domains, including working memory, response inhibition and emotion recognition. Our findings lend support to the developmental vulnerability hypothesis, in that, tobacco and cannabis use in adolescence, when the brain is undergoing critical development, may have neurotoxic effects. Better powered genetically informed studies are required to determine whether these associations are causal. In order to rule out the possibility of deficient cognitive functioning preceding substance in adolescence, future research should use an equally robust approach to examine the alternate hypothesis. This study lends support to public health strategies and interventions aimed at reducing tobacco and cannabis exposure in young people.In the last two decades and owing to serious adverse effects associated with the use of opiate medications and nonsteroidal anti-inflammatory drugs , the two major pharmacological groups used in pain management, more attention has been paid to cannabis-based extracts and cannabinoid-based products to fill the gap left by analgesics currently available in the clinic .
Increasing evidence from human clinical trials has demonstrated that cannabis-based therapeutics can minimize neuropathic pain intensity and provide effective remedies for chronic pain management . However, the clinical use of herbal cannabis is opposed by several limitations including the psychoactive adverse effects and associated harm to individuals and the public health, the complex and variable chemical content with the associated lack of consistency and standardization, possible microbial and pesticidal contamination, as well as the lack of solid evidence of effectiveness. Therefore, there is a critical need for robust research on herbal cannabis and its ingredients to evaluate the medical potential of the active ingredients alone and in complex mixtures . Potentially bioactive cannabis-derived compounds not only include cannabinoids but also terpenes, which comprise more than 150compounds out of the 500 plus constituents of Cannabis Sativa, the most commonly used cannabis species . Based on the fact that different cannabis varieties are able to induce various physiological effects, as has been observed among cannabis users, the socalled “entourage effect” has been proposed to refer to the additive or synergistic contribution of terpenes to the pharmacological effects shown by cannabinoids . In a recent study that investigated the ability of terpenes found in C. Sativa to activate TRPV1 in HEK cells, a mixture of terpenes was found to remarkably promote intracellular calcium influxes. In addition, betamyrcene and, to a lesser extent, nerolidol were identified as the major contributors to the calcium influx activity. MC activity was completely dependent upon the presence of TRPV1 protein and thus the TRPV1 antagonist capsazeine could effectively bloke MC-induced calcium influx. Furthermore, based on molecular docking data, MC binds to TRPV1 to via a hydrophobic, non-covalent interaction . MC , a monoterpene and the most abundant terpene in cannabis, and NL , a sesquiterpene, have both demonstrated anti-nociceptive and anti-inflammatory effects . In addition to these two terpenes, betacaryophyllene , a bicyclic sesquiterpene found in cannabis, has been reported to act as a specific agonist against cannabinoid receptor 2 , which presents in peripheral organs, outside the CNS . This interesting activity, beside other mechanisms of action, make CPh a potential therapeutic candidate in the management of neuropathic pain . However, the volatile and hydrophobic nature of these terpenes result in poor solubility and low bioavailability, limiting their in vivo pharmacological efficacy. To address these limitations, we have recently developed polymeric nanoparticles that successfully encapsulated these three terpenes . The new nanosystems were fabricated using poly-poly , a block co-polymer of a hydrophilic chain of PEG linked to PLGA a biocompatible, biodegradable, Food and Drug Administration -approved co-polymer based .
It is anticipated that the encapsulation of the cannabis-derived terpenes in PLGA NPs will equip them with a wide range of qualities such as enhanced solubility and stability, promoted absorption by biological membranes, sustained release, and ultimately improved therapeutic efficacy . In this work we sought to study the impact of the of MC, CPh, and NL encapsulation on their potential effectiveness in pain management. To this end, we tested the terpene-loaded PEG-PLGA NPs in HEK293 cells that express the nociceptive transient receptor potential vanilloid- 1 ion channel , a non-selective ligand-gated cation channel that is involved in the sensation of scalding heat and pain . This ion channel is a member of the TRPV subfamily of the transient receptor potential channels, a family of membrane calcium channels that are activated by a variety of exogenous and endogenous physical and chemical stimuli . TRPV1 is identified by its responsiveness to capsaicin and its analogues , but TRPV1 also responds to noxious temperatures , low pH , and to some endogenous compounds; in particular, the endocannabinoids. TRPV1 antagonists include capsazepine and ruthenium red . TRPV1 is expressed in both peripheral and central nervous systems, predominantly in primary sensory neurons involved in pain perception, in addition to several nonneuronal cells such as immune cells and smooth muscle cells . Both antagonism and agonism of TRPV1, and other TRP channels, can induce analgesia, via inactivation and chronic desensitization of this nociceptive ion channel making it a target for pain treatment . The activation of TRPV1 leads to an influx of Ca2+ via the plasma membrane, thus generating changes in intracellular Ca2+ concentration. It was also found that TRP channels are present in intracellular organelles and control the release of intracellular Ca2+ . Influxes of Ca2+ were monitored by the fluorescent indicator Fluo-4 acetoxymethyl. We used similar experimental conditions to acquire fluorescence images of cells treated with the terpenes-loaded NPs. Additionally, the cytotoxicity of the free and encapsulated terpenes was assessed. The terpene-loaded PEG-PLGA NPs were synthesized and tested in HEK cells that express TRPV1. The calcium signaling assay utilized in this work enables the measurement of calcium influx, as a result of TRPV1 activation. Fluorescence intensity changes were monitored using Fluo-4, which was used to measure intra-cellular free Ca2+ concentrations . Our work was based on the findings of Jansen et al. who demonstrated that cannabis-derived terpenes, predominantly MC, activate TRPV1 channels inducing calcium fluxes . We obtained the HEK TRPV1 cells from the same research group. However, we had to introduce some modifications to the calcium signaling assay developed by Jansen et al. in which a more sophisticated and sensitive instrument was employed. Thus, a higher count of cells and higher concentrations of terpenes were involved. Similar high concentrations of terpenes have previously been reported in the literature. For example, linalool has been found to activate human TRPA1 at an EC50 of 117 μM . In addition, the experiment duration was set to 1 h to provide enough time for drug release from NPs and trim tray for weed interaction with the receptors. Our results confirm the findings of Jansen et al. regarding free MC, and the relatively small effect of free NL on TRPV1. Moreover, this work demonstrates that the PLGA-based nano-formulations significantly enhance the calcium influx induced by the three terpenes.
In general, this improved effect may be explained by the solubilization of the lipophilic terpenes in the core of the nanocapsules, thus improving their ability to interact with the TRPV1 channels. Furthermore, the likely slow drug release, as generally observed for lipophilic substances encapsulated in PEG-PLGA NPs , may explain the time-dependent enhancement of fluorescence intensity shown by the terpene-loaded NPs. Interestingly, NL-loaded NPs demonstrated a two-phase response characterized by a small peak followed by a drastic logistic phase pattern reaching a calcium signal similar to that exhibited by ionomycin. In fact, the two-phase response suggests that the generation of calcium signal by the encapsulated NL may be provided by a more complex mechanism. The first small peak may result from the terpene that is released immediately by the nanoparticle formulation followed by a slower but extended release of the terpene. However, as this pattern was not observed by the other two terpenes, it is also possible that the high intercellular concentration of NL achieved by the nano-formulation induces other mechanisms for increasing calcium concentration in the cytosol. One possible mechanism is the mobilization of calcium from endoplasmic reticulum in which functional TRPV1 channels are located and serve as intracellular Ca2+ release channels . A more in-depth investigation is needed to clarify the mechanism underlying effect induced by NL-loaded NPs, which is not within thescope of the current work. Moreover, the high-level responses seen by the combinations of the three terpenes may provide an evidence of a synergistic effect. It is noteworthy that shifts in intracellular Ca2+ have been found to promote cell death, through apoptotic or necrotic pathways .