Effects of cannabis, one of the most widely used drugs, on HIV and particularly on biomarkers of inflammation and cognition, are largely unknown, diverse or anecdotal. By examining a large number of transcripts associated with neurological disorders and pathways of inflammation in peripheral leukocytes, we fill a gap on the understanding of how drugs of abuse impact cellular phenotypes, with the goals of identifying biomarkers of HIV neurocognitive disorders that are sensitive to interactions with substance use. In this study, we examined cells from102 subjects evenly distributed as HIVt/-and CANt/. In order to increase the power, the cohort was homogeneous in sex, age and education.The use of other substances was limited but not excluded, due to characteristics of the population. The sample size was a limitation for the identification of strong predictors. However, systems biology strategies helped us identify genes that exhibited interactive properties based on their co-involvement in highly overlapping molecular pathways. Visualization strategies helped identify gene networks with a concerted behavior in different groups, highlighting important trends in effects of cannabis use dependence.Our results show that cannabis has strong effects on the expression of a number of genes in peripheral leukocytes, which serve as reporters of biological processes that are relevant both to HIV infection as well as toneurological disorders. For instance, the pathway identified as viral host interactions included class II HLA-DRA, CCR5 and CCR2. While HIV in the context of cannabis developed to lower expression of HLA-DRA, cannabis did not lower the transcription of CCR5, suggesting a limited impact on viral entry . On the other hand, HIV in the context of cannabis and cannabis alone showed detectable decrease in the transcription of SIRT1, a histone deacetylase with epigenetic silencing properties .
We have previously reported that the transcriptional decrease of SIRT1 may be one factor in the dysregulation of the inflammatory environment and others have suggested that SIRT1 regulates viral transcription . Whether the effects of cannabis in this pathway have real implications to the infection remains to be addressed.In this cohort, we did not find correlations between the activation of these pathways in leukocytes and plasma or CSF viral load .Yet, the attenuating effects of cannabis observed in the context of HIV links and expands to pathways in inflammation and neurode generation,as well as to apoptosis, due to overlap in genes and transcriptional co-regulators .The actions of cannabis on the expression of genes involved in vascular adhesion and leukocyte transmigration have indicated that in HIVt cannabis grow tray users, peripheral leukocytes may be less likely to focally adhere to endothelial cells and migrate into tissues. This may be beneficial at preventing inflammation in end-organs including the brain,while potentially impairing surveillance, but also viral spread . The implications of this findings must be addressed using experimental models.Overall, the findings were consistent across pathways, suggesting that, like HIV alone, cannabis alone may increase the expression of a number of inflammation-associated genes, but this may differ in the context of HIV, where cannabis use was associated with attenuated or decreased expression of pathway components. In end organs, the actions of cannabis may differ due to effects via distinct receptors. Cannabinoid receptor 1 is largely expressed in CNS but also in several tissues with links to psychoactive and physiological effects, while CB2R is expressed mainly by immune cells with described anti-inflammatory and immuno suppressive properties . Given the differences in distribution and signaling between the receptors, effects of cannabis or cannabinoids may differ in the presence or absence of inflammatory cells, or in the context of infection, where pro-inflammatory signals are occurring.
Other less studied cannabinoid receptors and endocannabinoids may also play arole. Our data supports this idea that cannabis effects on molecular markers and biological processes is context-dependent, potentially driven by infection and inflammation, and the resulting differences in numbers and activation status of CB2R-expressing innate and adaptive immune cells.The examination of changes in expression patterns in kinase networks can inform mechanisms of action by cannabis in the context of HIV.Aberrant kinase activity is linked to a wide range of diseases including neoplastic diseases, central nervous system disorders, vascular disorders, and chronic inflammatory diseases. The analysis of a gene networks assigned to kinases indicated that cannabis in the context of HIV decreased transcription of components of the p38 MAPK pathway,which is involved in a diversity of biological functions . The blockage of p38 MAPK by cannabinoids has been previously reported in other models, with both suppressor and stimulating effects .Suppression of this pathway may be associated with blockage of oxidative stress.The anti-oxidant activity of cannabis and cannabinoid compounds has been previously acknowledged , although healthy cannabis users do not differ from non-users regarding oxidative stress markers.HIV infection promotes changes in the number of immune cells,quality and activation status of cell subsets. The infection and the broken homeostasis are likely critical in the determination of the effects of cannabis as a therapy or a complication. It has been suggested that the effects of cannabinoids on macrophages are critical to resulting T-cell mediated responses and may differ according to those cells activation status and to stimuli . Moreover, here we have shown by transcription factor usage predictions, that the effects of cannabis are associated with transcriptional co-regulation at the individual gene promoters, by multiple factors that may vary by context.Co-regulation by different transcription factors is a critical factor indetermination of transcriptional levels and kinetics , and is highly influenced by covariates and comorbidities.Cannabis use may be considered as a con-founder in biomarker investigations as it tended to mask the expression of molecules upregulated by HIV, particularly if cognitive function was not improved in parallel with markers, for instance when other drugs were present.
Cannabis users had better neurocognitive performance, overall and in learning and memory subdomains, particularly if they did not have a history of lifetime METH dependence. Such effect was stronger in METH users, but also observed in markers sensitive to HIV/alcohol and HIV/cocaine. This suggests differential effects of cannabis in the context of poly substanceuse and how the potentially beneficial effect of cannabis on HIV biomarkers may be relative when other drugs are also used.Overall, our work has screened effects of cannabis on an extensive number of transcript biomarkers of inflammation and neurological outcomes,which were peripherally expressed by uninfected and HIV infected subjects. Systems biology strategies have aided the identification of gene networks assigned to processes relevant to neuro HIV, which exhibited orchestrated behaviors in response to HIV or cannabis alone, or their interactions. Cannabis effects were largely dependent on context,with infection as the most significant interacting factor followed by poly substance use. Other factors not examined here may include cannabis use frequency and dose. The results suggest that cannabis maybe beneficial in the context of HIV when other substances are not concomitantly used. Despite an extensive history of use as a medicinal plant spanning ancient cultures , vertical grow systems for sale cannabis use is contentious in many jurisdictions as it has been considered a social drug of abuse since the mid-1930s . Over the last two decades, meaningful legal, sociocultural and economic change has led to the establishment of medicinal cannabis research programs in several countries, which have validated the therapeutic use of cannabis for indications including: chronic neuropathic pain, certain intractable epilepsies, the vomiting and spasticity of multiple sclerosis, and chemotherapy-induced nausea . Further to this, the use of medicinal cannabis has expanded into paediatric and vulnerable patient groups and regulated markets for recreational use have developed in some jurisdictions. Accordingly, quality control across the supply chain is increasingly important to ensure that cannabis products are safe and have well-defined chemical and therapeutic profiles. Critically, the complex relationship between chemical profiles and therapeutic activity requires further exploration. Presently, the activities of the three most abundant neutral cannabinoids – Δ9-THC, CBD, and CBG – have been studied closely, exhibiting properties including: analgesic, anticonvulsant, and anti-inflammatory . However, the full potential of medicinal cannabis may not be realised without leveraging the full diversity of cannabinoids. Over 140 cannabinoids have been identified, many of which have their own inherent pharmacological properties.This includes the acidic cannabinoids which have significant anticonvulsant activities, contrary to the historical perspective that they were inert precursors which only acquired activity after decarboxylating into the neutral cannabinoids.Furthermore, the complexity of cannabis increases geometrically under the ‘entourage effect’, which postulates that cannabinoids interact to modulate their therapeutic effects . An experimental basis for the entourage effect is provided by murine studies, which have demonstrated that binary combinations with acidic cannabinoids increase bio-availability, potency and efficacy of neutral cannabinoids in epilepsy models .
Clinical evidence is also mounting, with a recent meta-analysis on observational studies of epileptic patients concluding that crude cannabis extracts yielded a greater reduction in seizure frequency and had fewer side-effects than equivalent doses of purified CBD . However, as most extracts were only characterised to the extent of standardising the CBD dose, information about other cannabinoids was absent or based on inference. Consequently, the authors’ attribution of the differences between the extracts and purified CBD to the entourage effect was speculative. It was not possible to evaluate if the effects of the other cannabinoids added together, comparable to merely increasing the dose of CBD, or if they magnified the effect to surpass what CBD could achieve alone. Evidently, to progress beyond studies of binary combinations or poorly characterised extracts, routine analyses capable of quantifying panels of cannabinoids could help to better inform the design and interpretation of future studies that investigate the entourage effect. A clinical understanding of this effect might subsequently inform the extent to which cannabinoids are screened during cannabis product quality control. Several published methods are available for the separation and quantification of cannabinoids, with a variety of limitations which constrain their routine use. For the analysis of neutral cannabinoids, GC is simple, sensitive, and provides acceptable resolution . However, GC is not immediately suitable for acidic cannabinoids, as they are poorly volatilised and rapidly undergo thermal decarboxylation into neutral cannabinoids . Fortunately, this limitation can be surmounted by trimethylsilyl derivatisation of the labile acid group . Alternatively, some analysts have adopted LC for the separation of cannabinoids in medicinal cannabis. Following separation by LC, detection can be achieved by MS or by PDA. The MS detector enables the peak identity confirmation from their fragmentation patterns and relative ratios , and is sufficiently specific to recognise coeluting impurities in complex matrices . However, the required technical expertise, operation, and maintenance costs prohibit the use of MS for the routine analysis of cannabinoids. The UV-Vis PDA detectors are much cheaper, require less operator expertise, and are widely available. Since cannabinoids contain UV chromophores , they are amenable to PDA detection. Moreover, the UV spectra may assist with compound identity confirmation and the measurement of peak purity, which aids in quantification. Whichever detector is used, the elevated cost and limited availability of certified analytical reference standards for some cannabinoids remain impediments to their analysis. The cost can exceed $200 AUD per mg, and newly identified pharmacological leads in cannabis are possibly more expensive with significantly longer shipping times. To surmount this, some analysts have performed stereoselective microscales syntheses to obtain cannabinoids in a timelier manner , but this is beyond the remit of a typical QC lab. If the analysis of such cannabinoids is to become routine, the cost for their quantification must be mitigated. To this end, this study aimed to develop and validate a HPLC-PDA method for the determination of ten cannabinoids in medicinal cannabis inflorescence and oil and to explore the feasibility of using RRT for peak identification and RRF for their quantification. By this approach, an initial once-off purchase of all the standards was required to establish the RRT and RRF between the cannabinoids and the reference compounds: CBD as a reference for neutral cannabinoids, and CBDA as a reference for acidic cannabinoids; chosen as they are cheaper and available in many jurisdictions. Subsequently, the method may be routinely used in QC laboratories for the quantification of a panel of ten cannabinoids, requiring only sparing amounts of the reference compounds.