Removing barriers to accessing medical cannabis for paediatric patients.

Medical cannabis (MC) may offer therapeutic benefits for children with complex neurological conditions and chronic diseases. In Canada, parents, and caregivers frequently report encountering barriers when accessing MC for their children. These include negative preconceived notions about risks and benefits, challenges connecting with a knowledgeable healthcare provider (HCP), the high cost of MC products, and navigating MC product shortages. In this manuscript, we explore several of these barriers and provide recommendations to decision-makers to enable a family-centered and evidence-based approach to MC medicine and research for children.

Novel Caulobacter bacteriophages illustrate the diversity of the podovirus genus Rauchvirus.

The podovirus BPP-1 is currently the only member of the Podovirus genus Rauchvirus. Here, we describe three new Caulobacter bacteriophages (Jess A, SR18, and RW) that show genetic similarity to BPP-1 but have many different genetic and structural features that differentiate them from BPP-1. Jess A and SR18 are closely related to each other and should be considered two members of a new species. They share a similar gene order with BPP-1. However, they do not appear to form lysogens or have the tropism switching mechanism that has been described for BPP-1. Bacteriophage RW also exhibits some homology to BPP-1. However, it is quite different from the other three phages, and we propose that it should be considered a representative of a third species of the genus Rauchvirus. Taken together, the differences among these four members of the genus Rauchvirus indicate that this divergent genus has a long evolutionary history and that there are many more rauchviruses waiting to be discovered.

Dosage Related Efficacy and Tolerability of Cannabidiol in Children With Treatment-Resistant Epileptic Encephalopathy: Preliminary Results of the CARE-E Study.

Purpose: There is uncertainty regarding the appropriate dose of Cannabidiol (CBD) for childhood epilepsy. We present the preliminary data of seven participants from the Cannabidiol in Children with Refractory Epileptic Encephalopathy (CARE-E) study. Methods: The study is an open-label, prospective, dose-escalation trial. Participants received escalating doses of a Cannabis Herbal Extract (CHE) preparation of 1:20 Δ9-tetrahydrocannabinol (THC): CBD up to 10-12 mg CBD/kg/day. Seizure frequency was monitored in daily logs, participants underwent regular electroencephalograms, and parents filled out modified Quality of Life in Childhood Epilepsy (QOLCE) and Side Effect rating scale questionnaires. Steady-state trough levels (Css, Min) of selected cannabinoids were quantified. Results: All seven participants tolerated the CHE up to 10-12 mg CBD/kg/day and had improvements in seizure frequency and QOLCE scores. CSS, Min plasma levels for CBD, THC, and cannabichromene (CBC) showed dose-independent pharmacokinetics in all but one participant. CSS, Min CBD levels associated with a >50% reduction in seizures and seizure freedom were lower than those reported previously with purified CBD. In most patients, CSS, Min levels of THC remained lower than what would be expected to cause intoxication. Conclusion: The preliminary data suggest an initial CBD target dose of 5-6 mg/kg/day when a 1:20 THC:CBD CHE is used. Possible non-linear pharmacokinetics of CBD and CBC needs investigation. The reduction in seizure frequency seen suggests improved seizure control when a whole plant CHE is used. Plasma THC levels suggest a low risk of THC intoxication when a 1:20 THC:CBD CHE is used in doses up to 12 mg/kg CBD/kg/day.

The protocol for the Cannabidiol in children with refractory epileptic encephalopathy (CARE-E) study: a phase 1 dosage escalation study.

BACKGROUND: Initial studies suggest pharmaceutical grade cannabidiol (CBD) can reduce the frequency of convulsive seizures and lead to improvements in quality of life in children affected by epileptic encephalopathies. With limited access to pharmaceutical CBD, Cannabis extracts in oil are becoming increasingly available. Physicians show reluctance to recommend Cannabis extracts given the lack of high quality safety data especially regarding the potential for harm caused by other cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC). The primary aims of the study presented in this protocol are (i) To determine whether CBD enriched Cannabis extract is safe and well-tolerated for pediatric patients with refractory epilepsy, (ii) To monitor the effects of CBD-enriched Cannabis extract on the frequency and duration of seizure types and on quality of life. METHODS: Twenty-eight children with treatment resistant epileptic encephalopathy ranging in age from 1 to 10 years will be recruited in four Canadian cities into an open-label, dose-escalation phase 1 trial. The primary objectives for the study are (i) To determine if the CBD-enriched Cannabis herbal extract is safe and well-tolerated for pediatric patients with treatment resistant epileptic encephalopathy and (ii) To determine the effect of CBD-enriched Cannabis herbal extract on the frequency and duration of seizures. Secondary objectives include (i) To determine if CBD-enriched Cannabis herbal extracts alter steady-state levels of co-administered anticonvulsant medications. (ii) To assess the relation between dose escalation and quality of life measures, (iii) To determine the relation between dose escalation and steady state trough levels of bioactive cannabinoids. (iv) To determine the relation between dose escalation and incidence of adverse effects. DISCUSSION: This paper describes the study design of a phase 1 trial of CBD-enriched Cannabis herbal extract in children with treatment-resistant epileptic encephalopathy. This study will provide the first high quality analysis of safety of CBD-enriched Cannabis herbal extract in pediatric patients in relation to dosage and pharmacokinetics of the active cannabinoids. TRIAL REGISTRATION: http://clinicaltrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2016 Dec 16. Identifier NCT03024827, Cannabidiol in Children with Refractory Epileptic Encephalopathy: CARE-E; 2017 Jan 19 [cited 2017 Oct]; Available from: http://clinicaltrials.gov/ct2/show/NCT03024827.

Primary and secondary photodynamics of the violet/orange dual-cysteine NpF2164g3 cyanobacteriochrome domain from Nostoc punctiforme.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Like phytochromes, CBCRs photointerconvert between two photostates that accompany photoisomerization of their bilin chromophores. While phytochromes typically exhibit red/far-red photocycles, CBCR photocycles are much more diverse, spanning the near-ultraviolet and the entire visible region. All CBCRs described to date have a conserved Cys residue covalently attached to the linear tetrapyrrole (bilin) chromophore; two CBCR subfamilies also exploit a second thioether linkage to the chromophore for detection of near-ultraviolet to blue light. Here, we present the photodynamic analysis of the insert-Cys CBCR NpF2164g3, a representative of the second class of two-cysteine CBCRs. Using broadband transient absorption pump-probe spectroscopy, we characterize the primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics in both directions, examining photodynamics over nine decades of time. Primary isomerization dynamics occur on a ~10 ps time scale for both forward and reverse reactions. In contrast to previous studies on Tlr0924, a representative of the other class of two-cysteine CBCRs, formation and elimination of the second linkage are slower than the 1 ms experimental range probed here. These results extend our understanding of dual-cysteine CBCR photocycles in the phytochrome superfamily.

Chemical inhomogeneity in the ultrafast dynamics of the DXCF cyanobacteriochrome Tlr0924.

Cyanobacteriochromes (CBCRs) are diverse biliprotein photosensors distantly related to the red/far-red photoreceptors of the phytochrome family. There are several subfamilies of CBCRs, displaying varied spectral responses spanning the entire visible region. Tlr0924 belongs to the DXCF subfamily that utilizes the Cys residue in a conserved Asp-Xaa-Cys-Phe (DXCF) motif to form a second covalent linkage to the chromophore, resulting in a blue-absorbing dark state. Photoconversion leads to elimination of this linkage, resulting in a green-absorbing photoproduct. Tlr0924 initially incorporates phycocyanobilin (PCB) as a chromophore, exhibiting a blue/orange photocycle, but slowly isomerizes PCB to phycoviolobilin (PVB) to yield a blue/green photocycle. Ultrafast transient absorption spectroscopy was used to study both forward and reverse reaction photodynamics of the recombinant GAF domain of Tlr0924. Primary photoproducts were identified, as were subsequent intermediates at 1 ms. PCB and PVB population photodynamics were decomposed using global target analysis. PCB and PVB populations exhibit similar and parallel photocycles in Tlr0924, but the PVB population exhibits faster excited-state decay in both reaction directions. On the basis of longer time analysis, we show that the photochemical coordinate (15,16-isomerization) and second-linkage coordinate (elimination or bond formation at C10) are separate processes in both directions.

Note: a flexible light emitting diode-based broadband transient-absorption spectrometer.

This Note presents a simple and flexible ns-to-ms transient absorption spectrometer based on pulsed light emitting diode (LED) technology that can be incorporated into existing ultrafast transient absorption spectrometers or operate as a stand-alone instrument with fixed-wavelength laser sources. The LED probe pulses from this instrument exhibit excellent stability (∼0.5%) and are capable of producing high signal-to-noise long-time (>100 ns) transient absorption signals either in a broadband multiplexed (spanning 250 nm) or in tunable narrowband (20 ns) operation. The utility of the instrument is demonstrated by measuring the photoinduced ns-to-ms photodynamics of the red/green absorbing fourth GMP phosphodiesterase/adenylyl cyclase/FhlA domain of the NpR6012 locus of the nitrogen-fixing cyanobacterium Nostoc punctiforme.

The bilayer enhances rhodopsin kinetic stability in bovine rod outer segment disk membranes.

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-ß-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-ß-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (T(m)) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T(m) as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (E(act)) was calculated from the scan rate dependence of the T(m) and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E(act) as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E(act) to rhodopsin denaturation.

Assessment of bovine rod outer segment disk membrane heterogeneity utilizing flow cytometry.

The outer segment of rod photoreceptor cells is responsible for initiating visual signal transduction when light levels are low. It consists of stacked disk membranes surrounded by the plasma membrane and is under continuous renewal. Disk membranes are synthesized at the base of the outer segment and are progressively displaced toward the apical tip where they are phagocytosed by the pigment epithelium. This process takes approximately ten days during which time the lipid composition of the disk membrane is modified. Disk membranes become progressively lower in cholesterol and higher in unsaturated phospholipids during spatial displacement. The modification of disk composition is likely important for normal function of the rod cell. Flow cytometry is shown in this study to be a powerful tool to assess differences among rod outer segment disk membranes. First, the feasibility of flow cytometry to detect individual osmotically intact disks was established. Fluorescent beads of 1.0 µm, 0.5 µm, 0.2 µm, and 0.1 µm diameter were used to demonstrate that side-scatter intensity measured on a log scale corresponds to bead diameter. The intensity of disk side scatter predominantly corresponded to 0.1-0.2 µm diameter beads. We next applied this technique to detect caveolin-1, peripherin/rds, and GM1 in disk membranes. Caveolin-1 was detected with FITC-labeled anti-caveolin-1, peripherin/rds with Cy3 labeled anti-peripherin/rds, and GM1 with FITC-labeled cholera toxin subunit B. Fluorescence due to caveolin-1 and peripherin/rds binding was detected in 80% and 70% respectively of the events that corresponded to scattering attributed to disks. Fluorescence attributed to GM1 was detected in 60% of the events that corresponded to disk scattering. The intensity of fluorescence due to caveolin-1 and peripherin/rds labeling was directly proportional to the intensity of side scatter, indicating that both caveolin-1 and peripherin/rds concentrations are uniform among all the disks. Fluorescence intensity of GM1 labeled disks was independent of side-scatter intensity indicating GM1 is heterogeneously distributed among the disks. These results are consistent with integral membrane proteins such as caveolin-1, and peripherin/rds remaining in the disk bilayer as the disks are apically displaced. The GM1 distribution may be similar to the spatial distribution of cholesterol in the outer segment.

Clinical factors that influence response to treatment strategies in atrial fibrillation: the Atrial Fibrillation Follow-up Investigation of Rhythm Management (AFFIRM) study.

BACKGROUND: The AFFIRM Study was a randomized multicenter comparison of 2 treatment strategies, rate-control versus rhythm-control, in high-risk patients with atrial fibrillation (AF). The primary outcome of the trial showed no overall difference in survival between strategies. However, there may be important patient subgroups for which there are identifiable differences in outcome with 1 of the 2 strategies. METHODS AND RESULTS: Subgroups that were prespecified for analysis from the main AFFIRM Study were age, sex, coronary artery disease (CAD), hypertension, congestive heart failure (CHF), left ventricular ejection fraction (LVEF), rhythm at randomization, first episode of AF, and duration of the qualifying episode of AF. Baseline characteristics were analyzed for each subgroup. Adjusted hazard ratios for each subgroup and for each stratum were generated using Cox models, and these models were used to determine whether treatment strategy affected overall survival differentially by subgroup. Adjusted survival was worse for patients > or =65 years and for patients with a history of CHF, CAD, or an abnormal LVEF. In the adjusted analyses, the effect of treatment strategy was similar within all of the prespecified subgroups. When each subgroup stratum was analyzed separately, patients > or =65 years and patients without a history of CHF had significantly better outcome with rate-control therapy (each P < .01). CONCLUSIONS: Overall, treatment effect for rate control versus rhythm control was the same within each subgroup. However, certain selected patient categories may have better survival with one particular strategy for management of AF.

Factors affecting bleeding risk during anticoagulant therapy in patients with atrial fibrillation: observations from the Atrial Fibrillation Follow-up Investigation of Rhythm Management (AFFIRM) study.

BACKGROUND: Stroke and systemic thromboembolism are serious problems for patients with atrial fibrillation (AF), but their incidence can be substantially reduced by appropriate anticoagulation. Bleeding is the major complication of anticoagulant treatment, and the relative risks for bleeding vs stroke must be considered when starting anticoagulation. METHODS: The AFFIRM trial included patients with AF and at least one risk factor for stroke, randomly assigning them to either a rate-control or rhythm-control strategy. All patients were initially treated with warfarin. The incidence of protocol-defined major and minor bleeding was documented during follow-up. Variables associated with bleeding were determined using a Cox proportional hazards model, using baseline and time-dependent covariates. RESULTS: The 4060 patients in the AFFIRM trial were followed for an average of 3.5 years. Major bleeding occurred in 260 patients, an annual incidence of approximately 2% per year, with no significant difference between the rate-control and rhythm-control groups. Increased age, heart failure, hepatic or renal disease, diabetes, first AF episode, warfarin use, and aspirin use were significantly associated with major bleeding. Minor bleeding was common in both treatment arms, with 738 patients reporting this problem in one or more visits. CONCLUSIONS: Bleeding is a significant problem that complicates management of patients with AF. Risk factors for bleeding can be identified, and knowledge of these risk factors can be used to plan therapy.

Occurrence and characteristics of stroke events in the Atrial Fibrillation Follow-up Investigation of Sinus Rhythm Management (AFFIRM) study.

BACKGROUND: Atrial fibrillation (AF) is a risk factor for stroke, especially when accompanied by other high-risk cardiovascular predictors. The Atrial Fibrillation Follow-up Investigation of Rhythm Management (AFFIRM) Study was a multicenter comparison of high-risk patients with AF who were randomized to either a sinus rhythm control or a rate control strategy. METHODS: Physicians were encouraged to continue anticoagulation therapy for their patients. Patients in the sinus rhythm control group could stop warfarin sodium therapy after 4 (preferably a minimum of 12) weeks if they maintained sinus rhythm while receiving an antiarrhythmic drug. RESULTS: The AFFIRM Study enrolled 4060 patients. Mortality was the same in both groups. Two hundred eleven patients (8.2%) had a stroke event. Ischemic stroke occurred in 157 patients (6.3%), primary intraparenchymal hemorrhage in 34 (1.2%), and subdural or subarachnoid hemorrhage in 24 (0.8%). The most frequently determined ischemic stroke mechanism was cardioembolic (35/71 [49%]). Treatment assignment had no significant effect on the occurrence of ischemic stroke. Patients in AF at the time of the stroke event had a 60% greater chance of having an ischemic stroke, and those taking warfarin at the time of follow-up had a 69% decrease in the risk of having an ischemic stroke. CONCLUSIONS: In the AFFIRM Study, stroke rates were not significantly different in the rate control and sinus rhythm control arms. However, several clinical and therapeutic variables were associated with stroke risk. In patients with a history of AF at high risk for stroke or death, the presence of AF increases the risk of having a stroke, and warfarin therapy reduces the risk of having a stroke. The beneficial effect of warfarin therapy is seen not only in patients in AF but also in patients with a history of AF but who presumably remain in sinus rhythm.

Relationships between sinus rhythm, treatment, and survival in the Atrial Fibrillation Follow-Up Investigation of Rhythm Management (AFFIRM) Study.

BACKGROUND: The AFFIRM Study showed that treatment of patients with atrial fibrillation and a high risk for stroke or death with a rhythm-control strategy offered no survival advantage over a rate-control strategy in an intention-to-treat analysis. This article reports an "on-treatment" analysis of the relationship of survival to cardiac rhythm and treatment as they changed over time. METHODS AND RESULTS: Modeling techniques were used to determine the relationships among survival, baseline clinical variables, and time-dependent variables. The following baseline variables were significantly associated with an increased risk of death: increasing age, coronary artery disease, congestive heart failure, diabetes, stroke or transient ischemic attack, smoking, left ventricular dysfunction, and mitral regurgitation. Among the time-dependent variables, the presence of sinus rhythm (SR) was associated with a lower risk of death, as was warfarin use. Antiarrhythmic drugs (AADs) were associated with increased mortality only after adjustment for the presence of SR. Consistent with the original intention-to-treat analysis, AADs were no longer associated with mortality when SR was removed from the model. CONCLUSIONS: Warfarin use improves survival. SR is either an important determinant of survival or a marker for other factors associated with survival that were not recorded, determined, or included in the survival model. Currently available AADs are not associated with improved survival, which suggests that any beneficial antiarrhythmic effects of AADs are offset by their adverse effects. If an effective method for maintaining SR with fewer adverse effects were available, it might be beneficial.