Faculty Bibliography

Purpose: Evaluate the dose-ranging safety, tolerability, and pharmacokinetics (PK) of CBD in children with Dravet syndrome (DS). Method: Patients aged 4-10 years completed a 4-week baseline period and were randomised 4:1 to 1 of 3 CBD doses (5, 10, 20 mg/kg/day) or placebo as add-on therapy for 3 weeks. CBD (25 or 100 mg/mL oral solution) was administered BID starting at 2.5 mg/kg/day and increasing by 2.5 mg/kg QOD to randomised dose. On Days 1 and 22, PK exposures were expressed as AUC0-t. Dose proportionality was assessed on Day 22 by regression analysis. Adverse events (AEs) were recorded daily. Results: 34 patients were randomised to CBD 5 mg/kg/day (n = 10), 10 mg/kg/day (n = 8), 20 mg/kg/day (n = 9), or placebo (n = 7). Patients took a median 3 antiepileptic drugs (AEDs). On Day 22, exposures to CBD and major metabolites increased dose-proportionally; there was minimal change in clobazam levels, but concentrations of clobazam's metabolite, N-clobazam, increased independent of CBD dose, except in patients on stiripentol. There was no demonstrable effect on other AEDs (valproic acid, topiramate, stiripentol, levetiracetam). Most common AEs (CBD vs. placebo) were pyrexia (22% vs. 0%), somnolence (19% vs. 14%), decreased appetite (19% vs. 0%), and sedation (15% vs. 0%). Treatment-related serious AEs occurred in 2 patients on CBD and discontinuations due to AEs occurred in 2 patients on CBD. Increases in ALT or AST (levels >3 9 ULN) occurred in 6 patients on CBD, all on valproic acid; none had elevated bilirubin and all recovered. Conclusion: CBD was well tolerated and 20 mg/kg/day was chosen for further development. Exposure to CBD and its metabolites increased dose-proportionally. A PK interaction of CBD on N-clobazam was observed, likely mediated through CYP2C19 inhibition, except with stiripentol, presumably from prior saturated inhibition of CYP2C19 by stiripentol

Purpose: Assess the effect of CBD added to antiepileptic drug (AED) therapy for the treatment of drug-resistant seizures in Dravet syndrome. Method: This double-blind, placebo-controlled trial randomised 120 children aged 2-18 years with Dravet syndrome and drug-resistant seizures to receive CBD oral solution 20 mg/kg/day (n = 61) or placebo (n = 59) for 14 weeks (2 week titration; 12 week maintenance). The primary endpoint was the percentage change from baseline in convulsive seizures (tonic-clonic, tonic, clonic, and atonic) frequency over the 14-week treatment period. Results: The groups were well-balanced at baseline for demographics. Mean age was 10 years, with 29% of patients <6 years. Patients had previously tried a median 4 AEDs, and were currently taking a median 3 AEDs. Convulsive seizure frequency per month decreased from a median of 12.4 to 5.9 (median reduction of 39%) with CBD vs. 14.9 to 14.1 (median reduction of 13%) with placebo (difference between groups of 23%; p = 0.012). The proportion of patients with >=50% reduction in convulsive seizure frequency was 42.6% with CBD vs. 27.1% with placebo (OR=2.0; p = 0.078). Adverse events (AEs) occurred in 93.4% of CBD and 74.6% of placebo patients, and were mostly mild or moderate; the most common were somnolence, diarrhea, and decreased appetite. Serious AEs were reported in 16.4% of CBD and 5.1% of placebo patients, and were considered treatment-related in 8.2% of CBD patients, all of whom discontinued CBD. Some elevations in transaminases were noted without elevations of bilirubin; all were on concomitant valproate and all resolved. There were no deaths in the study. Conclusion: Results from this study suggest that CBD add-on therapy for drug-resistant seizures in Dravet syndrome may be efficacious, with more AEs than placebo but generally well tolerated

Objective: Assess the effect of adjunctive CBD for treatment of drug-resistant seizures in Dravet syndrome. Methods: This double-blind, placebo-controlled trial randomized 120 children aged 2-18 years with Dravet syndrome and drug-resistant seizures to receive CBD oral solution 20 mg/kg/ day (n=61) or placebo (n=59) for 14 weeks (2 week titration; 12 week maintenance). The primary endpoint was the percentage change from baseline in convulsive seizures (tonic-clonic, tonic, clonic, and atonic) frequency over the 14-week treatment period. Results: The groups were well-balanced at baseline for demographics. Mean age was 10 years, with 29% of patients <6 years. Patients had previously tried a median 4 AEDs, and were currently taking a median 3 AEDs. Convulsive seizure frequency per month decreased from a median of 12.4 to 5.9 (median reduction of 39%) with CBD versus 14.9 to 14.1 (median reduction of 13%) with placebo (difference between groups of 23%; p=0.012). The proportion of patients with e50% reduction in convulsive seizure frequency was 42.6% with CBD versus 27.1% with placebo (OR=2.0; p=0.078). Adverse events (AEs) occurred in 93.4% of CBD and 74.6% of placebo patients, and were mostly mild or moderate; the most common were somnolence, diarrhea, and decreased appetite. Serious AEs were reported in 16.4% of CBD and 5.1% of placebo patients, and were considered treatment-related in 8.2% of CBD patients, all of whom discontinued CBD. Some elevations in transaminases were noted without elevations of bilirubin; all were on concomitant valproate and all resolved. There were no deaths in the study. Conclusions: In this study, CBD resulted in a significantly greater reduction in convulsive seizure frequency than placebo; adverse events were more frequent with CBD, but it was generally well tolerated

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

BACKGROUND:Intensive care units (ICUs) are high-risk settings for the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). METHODS:In a cluster-randomized trial, we evaluated the effect of surveillance for MRSA and VRE colonization and of the expanded use of barrier precautions (intervention) as compared with existing practice (control) on the incidence of MRSA or VRE colonization or infection in adult ICUs. Surveillance cultures were obtained from patients in all participating ICUs; the results were reported only to ICUs assigned to the intervention. In intervention ICUs, patients who were colonized or infected with MRSA or VRE were assigned to care with contact precautions; all the other patients were assigned to care with universal gloving until their discharge or until surveillance cultures obtained at admission were reported to be negative. RESULTS:During a 6-month intervention period, there were 5434 admissions to 10 intervention ICUs, and 3705 admissions to 8 control ICUs. Patients who were colonized or infected with MRSA or VRE were assigned to barrier precautions more frequently in intervention ICUs than in control ICUs (a median of 92% of ICU days with either contact precautions or universal gloving [51% with contact precautions and 43% with universal gloving] in intervention ICUs vs. a median of 38% of ICU days with contact precautions in control ICUs, P<0.001). In intervention ICUs, health care providers used clean gloves, gowns, and hand hygiene less frequently than required for contacts with patients assigned to barrier precautions; when contact precautions were specified, gloves were used for a median of 82% of contacts, gowns for 77% of contacts, and hand hygiene after 69% of contacts, and when universal gloving was specified, gloves were used for a median of 72% of contacts and hand hygiene after 62% of contacts. The mean (±SE) ICU-level incidence of events of colonization or infection with MRSA or VRE per 1000 patient-days at risk, adjusted for baseline incidence, did not differ significantly between the intervention and control ICUs (40.4±3.3 and 35.6±3.7 in the two groups, respectively; P=0.35). CONCLUSIONS:The intervention was not effective in reducing the transmission of MRSA or VRE, although the use of barrier precautions by providers was less than what was required. (Funded by the National Institute of Allergy and Infectious Diseases and others; STAR*ICU ClinicalTrials.gov number, NCT00100386.).

The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.