Quantitation of physiological and biochemical barriers to siRNA liver delivery via lipid nanoparticle platform.

Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50's ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.

Negative and Positive Emotional Reactivity in Women With and Without a History of Self-Injury.

In trying to better understand why certain individuals self-injure, researchers have proposed high emotional reactivity for negative emotions may influence vulnerabilities and predispose individuals to react to stressful situations in a dysregulated manner, thus engaging in non-suicidal self-injury (NSSI). However, the role of emotional reactivity for positive emotions in those with a history of NSSI is still unclear. Thus, the present study sought to examine group differences in the reactivity of (a) negative and (b) positive emotions in young adults with and without a history of NSSI engagement, and (c) to evaluate whether the reactivity of positive emotions could predict NSSI engagement when controlling for reactivity of negative emotions. The sample consisted of 96 female students who reported engaging in NSSI within the past 2 years (Mage = 20.28 years, SD = 1.65) and an age-matched female comparison group with no NSSI history (Mage = 20.43 years, SD = 1.76). Results from separate MANOVAs indicated individuals with a history of NSSI reported higher negative reactivity across all aspects (emotional intensity, sensitivity, and persistence) than the comparison group, Wilk's λ = .86, F (3,188) = 10.65, p < .001, partial η2 = .145; however, no significant differences emerged for positive reactivity, Wilk's λ = .99, F (3,188) = 0.52, p = .669. Moreover, a logistic regression revealed that persistence of negative emotions was the only significant predictor of NSSI, Wald χ2 (1) = 4.54, p = .03. The present results highlight the importance of the persistence of negative emotions for individuals who engage in NSSI. Furthermore, the current study provides the first suggestion of no significant differences in positive emotional reactivity between individuals with and without NSSI; underlining the importance of focusing on negative emotional reactivity in clinical practice as well as using positive emotions to "undo" the effect of negative emotions.

Productive replication of Vif-chimeric HIV-1 in feline cells.

Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G-->A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1(VF) and HIV-1(VS)) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection.

Perceived vs. Actual Emotion Reactivity and Regulation in Individuals With and Without a History of NSSI.

Non-suicidal self-injury (NSSI) has consistently been associated with self-reported difficulties in emotion reactivity and the regulation of negative emotions; however, less is known about the accuracy of these self-reports or the reactivity and regulation of positive emotions. The present study sought to investigate differences between women with and without a history of NSSI on: (a) self-reported general tendencies of negative and positive emotion reactivity, (b) self-reported general tendencies of negative and positive emotion regulation, and (c) emotion regulation reported in response to a positive and negative mood induction. The sample consisted of 36 women with a recent history of NSSI within the last 2 years (Mage = 20.06; SD = 1.51) and a comparison group with no history of NSSI (n = 34; Mage = 20.15; SD = 1.54). Participants completed self-report measures of negative and positive emotion reactivity and regulation. In a separate session, participants underwent both a negative and positive mood induction using a counterbalanced design and reported their experienced emotions. Results from two-way MANOVAs and ANOVAs revealed those with a history of NSSI reported significantly greater difficulties in negative emotion reactivity and regulation than the no-NSSI comparison group; however, no group differences emerged in self-reported reactivity or regulation of positive emotions. In contrast, repeated measures ANOVAs on data from the mood induction task found no group differences in reactivity or regulation for either negative or positive emotions. These findings highlight the possibility that although individuals with a history of NSSI evaluate their ability to manage negative emotions as significantly worse than individuals with no history of self-injury, this may not reflect their actual emotion regulatory processes.

Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors.

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Phase I Study of the Novel Enhancer of Zeste Homolog 2 (EZH2) Inhibitor GSK2816126 in Patients with Advanced Hematologic and Solid Tumors.

PURPOSE: Enhancer of zeste homolog 2 (EZH2) activity is dysregulated in many cancers. PATIENTS AND METHODS: This phase I study determined the safety, maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of the intravenously administered, highly selective EZH2 inhibitor, GSK2816126, (NCT02082977). Doses of GSK2816126 ranged from 50 to 3,000 mg twice weekly, and GSK2816126 was given 3-weeks-on/1-week-off in 28-day cycles. Eligible patients had solid tumors or B-cell lymphomas with no available standard treatment regimen. RESULTS: Forty-one patients (21 solid tumors, 20 lymphoma) received treatment. All patients experienced ≥1 adverse event (AE). Fatigue [22 of 41 (53.7%)] and nausea [20 of 41 (48.8%)] were the most common toxicity. Twelve (32%) patients experienced a serious AE. Dose-limiting elevated liver transaminases occurred in 2 of 7 patients receiving 3,000 mg of GSK2816126; 2,400 mg was therefore established as the MTD. Following intravenous administration of 50 to 3,000 mg twice weekly, plasma GSK2816126 levels decreased biexponentially, with a mean terminal elimination half-life of approximately 27 hours. GSK2816126 exposure (maximum observed plasma concentration and area under the plasma-time curve) increased in a dose-proportional manner. No change from baseline in H3K27me3 was seen in peripheral blood mononuclear cells. Fourteen of 41 (34%) patients had radiological best response of stable disease, 1 patient with lymphoma achieved a partial response, 21 of 41 (51%) patients had progressive disease, and 5 patients were unevaluable for antitumor response. CONCLUSIONS: The MTD of GSK2816126 was established at 2,400 mg, but the dosing method and relatively short half-life limited effective exposure, and modest anticancer activity was observed at tolerable doses.

The Progression of End-of-Life Wishes and Concordance with End-of-Life Care.

Since 2013, Kaiser Permanente Northern California has engaged in a systematic effort to elicit, document, and honor the care preferences of patients as they near the end of life. This is done through its Advanced Steps program, in which selected patients discuss their preferences for future medical care with their healthcare agent during a structured conversation with a trained advance care planning facilitator. The facilitator then translates the patient's wishes into an actionable medical order set using a Physician's Order for Life-Sustaining Treatment (POLST) form. We wanted to know whether these patients' recorded wishes were concordant with care received at the end of life. To evaluate, we conducted an in-depth chart review of 300 patients who died in 2015 and had participated in the program. We determined that 290 patients received concordant care, whereas three patients received care discordant with their wishes before death. Seven patients did not have sufficient information in their record to determine concordance. Interestingly, we found care preferences often changed over time; ∼20% of patients revised their end-of-life preferences after having the facilitated conversation, with most of those patients opting for less intensive care. Most changes to preferences were made verbally in the final setting of care. While advance care planning and the POLST form provide invaluable tools for recording patients' wishes, our study highlights a need to track patients' wishes as they evolve over time and a need for ongoing, real-time conversations about goals of care, even after a POLST is completed.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.