A Multimodal Sepsis Quality-Improvement Initiative Including 24/7 Screening and a Dedicated Sepsis Response Team-Reduced Readmissions and Mortality.

OBJECTIVES: To evaluate if a hospitalwide sepsis performance improvement initiative improves compliance with the Centers for Medicare and Medicaid Services-mandated sepsis bundle interventions and patient outcomes. STUDY DESIGN: Retrospective analysis comparing 6 months before and 14 months after intervention. SETTING: Tertiary teaching hospital in Washington, DC. SUBJECTS: Patients admitted with a diagnosis of sepsis to a tertiary hospital. INTERVENTIONS: Implementation of a multimodal quality-improvement initiative. MEASUREMENTS AND MAIN RESULTS: A total of 4,102 patients were diagnosed with sepsis, severe sepsis, or septic shock during the study period, 861 patients (21%) were diagnosed during a 6-month preintervention period, and 3,241 (79%) were diagnosed in a 13-month postintervention period. Adjusted for patient case-mix, the prevalence of simple sepsis increased by 12%, but it decreased for severe sepsis and septic shock by 5.3% and 6.9%, respectively. Compliance with all sepsis bundle interventions increased by 31.1 percentage points (p < 0.01). All-cause hospital readmission and readmission due to infection were both reduced by 1.6% and 1.7 percentage points (p < 0.05). Death from any sepsis diagnosis was reduced 4.5% (p < 0.01). Death from severe sepsis and septic shock both was reduced by 5% (p < 0.01) and 6.5% (p < 0.01), respectively. CONCLUSIONS: After the implementation of multimodal sepsis performance initiatives, we observed a higher prevalence of sepsis secondary to screening but a lower prevalence of severe sepsis and septic shock, an improvement in compliance with the sepsis bundle interventions bundle, as well as reduction in hospital readmission and all- cause mortality rate.

Towards a TREK-1/2 (TWIK-Related K+ Channel 1 and 2) dual activator tool compound: Multi-dimensional optimization of BL-1249.

This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K2P potassium channels TREK-1 (K2P2.1) and TREK-2 (K2P10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds.

Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues.

To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.

Selective inhibition of M5 muscarinic acetylcholine receptors attenuates cocaine self-administration in rats.

Cocaine use disorder (CUD) remains a debilitating health problem in the United States for which there are no Food and Drug Administration-approved treatment options. Accumulating anatomical and electrophysiological evidence indicates that the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5 ) plays a critical role in the regulation of the mesolimbic dopaminergic reward circuitry, a major site of action for cocaine and other psychostimulants. In addition, M5 knockout mice exhibit reduced cocaine self-administration behaviors with no differences in sugar pellet-maintained responding relative to wild-type mice. These findings suggest that selective inhibition of M5 mAChR may provide a novel pharmacological approach for targeting CUD. Recently, we reported the synthesis and characterization of ML375, a selective negative allosteric modulator (NAM) for the rat and human M5 mAChR with optimized pharmacokinetic properties for systemic dosing in rodents. In the present study, male Sprague-Dawley rats were trained to self-administer intravenous cocaine (0.1-0.75 mg/kg/infusion) under a 10-response fixed ratio or a progressive ratio schedule of reinforcement. Under both schedules of reinforcement, ML375 produced dose-related reductions in cocaine self-administration. ML375 also modestly reduced sugar pellet-maintained responding on the 10-response, fixed ratio schedule but had no effect under a progressive ratio schedule of reinforcement. Further, ML375 did not affect general motor output as assessed by a rotarod test. Collectively, these results provide the first demonstration that selective inhibition of M5 using the M5 NAM ML375 can attenuate both the reinforcing effects and the relative strength of cocaine and suggest that M5 NAMs may represent a promising, novel treatment approach for CUD.

VU6010608, a Novel mGlu7 NAM from a Series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.

Herein, we report the structure-activity relationships within a series of mGlu7 NAMs based on an N-(2-(1H-1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamide core with excellent CNS penetration (Kp 1.9-5.8 and Kp,uu 0.4-1.4). Analogues in this series displayed steep SAR. Of these, VU6010608 (11a) emerged with robust efficacy in blocking high frequency stimulated long-term potentiation in electrophysiology studies.

Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5 NAM with high CNS penetration and a desired short half-life in rat for addiction studies.

This letter describes the continued optimization of M5 NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375has an excessively long elimination half-life in rat (t1/2=80h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M5 NAM of comparable potency to ML375, but with a rat t1/2 of less than 4h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M5 NAM, with high CNS penetration, excellent selectivity versus M1-4 and the desired short half-life (t1/2=2.3h) in rat.

Ligand-based virtual screen for the discovery of novel M5 inhibitor chemotypes.

This Letter describes a ligand-based virtual screening campaign utilizing SAR data around the M5 NAMs, ML375 and VU6000181. Both QSAR and shape scores were employed to virtually screen a 98,000-member compound library. Neither approach alone proved productive, but a consensus score of the two models identified a novel scaffold which proved to be a modestly selective, but weak inhibitor (VU0549108) of the M5 mAChR (M5 IC50=6.2µM, M1-4 IC50s>10µM) based on an unusual 8-((1,3,5-trimethyl-1H-pyrazol-4-yl)sulfonyl)-1-oxa-4-thia-8-azaspiro[4,5]decane scaffold. [(3)H]-NMS binding studies showed that VU0549108 interacts with the orthosteric site (Ki of 2.7µM), but it is not clear if this is negative cooperativity or orthosteric binding. Interestingly, analogs synthesized around VU0549108 proved weak, and SAR was very steep. However, this campaign validated the approach and warranted further expansion to identify additional novel chemotypes.

Synthesis and Biological Evaluation of Several Bryostatin Analogues Bearing a Diacylglycerol Lactone C-Ring.

As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings.

Biological activity of the bryostatin analog Merle 23 on mouse epidermal cells and mouse skin.

Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12-myristate 13-acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified bryostatin analogs that preserve the critical functional features of bryostatin 1, including its lack of tumor promoting activity. The degree to which bryostatin analogs maintain the unique pattern of biological behavior of bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified bryostatin analog that retains bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to bryostatin 1 or intermediate between bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.

Keratin 16 expression defines a subset of epithelial cells during skin morphogenesis and the hair cycle.

The morphogenesis of skin epithelia and adult hair follicle cycling both require integrated signaling between the epithelium and underlying mesenchyme. Because of their unique regulation, keratin intermediate filaments represent useful markers for the analysis of determination and differentiation processes in complex epithelia, such as the skin. In this study, we analyzed the distribution of mouse type I keratin 16 during skin morphogenesis, in the adult hair cycle, and in challenged epidermis. In mature hair follicles, we find keratin 16 along with its type II keratin partner keratin 6 in the companion layer of the outer root sheath during anagen and in the club hair sheath during catagen and telogen. During embryonic development, the distribution of keratin 16 is uncoupled from its presumed polymerization partner, keratin 6. Keratin 16 initially localizes within early hair germs, but rapidly shifts to a subset of cells at the interface of basal and suprabasal cells above and around the hair germ. The presence of keratin 16 at the transition between mitotically active and differentiating cells is recapitulated in primary keratinocytes cultured in vitro and in phorbol 12-myristate 13-acetate-treated back skin in vivo. We propose that keratin 16 marks cells in an intermediate state of cellular properties in which keratinocytes retain the flexibility required for activities such as cell migration and even mitosis but are resilient enough to provide the structural integrity required of the early suprabasal layers in the context of development, adult hair cycling, and wound repair.

Keratin 17 null mice exhibit age- and strain-dependent alopecia.

Onset of type I keratin 17 (K17) synthesis marks the adoption of an appendageal fate within embryonic ectoderm, and its expression persists in specific cell types within mature hair, glands, and nail. We report that K17 null mice develop severe alopecia during the first week postbirth, correlating with hair fragility, alterations in follicular histology, and apoptosis in matrix cells. These alterations are incompletely penetrant and normalize starting with the first postnatal cycle. Absence of a hair phenotype correlates with a genetic strain-dependent compensation by related keratins, including K16. These findings reveal a crucial role for K17 in the structural integrity of the first hair produced and the survival of hair-producing cells. Given that identical inherited mutations in this gene can cause either pachyonychia congenita or steatocystoma multiplex, the features of this mouse model suggest that this clinical heterogeneity arises from a cell type-specific, genetically determined compensation by related keratins.