Thoracic impedance pneumography in propofol-sedated patients undergoing percutaneous endoscopic gastrostomy (PEG) placement in gastrointestinal endoscopy: A prospective, randomized trial.

STUDY OBJECTIVE: To assess the efficacy of an ECG-based method called thoracic impedance pneumography to reduce hypoxic events in endoscopy. DESIGN: This was a single center, 1:1 randomized controlled trial. SETTING: The trial was conducted during the placement of percutaneous endoscopic gastrostomy (PEG). PATIENTS: 173 patients who underwent PEG placement were enrolled in the present trial. Indication was oncological in most patients (89%). 58% of patients were ASA class II and 42% of patients ASA class III. INTERVENTIONS: Patients were randomized in the standard monitoring group (SM) with pulse oximetry and automatic blood pressure measurement or in the intervention group with additional thoracic impedance pneumography (TIM). Sedation was performed with propofol by gastroenterologists or trained nurses. MEASUREMENTS: Hypoxic episodes defined as SpO2 < 90% for >15 s were the primary endpoint. Secondary endpoints were minimal SpO2, apnea >10s/>30s and incurred costs. MAIN RESULTS: Additional use of thoracic impedance pneumography reduced hypoxic episodes (TIM: 31% vs SM: 49%; p = 0.016; OR 0.47; NNT 5.6) and elevated minimal SpO2 per procedure (TIM: 90.0% ± 8.9; SM: 84.0% ± 17.6; p = 0.007) significantly. Apnea events >10s and > 30s were significantly more often detected in TIM (43%; 7%) compared to SM (1%; 0%; p < 0.001; p = 0.014) resulting in a time advantage of 17 s before the occurrence of hypoxic events. As a result, adjustments of oxygen flow were significantly more often necessary in SM than in TIM (p = 0.034) and assisted ventilation was less often needed in TIM (2%) compared with SM (9%; p = 0.053). Calculated costs for the additional use of thoracic impedance pneumography were 0.13$ (0.12 €/0.11 £) per procedure. CONCLUSIONS: Additional thoracic impedance pneumography reduced the quantity and extent of hypoxic events with less need of assisted ventilation. Supplemental costs per procedure were negligible. KEY WORDS: thoracic impedance pneumography, capnography, sedation, monitoring, gastrointestinal endoscopy, percutaneous endoscopic gastrostomy.

Optimization of a High-Throughput Cell-Based Screening Strategy to Identify Small-Molecule Inhibitors of IL-23 Signaling.

Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rß1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z' > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose-response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors.

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Prevalence of mental illness within families in a regional child-focussed mental health service.

Nearly 50% of all mental illnesses begin in childhood before the age of 14 years, and over 20% of parents have a mental illness. Few studies have examined the co-occurrence of mental illnesses in parents and children. In the present study, we examined the extent of mental illness within families of 152 clients attending an Australian regional child and adolescent mental health service (CAMHS). A cross-sectional study design was employed involving a case record review and clinician-completed questionnaire of the children and youth attending a CAMHS. It was found that 79% of these children were living with a parent with mental illness. The predominant diagnosis of both child and parent was an anxiety or mood disorder, and many families had co-occurring risk factors of domestic violence and limited social supports. The findings in this Australian cohort are similar to those of other international research. While novel in nature, the present study has highlighted the extent of both mental illness and scarce supports for both children and parents in the same family. The findings indicate the need for a coordinated multiservice delivery of appropriate and consistent family-focussed interventions, responding to both mental illness and social supports for children and parents. Further research should examine specific components of family need and support, as seen through the eyes of the child and their parent.

Mechanism of Hydrogen-Bonded Complex Formation between Ibuprofen and Nanocrystalline Hydroxyapatite.

Nanocrystalline hydroxyapatite (nanoHA) is the main hard component of bone and has the potential to be used to promote osseointegration of implants and to treat bone defects. Here, using active pharmaceutical ingredients (APIs) such as ibuprofen, we report on the prospects of combining nanoHA with biologically active compounds to improve the clinical performance of these treatments. In this study, we designed and investigated the possibility of API attachment to the surface of nanoHA crystals via the formation of a hydrogen-bonded complex. The mechanistic studies of an ibuprofen/nanoHA complex formation have been performed using a holistic approach encompassing spectroscopic (Fourier transform infrared (FTIR) and Raman) and X-ray diffraction techniques, as well as quantum chemistry calculations, while comparing the behavior of the ibuprofen/nanoHA complex with that of a physical mixture of the two components. Whereas ibuprofen exists in dimeric form both in solid and liquid state, our study showed that the formation of the ibuprofen/nanoHA complex most likely occurs via the dissociation of the ibuprofen dimer into monomeric species promoted by ethanol, with subsequent attachment of a monomer to the HA surface. An adsorption mode for this process is proposed; this includes hydrogen bonding of the hydroxyl group of ibuprofen to the hydroxyl group of the apatite, together with the interaction of the ibuprofen carbonyl group to an HA Ca center. Overall, this mechanistic study provides new insights into the molecular interactions between APIs and the surfaces of bioactive inorganic solids and sheds light on the relationship between the noncovalent bonding and drug release properties.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Succinic acid production on xylose-enriched biorefinery streams by Actinobacillus succinogenes in batch fermentation.

BACKGROUND: Co-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. However, SA production from biomass-derived hydrolysates has not yet been fully explored or developed. RESULTS: Here, we employ Actinobacillus succinogenes 130Z to produce succinate in batch fermentations from various substrates including (1) pure sugars to quantify substrate inhibition, (2) from mock hydrolysates similar to those from DAP containing single putative inhibitors, and (3) using the hydrolysate derived from two pilot-scale pretreatments: first, a mild alkaline wash (deacetylation) followed by DAP, and secondly a single DAP step, both with corn stover. These latter streams are both rich in xylose and contain different levels of inhibitors such as acetate, sugar dehydration products (furfural, 5-hydroxymethylfurfural), and lignin-derived products (ferulate, p-coumarate). In batch fermentations, we quantify succinate and co-product (acetate and formate) titers as well as succinate yields and productivities. We demonstrate yields of 0.74 g succinate/g sugars and 42.8 g/L succinate from deacetylated DAP hydrolysate, achieving maximum productivities of up to 1.27 g/L-h. Moreover, A. succinogenes is shown to detoxify furfural via reduction to furfuryl alcohol, although an initial lag in succinate production is observed when furans are present. Acetate seems to be the main inhibitor for this bacterium present in biomass hydrolysates. CONCLUSION: Overall, these results demonstrate that biomass-derived, xylose-enriched hydrolysates result in similar yields and titers but lower productivities compared to clean sugar streams, which can likely be improved via fermentation process developments and metabolic engineering. Overall, this study comprehensively examines the behavior of A. succinogenes on xylose-enriched hydrolysates on an industrially relevant, lignocellulosic feedstock, which will pave the way for future work toward eventual SA production in an integrated biorefinery.

Continuous succinic acid production from xylose by Actinobacillus succinogenes.

Continuous, anaerobic fermentations of D-xylose were performed by Actinobacillus succinogenes 130Z in a custom, biofilm reactor at dilution rates of 0.05, 0.10 and 0.30 h(-1). Succinic acid yields on xylose (0.55-0.68 g g(-1)), titres (10.9-29.4 g L(-1)) and productivities (1.5-3.4 g L(-1) h(-1)) were lower than those of a previous study on glucose, but product ratios (succinic acid/acetic acid = 3.0-5.0 g g(-1)) and carbohydrate consumption rates were similar. Also, mass balance closures on xylose were up to 18.2 % lower than those on glucose. A modified HPLC method revealed pyruvic acid excretion at appreciable concentrations (1.2-1.9 g L(-1)) which improved the mass balance closure by up to 16.8 %. Furthermore, redox balances based on the accounted xylose consumed and the excreted metabolites, indicated an overproduction of reducing power. The oxidative pentose phosphate pathway was shown to be a plausible source of the additional reducing power.

Continuous succinic acid production by Actinobacillus succinogenes on xylose-enriched hydrolysate.

BACKGROUND: Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported. RESULTS: Actinobacillus succinogenes 130Z was immobilised in a custom continuous fermentation setup to produce SA on the xylose-enriched fraction of a non-detoxified, xylose-rich corn stover hydrolysate stream produced from deacetylation and dilute acid pretreatment. Effective biofilm attachment, which serves as a natural cell retention strategy to increase cell densities, productivities and resistance to toxicity, was accomplished by means of a novel agitator fitting. A maximum SA titre, yield and productivity of 39.6 g L(-1), 0.78 g g(-1) and 1.77 g L(-1) h(-1) were achieved, respectively. Steady states were obtained at dilution rates of 0.02, 0.03, 0.04, and 0.05 h(-1) and the stirred biofilm reactor was stable over prolonged periods of operation with a combined fermentation time of 1550 h. Furthermore, it was found that a gradual increase in the dilution rate was required to facilitate adaptation of the culture to the hydrolysate, suggesting a strong evolutionary response to the toxic compounds in the hydrolysate. Moreover, the two primary suspected fermentation inhibitors, furfural and HMF, were metabolised during fermentation with the concentration of each remaining at zero across all steady states. CONCLUSIONS: The results demonstrate that immobilised A. succinogenes has the potential for effective conversion of an industrially relevant, biomass-derived feed stream to succinic acid. Furthermore, due to the attractive yields, productivities and titres achieved in this study, the process has the potential to serve as a means for value-added chemical manufacturing in the integrated biorefinery.

High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC50 values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel.

A novel series of substituted tetrahydropyrrolo[3,4-c]pyrazoles were investigated as blockers of the N-type calcium channel (Cav2.2 channels), a chronic pain target.

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors.

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Cav2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.

Novel, broad-spectrum anticonvulsants containing a sulfamide group: pharmacological properties of (S)-N-[(6-chloro-2,3-dihydrobenzo[1,4]dioxin-2-yl)methyl]sulfamide (JNJ-26489112).

Broad-spectrum anticonvulsants are of considerable interest as antiepileptic drugs, especially because of their potential for treating refractory patients. Such "neurostabilizers" have also been used to treat other neurological disorders, including migraine, bipolar disorder, and neuropathic pain. We synthesized a series of sulfamide derivatives (4-9, 10a-i, 11a, 11b, 12) and evaluated their anticonvulsant activity. Thus, we identified promising sulfamide 4 (JNJ-26489112) and explored its pharmacological properties. Compound 4 exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures. Mechanistically, 4 inhibited voltage-gated Na(+) channels and N-type Ca(2+) channels and was effective as a K(+) channel opener. The anticonvulsant profile of 4 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. On the basis of its pharmacology and other favorable characteristics, 4 was advanced into human clinical studies.

A novel series of pyrazolylpiperidine N-type calcium channel blockers.

Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series.

An integrated multiassay approach to the discovery of small-molecule N-type voltage-gated calcium channel antagonists.

Abstract The N-type voltage-gated calcium channel (Cav2.2) has been intensively explored as a target for novel, small-molecule analgesic drugs because of its distribution in the pain pathway and its role in nociceptive processing. For example, Cav2.2 is localized at presynaptic terminals of pain fibers in the dorsal horn, and it serves as a downstream effector of µ-opioid receptors. Most importantly, antagonism of the channel by the highly specific and potent Cav2.2 blocker ω-conotoxin MVIIA (ziconotide) produces clinical efficacy in the treatment of severe, intractable pain. To identify novel small-molecule Cav2.2 inhibitors, we developed new tools and screening methods critical to enhance the efficiency and probability of success. First, we established and characterized a new cell line stably expressing the three subunits of the Cav2.2, including an α-subunit splice variant that is uniquely expressed by dorsal root ganglion neurons. Second, using this cell line, we validated and employed a fluorescence-based calcium flux assay. Third, we developed a new "medium-throughput" electrophysiology assay using QPatch-HT to provide faster turnaround on high-content electrophysiology data that are critical for studying ion channel targets. Lastly, we used a therapeutically relevant, ex vivo spinal cord calcitonin gene-related peptide-release assay to confirm activities in the other assays. Using this approach we have identified compounds exhibiting single-digit nM IC50 values and with a positive correlation across assay methods. This integrated approach provides a more comprehensive evaluation of small-molecule N-type inhibitors that may lead to improved therapeutic pharmacology.

Novel, broad-spectrum anticonvulsants containing a sulfamide group: advancement of N-((benzo[b]thien-3-yl)methyl)sulfamide (JNJ-26990990) into human clinical studies.

In seeking broad-spectrum anticonvulsants to treat epilepsy and other neurological disorders, we synthesized and tested a group of sulfamide derivatives (4a-k, 5), which led to the clinical development of 4a (JNJ-26990990). This compound exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures, with very weak inhibition of human carbonic anhydrase-II (IC(50) = 110 microM). The pharmacological profile for 4a supports its potential in the treatment of multiple forms of epilepsy, including pharmacoresistant variants. Mechanistically, 4a inhibited voltage-gated Na(+) channels and N-type Ca(2+) channels but was not effective as a K(+) channel opener. The pharmacokinetics and metabolic properties of 4a are discussed.

SNAP-25 Ser187 does not mediate phorbol ester enhancement of hippocampal synaptic transmission.

Phorbol esters, activators of protein kinase C (PKC), have been shown to enhance synaptic transmission. One potential downstream target of PKC in the presynaptic terminal is the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) SNAP-25, which has a PKC phosphorylation site in its C-terminal coil centered at serine 187 (S187/Ser187). We examined the role of S187 in hippocampal synaptic transmission. After proteolytic cleavage of native SNAP-25 by botulinum neurotoxin E (BoNT/E), synaptic transmission was restored in a subset of transfected CA3 pyramidal cells with a toxin-resistant form of SNAP-25 containing unaltered S187 (Swt), S187 mutated to alanine (SA) or S187 mutated to glutamate (SE). We observed that phorbol-12,13-diacetate (PDAc, 10 microM) induced potentiation of neurotransmission to a similar degree for both Swt and SA (2.4-fold and 3.1-fold increase, respectively). Furthermore, basal levels of transmission mediated by SE were reduced relative to that of Swt (failure rates of 72% and 41%, respectively). Together, these data suggest that phosphorylation of SNAP-25 S187 does not mediate the observed enhancement of neurotransmission by phorbol esters at hippocampal synapses.

The core membrane fusion complex governs the probability of synaptic vesicle fusion but not transmitter release kinetics.

Synaptic vesicle fusion is driven by the formation of a four-helical bundle composed of soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors (SNAREs). Exactly how the structural interactions that lead to the formation of this complex relate to neurotransmitter release is not well understood. To address this question, we used a strategy to "rescue" synaptic transmission after proteolytic cleavage of the synaptosome-associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin E (BoNtE). Transfection of CA3 hippocampal pyramidal cells with BoNtE-resistant SNAP-25 restored synaptic transmission. Additional mutations that alter the interaction between SNAP-25 C-terminal coil and the other SNARE coils dramatically reduce transmitter release probability but leave the kinetics of synaptic responses unaltered. These data indicate that at synapses, SNARE interactions are necessary for fusion but are not the rate-limiting step of neurotransmission.