Downregulation of Ubiquitin-Specific Protease 15 (USP15) Does Not Provide Therapeutic Benefit in Experimental Mesial Temporal Lobe Epilepsy.

Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.

Planning experiments: Updated guidance on experimental design and analysis and their reporting III.

Scientists who plan to publish in British Journal of Pharmacology (BJP) must read this article before undertaking a study. This editorial provides guidance for the design of experiments. We have published previously two guidance documents on experimental design and analysis (Curtis et al., 2015; Curtis et al., 2018). This update clarifies and simplifies the requirements on design and analysis for BJP manuscripts. This editorial also details updated requirements following an audit and discussion on best practice by the BJP editorial board. Explanations for the requirements are provided in the previous articles. Here, we address new issues that have arisen in the course of handling manuscripts and emphasise three aspects of design that continue to present the greatest challenge to authors: randomisation, blinded analysis and balance of group sizes.

The Chemical Synthesis of Knob Domain Antibody Fragments.

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

An Analysis of Clostridium difficile Environmental Contamination During and After Treatment for C difficile Infection.

BACKGROUND: Lower Clostridium difficile spore counts in feces from C difficile infection (CDI) patients treated with fidaxomicin versus vancomycin have been observed. We aimed to determine whether environmental contamination is lower in patients treated with fidaxomicin compared with those treated with vancomycin/metronidazole. METHODS: The CDI cases were recruited at 4 UK hospitals (Leeds, Bradford, and London [2 centers]). Environmental samples (5 room sites) were taken pretreatment and at 2-3, 4-5, 6-8, and 9-12 days of treatment, end of treatment (EOT), and post-EOT. Fecal samples were collected at diagnosis and as often as produced thereafter. Swabs/feces were cultured for C difficile; percentage of C difficile-positive samples and C difficile bioburden were compared between different treatment arms at each time point. RESULTS: Pre-EOT (n = 244), there was a significant reduction in environmental contamination (≥1 site positive) around fidaxomicin versus vancomycin/metronidazole recipients at days 4-5 (30% vs 50% recipients, P = .04) and at days 9-12 (22% vs 49%, P = .005). This trend was consistently seen at all other timepoints, but it was not statistically significant. No differences were seen between treatment groups post-EOT (n = 76). Fidaxomicin-associated fecal positivity rates and colony counts were consistently lower than those for vancomycin/metronidazole from days 4 to 5 of treatment (including post-EOT); however, the only significant difference was in positivity rate at days 9-12 (15% vs 55%, P = .03). CONCLUSIONS: There were significant reductions in C difficile recovery from both feces and the environment around fidaxomicin versus vancomycin/metronidazole recipients. Therefore, fidaxomicin treatment may lower the C difficile transmission risk by reducing excretion and environmental contamination.

Helping to drive the robustness of preclinical research - the assay capability tool.

Numerous articles in Nature, Science, Pharmacology Research and Perspectives, and other biomedical research journals over the past decade have highlighted that research is plagued by findings that are not reliable and cannot be reproduced. Poor experiments can occur, in part, as a consequence of inadequate statistical thinking in the experimental design, conduct and analysis. As it is not feasible for statisticians to be involved in every preclinical experiment many of the same journals have published guidelines on good statistical practice. Here, we outline a tool that addresses the root causes of irreproducibility in preclinical research in the pharmaceutical industry. The Assay Capability Tool uses 13 questions to guide scientists and statisticians during the development of in vitro and in vivo assays. It promotes the absolutely essential experimental design and analysis strategies and documents the strengths, weaknesses, and precision of an assay. However, what differentiates it from other proposed solutions is the emphasis on how the resulting data will be used. An assay can be assigned a low, medium, or high rating to indicate the level of confidence that can be afforded when making important decisions using data from that assay. This provides transparency on the appropriate interpretation of the assay's results in the light of its current capability. We suggest that following a well-defined process during assay development and use such as that laid out within the Assay Capability Tool means that whatever the results, positive or negative, a researcher can have confidence to make decisions upon and publish their findings.

A preclinical physiological assay to test modulation of knee joint pain in the spinal cord: effects of oxycodone and naproxen.

Sensory processing in the spinal cord during disease states can reveal mechanisms for novel treatments, yet very little is known about pain processing at this level in the most commonly used animal models of articular pain. Here we report a test of the prediction that two clinically effective compounds, naproxen (an NSAID) and oxycodone (an opiate), are efficacious in reducing the response of spinal dorsal horn neurons to noxious knee joint rotation in the monosodium iodoacetate (MIA) sensitized rat. The overall objective for these experiments was to develop a high quality in vivo electrophysiology assay to confidently test novel compounds for efficacy against pain. Given the recent calls for improved preclinical experimental quality we also developed and implemented an Assay Capability Tool to determine the quality of our assay and ensure the quality of our results. Spinal dorsal horn neurons receiving input from the hind limb knee joint were recorded in anesthetized rats 14 days after they were sensitized with 1 mg of MIA. Intravenous administered oxycodone and naproxen were each tested separately for their effects on phasic, tonic, ongoing and afterdischarge action potential counts in response to innocuous and noxious knee joint rotation. Oxycodone reduced tonic spike counts more than the other measures, doing so by up to 85%. Tonic counts were therefore designated the primary endpoint when testing naproxen which reduced counts by up to 81%. Both reductions occurred at doses consistent with clinically effective doses for osteoarthritis. These results demonstrate that clinically effective doses of standard treatments for osteoarthritis reduce pain processing measured at the level of the spinal cord for two different mechanisms. The Assay Capability Tool helped to guide experimental design leading to a high quality and robust preclinical assay to use in discovering novel treatments for pain.

Simulation of human intravenous and oral pharmacokinetics of 21 diverse compounds using physiologically based pharmacokinetic modelling.

BACKGROUND: The importance of predicting human pharmacokinetics during compound selection has been recognized in the pharmaceutical industry. To this end there are many different approaches that are applied. METHODS: In this study we compared the accuracy of physiologically based pharmacokinetic (PBPK) methodologies implemented in GastroPlus™ with the one-compartment approach routinely used at Pfizer for human pharmacokinetic plasma concentration-time profile prediction. Twenty-one Pfizer compounds were selected based on the availability of relevant preclinical and clinical data. Intravenous and oral human simulations were performed for each compound. To understand any mispredictions, simulations were also performed using the observed clearance (CL) value as input into the model. RESULTS: The simulation results using PBPK were shown to be superior to those obtained via traditional one-compartment analyses. In many cases, this difference was statistically significant. Specifically, the results showed that the PBPK approach was able to accurately predict passive distribution and absorption processes. Some issues and limitations remain with respect to the prediction of CL and active transport processes and these need to be improved to further increase the utility of PBPK modelling. A particular advantage of the PBPK approach is its ability to accurately predict the multiphasic shape of the pharmacokinetic profiles for many of the compounds tested. CONCLUSION: The results from this evaluation demonstrate the utility of PBPK methodology for the prediction of human pharmacokinetics. This methodology can be applied at different stages to enhance the understanding of the compounds in a particular chemical series, guide experiments, aid candidate selection and inform clinical trial design.