Jon Tinsley

Iminosugars past, present and future: medicines for tomorrow.

Iminosugars comprise the most attractive class of carbohydrate mimetics reported to date and are ideally positioned to take advantage of our increasing understanding of glycobiology in the search for new medicines. First-generation iminosugar drugs suffered from lack of adequate selectivity, resulting in considerable side-effects in the clinic. Current efforts directed towards second-generation compounds, encompassing a much greater range of structures and addressing a wider selection of biochemical targets, are enabling the identification and development of suitable candidates that benefit from improved activity and selectivity. Furthermore, second-generation compounds can address a variety of established targets that have previously proved refractory to other compound classes. This review focuses on the breadth of opportunities provided by second-generation leads from iminosugars (Seglins™).

Second-generation compound for the modulation of utrophin in the therapy of DMD

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches are progressing through human clinical trials. By pharmacologically modulating the expression of the dystrophin-related protein utrophin, we have previously demonstrated in dystrophin-deficient mdx studies, daily SMT C1100 treatment significantly reduced muscle degeneration leading to improved muscle function. This manuscript describes the significant disease modifying benefits associated with daily dosing of SMT022357, a second-generation compound in this drug series with improved physicochemical properties and a more robust metabolism profile. These studies in the mdx mouse demonstrate that oral administration of SMT022357 leads to increased utrophin expression in skeletal, respiratory and cardiac muscles. Significantly, utrophin expression is localized along the length of the muscle fibre, not just at the synapse, and is fibre-type independent, suggesting that drug treatment is modulating utrophin transcription in extra-synaptic myonuclei. This results in improved sarcolemmal stability and prevents dystrophic pathology through a significant reduction of regeneration, necrosis and fibrosis. All these improvements combine to protect the mdx muscle from contraction induced damage and enhance physiological function. This detailed evaluation of the SMT C1100 drug series strongly endorses the therapeutic potential of utrophin modulation as a disease modifying therapeutic strategy for all DMD patients irrespective of their dystrophin mutation.

Safety, tolerability, and pharmacokinetics of SMT C1100, a 2-arylbenzoxazole utrophin modulator, following single- and multiple-dose administration to healthy male adult volunteers.

SMT C1100 is a small molecule utrophin modulator in development to treat Duchenne muscular dystrophy. This study evaluated the safety, tolerability, and pharmacokinetics of SMT C1100 in healthy volunteers. This double‐blind, placebo‐controlled Phase 1 study comprised: Part 1, an escalating, single‐dose with/without fasting involving 50 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg doses; and Part 2, a multiple 10 day dose evaluation involving 100 mg/kg bid and 200 mg/kg bid doses. Adverse events were recorded. SMT C1100 was absorbed rapidly following single and multiple oral doses, with median tmax attained within 2–3.5 hour across all doses. Considerable variability of pharmacokinetic parameters was noted among subjects. Following single doses, systemic exposure increased in a sub‐proportional manner, with the 8.0‐fold dose increment resulting in 2.7‐ and 2.4‐fold increases in AUC0‐∞ and Cmax, respectively. AUC0‐∞ and Cmax were estimated as 4.2‐ and 4.8‐fold greater, respectively, following food. Systemic exposure reduced upon repeat dosing with steady‐state concentrations achieved within 3–5 days of multiple bid dosing. No serious or severe adverse events were reported. SMT C1100 was safe and well tolerated with plasma concentrations achieved sufficient to cause a 50% increase in concentrations of utrophin in cells in vitro.

Imaging-based chemical screens using normal and glioma-derived neural stem cells

The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.

Automated Large-Scale Culture and Medium-Throughput Chemical Screen for Modulators of Proliferation and Viability of Human Induced Pluripotent Stem Cell–Derived Neuroepithelial-like Stem Cells

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell–derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture (“scale-up”) of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated “scale-out” into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.

226th ENMC International Workshop: . Towards validated and qualified biomarkers for therapy development for Duchenne muscular dystrophy 20-22 January 2017, Heemskerk, The Netherlands

Twenty-three participants from 6 countries (England; Germany; Italy; Sweden, The Netherlands; USA) attended the 226th ENMC workshop on Duchenne biomarkers “Towards validated and qualified biomarkers for therapy development for Duchenne Muscular Dystrophy.” The meeting was a follow-up of the 204th ENMC workshop on Duchenne muscular dystrophy biomarkers. The workshop was organized with the support of Parent Project Muscular Dystrophy (PPMD) and Marathon Pharmaceuticals, which provided travel support for participants from the US via an unrestricted grant to PPMD in addition to ENMC support. It was attended by representatives of academic institutions, industry working in the Duchenne muscular dystrophy field and patient representatives. 1.1. Background to the workshop 1.1.1. Biomarkers Biomarkers are defined as biological, measurable and quantifiable indicators of underlying biological processes. Different types of biomarkers can be distinguished: diagnostic biomarkers indicate the presence of disease, prognostic biomarkers correlate with predicted disease course, and therapeutic biomarkers are designed to predict or measure response to treatment [1]. Therapeutic biomarkers can indicate whether a therapy is having an effect. This type of biomarker is called a pharmacodynamics biomarker and can be used to e.g. show that a missing protein is restored after a therapy. Safety biomarkers assess likelihood, presence, or extent of toxicity as an adverse effect, e.g. through monitoring blood markers indicative of liver or kidney damage. Sometimes biomarkers can also be used as primary endpoints in clinical trials instead of functional outcome measures, and these are termed “surrogate endpoints”. In Europe [2,3] biomarkers can only be used as surrogate endpoints after going through a rigorous regulatory process to officially qualify them for this purpose. Similar pathways exist in the US, where the Food and Drug Administration (FDA) also supplies a process for qualification of biomarkers for other contexts of use.