Synthesis of poly(vinyl alcohol) by blue light bismuth oxide photocatalysed RAFT. Evaluation of the impact of freeze/thaw cycling on ice recrystallisation inhibition.

Poly(vinyl alcohol), PVA, is the most potent polymeric ice recrystallisation inhibitor (IRI), mimicking a complex function of ice binding proteins. The IRI activity of PVA scales with its molecular weight and hence broad molecular weight distributions in free radical-derived PVAs lead to activity measurements dominated by small amounts of heavier fractions. Well-defined PVA can be prepared by thermally initiated RAFT/MADIX polymerization using xanthates by the polymerization of the less activated monomer vinyl acetate. The low conversions and molecular weights obtained during this approach, often requires feeding of additional initiator and bulk polymerization. Here we employ bismuth oxide photo-RAFT in solution, using blue light (450 nm), rather than previously reported white light, to obtain a library of PVA's. The use of blue light enabled quantitative conversion and acceptable dispersities. Purple light (380 nm) was also used, but asymmetric molecular weight distributions were obtained in some cases. High concentrations of high molecular weight PVA is known to form cryogels during freeze/thaw which has led to speculation this might limit the use of PVA in environments where the temperature cycles e.g. the construction industry. After 4 freeze/thaw cycles there was only small changes in observable IRI for all synthesised PVAs and two commercial standards. In an extended test, activity was retained after 100 freeze/thaw cycles, mitigating concerns that PVA could not be used in situations where freeze/thaw cycles occur. This work presents a convenient method to obtain well-defined PVAs for cryoscience studies compared to conventional thermal-RAFT and indicates that cryogelation concerns may not prevent their use.

Macular thickness measurements of healthy, naïve cynomolgus monkeys assessed with spectral-domain optical coherence tomography (SD-OCT).

The purpose of this study was to measure central macular thickness in an unprecedented number of cynomolgus monkeys. Macular thickness was measured with Heidelberg spectral-domain OCT in 320 eyes of healthy and treatment-naïve cynomolgus monkeys (80 males and 80 females). The macula was successfully measured in all 320 eyes. Macular thickness was not significantly different between the sexes. The mean central macular thickness was 244 µm (+/- 21 µm). Macular thicknesses in the quadrants were 327 +/-17 µm (temporal inner), 339 +/- 17 µm (inferior inner), 341 +/- 14 µm (superior inner), 341 +/-18 µm (nasal inner), and 299 +/- 20 µm (temporal outer), 320 +/- 16 µm (superior outer), 332 +/-23 µm (inferior outer), and 337 +/-18 µm (nasal outer). Highly significant differences between the nasal and temporal quadrants were detected. This study successfully demonstrated the feasibility of retinal thickness measurements in healthy cynomolgus monkeys. The present findings indicate that the macula is thicker in cynomolgus monkeys than in humans and provide important normative data for future studies.

Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

Application of Imaging Techniques to Cases of Drug-Induced Crystal Nephropathy in Preclinical Studies.

A number of drugs can cause precipitates within renal tubules leading to crystal nephropathy. Crystal nephropathy is usually an exposure-related finding and is not uncommon in preclinical studies, where high doses are tested. An understanding of the nature of precipitates is important for human risk assessment and further development. Our aim was to investigate the ability of various imaging techniques to detect the presence of drugs or metabolites in renal crystals. We applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) imaging, Raman and infrared microspectroscopy, scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and standard histopathology to cases of drug-induced crystal nephropathy, induced in rodents and primates by 4 compounds. MALDI-FTICR MS imaging enabled the identification of the drug-related crystal content in all 4 cases of nephropathy, without reference material and with high accuracy. Crystals were composed of unchanged parent drug and/or metabolites. Similar results were obtained using Raman and infrared microspectroscopy for 2 compounds. In the absence of reference standards of metabolites, Raman and infrared microspectroscopy showed that the crystals consisted of components similar, but not identical, to the administered drug for the other compounds, a limitation for these techniques. SEM/EDX showed which counter ions were colocalized with the identified drug-related material, complementing the MALDI-FTICR MS findings. Therefore, we recommend MALDI-FTICR MS as a first-line methodology to characterize crystal nephropathies. Raman and infrared microspectroscopy may be useful when MALDI-FTICR MS imaging cannot be applied. SEM/EDX could be considered as a complementary technology.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro.

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

Controlled aqueous polymerization of acrylamides and acrylates and "in situ" depolymerization in the presence of dissolved CO2.

Aqueous copper-mediated radical polymerization of acrylamides and acrylates in carbonated water resulted in high monomer conversions (t < 10 min) before undergoing depolymerization (60 min > t > 10 min). The regenerated monomer was characterized and repolymerized following deoxygenation of the resulting solutions to reyield polymers in high conversions that exhibit low dispersities.

A high pressure cell for supercritical CO2 on-line chemical reactions studied with X-ray techniques.

A versatile high pressure X-ray sample cell has been developed for conducting in situ time-resolved X-ray scattering experiments in the pressure and temperature regime required (pressures up to 210 bars and temperatures up to 120 °C) for chemical reactions in supercritical fluids. The large exit opening angle of the cell allows simultaneous performance of SAXS-WAXS experiments. Diamond windows are used in order to benefit from the combination of maximum strength, minimal X-ray absorption and chemical inertia. The sample cell can also be utilised for X-ray spectroscopy experiments over a wide range of photon energies. Results of the online synthesis of a block copolymer, poly(methyl methacrylate-block-poly(benzyl methacrylate), by Reversible Addition-Fragmentation Chain Transfer (RAFT) in a supercritical CO2 dispersion polymerisation will be discussed. The contribution of the density fluctuations, as function of temperature, to the X-ray scattering signal has been quantified in order to allow appropriate background subtractions.