Wilfried Frieauff

A novel BACE inhibitor NB-360 shows a superior pharmacological profile and robust reduction of amyloid-β and neuroinflammation in APP transgenic mice

BackgroundAlzheimer’s disease (AD) is the most common form of dementia, the number of affected individuals is rising, with significant impacts for healthcare systems. Current symptomatic treatments delay, but do not halt, disease progression. Genetic evidence points to aggregation and deposition of amyloid-β (Aβ) in the brain being causal for the neurodegeneration and dementia typical of AD. Approaches to target Aβ via inhibition of γ-secretase or passive antibody therapy have not yet resulted in substantial clinical benefits. Inhibition of BACE1 (β-secretase) has proven a challenging concept, but recent BACE1inhibitors can enter the brain sufficiently well to lower Aβ. However, failures with the first clinical BACE1 inhibitors have highlighted the need to generate compounds with appropriate efficacy and safety profiles, since long treatment periods are expected to be necessary in humans.ResultsTreatment with NB-360, a potent and brain penetrable BACE-1 inhibitor can completely block the progression of Aβ deposition in the brains of APP transgenic mice, a model for amyloid pathology. We furthermore show that almost complete reduction of Aβ was achieved also in rats and in dogs, suggesting that these findings are translational across species and can be extrapolated to humans. Amyloid pathology may be an initial step in a complex pathological cascade; therefore we investigated the effect of BACE-1 inhibition on neuroinflammation, a prominent downstream feature of the disease. NB-360 stopped accumulation of activated inflammatory cells in the brains of APP transgenic mice. Upon chronic treatment of APP transgenic mice, patches of grey hairs appeared.ConclusionsIn a rapidly developing field, the data on NB-360 broaden the chemical space and expand knowledge on the properties that are needed to make a BACE-1 inhibitor potent and safe enough for long-term use in patients. Due to its excellent brain penetration, reasonable oral doses of NB-360 were sufficient to completely block amyloid-β deposition in an APP transgenic mouse model. Data across species suggest similar treatment effects can possibly be achieved in humans. The reduced neuroinflammation upon amyloid reduction by NB-360 treatment supports the notion that targeting amyloid-β pathology can have beneficial downstream effects on the progression of Alzheimer’s disease.

Automatic analysis of the in vitro micronucleus test on V79 cells

The in vitro micronucleus test is a well established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and specific parameter. As a measure for numerical and structural chromosome aberrations, the in vitro micronucleus test consists of determining the frequency of micronucleated cells in a representative fraction of cells in a culture. So far, manual counting has been the only method for evaluating microscopic V79 Chinese hamster cell preparations. To replace this tedious and time consuming procedure, a fully automatic system for micronucleus scoring in V79 cells by image analysis has been developed and introduced into the routine genotoxicity screening of drug candidates. The comparison of manual and automatic micronucleus analysis showed a high degree of concordance between the results obtained by the two techniques. For concentration series of cyclophosphamide (CP) and ethyl-methanesulphonate (EMS) as test compounds, the frequency of erroneously missed micronuclei through automatic scoring proved to be below 15% in comparison with manual scoring. Generally, false positive micronucleus decisions could be controlled easily by fast and simple relocation of the automatically detected patterns. The possibility to analyze 24 slides within 1 day by fully automatic overnight analysis and the high reproducibility of the results make automatic image processing a powerful tool for the in vitro micronucleus analysis.

Clinical Classification of Cancer Cachexia: Phenotypic Correlates in Human Skeletal Muscle

Background Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system. Methods 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation. Findings Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (p = 0.022) and with >10%WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups. Conclusion Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.

Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS). While multiple effective immunomodulatory therapies for MS exist today, they lack the scope of promoting CNS repair, in particular remyelination. Microglia play a pivotal role in regulating myelination processes, and the colony-stimulating factor 1 (CSF-1) pathway is a key regulator for microglia differentiation and survival. Here, we investigated the effects of the CSF-1 receptor kinase inhibitor, BLZ945, on central myelination processes in the 5-week murine cuprizone model by non-invasive and longitudinal magnetic resonance imaging (MRI) and histology. Therapeutic 2-week BLZ945 treatment caused a brain region-specific enhancement of remyelination in the striatum/cortex, which was absent in the corpus callosum/external capsule. This beneficial effect correlated positively with microglia reduction, increased oligodendrocytes and astrogliosis. Prophylactic BLZ945 treatment prevented excessive demyelination in the corpus callosum by reducing microglia and increasing oligondendrocytes. In the external capsule oligodendrocytes were depleted but not microglia and a buildup of myelin debris and axonal damage was observed. A similar microglial dysfunction in the external capsule with an increase of myelin debris was obvious in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice treated with cuprizone. Finally, therapeutic BLZ945 treatment did not change the disease course in experimental autoimmune encephalomyelitis mice, a peripherally driven neuroinflammation model. Taken together, our data suggest that a short-term therapeutic inhibition of the CSF-1 receptor pathway by BLZ945 in the murine cuprizone model enhances central remyelination by modulating neuroinflammation. Thus, microglia-modulating therapies could be considered clinically for promoting myelination in combination with standard-of-care treatments in MS patients.

The Bace-1 inhibitor CNP520 for prevention trials in Alzheimer's disease

The beta‐site amyloid precursor protein cleaving enzyme‐1 (BACE‐1) initiates the generation of amyloid‐β (Aβ), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimers disease (AD). Clinical failures of anti‐Aβ therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE‐1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aβ in rats and dogs, and Aβ plaque deposition in APP‐transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose‐dependent Aβ reduction in the cerebrospinal fluid. Thus, long‐term, pivotal studies with CNP520 have been initiated in the Generation Program.

Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo : Gamma-H2AX Tissue-specific Genotoxicity

The phosphorylation of histone H2AX in Serine 139 (gamma‐H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma‐H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma‐H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma‐H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma‐H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma‐H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma‐H2AX staining allows the detection of tissue‐specific effects in situ. Moreover, since gamma‐H2AX staining can be performed on formalin‐fixed and paraffin‐embedded tissue sections generated during repeated‐dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4–16, 2019.

PRECLINICAL PHARMACOLOGY OF BACE INHIBITOR CNP520

neuronal culture assay with an EC50of approximately 250 pM. Following acute oral treatment with 0, 0.3, 1.0, or 3.0 mg/kg LY3202626, dose-dependent reductions in Ab, sAPPb, and C99 were observed in cortex and hippocampus of PDAPPmice. In beagle dog, acute oral dosing of 1.5 mg/kg LY3202626 resulted in lowering of plasma and CSF Ab 1-x by approximately 80% at the nadir; the CSF Ab 1-x was still reduced by approximately 75% at 24 hours post-dosing. Exposure of LY3202626 in plasma and CSF correlated significantly with pharmacodynamic effects upon Ab in both PDAPP mice and beagle dogs. Conclusions:LY3202626 is a potent and selective inhibitor of the BACE1 and BACE2 enzymes. The robust in vivo effects of LY3202626 are consistent with its in vitro potency and exposure in target compartments. These data support further clinical development of LY3202626 for the treatment of AD.

THE BACE INHIBITOR NB-360 HAS EXCELLENT BRAIN PENETRATION AND EFFICACY ON AMYLOID-B LOAD IN ANIMAL MODELS

human APOE3 or APOE4) were treated with Bex, LG268 (a more selective RXR agonist), or vehicle control in 3 treatment paradigms: T1) 7-day oral gavage (5.75-6M); T2) 7-day hydrogel (5.75-6M); and T3) 30-day hydrogel (5-6M). Hydrogel provides a steady dosage of drug throughout the awake period of the mice. Brains were harvested, dissected, and homogenized by 3-step serial extraction.Results: In brain regions with lowAb levels at treatment, RXR agonists did not change soluble levels of Ab 42 and oAb in E3FAD or E4FAD mice. In brain regions with intermediate Ab levels, RXR agonist treatment induced an increase in soluble Ab 42 and oAb levels in E3FAD and E4FADmice. However, in the hippocampus of E4FADmice, with high Ab levels at treatment, RXR agonists induced a decrease in soluble Ab 42 and oAb levels and an increase in synaptic proteins. Importantly, total apoE levels were unaffected for all treatment groups, suggesting an alternate mechanism of action for RXR agonists. Our data further demonstrate that the beneficial effects of RXR agonists in E4FAD mice are mediated via: increased ABCA1 and ABCG1 expression, increased apoE4 association with lipoproteins, increased apoE/Ab complex levels, reduced oAb levels and enhanced synaptic viability. Conclusions: Collectively, our data demonstrate that RXR agonist efficacy is determined by the levels of Ab pathology at time of treatment, exhibiting no effect, or even an increase the levels of neurotoxic Ab in prevention paradigms where Ab levels are likely sub-pathological. However, in later stages of AD, RXR agonists may address the loss of function associated with APOE4 by increasing apoE4 lipidation and apoE4/Ab complex formation. Future studies are necessary to determine whether this pathway is relevant for APOE3 carriers with high Ab pathology, or if RXR agonists are an APOE4specific AD therapeutic.