Nonclinical safety evaluation, pharmacokinetics, and target engagement of Lu AF82422, a monoclonal IgG1 antibody against alpha-synuclein in development for treatment of synucleinopathies.

Alpha-synuclein is a 15 kDa protein associated with neurodegenerative diseases such as Parkinson disease and multiple-system atrophy where pathological forms of alpha-synuclein aggregate and become neurotoxic. Here we describe the nonclinical program to support a first-in-human (FIH) single ascending dose (SAD) study for Lu AF82422, a human recombinant, anti-alpha-synuclein monoclonal antibody (mAb) in development for treatment of synucleinopathies. Alpha-synuclein is primarily expressed in brain, peripheral nerves and in blood cells. A tissue cross-reactivity assessment showed that Lu AF82422 binding was generally restricted to nervous tissues. Flow cytometry analysis did not show extracellular surface binding of Lu AF82422 to human platelets, erythrocytes, granulocytes, or lymphocytes, but to a low fraction of monocytes, without any functional consequences on activation or phagocytic capacity. A single dose pharmacokinetic (PK) study in cynomolgus monkeys with dose levels of 1-30 mg/kg confirmed PK properties in the expected range for a mAb with a soluble target, and target engagement was shown as a decrease in free alpha-synuclein in plasma. Four-week repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys at doses up to 600 mg/kg administered intravenously every 10 days. Results showed no treatment-related adverse findings and the no-observed-adverse-effect-level was the highest dose tested. Target engagement was shown in plasma and cerebrospinal fluid. Taken together, the nonclinical data indicated no safety signal of concern and provided adequate safety margins between observed safe doses in animals and the planned dose levels in the FIH SAD study.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.

Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells.

Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96-well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell-based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell-based genotoxicity assays. The resulting dataset was used to set the relevant cut-off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well-suited for use in early drug discovery projects. Environ.