Lutz Mueller

Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine To Treat Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.

MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages

Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by macrophages.

Summary of major conclusions from the 6th International Workshop on Genotoxicity Testing (IWGT), Foz do Iguaçu, Brazil.

a Novartis Institutes for BioMedical Research, Preclinical Safety, 4002 Basel, Switzerland b Public Interest Incorporated Foundation BioSafety Research Center, Iwata, Japan c National Institute of Health Sciences, Division of Genetics & Mutagenesis, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan d Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany e Exponent, Inc., Center for Toxicology & Mechanistic Biology, Midland, MI 48640, USA f Pharmaceutical Sciences, Roche Innovation Center, F Hoffmann-La Roche AG, CH 4070 Basel, Switzerland g Office of Science Policy, Office of Research and Development, U.S. Environmental Protection Agency, Room 51134 RRB, 1200 Pennsylvania Avenue NW (8104R), Washington DC 20460-0001, USA h Safety Research Laboratories, Mitsubishi Tanabe Pharma Co., Chiba 292-0818, Japan i Kirkland Consulting, PO Box 79, Tadcaster LS24 0AS, UK

GADD45α induction in the greenscreen HC indicator assay does not occur independently of cytotoxicity

Mammalian chromosomal integrity assays are influenced by cytotoxicity, a phenomenon which impacts data interpretation, assay specificity, and regulatory testing guidelines. Concordance of the GADD45α GreenScreen HC indicator assay to established in vitro and in vivo genetic toxicological assays has previously been described, yet a detailed description in the manner by which cytotoxicity influences its performance has not. Here we present a post‐hoc analysis of a previously tested set of 91 proprietary and nonproprietary compounds investigating the influence of cytotoxicity on GADD45α induction and how varying assay cutoff criteria impacts assay performance. Significant cytotoxicity was identified to accompany the majority (72%) of compounds classified as genotoxic by GADD45α induction. Decreasing the GADD45α genotoxic induction criteria (from a 50 to a 30% increase over solvent controls) resulted in an increased assay sensitivity (from 30 to 68%) and concordance (from 55 to 68%), though a concomitant decrease in specificity was also observed (from 97 to 68%). We conclude that GADD45α induction in the GreenScreen HC indicator assay is influenced by cytotoxicity and that assay performance can be improved if different cutoff criteria are implemented. Environ. Mol. Mutagen. 52:28–34, 2011.

International Workshop on Qualification and Control of Impurities

During the DIA International Workshop “Impurities and Degradation Products—Biological Qualification, Process Capability, and Analytical Control,” the EU Committee for Human Medicinal Products draft guideline on genotoxic impurities was presented. About half of the attendees expressed their support for the use of the threshold of toxicological concern concept as a pragmatic solution. The Food and Drug Administration guidance is under development. The process of qualification of impurities starts with identification. For genotoxic impurities, identification should start with an evaluation of the starting materials and the routes of synthesis to compile a list of impurities that can reasonably be expected to be present in the drug substance. Among this list, the search for alerting structures and classification of impurities as highly toxic can be facilitated by use of structure activity relationship databases. Relevant impurities that were identified as potentially genotoxic should be tested as isolated impurities because these impurities are usually present below the detection level of the genotoxicity assay when the drug substance is used in the assay. Knowledge of the impurity and its source is also the basis for a reduction of these compounds to the lowest possible level that can be achieved with reasonable effort. Trace analytical methods are usually required to monitor process optimization activities. Avoiding a hazardous impurity or a reduction of its levels may require a substantial effort and time in process chemistry or formulation development. The allowance of higher limits for short-term exposure is therefore seen as a pragmatic way to define reasonable effort, especially for batches used in the early clinical development. This remains an important issue that should be further addressed.

Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 (SMN2) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA)

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.