Sock-Cheng Lewin-Koh

Effect of selective LRRK2 kinase inhibition on nonhuman primate lung

LRRK2 kinase inhibitors, under development for Parkinson’s disease, have an effect on type II pneumocytes in nonhuman primate lung, suggesting that pulmonary toxicity may be a critical safety liability. A lung phenotype for LRRK2 inhibitors Human genetic evidence implicates leucine-rich repeat kinase 2 (LRRK2) as a high-priority drug target for Parkinson’s disease. However, the benefit and risk of inhibiting the kinase activity of LRRK2 is unknown and is currently untested in humans. Using two selective LRRK2 kinase inhibitors, Fuji et al. report a safety liability in nonhuman primates characterized by morphological changes in lung. These results are consistent with observations in mice lacking LRRK2. These safety observations offer a cautionary note for pharmacological modulation of LRRK2 in humans. Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson’s disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

A novel homogeneous Biotin–digoxigenin based assay for the detection of human anti-therapeutic antibodies in autoimmune serum

Electrochemiluminescence (ECL) assays have been widely used for the detection of anti-therapeutic antibodies (ATAs) against biotherapeutics. With the discontinuation of BioVeris (BV) ECL platform, an alternative technology was needed to replace BV assays to ensure continuous support of multi-year clinical studies. After evaluation of several immunoassay platforms, a novel homogeneous Biotin-digoxigenin (DIG) based bridging ELISA format was selected to develop an anti-rhuMAbX antibody screening assay to test serum samples from rheumatoid arthritis (RA) patients. With a homogeneous overnight sample incubation, the Biotin-DIG ELISA achieved comparable relative sensitivity and free drug tolerance to the previous BV ATA assay for rhuMAbX. To abrogate potential auto-antibody interference in RA sera, various assay conditions were thoroughly evaluated and a horseradish peroxidase (HRP)-conjugated chicken anti-DIG antibody was selected as the detection conjugate. Other potential interferences from serum Biotin, naturally occurring anti-avidin antibodies, and concomitant medications such as digoxin and hydrocortisone, which have similar structures to digoxigenin, were also investigated. Under optimized final assay conditions, the Biotin-DIG assay showed a relative sensitivity of approximately 11 ng/mL using a polyclonal anti-complementarity determining region (CDR) enriched positive control; the assay could detect 500 ng/mL of the positive control in the presence of approximately 27 μg/mL of rhuMAbX in RA serum. In addition, a confirmatory step was optimized for the assay based upon pre-incubating serum samples with an excess of free drug. Overall, the Biotin-DIG assay met the performance requirements for an ATA screening assay and had comparable sensitivity and drug tolerance to the BV assay; therefore this assay was a suitable replacement for the BV assay used for previous clinical studies of rhuMAbX. The Biotin-DIG based assay format can be broadly used as an effective screening platform for the detection of anti-therapeutic antibodies.

Toxicity Profile of Small-Molecule IAP Antagonist GDC-0152 Is Linked to TNF-α Pharmacology

Inhibitor-of-apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. Small-molecule IAP antagonists are currently being tested in clinical trials as novel cancer therapeutics. GDC-0152 is a small-molecule drug that triggers tumor cell apoptosis by selectively antagonizing IAPs. GDC-0152 induces NF-κB transcriptional activity leading to expression of several chemokines and cytokines, of which tumor necrosis factor alpha (TNF-α) is the most important for single-agent tumor activity. TNF-α is a pleiotropic cytokine that drives a variety of cellular responses, comprising inflammation, proliferation, and cell survival or death depending on the cellular context. As malignant and normal cells produce TNF-α upon IAP antagonism, increased TNF-α could drive both efficacy and toxicity. The toxicity profile of GDC-0152 in dogs and rats was characterized after iv dose administration once every 2 weeks for four doses. Findings in both species consisted of a dose-related, acute, systemic inflammatory response, and hepatic injury. Laboratory findings included elevated plasma cytokines, an inflammatory leukogram, and increased liver transaminases with histopathological findings of inflammatory infiltrates and apoptosis/necrosis in multiple tissues; a toxicology profile consistent with TNF-α-mediated toxicity. Dogs exhibited more severe findings than rats, and humans did not exhibit these findings, at comparable exposures across species. Furthermore, elevations in blood neutrophil count, serum monocyte chemoattractant protein-1, and other markers of inflammation corresponded to GDC-0152 exposure and toxicity and thus may have utility as safety biomarkers.

Phosphorous Dysregulation Induced by MEK Small Molecule Inhibitors in the Rat Involves Blockade of FGF-23 Signaling in the Kidney

MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.

Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

Influence of the compound selection process on the performance of human clearance prediction methods.

This is a commentary on the series of five manuscripts written as part of the Pharmaceutical Research and Manufacturers of America Clinical and Preclinical Development Committee initiative on predictive models of human pharmacokinetics (PK). In particular, we wish to comment on the third paper in the series, which describes the performance of prediction methods of human clearance (CL). Human CL prediction methods described in the third manuscript are fundamental to the work presented in manuscripts four and five on the prediction of human PK profiles. In this commentary, we examine the influence of the compound selection process by performing a probability analysis and examining the CL properties of compounds that are selected using an idealized drug discovery screening process focused on PK optimization. The results of the analysis suggest that the selection of screening species can influence the performance of various predictive models of human CL.

Bruton’s Tyrosine Kinase Small Molecule Inhibitors Induce a Distinct Pancreatic Toxicity in Rats

Bruton’s tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3′-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4′-bipyridin]-2′-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.

Comparative evaluation of 11 in silico models for the prediction of small molecule mutagenicity: role of steric hindrance and electron-withdrawing groups

Abstract The goal of this investigation was to perform a comparative analysis on how accurately 11 routinely-used in silico programs correctly predicted the mutagenicity of test compounds that contained either bulky or electron-withdrawing substituents. To our knowledge this is the first study of its kind in the literature. Such substituents are common in many pharmaceutical agents so there is a significant need for reliable in silico programs to predict precisely whether they truly pose a risk for mutagenicity. The predictions from each program were compared to experimental data derived from the Ames II test, a rapid reverse mutagenicity assay with a high degree of agreement with the traditional Ames assay. Eleven in silico programs were evaluated and compared: Derek for Windows, Derek Nexus, Leadscope Model Applier (LSMA), LSMA featuring the in vitro microbial Escherichia coli–Salmonella typhimurium TA102 A-T Suite (LSMA+), TOPKAT, CAESAR, TEST, ChemSilico (±S9 suites), MC4PC and a novel DNA docking model. The presence of bulky or electron-withdrawing functional groups in the vicinity of a mutagenic toxicophore in the test compounds clearly affected the ability of each in silico model to predict non-mutagenicity correctly. This was because of an over reliance on the part of the programs to provide mutagenicity alerts when a particular toxicophore is present irrespective of the structural environment surrounding the toxicophore. From this investigation it can be concluded that these models provide a high degree of specificity (ranging from 71% to 100%) and are generally conservative in their predictions in terms of sensitivity (ranging from 5% t o 78%). These values are in general agreement with most other comparative studies in the literature. Interestingly, the DNA docking model was the most sensitive model evaluated, suggesting a potentially useful new mode of screening for mutagens. Another important finding was that the combination of a quantitative structure–activity relationship and an expert rules system appeared to offer little advantage in terms of sensitivity, despite of the requirement for such a screening paradigm under the ICH M7 regulatory guideline.

Development of a safety biomarker signature to detect hepatic sinusoidal dilation associated with an anti-DLL4 biotherapeutic

Abstract Context: Biomarkers of lesion-specific drug induced liver toxicity are currently lacking. Objective: To develop a biomarker signature using routine clinical pathology parameters that predict hepatic sinusoidal dilation related to anti-DLL4 biotherapeutics. Methods: Random forest and factor analysis was used to construct a signature of routine laboratory tests to detect microscopically confirmed sinusoidal dilation of the liver. Results: A biomarker signature was developed comprising two scores (S1 and S2) with area under the curve (AUC) for sinusoidal dilation prediction of 0.81, 0.85 and 0.96 in three rat studies and 0.48 and 0.81 in two monkey studies. Conclusion: A unique, two-dimensional signature of liver parameters and red blood cell parameters could detect sinusoidal dilation in multiple preclinical species.