Retrospective analysis of model-based predictivity of human pharmacokinetics for anti-IL-36R monoclonal antibody MAB92 using a rat anti-mouse IL-36R monoclonal antibody and RNA expression data (FANTOM5).

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1 κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.

Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities.

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on "macro" impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor.

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

SNARE complex-mediated degranulation in mast cells.

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.

5-Aminomethylbenzimidazoles as potent ITK antagonists.

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.

Inhibition of pro-inflammatory cytokine production by the dual p38/JNK2 inhibitor BIRB796 correlates with the inhibition of p38 signaling.

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.24) has complicated the interpretation of its reported anti-inflammatory activity. To better understand the contribution of JNK2 inhibition to the anti-inflammatory activities of BIRB796, we explored the relationship between the effects of BIRB796 and analogues on cytokine production and on cellular p38 and JNK signaling. We determined the binding affinity for BIRB796 and structural analogues to p38alpha and JNK2 and characterized compound 2 as a p38 inhibitor that binds to p38alpha with an affinity equivalent to BIRB796 but does not bind to any of the JNK isoforms. High-content imaging enabled us to show that the inhibition of p38 signaling by BIRB796 and analogues correlates with the ability of these compounds to inhibit the lipopolysaccharide (LPS)-induced TNF-alpha production in THP-1 monocytes. This finding was extended to cytokine release by disease-relevant human primary cells: to the production of TNF-alpha by peripheral blood mononuclear cells, and of IL-8 by neutrophils. Furthermore, BIRB796 and compound 2 inhibited the production of TNF-alpha in THP-1 monocytes and the IL-12/IL-18-induced production of interferon-gamma in human T-cells with similar potencies. In contrast, cellular JNK signaling in response to cytokines or stress stimuli was only weakly inhibited by BIRB796 and analogues and not affected by compound 2. In summary, our data suggest that p38 inhibition alone is sufficient to completely suppress cytokine production and that the added inhibition of JNK2 does not significantly contribute to the effects of BIRB796 on cytokine production.

2-Aminobenzimidazoles as potent ITK antagonists: trans-stilbene-like moieties targeting the kinase specificity pocket.

Based on the information from molecular modeling and X-ray crystal structures, the kinase specificity pocket of ITK could be occupied upon extension of the right-hand-side of the 2-benzimidazole core of the inhibitors. 2-Aminobenzimidazoles with a trans-stilbene-like extension were designed and synthesized as novel ITK antagonists. Significant improvement on binding affinity and cellular activity were obtained through the trans-stilbene-like antagonists. Several compounds showed inhibitory activity in an IL-2 functional assay.

5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of inducible T-cell kinase (Itk).

A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.

Discovery, SAR and X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-amides as inhibitors of inducible T-cell kinase (Itk).

A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.

The determination and correlation of molecular and cellular equilibrium Kd and kinetic k(off) values for small molecule allosteric antagonists of LFA-1.

Molecular K(d) and k(off) parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA (K(d)), SPR (k(off)), and flow cytometry (k(off)) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the alpha=0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K(d) and k(off)'s determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.

Discovery of potent and selective PKC-theta inhibitors.

An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.

Overview of approaches to the identification of inhibitors of cytokine action.

Cytokines are key regulatory molecules that serve as critical mediators of cell-to-cell communication. They play a central role in immune function and are important molecular targets for drug discovery because of their dysregulation in immune disease. Initial attempts to develop agents that block the actions of cytokines focused on identifying small-molecule antagonists that would directly compete with these cytokines for their cognate receptors. These efforts were, for the most part, unsuccessful. Outlined in this unit are strategies for developing new approaches. The first section describes strategies for how one might build a conceptual framework to approach this problem. The second section focuses on the technical approaches that can be utilized to accomplish the strategies outlined in the first section. These concepts are illustrated using interleukin-2 (IL-2) as a prototype, since there is a substantial body of knowledge regarding this cytokine, including information on its functions, signaling pathways, and physiological effects in normal and diseased tissue.

Second-generation lymphocyte function-associated antigen-1 inhibitors: 1H-imidazo[1,2-alpha]imidazol-2-one derivatives.

A novel class of lymphocyte function-associated antigen-1 (LFA-1) inhibitors is described. Discovered during the process to improve the physicochemical and metabolic properties of BIRT377 (1, Figure 1), a previously reported hydantoin-based LFA-1 inhibitor, these compounds are characterized by an imidazole-based 5,5-bicyclic scaffold, the 1,3,3-trisubstituted 1H-imidazo[1,2-alpha]imidazol-2-one (i.e. structure 3). The structure-activity relationship (SAR) shows that electron-withdrawing groups at C5 on the imidazole ring benefit potency and that oxygen-containing functional groups attached to a C5-sulfonyl or sulfonamide group further improve potency. This latter gain in potency is attributed to the interaction(s) of the functionalized sulfonyl/sulfonamide groups with the protein, likely polar-polar in nature, as suggested by SAR data. X-ray studies revealed that these bicyclic inhibitors bind to the I-domain of LFA-1 in a pattern similar to that of compound 1.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.