Anti-Drug Antibody Incidence Comparison of Therapeutic Proteins Administered Via Subcutaneous vs. Intravenous Route.

Subcutaneous (SC) administration of therapeutic proteins is perceived to pose higher risk of immunogenicity when compared with intravenous (IV) route of administration (RoA). However, systematic evaluations of clinical data to support this claim are lacking. This meta-analysis was conducted to compare the immunogenicity of the same therapeutic protein by IV and SC RoA. Anti-drug antibody (ADA) data and controlling variables for 7 therapeutic proteins administered by both IV and SC routes across 48 treatment groups were analyzed. RoA was the primary independent variable of interest while therapeutic protein, patient population, adjusted dose, and number of ADA samples were controlling variables. Analysis of variance was used to compare the ADA incidence between IV and SC RoA, while accounting for controlling variables and potential interactions. Subsequently, 10 additional therapeutic proteins with ADA data published for both IV and SC administration were added to the above 7 therapeutic proteins and were evaluated for ADA incidence. RoA had no statistically significant effect on ADA incidence for the initial dataset of 7 therapeutic proteins (p = 0.55). The only variable with a significant effect on ADA incidence was the therapeutic protein. None of the other controlling variables, including their interactions with RoA, was significant. When all data from the 17 therapeutic proteins were pooled, there was no statistically significant effect of RoA on ADA incidence (p = 0.81). In conclusion, there is no significant difference in ADA incidence between the IV and SC RoA, based on analysis of clinical ADA data from 17 therapeutic proteins.

Studies with neutralizing antibodies suggest CXCL8-mediated neutrophil activation is independent of C-C motif chemokine receptor-like 2 (CCRL2) ligand binding function.

C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.

Application of blood microsampling in cynomolgus monkey and demonstration of equivalent monoclonal antibody PK parameters compared to conventional sampling.

PURPOSE: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. METHOD: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. RESULTS: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). CONCLUSION: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments.

Safety, Tolerability, and Pharmacokinetics of PF-06823859, an Anti-Interferon ß Monoclonal Antibody: A Randomized, Phase I, Single- and Multiple-Ascending-Dose Study.

This double-blind, randomized, placebo-controlled, dose-ascending, first-in-human study (NCT02766621) assessed the safety, tolerability, and pharmacokinetics (PK) of PF-06823859, an anti-interferon ß monoclonal antibody. Healthy subjects were randomized to single ascending doses (SADs) of intravenous PF-06823859 30, 100, 300, 900, or 2000 mg or placebo; to multiple ascending doses (MADs) of subcutaneous PF-06823859 100 or 300 mg or placebo (once every 2 weeks for a total of 3 doses); or to MAD of intravenous PF-06823859 600 mg or placebo (once every 3 weeks or once every 4 weeks for a total of 2 doses). The incidence, severity, and causal relationship of adverse events (AEs) were assessed, along with immunogenicity and PK. In total, 62 subjects were randomized to treatment (SAD, n = 35; MAD, n = 27). There were 76 treatment-emergent all-causality AEs in the SAD (PF-06823859: n = 25; placebo: n = 4) and MAD (PF-06823859: n = 40; placebo: n = 7) cohorts. In the SAD cohorts, all treatment-emergent all-causality AEs were mild in severity; 4 AEs of moderate severity were identified in the MAD cohorts. No dose-limiting AEs, serious AEs, treatment-related discontinuations, dose reductions, or deaths occurred. PF-06823859 exposure increased dose-proportionally, with half-life values ranging between 23 and 35 days. The estimated subcutaneous bioavailability was 43% to 44%. Immunogenicity incidence rates were low (antidrug antibodies, 12.5%; neutralizing antibodies, 2.1%). No immunogenically related clinical responses of concern were observed. In conclusion, PF-06823859 demonstrated an acceptable safety, tolerability, and PK profile that supports clinical development for treating disorders associated with increased interferon ß levels, such as dermatomyositis or systemic lupus erythematosus.

A Physiologically-Based Pharmacokinetic Model for the Prediction of "Half-Life Extension" and "Catch and Release" Monoclonal Antibody Pharmacokinetics.

Monoclonal antibodies (mAbs) can be engineered to have "extended half-life" and "catch and release" properties to improve target coverage. We have developed a mAb physiologically-based pharmacokinetic model that describes intracellular trafficking, neonatal Fc receptor (FcRn) recycling, and nonspecific clearance of mAbs. We extended this model to capture target binding as a function of target affinity, expression, and turnover. For mAbs engineered to have an extended half-life, the model was able to accurately predict the terminal half-life (82% within 2-fold error of the observed value) in the human FcRn transgenic (Tg32) homozygous mouse and human. The model also accurately captures the trend in pharmacokinetic and target coverage data for a set of mAbs with differing catch and release properties in the Tg32 mouse. The mechanistic nature of this model allows us to explore different engineering techniques early in drug discovery, potentially expanding the number of "druggable" targets.

Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.

Modeling the clinical phenotype of BTK inhibition in the mature murine immune system.

Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

Selective inhibition of BTK prevents murine lupus and antibody-mediated glomerulonephritis.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: optimization and data analysis.

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.

Discovery and characterization of atropisomer PH-797804, a p38 MAP kinase inhibitor, as a clinical drug candidate.

PH-797804 ((aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamde) is a diarylpyridinone inhibitor of p38 mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. Due to steric constraints imposed by the pyridinone carbonyl group and the 6- and 6'-methyl substituents of PH-797804, rotation around the connecting bond of the pyridinone and the N-phenyl ring is restricted. Density functional theory predicts a remarkably high rotational energy barrier of >30 kcal mol(-1), corresponding to a half-life of more than one hundred years at room temperature. This gives rise to discrete conformational spaces for the N-phenylpyridinone group, and as a result, two atropic isomers that do not interconvert under ambient conditions. Molecular modeling studies predict that the two isomers should differ in their binding affinity for p38α kinase; whereas the atropic S (aS) isomer binds favorably, the opposite aR isomer incurs significant steric interference with p38α kinase. The two isomers were subsequently identified and separated by chiral chromatography. IC50 values from p38α kinase assays confirm that one atropisomer is >100-fold more potent than the other. It was ultimately confirmed by small-molecule X-ray diffraction that the more potent atropisomer, PH-797804, is the aS isomer of the racemic pair. Extensive pharmacological characterization supports that PH-797804 carries most activity both in vitro and in vivo, and it has a stability profile compatible with oral formulation and delivery options.

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase.

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase.

The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.

Substituted N-aryl-6-pyrimidinones: a new class of potent, selective, and orally active p38 MAP kinase inhibitors.

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

An improved algorithm for quantifying real-time impedance biosensor signals.

In previously published work [1] we presented a real-time electrochemical impedance biosensor prototype system and a state-space estimation algorithm for signal quantification. Experiments in the interim have revealed some algorithm failure modes which reduced the reliability and repeatability of quantification. The present work describes a related algorithm that introduces constraints based on a priori knowledge of the expected signals predicted by the biosensor signal model. The improvements in reliability and repeatability bring the system close to deployment for real-world trials.

Impedimetric biosignal analysis and quantification in a real-time biosensor system.

We describe our real-time, label-free, electrochemical impedance biosensor system with an emphasis on the use of an impedance response signal model to quantify assays. The signal processing for estimating model parameters from noisy data and the quantitative verification against target concentration and affinity are also presented.

Cartilage degradation biomarkers predict efficacy of a novel, highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: confirmation by multivariate analysis that modulation of type II collagen and aggrecan degradation peptides parallels pathologic changes.

OBJECTIVE: To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP-13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation. METHODS: The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS-4 and ADAMTS-5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis. RESULTS: The potent and selective MMP-13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose-dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results. CONCLUSION: This highly selective MMP-13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP-13 inhibitor. These data support the potential therapeutic value of selective MMP-13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.