Mini-PBPK-Based Population Model and Covariate Analysis to Assess the Complex Pharmacokinetics and Pharmacodynamics of RO7449135, an Anti-KLK5/KLK7 Bispecific Antibody in Cynomolgus Monkeys.

RO7449135, an anti-kallikrein (KLK)5/KLK7 bispecific antibody, is in development as a potential therapy against Netherton's syndrome (NS). In cynomolgus monkey studies, RO7449135 bound to KLK5 and KLK7, causing considerable accumulation of total KLKs, but with non-dose-proportional increase. To understand the complex PKPD, a population model with covariate analysis was developed accounting for target binding in skin and migration of bound targets from skin to blood. The covariate analysis suggested the animal batch as the categorical covariate impacting the different KLK5 synthesis rates between the repeat-dose study and single-dose study, and the dose as continuous covariate impacting the internalization rate of the binary and ternary complexes containing KLK7. To comprehend the mechanism underlying, we hypothesized that inhibition of KLK5 by RO7449135 prevented its cleavage of the pro-enzyme of KLK7 (pro-KLK7) and altered the proportion between pro-KLK7 and KLK7. Besides the pro-KLK7, RO7449135 can interact with other proteins like LEKTI through KLK7 connection in a dose-dependent manner. The different high-order complexes formed by RO7449135 interacting with pro-KLK7 or LEKTI-like proteins can be subject to faster internalization rate. Accounting for the dose and animal batch as covariates, the model-predicted free target suppression is well aligned with the visual target engagement check. The population PKPD model with covariate analysis provides the scientific input for the complex PKPD analysis, successfully predicts the target suppression in cynomolgus monkeys, and thereby can be used for the human dose projection of RO7449135.

Dose-dependent inactivation of airway tryptase with a novel dissociating anti-tryptase antibody (MTPS9579A) in healthy participants: A randomized trial.

Tryptase is the most abundant secretory granule protein in human lung mast cells and plays an important role in asthma pathogenesis. MTPS9579A is a novel monoclonal antibody that selectively inhibits tryptase activity by dissociating active tetramers into inactive monomers. The safety, tolerability, pharmacokinetics (PKs), and systemic and airway pharmacodynamics (PDs) of MTPS9579A were assessed in healthy participants. In this phase I single-center, randomized, observer-blinded, and placebo-controlled study, single and multiple ascending doses of MTPS9579A were administered subcutaneously (s.c.) or intravenously (i.v.) in healthy participants. In addition to monitoring safety and tolerability, the concentrations of MTPS9579A, total tryptase, and active tryptase were quantified. This study included 106 healthy participants (82 on active treatment). Overall, MTPS9579A was well-tolerated with no serious or severe adverse events. Serum MTPS9579A showed a dose-proportional increase in maximum serum concentration (Cmax ) values at high doses, and a nonlinear increase in area under the curve (AUC) values at low concentrations consistent with target-mediated clearance were observed. Rapid and dose-dependent reduction in nasosorption active tryptase was observed postdose, confirming activity and the PK/PD relationship of MTPS9579A in the airway. A novel biomarker assay was used to demonstrate for the first time that an investigative antibody therapeutic (MTPS9579A) can inhibit tryptase activity in the upper airway. A favorable safety and tolerability profile supports further assessment of MTPS9579A in asthma. Understanding the exposure-response relationships using the novel PD biomarker will help inform clinical development, such as dose selection or defining patient subgroups.

Development of a specific immunoassay to selectively measure active tryptase in airway samples.

Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.

An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

IL-17A is associated with the breakdown of the blood-brain barrier in relapsing-remitting multiple sclerosis.

IL-17 has been implicated in the pathogenesis of multiple sclerosis (MS). Here, we show that blockade of IL-17A, but not IL-17F, attenuated experimental autoimmune encephalomyelitis (EAE). We further show that IL-17A levels were elevated in the CSF of relapsing-remitting MS (RRMS) patients and that they correlated with the CSF/serum albumin quotient (Qalb), a measure of blood-brain barrier (BBB) dysfunction. We then demonstrated that the combination of IL-17A and IL-6 reduced the expression of tight junction (TJ)-associated genes and disrupted monolayer integrity in the BBB cell line hCMEC/D3. However, unlike IL-17A, IL-6 in the CSF from RRMS patients did not correlate with Qalb. These data highlight the potential importance of targeting IL-17A in preserving BBB integrity in RRMS.

Safety, Pharmacokinetics, and Pharmacodynamics in Healthy Volunteers Treated With GDC-0853, a Selective Reversible Bruton's Tyrosine Kinase Inhibitor.

GDC-0853 is a small molecule inhibitor of Bruton's tyrosine kinase (BTK) that is highly selective and noncovalent, leading to reversible binding. In double-blind, randomized, and placebo-controlled phase I healthy volunteer studies, GDC-0853 was well tolerated, with no dose-limiting adverse events (AEs) or serious AEs. The maximum tolerated dose was not reached during dose escalation (≤600 mg, single ascending dose (SAD) study; ≤250 mg twice daily (b.i.d.) and ≤500 mg once daily, 14-day multiple ascending dose (MAD) study). Plasma concentrations peaked 1-3 hours after oral administration and declined thereafter, with a steady-state half-life ranging from 4.2-9.9 hours. Independent assays demonstrated dose-dependent BTK target engagement. Based on pharmacokinetic/pharmacodynamic (PK/PD) simulations, a once-daily dosing regimen (e.g., 100 mg, q.d.) is expected to maintain a high level of BTK inhibition over the dosing interval. Taken together, the safety and PK/PD data support GDC-0853 evaluation in rheumatoid arthritis, lupus, and other autoimmune or inflammatory indications.

Discovery of Potent and Selective Tricyclic Inhibitors of Bruton's Tyrosine Kinase with Improved Druglike Properties.

In our continued effort to discover and develop best-in-class Bruton's tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis.

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Development of a multi-matrix LC-MS/MS method for urea quantitation and its application in human respiratory disease studies.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

Preclinical Safety Profile of a Depleting Antibody against CRTh2 for Asthma: Well Tolerated Despite Unexpected CRTh2 Expression on Vascular Pericytes in the Central Nervous System and Gastric Mucosa.

CRTh2 is expressed on immune cells that drive asthma pathophysiology. Current treatment options for severe asthma are inadequate and therapeutic antibody-mediated depletion of CRTh2-expressing cells represents a promising new therapeutic strategy. Here we report for the first time that CRTh2 is not only expressed on immune cells, but also on microvasculature in the central nervous system (CNS) and gastric mucosa in humans. Microvascular expression of CRTh2 raises a safety concern because a therapeutic antiCRTh2 antibody with enhanced depletion capacity could lead to vascular damage. To evaluate this safety risk, we characterized microvascular expression in human and in transgenic mice expressing human CRTh2 protein (hCRTh2.BAC.Tg) and found that CRTh2 is not localized to microvascular endothelium that is directly exposed to circulating therapeutic antibody, but rather, to pericytes that in the CNS are shielded from direct circulatory exposure by the blood-brain barrier. Immunohistochemical visualization of an intravenously administered antiCRTh2 antibody in transgenic mice revealed localization to microvascular pericytes in the gastric mucosa but not in the CNS, suggesting the blood-brain barrier effectively limits pericyte exposure to circulating therapeutic antibody in the CNS. Repeated dosing with a depleting antiCRTh2 antibody in hCRTh2.BAC.Tg mice revealed linear pharmacokinetics and no drug-related adverse findings in any tissues, including the CNS and gastric mucosa, despite complete depletion of CRTh2 expressing circulating eosinophils and basophils. Collectively, these studies demonstrate that the likelihood of drug-related CNS or gastrointestinal toxicity in humans treated with a therapeutic depleting antiCRTh2 antibody is low despite pericyte expression of CRTh2 in these tissues.

Discovery of highly potent and selective Bruton's tyrosine kinase inhibitors: Pyridazinone analogs with improved metabolic stability.

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase alpha is mediated by hydrogen peroxide.

Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.

The minimal essential core of a cysteine-based protein-tyrosine phosphatase revealed by a novel 16-kDa VH1-like phosphatase, VHZ.

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process.

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX.

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.