Characterization of the Pharmacokinetic and Pharmacodynamic Profile of Apraglutide, a Glucagon-Like Peptide-2 Analog, in Healthy Volunteers.

Apraglutide (FE 203799) is a glucagon-like peptide-2 (GLP-2) analog under development for the treatment of intestinal failure associated with short bowel syndrome (SBS-IF) and graft-versus-host disease (GvHD). Compared with native GLP-2, apraglutide has slower absorption, reduced clearance, and higher protein binding, enabling once-weekly dosing. This study evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) profile of apraglutide in healthy adults. Healthy volunteers were randomized to receive 6 weekly subcutaneous administrations of 1, 5, or 10 mg apraglutide or placebo. PK and citrulline (an enterocyte mass PD marker) samples were collected at multiple time points. Kinetic parameters of apraglutide and citrulline were calculated using noncompartmental analysis; repeated PD measures were analyzed with a mixed model of covariance. A population PK/PD model was developed that also included data from a previous phase 1 study in healthy volunteers. Twenty-four subjects were randomized; 23 received all study drug administrations. Mean estimated apraglutide clearance was 16.5-20.7 l/day, and mean volume of distribution was 55.4-105.0 liters. A dose-dependent increase in citrulline plasma concentration was observed, with 5-mg and 10-mg doses inducing higher citrulline levels than 1-mg doses and placebo. PK/PD analysis showed that weekly 5-mg apraglutide induced the maximal citrulline response. Increased plasma citrulline levels were sustained for 10-17 days after the final apraglutide administration. Apraglutide displays predictable dose-dependent PK and PD profiles, with a 5-mg dose showing significant PD effects. Results suggest that apraglutide has early and enduring effects on enterocyte mass and supports the continued development of weekly subcutaneous apraglutide for SBS-IF and GvHD patient populations. SIGNIFICANCE STATEMENT: Once-weekly subcutaneous apraglutide results in dose-dependent elevations of plasma citrulline (an enterocyte mass pharmacodynamic marker) with parameters suggesting that apraglutide has lasting effects on enterocyte mass and the potential to provide therapeutic benefits. This is the first report of a model relating glucagon-like peptide-2 (GLP-2) agonism and its effects in intestinal mucosa, affording not only the ability to predict pharmacologic effects of GLP-2 analogs but also the exploration of optimal dosing regimens for this drug class across populations with different body weights.

Modeling the activation of the alternative complement pathway and its effects on hemolysis in health and disease.

The complement system is a powerful mechanism of innate immunity poised to eliminate foreign cells and pathogens. It is an intricate network of >35 proteins, which, once activated, leads to the tagging of the surface to be eliminated, produces potent chemoattractants to recruit immune cells, and inserts cytotoxic pores into nearby lipid surfaces. Although it can be triggered via different pathways, its net output is largely based on the direct or indirect activation of the alternative pathway. Complement dysregulation or deficiencies may cause severe pathologies, such as paroxysmal nocturnal hemoglobinuria (PNH), where a lack of complement control proteins leads to hemolysis and life-threatening anemia. The complexity of the system poses a challenge for the interpretation of experimental data and the design of effective pharmacological therapies. To address this issue, we developed a mathematical model of the alternative complement pathway building on previous modelling efforts. The model links complement activation to the hemolytic activity of the terminal alternative pathway, providing an accurate description of pathway activity as observed in vitro and in vivo, in health and disease. Through adjustment of the parameters describing experimental conditions, the model was capable of reproducing the results of an array of standard assays used in complement research. To demonstrate its clinical applicability, we compared model predictions with clinical observations of the recovery of hematological biomarkers in PNH patients treated with the complement inhibiting anti-C5 antibody eculizumab. In conclusion, the model can enhance the understanding of complement biology and its role in disease pathogenesis, help identifying promising targets for pharmacological intervention, and predict the outcome of complement-targeting pharmacological interventions.

Pharmacokinetics-Pharmacodynamics of Enmetazobactam Combined with Cefepime in a Neutropenic Murine Thigh Infection Model.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum ß-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > CT ) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 ß-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 µg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log10 bioburden reduction of 8% and 44% fT > 2 µg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.

Safety and Tolerability, Pharmacokinetics, and Pharmacodynamics of ACT017, an Antiplatelet GPVI (Glycoprotein VI) Fab.

Objective- ACT017 is a novel, first in class, therapeutic antibody to platelet GPVI (glycoprotein VI) with potent and selective antiplatelet effects. This first-in-human, randomized, placebo-controlled phase 1 study was conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ACT017 in healthy subjects. Approach and Results- Six cohorts of 8 healthy male and female subjects each received ascending single doses of ACT017 (n=6) or placebo (n=2) as a 6-hour intravenous infusion, with » of the total dose administered within 15 minutes and the rest of the dose (¾ of the total dose) administered within 5 hours and 45 minutes. The 6 investigated doses ranged from 62.5 to 2000 mg. All doses of ACT017 were well tolerated, and no serious adverse events occurred during the study. None of the subjects reported an infusion site reaction. Template bleeding time was not affected in a clinically significant manner by any of the ACT017 doses. Plasma concentrations, determined by liquid chromatography-tandem mass spectrometry, increased linearly with the dose received as were the established pharmacokinetics values. There was no change in the platelet count, platelet GPVI expression assessed by flow cytometry, or plasma levels of soluble GPVI assessed by ELISA. In contrast, administration of ACT017 inhibited collagen-induced platelet aggregation measured by light transmission aggregometry on platelet-rich plasma, and the extent and duration of the effect were dose-dependent. Conclusions- The novel antiplatelet agent ACT017 has consistent pharmacokinetic/pharmacodynamic properties and favorable safety and tolerability profiles warranting further clinical development.

Coordination of Rho GTPase activities during cell protrusion.

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks.

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals.

Protrusion and actin assembly are coupled to the organization of lamellar contractile structures.

Directed cell migration requires continuous cycles of protrusion of the leading edge and contraction to pull up the cell rear. How these spatially distributed processes are coordinated to maintain a state of persistent protrusion remains unknown. During wound healing responses of epithelial sheets, cells along the wound edge display two distinct morphologies: 'leader cells' exhibit persistent edge protrusions, while the greater majority of 'follower cells' randomly cycle between protrusion and retraction. Here, we exploit the heterogeneity in cell morphodynamic behaviors to deduce the requirements in terms of cytoskeleton dynamics for persistent and sporadic protrusion events. We used quantitative Fluorescent Speckle Microscopy (qFSM) to compare rates of F-actin assembly and flow relative to the local protrusion and retraction dynamics of the leading edge. Persistently protruding cells are characterized by contractile actomyosin structures that align with the direction of migration, with converging F-actin flows interpenetrating over a wide band in the lamella. Conversely, non-persistent protruders have their actomyosin structures aligned perpendicular to the axis of migration, and are characterized by prominent F-actin retrograde flows that end into transverse arcs. Analysis of F-actin kinetics in the lamellipodia showed that leader cells have three-fold higher assembly rates when compared to followers. To further investigate a putative relationship between actomyosin contraction and F-actin assembly, myosin II was inhibited by blebbistatin. Treated cells at the wound edge adopted a homogeneously persistent protrusion behavior, with rates matching those of leader cells. Surprisingly, we found that disintegration of actomyosin structures led to a significant decrease in F-actin assembly. Our data suggests that persistent protrusion in these cells is achieved by a reduction in overall F-actin retrograde flow, with lower assembly rates now sufficient to propel forward the leading edge. Based on our data we propose that differences in the protrusion persistence of leaders and followers originate in the distinct actomyosin contraction modules that differentially regulate leading edge protrusion-promoting F-actin assembly, and retraction-promoting retrograde flow.

Population Pharmacokinetic/Pharmacodynamic Modeling of Glenzocimab (ACT017) a Glycoprotein VI Inhibitor of Collagen-Induced Platelet Aggregation.

Glenzocimab (ACT017) is a humanized monoclonal antigen-binding fragment (Fab) directed against the human platelet glycoprotein VI, a key receptor for collagen and fibrin that plays a major role in thrombus growth and stability. Glenzocimab is being developed as an antiplatelet agent to treat the acute phase of ischemic stroke. During a phase I study in healthy volunteers, the population pharmacokinetics (PK) and pharmacodynamics (PD) of glenzocimab were modeled using Monolix software. The PK/PD model thus described glenzocimab plasma concentrations and its effects on ex vivo collagen-induced platelet aggregation. Glenzocimab was found to have dose-proportional, 2-compartmental PK with a central distribution volume of 4.1 L, and first and second half-lives of 0.84 and 9.6 hours. Interindividual variability in clearance in healthy volunteers was mainly explained by its dependence on body weight. The glenzocimab effect was described using an immediate effect model with a dose-dependent half maximal inhibitory concentration: Larger doses resulted in a stronger effect at the same glenzocimab plasma concentration. The mechanism of the overproportional concentration effect at higher doses remained unexplained. PK/PD simulations predicted that 1000-mg glenzocimab given as a 6-hour infusion reduced platelet aggregation to 20% in 100% of subjects at 6 hours and in 60% of subjects at 12 hours after dosing. Simulations revealed a limited impact of creatinine clearance on exposure, suggesting that no dose adjustments were required with respect to renal function. Future studies in patients with ischemic stroke are now needed to establish the relationship between ex vivo platelet aggregation and the clinical effect.

Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4.

Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.

Photogrammetric and image processing aspects in quantitative flow visualization.

The development of a measurement system for the visualization, topological classification, and quantitative analysis of complex flows in large-scale wind tunnel experiments is described. A new approach was sought in which the topological features of the flow (e.g., stream lines, separation and reattachment regions, stagnation points, and vortex lines) were extracted directly and preferably visualized in real-time in a virtual wind tunnel environment. The system was based on a stereo arrangement of two CCD cameras. A frame rate of 120 fps allowed measurements at high flow velocities. The paper focuses on the problem of fast and accurate reconstruction of path lines of helium filled soap bubbles in three dimensions (3D). A series of simple algorithmic steps was employed to ensure fast data processing. These included fast image segmentation, a spline approximation of the path lines, a camera model, point correspondence building, calculation of path line points in 3D and creation of a three-dimensional spline representation. The path lines, which contained both velocity and topological information, were analyzed to extract the relevant information.

Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation.

The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that do not require fluorescence tagging and that automatically track and characterize three aspects of scattering in live cells: increase in cell motility, loss of cell-cell adhesion, and spatial dispersion of cells (the redistribution of cells during scattering). We used these tools to screen a library of drugs, and we identified several efficient inhibitors of scattering, which we classified as selective inhibitors of either motility or loss of cell-cell adhesion, or as nonselective inhibitors. We validated the inhibitors and putative targets from this screen in two unrelated model cell lines. Using pharmacological treatments and RNA interference (RNAi), we found that nonsteroidal anti-inflammatory drugs inhibited cell-cell dissociation, that indirubins inhibited cell motility, and that cyclin-dependent kinase 1 and ribosomal S6 kinase were signaling intermediates in HGF-induced cell scattering. This assay is suitable for larger-scale screenings of chemical compounds or RNAi libraries.

Protein kinase A governs a RhoA-RhoGDI protrusion-retraction pacemaker in migrating cells.

The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation and tumorigenesis. The molecular identity of the signalling mechanisms that control these cycles has remained unknown. Here, we used live-cell imaging of biosensors to monitor spontaneous morphodynamic and signalling activities, and employed correlative image analysis to examine the role of cyclic-AMP-activated protein kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI (Rho GDP-dissociation inhibitor) establish a negative feedback mechanism that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA and RhoGDI forms a pacemaker that governs the morphodynamic behaviour of migrating cells.