Clinical Evaluation of a Novel Electromechanical Topical Ocular Drug Delivery System: Two Phase 1 Proof of Concept Studies.

PURPOSE: Self-administration of topical ophthalmic therapies remains challenging for many patients as errors due to improper technique are common. The aim of the current studies was to evaluate a novel electromechanical topical ocular drug delivery device designed to facilitate precise dosing and accurate delivery with substantially lower drug exposure than conventional eye drops. PATIENTS AND METHODS: Two randomized Phase 1 studies were performed to evaluate the efficacy and safety of a single dose of a topical ophthalmic solution administered as a ~9 µL microfluid stream via the test device compared with a ~30-40 µL drop delivered via conventional dropper in healthy subjects (Trial 1) and glaucoma patients (Trial 2). In Trial 1, a 1% tropicamide/2.5% phenylephrine solution was administered via the test device in one eye and by conventional dropper in the contralateral eye. Pupil dilation was measured at 30 min intervals post-instillation and subject comfort was assessed using a visual analogue scale (range, 0-100). In Trial 2, patients were randomized to receive latanoprost 0.005% via the test device or conventional dropper. Intraocular pressure was measured at baseline and 4-8 hrs post-instillation. RESULTS: In Trial 1 (N=20), mean (SD) pupil diameter 30 mins post-instillation increased by 3.4 (0.9) and 3.5 (1.0) mm in the test and control eyes, respectively. The mean comfort score was 81.7 for the test device versus 57.3 for conventional dropper delivery. In Trial 2 (N=18), the mean change in intraocular pressure following administration of latanoprost was -5.0 (1.8) and -4.3 (3.3) mm Hg in the test and control groups, respectively. No serious adverse events were observed in either study. CONCLUSION: Administration of a single dose of topical ophthalmic therapy via an electromechanical drug delivery device resulted in comparable effects on pupil dilation and intraocular pressure with lower drug exposure and increased patient comfort compared with conventional dropper delivery.

6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6c(hi) Ly6g(-) monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6c(lo) Ly6g(+) granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1(int) Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6c(hi) macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8(+) T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.

Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice.

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet ß cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation.

Vaccines aiming to activate cytotoxic T cells require cross-presentation of exogenous antigen by antigen-presenting cells (APCs). We recently developed a synthetic nanoparticle vaccine platform that targets lymph node-resident dendritic cells (DCs), capable of mounting an immune response to conjugated antigen. Here, we explore routes of processing and the efficiency of MHC I cross-presentation of OVA peptides conjugated using both reducible and non-reducible linkages, exploring the hypothesis that reduction-sensitive conjugation will lead to better antigen cross-presentation. Both clathrin and macropinocytic pathways were implicated in nanoparticle uptake by colocalization and inhibitor studies. Cross-presentation by DCs was demonstrated by direct antibody staining and in vitro stimulation of CD8(+) T cells from OT-I mice and was indeed most efficient with the reduction-sensitive conjugation. Similarly, we observed IFN-γ production by CD4(+) T cells from OT-II mice. Finally, immunization with the OVA peptide-bearing nanoparticles resulted in in vivo proliferation and IFN-γ production by adoptively transferred CD8(+) OT-I T cells and was also most efficient with reduction-sensitive linking of the peptide antigen. These results demonstrate the relevance of the poly(propylene sulfide) nanoparticle vaccine platform and antigen conjugation scheme for activating both cytotoxic and helper T cell responses.

Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21.

Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoid-like reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and even nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, which facilitates tumor progression.

ANGIOCARE: an automated system for fast three-dimensional coronary reconstruction by integrating angiographic and intracoronary ultrasound data.

OBJECTIVES: The development of an automated, user-friendly system (ANGIOCARE), for rapid three-dimensional (3D) coronary reconstruction, integrating angiographic and, intracoronary ultrasound (ICUS) data. METHODS: Biplane angiographic and ICUS sequence images are imported into the system where a prevalidated method is used for coronary reconstruction. This incorporates extraction of the catheter path from two end-diastolic X-ray images and detection of regions of interest (lumen, outer vessel wall) in the ICUS sequence by an automated border detection algorithm. The detected borders are placed perpendicular to the catheter path and established algorithms used to estimate their absolute orientation. The resulting 3D object is imported into an advanced visualization module with which the operator can interact, examine plaque distribution (depicted as a color coded map) and assess plaque burden by virtual endoscopy. RESULTS: Data from 19 patients (27 vessels) undergoing biplane angiography and ICUS were examined. The reconstructed vessels were 21.3-80.2 mm long. The mean difference was 0.9 +/- 2.9% between the plaque volumes measured using linear 3D ICUS analysis and the volumes, estimated by taking into account the curvature of the vessel. The time required to reconstruct a luminal narrowing of 25 mm was approximately 10 min. CONCLUSION: The ANGIOCARE system provides rapid coronary reconstruction allowing the operator accurately to estimate the length of the lesion and determine plaque distribution and volume.

Three-dimensional finite element modeling of guided ultrasound wave propagation in intact and healing long bones.

The use of guided waves has recently drawn significant interest in the ultrasonic characterization of bone aiming at supplementing the information provided by traditional velocity measurements. This work presents a three-dimensional finite element study of guided wave propagation in intact and healing bones. A model of the fracture callus was constructed and the healing course was simulated as a three-stage process. The dispersion of guided modes generated by a broadband 1-MHz excitation was represented in the time-frequency domain. Wave propagation in the intact bone model was first investigated and comparisons were then made with a simplified geometry using analytical dispersion curves of the tube modes. Then, the effect of callus consolidation on the propagation characteristics was examined. It was shown that the dispersion of guided waves was significantly influenced by the irregularity and anisotropy of the bone. Also, guided waves were sensitive to material and geometrical changes that take place during healing. Conversely, when the first-arriving signal at the receiver corresponded to a nondispersive lateral wave, its propagation velocity was almost unaffected by the elastic symmetry and geometry of the bone and also could not characterize the callus tissue throughout its thickness. In conclusion, guided waves can enhance the capabilities of ultrasonic evaluation.

A method for 3D reconstruction of coronary arteries using biplane angiography and intravascular ultrasound images.

The aim of this study is to describe a new method for the three-dimensional reconstruction of coronary arteries and its quantitative validation. Our approach is based on the fusion of the data provided by intravascular ultrasound images (IVUS) and biplane angiographies. A specific segmentation algorithm is used for the detection of the regions of interest in intravascular ultrasound images. A new methodology is also introduced for the accurate extraction of the catheter path. In detail, a cubic B-spline is used for approximating the catheter path in each biplane projection. Each B-spline curve is swept along the normal direction of its X-ray angiographic plane forming a surface. The intersection of the two surfaces is a 3D curve, which represents the reconstructed path. The detected regions of interest in the IVUS images are placed perpendicularly onto the path and their relative axial twist is computed using the sequential triangulation algorithm. Then, an efficient algorithm is applied to estimate the absolute orientation of the first IVUS frame. In order to obtain 3D visualization the commercial package Geomagic Studio 4.0 is used. The performance of the proposed method is assessed using a validation methodology which addresses the separate validation of each step followed for obtaining the coronary reconstruction. The performance of the segmentation algorithm was examined in 80 IVUS images. The reliability of the path extraction method was studied in vitro using a metal wire model and in vivo in a dataset of 11 patients. The performance of the sequential triangulation algorithm was tested in two gutter models and in the coronary arteries (marked with metal clips) of six cadaveric sheep hearts. Finally, the accuracy in the estimation of the first IVUS frame absolute orientation was examined in the same set of cadaveric sheep hearts. The obtained results demonstrate that the proposed reconstruction method is reliable and capable of depicting the morphology of coronary arteries.