Selective block of Na1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several Na1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.
ISO 10993-4 in vivo thrombogenicity testing is frequently performed for regulatory approval of many blood-contacting medical devices and is often a key part of submission packages. Given the current state of in vivo thrombogenicity assays, a more robust and reproducible assay design, including in vitro models, is needed. This study describes an in vitro assay that integrates freshly harvested ovine blood containing minimal heparin in a closed pumped loop. To confirm the reproducibility of this assay, control materials were identified that elicited either a positive or a negative thrombogenic response. These controls demonstrated reproducibility in the resulting thrombogenicity scores with median scores of 5 and 0 for the positive and negative controls, respectively, which also demonstrated a significant difference (p < 0.0001). For a direct comparison of the in vitro blood loop assay to the traditional in vivo nonanticoagulated venous implant (NAVI) assay, seven sheep were used as blood donors for the loop and then as subjects for an NAVI assay. In each assay—loop or NAVI—three study articles were used: the positive and negative controls and a marketed, approved catheter. The resulting thrombogenicity scores were similar when comparing the loop to the NAVI results. For each study article, the median thrombogenicity scores were the same in these two different assays, being 0, 1, and 5 for the negative control, the marketed catheter, and the positive control, respectively. These data suggest that the in vitro assay performs similarly to the in vivo NAVI assay. This in vitro blood loop method has the potential to predict a materials' in vivo thrombogenicity, can substantially de-risk the materials or coating selection process, and may eventually be able to replace the in vivo models currently in use.
Most blood-contacting medical devices must be assessed for potential thrombogenicity prior to regulatory approval. A common assay for screening and qualifying devices involves monitoring the reduction of platelet and leukocyte (P&L) counts in whole blood exposed to the device. We have validated an improved method for assessing a device's effect on platelet activation and surface adhesion, offering significant improvement over the current ASTM F2888-13 method, which uses blood fully anticoagulated by acidified citrate (known to significantly inhibit platelet responsiveness). Our method uses minimal heparinization (final concentration 1 IU/mL) to optimize the response to commonly used control materials: latex, black rubber, and high-density polyethylene (HDPE). We also have shown the assay's capacity to appropriately assess a legally marketed comparator device (LMCD) with a documented clinical history. The test materials were prepared for incubation and allowed to remain in contact with the citrated or heparinized blood for ∼1 h at 37 °C. A complete blood count was performed prior to exposure, and at the end of the incubation period, reductions in P&L counts were recorded. Results from citrate-anticoagulated assay showed only a marginal response to the positive control, black rubber. Using heparinized blood, the assay generated a robust response to the positive controls, the “intermediate scoring” controls, and also assessed a legally marketed and approved device as clearly nonthrombogenic. This modification adds robustness and sensitivity to this quick and inexpensive thrombogenicity assay and should be incorporated into the next ASTM standards.
Thrombogenicity testing is often a requirement for regulatory approval of many types of blood-contacting medical devices [1, 2]. This study describes the continuing improvement in design and characterization of a minimally-heparinized in vitro blood-loop assay which utilizes freshly drawn ovine blood. These modifications were made after studies using this in vitro model were submitted to the FDA in lieu of the in vivo nonanticoagulated venous implant (NAVI) thrombogenicity test. After extensive discussions with FDA reviewers, several modifications which further characterize and improve the assay have been included: 1). Improved temperature control of the blood before and during the incubation period, 2). Improved uniformity and reproducibility of loop geometry, specifically the length of working space for device deployment and a fixed curvature for the radius of the return segment of the loop, 3). Additional measurement of blood parameters prior to and during the incubation period, complete blood counts and activated clotting time (ACT), 4). More rigorous management of ACT, 5). Measurement of non-adherent thrombus formation in the blood, 6).Incorporation of a legally marketed predicate comparator device in all the assays, and 7).Physical characterization of the positive controls. This validated method with enhanced characterization and more reproducible methods allows for a more robust and reliable assay. These results continue to support the premise that this in vitro blood loop assay may eventually supplant the NAVI model for routine hemocompatibility testing for catheter-like blood contacting medical devices.
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