Pharmacokinetics of Tranexamic Acid (TXA) Delivered by Expeditious Routes in a Swine Model of Polytrauma and Hemorrhagic Shock.

OBJECTIVE: Hemorrhage is the leading cause of preventable death in civilian trauma centers and on the battlefield. One of the emerging treatment options for hemorrhage in austere environments is tranexamic acid (TXA). However, the landscape is not amenable to the current delivery standard. This study compared the pharmacokinetics of TXA via a standard 10-minute intravenous infusion (IV infusion), intravenous rapid push over 10 s (IV push), and intramuscular injection (IM) in a swine polytrauma and hemorrhagic shock model (trauma group) compared to uninjured controls (control group). METHODS: Thirty swine were randomized to the trauma or control group. Following anesthesia, the trauma group experienced a simulated blast injury and 40% controlled hemorrhage. Subjects in both groups were then randomized to receive 1 g/10 mL TXA via IV infusion, IV push, or IM. Animals were monitored for four hours with serial blood sampling. Serum TXA concentrations were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and analyzed. RESULTS: The time to maximum TXA concentration (Tmax) was not affected by trauma in IV infusion or IV push, but was affected in the IM administration with Tmax significantly slower than the control group (p = 0.016). The minimum effective serum concentration of TXA (Ceff, 10 µg/mL) was reached in less than one minute with IV infusion and instantaneously with IV push. Despite lower bioavailability, the time to reach Ceff (Teff) was achieved via IM administration in less than 10 min for both groups (6.4 min trauma vs. 2.1 min control). CONCLUSIONS: In austere prehospital environments, an alternative to intravenous infusion of a life-saving medication is desired. Administration of TXA via all three methods reached the level needed to cause substantial inhibition of fibrinolysis within 10 min. The IV push method showed similar pharmacokinetics to IV infusion of TXA but can be delivered quickly without sacrificing an access site for 10 min.

Defining the Canadian rural general surgeon.

BACKGROUND: A total of 18%-30% of Canadians live in a rural area and are served by 8% of the country's general surgeons. The demographic characteristics of Canada's population and its geography greatly affect the health outcomes and needs of the population living in rural areas, and rural general surgeons hold a unique role in meeting the surgical needs of these communities. Rural general surgery is a distinct area of practice that is not well understood. We aimed to define the Canadian rural general surgeon to inform rural health human resource planning. METHODS: A scoping review of the literature was undertaken of Ovid, MEDLINE, and Embase using the terms "rural," "general surgery," and "workforce." We limited our review to articles from North America and Australia. RESULTS: The search yielded 425 titles, and 110 articles underwent full-text review. A definition of rural general surgery was not identified in the Canadian literature. Rurality was defined by population cut-offs or combining community size and proximity to larger centres. The literature highlighted the unique challenges and broad scope of rural general surgical practice. CONCLUSION: Rural general surgeons in Canada can be defined as specialists who work in a small community with limited metropolitan influence. They apply core general surgery skills and skills from other specialties to serve the unique needs of their community. Surgical training programs and health systems planning must recognize and support the unique skill set required of rural general surgeons and the critical role they play in the health and sustainability of rural communities.

Ultrasound Localization of Resuscitative Endovascular Balloon Occlusion of the Aorta in a Human Cadaver Model.

OBJECTIVE: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a method of gaining proximal control of noncompressible torso hemorrhage (NCTH). Catheter placement is traditionally confirmed with fluoroscopy, but few studies have evaluated whether ultrasound (US) can be used. METHODS: Using a pressurized human cadaver model, a certified REBOA placer was shown one of four randomized cards that instructed them to place the REBOA either correctly or incorrectly in Zone 1 (the distal thoracic aorta extending from the celiac artery to the left subclavian artery) or Zone 3 (in the distal abdominal aorta, from the aortic bifurcation to the lowest renal artery). Once the REBOA was placed, 10 US-trained locators were asked to confirm balloon placement via US. The participants were given 3 minutes to determine whether the catheter had been correctly placed, repeating this 20 times on two cadavers. RESULTS: Overall, US exhibited an average sensitivity of 83%, specificity of 76%, and accuracy of 80%. For Zone 1, US showed a sensitivity of 78% and specificity of 83%, and for Zone 3, a sensitivity of 88% and specificity of 76%. In addition, US exhibited a likelihood positive ratio (LR+) of 3.73 and a likelihood negative ratio (LR-) of 0.22 for either position, with similar numbers for Zone 1 (+4.57, -0.26) and Zone 3 (+3.16, -0.16). CONCLUSION: Ultrasound could prove to be a useful tool for confirming placement of a REBOA catheter, especially in austere environments.

Cric in the Dark: Surgical Cricothyrotomy in Low Light Tactical Environments.

BACKGROUND: Surgical cricothyrotomy (SC) is a difficult procedure with high failure rates in the battlefield environment. The difficulty of this procedure is compounded in a low-light tactical environment in which white light cannot be used. This study compared the use of red-green (RG) light and red (R) light in the performance of SC in a low-light environment. MATERIALS AND METHODS: Tactical Combat Casualty Care-certified navy corpsmen (n = 33) were provided 15 minutes of standardized instruction followed by hands-on practice with the Tactical CricKit and the H&H bougie-assisted Emergency Cricothyrotomy Kit. Participants acclimated to a dark environment for 30 minutes before performing SC on a mannequin with both devices using both R and RG light in a randomized order. Application time, success, participant preference, and participant confidence were analyzed. RESULTS: There were similarly high levels of successful placement (>87.5%) in all four cohorts. Light choice did not appear to affect placement time with either of the two kits. On Likert-scale surveys, participants reported that RG decreased difficulty (p < .0001) and increased confidence (p < .0001) in performing the procedure. CONCLUSION: RG light increased confidence and decreased perceived difficulty when performing SC, though no differences in placement time or success were observed.

iTClamp-Mediated Wound Closure Speeds Control of Arterial Hemorrhage With or Without Additional Hemostatic Agents.

BACKGROUND: Exsanguination is the leading cause of preventable posttraumatic death, especially in the prehospital arena. Traditional hemorrhage control methods involve packing the wound with hemostatic agents, providing manual pressure, and then applying a pressure dressing to stabilize the treatment. This is a lengthy process that frequently destabilizes upon patient transport. Conversely, the iTClamp, a compact wound closure device, is designed to rapidly seal wound edges mechanically, expediting clot formation at the site of injury. OBJECTIVES: To determine the efficacy of the iTClamp with and without wound packing in the control of a lethal junction hemorrhage. METHODS: Given the limited available information regarding the efficacy of the iTClamp in conjunction with traditional hemostatic agents, this study used a swine model of severe junctional hemorrhage. The goal was to compare a multiagent strategy using the iTClamp in conjunction with XSTAT to the traditional method of Combat Gauze packing with pressure dressing application. Readouts include application time, blood loss, and rebleed occurrence. RESULTS: Mean application times of the iTClamp treatment alone or in conjunction with other hemostatic agents were at least 75% faster than the application time of Combat Gauze with pressure dressing. Percent blood loss was not significantly different between groups but trended the highest for Combat Gauze treated swine, followed by iTClamp plus XSTAT, iTClamp alone and finally iTClamp plus Combat Gauze. CONCLUSION: The results from this study demonstrate that the iTClamp can be effectively utilized in conjunction with hemostatic packing to control junctional hemorrhages.

Speed, Skill Retention, and End User Perceptions of iTClamp Application by Navy Corpsmen on a Manikin Model of Femoral Hemorrhage.

INTRODUCTION: Tactical Combat Casualty Care guidelines recommend packing junctional wounds with gauze, applying direct pressure for 3 minutes, and then securing with an external pressure dressing. This method is time-consuming, which can be problematic in a combat environment. Alternatively, the iTClamp has documented efficacy and rapid application. However, no studies have evaluated device application by military prehospital medical providers, such as Navy corpsmen, or their user experience with the device. MATERIALS AND METHODS: Research data derived from a protocol were approved by the Naval Medical Center Portsmouth's Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects. Navy corpsmen with the current Tactical Combat Casualty Care certification applied the iTClamp or standard pressure dressing on a manikin model of femoral hemorrhage in a crossover study design. Each participant used both devices in a randomized fashion. Time to application was recorded, and participants completed Likert scale surveys to evaluate both devices for preference, ease of use, and physical assessment. A repeat assessment was performed 1 month later to assess skill atrophy. Repeated-measures ANOVA was used to compare application time. Likert scale survey data were analyzed using Mann-Whitney and Wilcoxon tests to compare survey data within and between time points, respectively. RESULTS: The application of the iTClamp was more than twice as fast as the application of pressure dressings at both the initial and follow-up evaluations. There was no statistically significant difference in application times between the first evaluation and the 30-day assessment of either device, indicating no atrophy in skill. While 65% and 52% of the participants expressed preference in for the iTClamp in their surveys during the initial and follow-up respective visits, the difference in preference was not statistically significant for either the initial or the follow-up survey. Open-ended survey responses yielded both perceived advantages and disadvantages for each treatment option. CONCLUSIONS: In austere or hostile environments, speed of treatment and extrication can have significant implications for the safety of both the patient and the medical providers. Hemorrhage control interventions must be both effective and easy to use for a prehospital provider to ensure its efficacy in a live battlefield situation. The iTClamp is small, simple, and fast to use, but its wide adoption in the field may be based on limitations perceived by participants, including narrow indications for use. However, based on our findings, it is reasonable to field the iTClamp depending on provider preference.

A comparison of efficacy, efficiency, and durability in novel tourniquet designs.

BACKGROUND: Exsanguination due to extremity hemorrhage is a major cause of preventable traumatic deaths. Extremity tourniquet use has been shown to be safe and improve survival. The purpose of this study was to compare the efficacy, efficiency, and durability of the Generation 7 Combat Application Tourniquet (CAT; North American Rescue, Greer, SC), the Tactical Mechanical Tourniquet (TMT; Combat Medical Systems, Harrisburg, NC), and the SOF Tactical Tourniquet-Wide (SOFTT-W; Tactical Medical Solutions, Anderson, SC). METHODS: This study was a three-phase randomized, cross-over trial. In successive trials, subjects were timed during the application of each tourniquet to the upper and lower extremity. Following successful lower extremity application, subjects low crawled 25 ft and then were dragged 25 ft, after which effectiveness was reassessed, as defined by the cessation of distal pulses by Doppler ultrasound. RESULTS: In arm application, both the CAT and TMT had significantly less failure rates than the SOFTT-W (5.56%, 19.44%, 58.33%), with the CAT being the fastest tourniquet when compared with TMT and SOFTT-W (37.8 seconds, 65.01 seconds, 63.07 seconds). In leg application, the CAT had significantly less rates of failure when compared with the SOFTT-W, but there was no other significant difference between the tourniquets (27.78%, 44.44%, 61.11%). In addition, the CAT was significantly faster than both the TMT and SOFTT-W when applied to the leg (8.33 seconds, 40.96 seconds, 34.5 seconds). There was no significant difference in tourniquet failure rates between the three tourniquets after subject maneuvers in phase 3 (34.29%, 42.86%, 45.45%). DISCUSSION: The CAT is as effective as the TMT and significantly more effective than the SOFTT-W. In addition, the CAT demonstrated shorter application times than either the TMT or SOFTT-W. However, there was no significant difference between the three tourniquets in their ability to maintain pulselessness after subject maneuvers. LEVEL OF EVIDENCE: Care management, level II.

PV1 in Caveolae Controls Lung Endothelial Permeability.

Caveolae are prominent plasmalemmal invaginations in endothelial cells, especially in the lung vasculature, which comprises a vast surface area. PV1 (plasmalemmal vesicle-associated protein-1), a 60-kD glycoprotein expressed in endothelial cells, is essential for generating spoke-like diaphragmatic structures that span the neck region of endothelial caveolae. However, their role in caveolae-mediated uptake and endothelial-barrier function is unknown. Here, we generated mice with endothelial cell-specific deletion of PV1 through tamoxifen-induced Cdh5.Cre.ERT2 (endothelial-specific vascular cadherin.Cre.estrogen receptor 2)-mediated excision of the floxed PV1 allele. We observed that loss of PV1 specifically in endothelial cells increased lung vascular permeability of fluid and protein, indicating that PV1 is required for maintenance of lung vascular-barrier integrity. Endothelial-specific PV1 deletion also increased caveolae-mediated uptake of tracer albumin compared with controls, promoted Au-albumin accumulation in the bulb of caveolae, and induced caveolar swelling. In addition, we observed the progressive loss of plasma proteins from the circulation and reduced arterial pressure resulting from transudation of water and protein as well as edema formation in multiple tissues, including lungs. These changes seen after endothelial-specific PV1 deletion occurred in the absence of disruption of endothelial junctions. We demonstrated that exposure of wild-type mice to endotoxin, which is known to cause acute lung injury and increase protein permeability, also significantly reduced PV1 protein expression. We conclude that the key function of PV1 is to regulate lung endothelial permeability through its ability to restrict the entry of plasma proteins such as albumin into caveolae and their transport through the endothelial barrier.

Knockdown of sodium channel Nax reduces dermatitis symptoms in rabbit skin.

The skin plays a critical role in maintenance of water homeostasis. Dysfunction of the skin barrier causes not only delayed wound healing and hypertrophic scarring, but it also contributes to the development of various skin diseases. Dermatitis is a chronic inflammatory skin disorder that has several different subtypes. Skin of contact dermatitis and atopic dermatitis (AD) show epidermal barrier dysfunction. Nax is a sodium channel that regulates inflammatory gene expression in response to perturbation of barrier function of the skin. We found that in vivo knockdown of Nax using RNAi reduced hyperkeratosis and keratinocyte hyperproliferation in rabbit ear dermatitic skin. Increased infiltration of inflammatory cells (mast cells, eosinophils, T cells, and macrophages), a characteristic of dermatitis, was reduced by Nax knockdown. Upregulation of PAR-2 and thymic stromal lymphopoietin (TSLP), which induce Th2-mediated allergic responses, was inhibited by Nax knockdown. In addition, expression of COX-2, IL-1ß, IL-8, and S100A9, which are downstream genes of Nax and are involved in dermatitis pathogenesis, were also decreased by Nax knockdown. Our data show that knockdown of Nax relieved dermatitis symptoms in vivo and indicate that Nax is a novel therapeutic target for dermatitis, which currently has limited therapeutic options.

Endothelial cell Piezo1 mediates pressure-induced lung vascular hyperpermeability via disruption of adherens junctions.

Increased pulmonary microvessel pressure experienced in left heart failure, head trauma, or high altitude can lead to endothelial barrier disruption referred to as capillary "stress failure" that causes leakage of protein-rich plasma and pulmonary edema. However, little is known about vascular endothelial sensing and transduction of mechanical stimuli inducing endothelial barrier disruption. Piezo1, a mechanosensing ion channel expressed in endothelial cells (ECs), is activated by elevated pressure and other mechanical stimuli. Here, we demonstrate the involvement of Piezo1 in sensing increased lung microvessel pressure and mediating endothelial barrier disruption. Studies were made in mice in which Piezo1 was deleted conditionally in ECs (Piezo1iΔEC ), and lung microvessel pressure was increased either by raising left atrial pressure or by aortic constriction. We observed that lung endothelial barrier leakiness and edema induced by raising pulmonary microvessel pressure were abrogated in Piezo1iΔEC mice. Piezo1 signaled lung vascular hyperpermeability by promoting the internalization and degradation of the endothelial adherens junction (AJ) protein VE-cadherin. Breakdown of AJs was the result of activation of the calcium-dependent protease calpain and degradation of the AJ proteins VE-cadherin, ß-catenin, and p120-catenin. Deletion of Piezo1 in ECs or inhibition of calpain similarly prevented reduction in the AJ proteins. Thus, Piezo1 activation in ECs induced by elevated lung microvessel pressure mediates capillary stress failure and edema formation secondary to calpain-induced disruption of VE-cadherin adhesion. Inhibiting Piezo1 signaling may be a useful strategy to limit lung capillary stress failure injury in response to elevated vascular pressures.

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo.

Detergents such as sodium dodecyl sulfate (SDS) are commonly used to extract cells from tissues in a process called "decellularization". Residual SDS is difficult to completely remove and may lead to an undesirable host response towards an implanted biomaterial. In this study, we developed a modification for SDS cell extraction from muscle equally efficient to previous methods but leading to significantly less residual SDS remnants in the matrices. Muscle-derived matrices were prepared via 2 SDS-based decellularization methods, which led to removal of either 81.4% or 98.4% of the SDS. In vitro, matrices were seeded with thp1 macrophages and primary human foreskin fibroblasts. By Day 2, both matrices demonstrated similar macrophage polarization; however, fibroblasts cultured on matrices with greater residual SDS expressed higher levels of mRNA associated with fibroblast activation: α-smooth muscle actin and connective tissue growth factor. In vivo, Collagen I gels spiked with increasing concentrations of SDS displayed a corresponding decrease in cell infiltration when implanted subcutaneously in rats after 4 days. Finally, as a model for muscle regeneration, matrices produced by each method were implanted in rat latissimus dorsi defects. At POD 30 greater levels of IL-1ß mRNA were present in defects treated with matrices containing higher levels of SDS, indicating a more severe inflammatory response. Although matrices containing higher levels of residual SDS became encapsulated by POD 30 and showed evidence of a foreign body response, matrices with the lower levels of SDS integrated into the defect area with lower levels of inflammatory and fibrosis-related gene expression.

FOXA1 expression is a strong independent predictor of early PSA recurrence in ERG negative prostate cancers treated by radical prostatectomy.

FOXA1 (Fork-head box protein A1, HNF-3a) is a transcription factor involved in androgen signaling with relevance for lineage-specific gene expression of the prostate. The expression was analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with tumor phenotype, biochemical recurrence, androgen receptor expression, ETS-related gene (ERG) status and other recurrent genomic alterations. FOXA1 expression was detectable in 97.6% of 8227 interpretable cancers and considered strong in 28.5%, moderate in 46.2% and weak in 22.9% of cases. High FOXA1 expression was associated with TMPRSS2:ERG rearrangement and ERG expression (P < 0.0001). High FOXA1 expression was linked to high Gleason grade, advanced pathological tumor (pT) stage and early PSA recurrence in ERG negative cancers (P < 0.0001), while these associations were either weak or absent in ERG positive cancers. In ERG negative cancers, the prognostic role of FOXA1 expression was independent of Gleason grade, pathological tumor stage, lymph node stage, surgical margin status and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features such as biopsy Gleason grade, preoperative PSA, cT stage and FOXA1 expression (P < 0.0001). Within ERG negative cancers, FOXA1 expression was also strongly associated with PTEN and 5q21 deletions (P < 0.0001). High expression of FOXA1 is an independent prognostic parameter in ERG negative prostate cancer. Thus, FOXA1 measurement might provide clinically useful information in prostate cancer.

High-Level Glyoxalase 1 (GLO1) expression is linked to poor prognosis in prostate cancer.

BACKGROUND: Glyoxalase 1 (GLO1) is an enzyme involved in removal of toxic byproducts accumulating during glycolysis from the cell. GLO1 is up regulated in many cancer types but its role in prostate cancer is largely unknown. METHODS: Here, we employed GLO1 immunohistochemistry on a tissue microarray including 11 152 tumors and an attached clinical and molecular database. RESULTS: Normal prostate epithelium was negative for GLO1, whereas 2059 (27.3%) of 7552 interpretable cancers showed cytoplasmic GLO1 staining, which was considered weak in 8.8%, moderate in 12.5%, and strong in 6.1% of tumors. Up regulation of GLO1 was significantly linked to high original Gleason grade, advanced pathological tumor stage and positive lymph node status (P < 0.0001 each). Comparison of GLO1 staining with several common genomic alterations of prostate cancers revealed a strong link between GLO1 up regulation and TMPRSS2:ERG fusion (P < 0.0001) and an ERG-independent association with PTEN deletion (P < 0.0001). GLO1 up regulation was strongly linked to early biochemical recurrence in univariate analysis (P < 0.0001) and predicted poor prognosis independent from most (except from nodal stage) established prognostic parameters in multivariate analysis (P ≤ 0.03). CONCLUSIONS: GLO1 upregulation is linked to aggressive prostate cancers characterized by ERG fusion and PTEN deletion. The strong and independent prognostic value makes it a promising candidate for routine diagnostic applications either alone or in combination with other markers.

Thermal injury model in the rabbit ear with quantifiable burn progression and hypertrophic scar.

Hypertrophic scar is a major clinical outcome of deep-partial thickness to full thickness thermal burn injury. Appropriate animal models are a limitation to burn research due to the lack of, or access to, animal models which address the endpoint of hypertrophic scar. Lower species, such as rodents, heal mainly by contracture, which limits the duration of study. Higher species, such as pigs, heal more similarly to humans, but are associated with high cost, long duration for scar development, challenges in quantifying scar hypertrophy, and poor manageability. Here, we present a quantifiable deep-partial thickness burn model in the rabbit ear. Burns were created using a dry-heated brass rod for 10 and 20 seconds at 90 °C. At the time of eschar excision on day 3, excisional wounds were made on the contralateral ear for comparison. Burn wound progression, in which the wound size expands over time is a major distinction between excisional and thermal injuries, was quantified at 1 hour and 3 days after the injuries using calibrated photographs and histology and the size of the wounds was found to be unchanged from the initial wound size at 1 hour, but 10% in the 20 seconds burn wounds at 3 days. A quantifiable hypertrophic scar, measured by histology as the scar elevation index, was present in both 20 seconds burn wounds and excisional wounds at day 35. ImageJ measurements revealed that the 20 seconds burn wound scars were 22% larger than the excisional wound scars and the 20 seconds burn scar area measurements from histology were 26% greater than in the excisional wound scar. The ability to measure both burn progression and scar hypertrophy over a 35-day time frame suits this model to screening early intervention burn wound therapeutics or scar treatments in a burn-specific scar model.

Transport patterns of anti-TNF-α in burn wounds: Therapeutic implications of hyaluronic acid conjugation.

A central complication in burn injuries is progression of the zone of necrosis, which is associated with intense inflammatory responses. Conjugation of monoclonal antibodies against tumor necrosis factor-α (TNF-α), a central mediator of inflammation, to high molecular weight hyaluronic acid (HA) has been shown to be an effective treatment in reducing secondary necrosis in rodent models of deep partial-thickness burns. Here the transport of conjugated and non-conjugated antibodies in burn injuries was investigated to explore the effects of antibody tethering on the spatiotemporal distribution of anti-TNF-α. Diffusion constants were measured in solution and in type I collagen gels in vitro using fluorescence correlation spectroscopy to provide quantitative comparisons of the effects of conjugation. It is shown that the HA significantly increased the antibody residence time in the superficial region at 24 h in burn injuries, which strongly correlated with the pattern of inflammatory cell infiltrate in the tissue. A transport model was used to fit the results of antibody distribution in the tissue based on fluorescence correlation spectroscopy measurements, resulting in estimates for effective diffusion constants that demonstrate the effects of HA conjugation on the biodistribution of therapeutic proteins. These results demonstrate that tuning residence time of therapeutic proteins can be an effective strategy in regulating the inflammatory response associated with acute injuries.

S100A12 Induced in the Epidermis by Reduced Hydration Activates Dermal Fibroblasts and Causes Dermal Fibrosis.

Disruption of the barrier function of skin increases transepidermal water loss and up-regulates inflammatory pathways in the epidermis. Consequently, sustained expression of proinflammatory cytokines from the epidermis is associated with dermal scarring. We found increased expression of S100A12 in the epidermis of human hypertrophic and keloid scar. Exposing a stratified keratinocyte culture to a reduced-hydration environment increased the expression and secretion of S100A12 by nearly 70%, which in turn activated dermal fibroblasts in vitro. Direct treatment of fibroblasts with conditioned medium collected from stratified keratinocyte culture under reduced-hydration conditions activated fibroblasts, shown by up-regulation of α-smooth muscle actin, pro-collagen 1, and F-actin expression. However, this fibroblast activation was not found when S100A12 was knocked down by RNA interference in keratinocytes. Pharmacological blockade of S100A12 receptors, RAGE, or TLR4 inhibited S100A12-induced fibroblast activation. Local delivery of S100A12 resulted in a marked hypertrophic scar formation in a validated rabbit hypertrophic scar model compared with saline control. Our findings indicate that S100A12 functions as a proinflammatory cytokine and suggest that S100A12 is a potential therapeutic target for dermal scarring.

Tip-Loaded Dissolvable Microneedle Arrays Effectively Deliver Polymer-Conjugated Antibody Inhibitors of Tumor-Necrosis-Factor-Alpha Into Human Skin.

Autoinflammatory skin diseases are characterized by a disequilibrium of cytokines in the local skin microenvironment, suggesting that local delivery of therapeutics, including anticytokine antibodies, may provide benefit without the unwanted off-target effects of systemically delivered therapies. Rapid diffusion of therapeutics away from the target site has been a challenge to the development of local therapies. Conjugation of high molecular weight hydrophilic polymers to cytokine neutralizing mAbs has been shown to be an effective strategy for local control of inflammation in healing burn wounds. However, the burn application is unique because the skin barrier is already breached. For the treatment of autoinflammatory skin diseases, the major challenge for local delivery lies in penetrating the stratum corneum. Here, we investigate a new therapeutic approach combining the use of tip-loaded dissolvable microneedle arrays (TL-dMNAs) for local application of polymer-conjugated antibody inhibitors of tumor-necrosis-factor-alpha (TNF-α). Specifically, intradermal delivery and pharmacokinetics of (anti-TNF-α-Ab)-(high molecular weight hyaluronic acid [HA]) conjugates from tip-loaded, obelisk-shaped dissolvable microneedle arrays were investigated in living human skin. The results indicate (1) TL-dMNAs can be successfully fabricated to integrate (anti-TNF-α-Ab)-HA at the tip portion of the microneedles while preserving the biological activity necessary for antibody ligand binding; (2) (anti-TNF-α-Ab)-HA can be effectively delivered into human skin using obelisk-shaped TL-dMNAs; and (3) polymer conjugation effectively inhibits antibody diffusion from the delivery site. Taken together, these results support the evaluation of microneedle array-based delivery of varying polymer-antibody conjugates for the treatment of inflammatory skin diseases.

Therapeutic intradermal delivery of tumor necrosis factor-alpha antibodies using tip-loaded dissolvable microneedle arrays.

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1ß expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications. STATEMENT OF SIGNIFICANCE: Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

Local delivery of antitumor necrosis factor-α through conjugation to hyaluronic acid: dosing strategies and early healing effects in a rat burn model.

The objective of this study was to measure dose-response effects of topical delivery of inhibitors of tumor necrosis factor-α (TNF-α) through conjugation to hyaluronic acid in a rat burn model to determine effects on inflammatory responses, burn progression, and early stages of healing. Monoclonal antibodies against TNF-α were conjugated to hyaluronic acid and applied topically in a rat partial-thickness burn model. Metrics of inflammatory responses and tissue necrosis were measured as well as the quantitative analysis of collagen composition and organization. The minimum effective conjugated antibody dose was found to be 100 µg with three applications 48 hours apart. Nonviable tissue thicknesses decreased with increasing dose and dose frequency. Free antibody retarded macrophage infiltration in the periphery but not at the surface, while the conjugated antibody was able to hinder macrophage infiltration at both the periphery and the surface. Quantification of collagen I and III staining ratios at days 4, 7, and 14 and quantitative image analysis of collagen organization at day 14 demonstrated differences between saline and conjugate treatment. This correlated with increases in re-epithelialization observed in conjugate-treated sites. Reductions in inflammatory markers and secondary tissue necrosis under treatment with the conjugates were understood in terms of differences in antibody transport compared to nonconjugated antibody. Differences in collagen composition and organization at Day 14 suggested that the reductions in inflammatory responses altered early healing responses. These results indicate anti-TNF-α conjugated to hyaluronic acid can be an effective treatment for reducing secondary necrosis and improving healing outcomes in burns.

Effects of hyaluronic acid conjugation on anti-TNF-α inhibition of inflammation in burns.

Biomaterials capable of neutralizing specific cytokines could form the basis for treating a broad range of conditions characterized by intense, local inflammation. Severe burns, spanning partial- to full-thickness of the dermis, can result in complications due to acute inflammation that contributes to burn progression, and early mediation may be a key factor in rescuing thermally injured tissue from secondary necrosis to improve healing outcomes. In this work, we examined the effects on burn progression and influence on the inflammatory microenvironment of topical application of anti-tumor necrosis factor-α (anti-TNF-α) alone, mixed with hyaluronic acid (HA) or conjugated to HA. We found that non-conjugated anti-TNF-α decreased macrophage infiltration to a greater extent than that conjugated to HA; however, there was little effect on the degree of progression or IL-1ß levels. A simple transport model is proposed to analyze the results, which predicts qualitative and quantitative differences between untreated burn sites and those treated with the conjugates. Our results indicate that conjugation of anti-TNF-α to high molecular weight HA provides sustained, local modulation of the post-injury inflammatory responses compared to direct administration of non-conjugated antibodies.