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.

Peptide inhibition of acute lung injury in a novel two-hit rat model.

Acute lung injury (ALI) often causes severe trauma that may progress to significant morbidity and mortality. ALI results from a combination of the underlying clinical condition of the patient (e.g., inflammation) with a secondary insult such as viral pneumonia or a blood transfusion. While the secondary insult may be variable, the rapidly progressive disease process leading to pulmonary failure is typically mediated by an overwhelming innate immunological or inflammatory reaction driven by excessive complement and neutrophil-mediated inflammatory responses. We recently developed a 'two-hit' ALI rat model mediated by lipopolysaccharide followed by transfusion of incompatible human erythrocytes resulting in complement activation, neutrophil-mediated ALI and free DNA in the blood indicative of neutrophil extracellular trap formation. The objective of this study was to evaluate the role of peptide inhibitor of complement C1 (RLS-0071), a classical complement pathway inhibitor and neutrophil modulator in this animal model. Adolescent male Wistar rats were infused with lipopolysaccharide followed by transfusion of incompatible erythrocytes in the presence or absence of RLS-0071. Blood was collected at various time points to assess complement C5a levels, free DNA and cytokines in isolated plasma. Four hours following erythrocyte transfusion, lung tissue was recovered and assayed for ALI by histology. Compared to animals not receiving RLS-0071, lungs of animals treated with a single dose of RLS-0071 showed significant reduction in ALI as well as reduced levels of C5a, free DNA and inflammatory cytokines in the blood. These results demonstrate that RLS-0071 can modulate neutrophil-mediated ALI in this novel rat model.

Peptide Inhibitor of Complement C1, RLS-0071, Reduces Zosteriform Spread of Herpes Simplex Virus Type 1 Skin Infection and Promotes Survival in Infected Mice.

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen primarily transmitted through skin-to-skin contact, especially on and around mucosal surfaces where there is contact with contaminated saliva during periods of viral shedding. It is estimated that 90% of adults worldwide have HSV-1 antibodies. Cutaneous HSV-1 infections are characterized by a sensation of tingling or numbness at the initial infection site followed by an eruption of vesicles and then painful ulcers with crusting. These symptoms can take ten days to several weeks to heal, leading to significant morbidity. Histologically, infections cause ballooning degeneration of keratinocytes and formation of multinucleated giant cells, ultimately resulting in a localized immune response. Commonly prescribed treatments against HSV-1 infections are nucleoside analogs, such as acyclovir (ACV). However, the emergence of ACV-resistant HSV (ACVR-HSV) clinical isolates has created an urgent need for the development of compounds to control symptoms of cutaneous infections. RLS-0071, also known as peptide inhibitor of complement C1 (PIC1), is a 15-amino-acid anti-inflammatory peptide that inhibits classical complement pathway activation and modulates neutrophil activation. It has been previously shown to aid in the healing of chronic diabetic wounds by inhibiting the excessive activation of complement component C1 and infiltration of leukocytes. Here, we report that treatment of cutaneous infections of HSV-1 and ACVR-HSV-1 in BALB/cJ mice with RLS-0071 significantly reduced the rate of mortality, decreased zosteriform spread, and enhanced the healing of the infection-associated lesions compared to control-treated animals. Therefore, RLS-0071 may work synergistically with other antiviral drugs to aid in wound healing of HSV-1 cutaneous infection and may potentially aid in rapid wound healing of other pathology not limited to HSV-1.

Peptide inhibition of neutrophil-mediated injury after in vivo challenge with supernatant of Pseudomonas aeruginosa and immune-complexes.

Neutrophils are recognized for their role in host defense against pathogens as well as inflammatory conditions mediated through many mechanisms including neutrophil extracellular trap (NET) formation and generation of reactive oxygen species (ROS). NETs are increasingly appreciated as a major contributor in autoimmune and inflammatory diseases such as cystic fibrosis. Myeloperoxidase (MPO), a key neutrophil granule enzyme mediates generation of hypochlorous acid which, when extracellular, can cause host tissue damage. To better understand the role played by neutrophils in inflammatory diseases, we measured and modulated myeloperoxidase activity and NETs in vivo, utilizing a rat peritonitis model. RLS-0071 is a 15 amino acid peptide that has been shown to inhibit myeloperoxidase activity and NET formation in vitro. The rat model of inflammatory peritonitis was induced with intraperitoneal injection of either P. aeruginosa supernatant or immune-complexes. After euthanasia, a peritoneal wash was performed and measured for myeloperoxidase activity and free DNA as a surrogate for measurement of NETs. P. aeruginosa supernatant caused a 2-fold increase in MPO activity and free DNA when injected IP. Immune-complexes injected IP increased myeloperoxidase activity and free DNA 2- fold. RLS-0071 injection decreased myeloperoxidase activity and NETs in the peritoneal fluid generally to baseline levels in the presence of P. aeruginosa supernatant or immune-complexes. Taken together, RLS-0071 demonstrated the ability to inhibit myeloperoxidase activity and NET formation in vivo when initiated by different inflammatory stimuli including shed or secreted bacterial constituents as well as immune-complexes.

Polynuclear ruthenium organometallic complexes containing a 1,3,5-triazine ligand: synthesis, DNA interaction, and biological activity.

It is now well established that ruthenium complexes are attractive alternatives to platinum-based anticancer agents. Most of the ruthenium compounds currently under investigation contain a single metal center. The synthesis of multinuclear analogues may provide access to novel complexes with enhanced biological activity. In this work, we have synthesized a set of three trinuclear complexes containing organometallic ruthenium fragments-(arene)RuCl-coordinated to a 2,4,6-tris(di-2-pyridylamino)-1,3,5-triazine core [(Arene = benzene (2), p-cymene (1), or hexamethylbenzene (3)]. The interaction of the complexes with DNA was extensively studied using a variety of biophysical probes as well as by molecular docking. The complexes bind strongly to DNA with apparent binding constants ranging from 2.20 to 4.79 × 104 M-1. The binding constants from electronic absorption titrations were an order of magnitude greater. The mode of binding to the nucleic acid was not definitively determined, but the evidence pointed to some kind of non-specific electrostatic interaction. None of the complexes displayed any significant antimicrobial activity against the organisms that were studied and exhibited anticancer activity only at high (> 100 µM) concentration.

Nano-Pulse Stimulation Ablates Orthotopic Rat Hepatocellular Carcinoma and Induces Innate and Adaptive Memory Immune Mechanisms that Prevent Recurrence.

Nano-pulse stimulation (NPS), previously called nsPEFs, induced a vaccine-like effect after ablation of orthotopic N1-S1 hepatocellular carcinoma (HCC), protecting rats from subsequent challenges with N1-S1 cells. To determine immunity, immune cell phenotypes were analyzed in naïve, treated and protected rats. NPS provides a positive, post-ablation immuno-therapeutic outcome by alleviating immunosuppressive T regulatory cells (Treg) in the tumor microenvironment (TME), allowing dendritic cell influx and inducing dynamic changes in natural killer cells (NKs), NKT-cells and T-lymphocytes in blood, spleen and liver. NPS induced specific increases in NKs and NKT-cells expressing CD8 and activation receptors CD314-NKG2D and CD161 (NK1.1) in the TME after treatment, as well as some variable changes in CD4+ and CD8+ effector (Tem) and central memory (Tem) lymphocytes in blood and spleen. After orthotopic challenge, CD8+ T-cells were cytotoxic, inducing apoptosis in N1-S1 cells; additionally, in contrast to post-treatment immune responses, CD4+ and CD8+ memory precursor effector cells (MPECs) and short-lived effector cells (SLECs) were present, while still including CD8+ CD161 NK cells, but not involving CD8+ CD314-NKG2D+ NKs. This immunity was N1-S1-specific and was sustained for at least 8 months. NPS vaccinates rats in vivo against HCC by activating innate and adaptive immune memory mechanisms that prevent HCC recurrence.

Nanopulse Stimulation (NPS) Induces Tumor Ablation and Immunity in Orthotopic 4T1 Mouse Breast Cancer: A Review.

Nanopulse Stimulation (NPS) eliminates mouse and rat tumor types in several different animal models. NPS induces protective, vaccine-like effects after ablation of orthotopic rat N1-S1 hepatocellular carcinoma. Here we review some general concepts of NPS in the context of studies with mouse metastatic 4T1 mammary cancer showing that the postablation, vaccine-like effect is initiated by dynamic, multilayered immune mechanisms. NPS eliminates primary 4T1 tumors by inducing immunogenic, caspase-independent programmed cell death (PCD). With lower electric fields, like those peripheral to the primary treatment zone, NPS can activate dendritic cells (DCs). The activation of DCs by dead/dying cells leads to increases in memory effector and central memory T-lymphocytes in the blood and spleen. NPS also eliminates immunosuppressive cells in the tumor microenvironment and blood. Finally, NPS treatment of 4T1 breast cancer exhibits an abscopal effect and largely prevents spontaneous metastases to distant organs. NPS with fast rise-fall times and pulse durations near the plasma membrane charging time constant, which exhibits transient, high-frequency components (1/time = Hz), induce responses from mitochondria, endoplasmic reticulum, and nucleus. Such effects may be responsible for release of danger-associated molecular patterns, including ATP, calreticulin, and high mobility group box 1 (HMBG1) from 4T1-Luc cells to induce immunogenic cell death (ICD). This likely leads to immunity and the vaccine-like response. In this way, NPS acts as a unique onco-immunotherapy providing distinct therapeutic advantages showing possible clinical utility for breast cancers as well as for other malignancies.

Nano-pulse stimulation induces potent immune responses, eradicating local breast cancer while reducing distant metastases.

Nano-pulse stimulation (NPS) as a developing technology has been studied for minimally invasive, nonthermal local cancer elimination for more than a decade. Here we show that a single NPS treatment results in complete regression of the poorly immunogenic, metastatic 4T1-Luc mouse mammary carcinoma. Impressively, spontaneous distant organ metastases were largely prevented, even in those animals with incomplete tumor regression. All tumor-free mice were protected from secondary tumor cell challenge, demonstrating a vaccine-like effect. NPS treatment induced antitumor immunity, long-term memory T cells, destruction of tumor microenvironment and reversal of the massive increase of immune suppressor cells in the tumor microenvironment and blood. NPS-treated 4T1 cells exhibited release of damage-associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, and activated dendritic cells. Those findings suggest that NPS is a potent immunogenic cell death inducer that elicits antitumor immunity to prevent distant metastases in addition to local tumor eradication.

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. CONTROLS: The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.