The Role of IL-13, IL-15 and Granulysin in the Pathogenesis of Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis.

Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) are Severe Cutaneous Adverse Reactions (SCARS) characterized by fever and mucocutaneous lesions leading to necrosis and sloughing of the epidermis. Conjunctival lesions are reported in 85% of patients. The pathogenesis of SJS/TEN/SCARS is not completely understood. It is hypothesized that IL-13, IL-15 and Granulysin expressed in plasma and skin may play a role. We measured the circulating levels of these cytokines in the plasma using ELISA and their expression in the skin using immunofluorescence microscopy. A total of 12 SJS/TEN skin biopsy samples (8 SJS, 2 SJS/TEN overlap and 2 TEN) were analyzed. Biopsy samples from patients with Lichen Planus (an inflammatory condition of the skin and mucous membranes) served as controls. Studies were also performed in human corneal epithelial cells where expression of these cytokines were measured following a challenge with TNF-α (0, 1, 10 and 100 ng/ml). The intensity of immunofluorescence was measured Using Imaris® software. The results showed significantly increased expression of these cytokines in the skin biopsy samples as measured by the average intensities of IL-13 (6.1 x 133.0 ± 4.231 x 10^8), and Granulysin (4.2 x 123.0 ± 4.231 x 10^8) compared to Lichen planus control (3.0 x 123.0 ±1.62 x 10^5). Increased expression of IL-13 and IL-15 were noted in cell culture studies and in the plasma samples when compared to Normal Human Plasma as controls. It is concluded that IL-13, IL-15 and Granulysin play a role in the pathogenesis of SJS/TEN.

Comparative Anticoagulant and Thrombin Generation Inhibitory Profile of Heparin, Sulodexide and Its Components.

INTRODUCTION: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. MATERIALS AND METHOD: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. RESULT: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. CONCLUSION: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.

Bovine Mucosal Heparins Are Comparable to Porcine Mucosal Heparin at USP Potency Adjusted Levels.

Introduction: Bovine mucosal heparins (BMH) are currently being developed for re-introduction for both medical and surgical indications. BMH active pharmaceutical ingredient (API) exhibits a somewhat weaker USP potency when compared to PMHs. We hypothesized that when dosages are normalized based on the USP reference heparin, BMH will exhibit comparable in vitro and in vivo effects to those produced by PMH. Therefore, studies were developed to compare the APIs of bovine and porcine heparin. Materials and Methods: API versions of PMH were obtained from Celsus Laboratories (Franklin, OH) and Medefil (Glen Ellen, IL). API versions of BMH were obtained from Kin Master (Passo Fundo, Brazil). Each of these heparins was assayed for their molecular weight profile, AT affinity, USP potency, and anticoagulant/antiprotease profiles using standard laboratory methods. In vitro protamine neutralization studies were carried out. Antithrombotic and hemorrhagic effects were measured in rats and pharmacodynamic profiles were assessed in primates. Results: Size exclusion chromatography demonstrated that the mean molecular weight of BMH was ~15% higher than that of PMH (BMH: 20.1 ± 0.8 kDa and PMH: 17.5 ± 0.7 kDa). BMH exhibited an anti-Xa potency of 130 U/mg while PMH had an anti-Xa potency of 185 U/mg. In the anticoagulant and antiprotease assays, the BMH exhibited lower functionality which was proportional to USP potency. When the BMH was compared with PMH at potency adjusted concentrations, it showed identical concentration-response curves in the aPTT and anti-protease assays. However, in the protamine neutralization studies, BMH required slightly higher amounts of protamine in contrast to PMH. BMH and PMH administered to rats at equivalent anti-Xa unit dosages resulted in comparable antithrombotic activity and prolongation of bleeding time. Similar pharmacodynamic profiles were observed in primates when BMH and PMH were dosed on an anti-Xa U/kg basis. Conclusion: BMH, when used at comparable anti-Xa unit levels, is comparable to PMH, however, it requires proportionally higher amount of protamine due to the increased mass for adjusting to higher potency. Additional studies on the structural characterization, interactions with PF4 and in vivo neutralization studies are ongoing.