Diethyl Azelate for the Treatment of Brown Recluse Spider Bite, a Neglected Orphan Indication

Abstract

Background/aim: Brown recluse spider bite releases hemolytic and cytotoxic phospholipase D to the wound that may cause necrosis or even death. We examined diethyl azelate (DEA), a plasma membrane fluidizer with a broad range of immunomodulatory activities, as a potential treatment for the brown recluse spider bite.

Materials and methods: Topical DEA was used in emergency to treat brown recluse spider bites in a human subject. We subsequently evaluated the effects of DEA on hemolysis induced by the brown recluse spider venom, recluse recombinant phospholipase D (rPLD), and venoms from honey bee and moccasin snake, and on phospholipase A2 activity in the bee and snake venoms and in human urine.

Results: Topical DEA resolved the consequences of human brown recluse spider envenomation in two weeks. In vitro, DEA inhibited hemolysis caused by the brown recluse spider venom and rPLD and suppressed phospholipase A2 activity in a dose-dependent manner.

Conclusion: DEA is a promising novel therapy for the brown recluse spider bite and perhaps even unrelated envenomations involving PLDs.

Keywords: Azelate; brown recluse; fatty acid ester; spider bite.

Figure 1. Time-dependent resolution of a brown recluse spider bite in the right forearm by topical DEA. The pictures obtained between 24 h (Day1) and 156 h (Day 7) are shown as color images (left column) and inverted color images (right column). The site of the spider bite is marked with an arrow.

Figure 2. Time-dependent resolution of a brown recluse spider bite in the neck by topical DEA. A) Magnified image of the neck area with two bites on Day 1 (D1) hour 1 (H1). Arrows point out to the bite sites. B) Time course images of the bite area and time-dependent resolution of a brown recluse spider bite over a treatment course with topical DEA. C) Quantitative analysis of the affected bite area. A bar chart shows relative size of the arm area at the bite site over the treatment with topical DEA for 14 days. The x-axis shows the elapsed time since the envenomation event in days and hours as applicable.

Figure 3. DEA inhibits hemolysis induced by brown recluse spider venom and recombinant phospholipase D. A) DEA dose effect on hemolysis induced by rPLD or brown recluse spider venom (LOX) compared to Triton X-100 control. Insets show the same data plotted in a linear scale of concentrations of DEA or Triton to underscore differences in the dose responses. Dotted line: hemolysis in negative control (PBS). B) The order of reagents added to the hemolysis assay reaction mixture affects the extent of hemolysis. Dotted line: hemolysis in negative control (PBS).

Figure 4. Effects of DEA and related azelates on hemolysis and enzymatic activity of secretory phospholipase A2 activity in bee venom. Hemolysis induced by bee venom in human erythrocytes was measured in the presence DEA and related azelates (all at 5%). DMA: Dimethyl azelate; DiPA: di-isopropyl azelate; DiBU: di-isobutyl azelate; D1PA: di-(1-pentyl) azelate; D3PA: di-(3-pentyl) azelate; DCHA: dicyclohexyl azelate. The extent of hemolysis measured in the presence of DEA was defined as 100%.

Figure 5. Effects of DEA on enzymatic activity of secreted phospholipase A2 activity in bee and snake venoms. A): Initial velocities of sPLA2 in snake venom in the presence of DEA measured as relative absorbance at 400 nm. Solid line; untreated control. Dashed lines of increasing density correspond to 2, 10, and 50% DEA. B) initial velocities of sPLA2 activity in snake venom are plotted against DEA concentration. C) Initial velocities of sPLA2 in bee venom in the presence of DEA measured as relative absorbance at 400 nm. Solid line; untreated control. Dashed lines of increasing density starting from the top correspond to 2, 10, and 50% DEA. D) initial velocities of sPLA2 activity in bee venom are plotted against DEA concentration.