Figure 1. Molecular-modeling derived geometry of the complex between LPS and EVK-203. The atomic coordinates of LPS were derived from its crystal structure (PDB code: 1FCP).⁸² The lipid A moiety of LPS is depicted in ball-and-stick, the KDO sugars in spacefill, and the inner core glycolipid region as sticks. The mono-homologated spermine backbone is predicted to form salt-bridges (dotted lines) with both phosphate groups on lipid A, as well as participate in additional ionic H-bonds with the inner core KDO sugars as determined by docking studies.

Scheme 1. Reagents and conditions: (a) A1C1₃, py, reflux; (b) MgSO₄, THF–MeOH, rt, followed by, NaBH₄, MeOH; (c) CF₃CO₂H (excess), rt.

Scheme 2. Reagents and conditions: (a) 10% Pd(OH)₂–C, H₂, MeOH, rt; (b) CF₃CO₂H (excess), rt.

Scheme 3. Reagents and conditions: (a) (H₃₁C₁₅)₂CO, AcOH (cat.), C₆H₆, reflux, followed by, NaBH₄, MeOH, rt; (b) CF₃CO₂H (excess), rt; (c) H₃₁C₁₅CHO, NaBH₄, MeOH, AcOH, rt; (d) H₃₁C₁₅CH₂I, C₆H₆, reflux.

Scheme 4. Reagents and conditions: (a) Aldehyde 2 (excess), MgSO₄, THF–MeOH, rt, then, NaBH₄, MeOH; (b) CF₃CO₂H, rt.

Scheme 5. Reagents and conditions: (a) (H₃₁C₁₅)₂CO (excess), AcOH (cat.), C₆H₆, reflux, then, NaBH₄, MeOH, rt; (b) CF₃CO₂H, rt.

Figure 2. Comparison of the inhibition profiles of LPS (100 ng/ml)-induced NF-κB induction in HEK-293 cells stably transfected with Tlr4, MD-2, CD14, and an NF-κB-secreted alkaline phosphatase reporter gene construct. The structures of the alkylpolyamines and the corresponding IC₅₀ values are shown.

Figure 3. (a) Hemolytic activity of the alkylpolyamines. A suspension of 1:1000 diluted, washed, human erythrocytes was used. Hemolysis was determined by automated video microscopy. (b) Cytotoxic/cytostatic activity measured by XTT assay.

Figure 4. (a) Binding affinity of EVK-203 and PMB (reference) to LPS determined by BODIPY^®-cadaverine displacement assay. (b) Inhibitory activity of EVK-203 and PMB on nitric oxide production (measured spectrophotometrically as nitrite using Griess assay) in murine J774 macrophage cells stimulated with 100 ng/ml LPS. Shown on the left are negative and positive (LPS alone and medium alone) controls.

Figure 5. (a) Inhibition of phosphorylation of p38 MAP kinase in neutrophils in whole human blood (ex vivo), stimulated with either LPS or hTNF-α (100 ng/ml) for 15 min in the presence of graded concentrations of EVK-203 or PMB. Quantification of p38 MAPK was performed using flow cytometry. Panels (b) and (d) show forward scatter/side scatter profile and the gating for p38 MAPK-negative and -positive gates obtained on unstimulated cells (negative control), respectively. Panels (c) and (e) are corresponding results obtained from LPS stimulated cells (positive control). Back-gating on the p38 MAPK-positive cells (panel e: heavily-shaded peak) maps to the polymorphonuclear population (panel c).

Figure 6. (a)–(c) Dose-dependent inhibition of LPS-induced proinflammatory cytokine production in ex vivo whole human blood. Whole human blood was stimulated with LPS and graded concentrations of either EVK-203 or PMB. Cytokine levels were quantified using a multiplexed flow-cytometric bead array system (CBA).

Figure 7. (a) Comparison of in vivo potency of PMB and EVK-203 in a d-galactosamine-primed murine model of lethal endotoxic shock. Dose-dependent increase in survival in mice challenged with a supralethal (200 ng/animal; 2× LD₁₀₀) dose of LPS. The difference in dose-dependent survival rates between animals receiving PMB and EVK-203 was statistically significant (p < 0.005; Fisher one-tailed exact probability). (b) Time-course (pharmacodynamics) of protection conferred by 8 mg/kg of EVK-203 administered at various times prior to, and following, supralethal LPS challenge in a d-galactosamine-primed murine model of lethal endotoxic shock. LPS (200 ng/animal; 2× LD₁₀₀) was administered at time = 0 h.

Figure 8. Time-course of cytokine profiles in plasma of mice receiving graded doses of EVK-203 and challenged with a lethal dose of LPS. Cohorts of five animals per group were administered graded doses of EVK-203 (or vehicle) subcutaneously at time = −1 h. Following lethal LPS challenge (200 ng/animal, administered intraperitoneally at t = 0 h), blood samples were obtained at t = 1, 2, and 3 h. Plasma samples were processed for cytokine analyses using the CBA assay.