SAGEs can vary in molecular size, and in extent of alkylation and sulfation. GM-1111 is a low-molecular weight SAGE with an average molecular weight of 5.5 kDa.

Data points in each figure represent the average value ± standard deviation of quadruplicate wells for each concentration of SAGE. A. SAGEs inhibit P-selectin. Inhibition of P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin by SAGEs was studied using calcein-labeled U937 cells incubated in microwells coated with P-selectin. After 1 h, plates were washed, bound cells were lysed with Triton-X100 buffer and bound cells were quantified using an excitation of 494 nm and emission of 517 nm. GM-1111 inhibits PSGL-1 attachment to P-selectin with an IC₅₀ of 25 nM. B. SAGEs inhibit HLE. HLE (100 nM) was incubated with GM-1111 at 1–100 nm concentrations in 0.5 M HEPES buffer for 15 min. Following incubation, the elastase substrate, Suc-Ala-Ala-Val-pNA was added to the reaction mixture to the final concentration of 0.3 mM. Absorbance due to p-NA hydrolysis was monitored for 15 min at absorbance of 405 nm. GM-1111 inhibits HLE with an IC₅₀ of 45 nM. C–E. SAGEs inhibit interaction of the AGE product CML-BSA, the calgranulin S100b and the alarmin HMGB-1 with RAGE. Microwell plates coated with CML-BSA (C), S100b calgranulin (D) or HMGB-1 (E) were incubated with RAGE-Fc chimera with or without GM-1111 for 2 h. Plates were washed, incubated with anti-RAGE antibody, incubated for 1 h, washed again four times and incubated with horse-radish peroxidase conjugated secondary for 1 h. A colorimetric reaction was produced by addition of tetramethyl benzidine chromogen (TMB) and quantified by absorbance at 450 nm. GM-1111 inhibits interaction of RAGE with CM-BSA, S100b and HMGB-1 with IC₅₀ values of 413, 275 and 80 nM, respectively. F. SAGEs inhibit function of RAGE as an adhesion ligand. Inhibition of Mac-1-dependent ligation of RAGE by U937 cells was studied as in A, but using microwells coated with RAGE. GM-1111 inhibits Mac-1 attachment to RAGE with an IC₅₀ of 7.6 nM.

Balb/c mice were shaved to expose an area of skin on the back. Twenty-four hours later, mice were intradermally injected with 40 µl of vehicle (nanopure water), LL-37 (at 320 µM concentration in water), SAGE GM-1111 (1,280 µM in water), or LL-37+ GM-1111 mix (peptide +4 molar equivalents of SAGE) placed intradermally into the shaved skin using a 31-gauge needle and 0.5 ml insulin syringe. Injections were repeated every 12 h thereafter. Forty-eight hours after the initial injection (four injections in total), animals were lightly anesthetized with isoflurane, the area of injected skin was photographed, the intensity of erythema was assessed as a redness score (from 1 to 5, with 5 as the most red), and the area of erythema was measured with micrometer calipers. The injected skin was then biopsied for histopathologic staining and to assess PMN infiltration through measurement of myeloperoxidase (MPO) activity. A. Gross picture of LL-37 injected skin region. B. Co-injection model of LL-37 and SAGE GM-1111. C. H&E-stained cross-sectional view of a LL-37 injected skin sample. D. H&E-stained cross-sectional view of a LL-37 mixed with GM-1111- injected skin region. E. MPO activity measurement of LL-37 injection with different treatments. F. Area of erythema from LL-37 injection. *P<0.05 vs intradermal LL-37 alone; n = 6 per group.

Balb/c mice were injected intradermally as described in Figure 5, and treated topically with GM-1111-1 (1% w/w) in a triglyceride-based emollient. A–C. Gross pictures of LL-37 injected skin region: A, no treatment; B, GM-1111 treatment immediately (t = 0) after LL-37 injection; C, GM-1111 treatment beginning at t = 12 h after LL-37 injection. D. H&E-stained cross-sectional view of a LL-37 injected skin sample. E. H&E-stained cross-sectional view of GM-1111 treatment at t = 0 in LL-37 injected skin region. F. H&E-stained cross-sectional view of GM-1111 at t = 12 h treatment in LL-37 injected skin region. G. MPO activity measurement of LL-37 injection with different treatments. H. Area of erythema from LL-37 injection. *P<0.05 vs intradermal LL-37 alone; n = 6 per group.

Balb/c mice were injected intradermally with LL-37 every 12 h for 48 h as in Figure 7. Control emollient [3% (w/w) methylcellulose] or emollient containing Alexa Fluor 633-labeled GM-1111 was applied topically to the injected skin after the last two LL-37 injections. Under low light conditions, skin was harvested 12 h after the last emollient application, fixed in paraformaldehyde and embedded in paraffin. Slides were deparaffinized with xylene, stained with DAPI (1∶100 dilution in fluorescent mounting medium) and imaged using an FV1000 confocal microscope. The skin surface is oriented at the top of each image. A. Skin from controls demonstrates blue DAPI (λ_ex = 358 nm; λ_em = 461 nm) image but no SAGE fluorescence. B. Skin treated with Alexa Fluor 633-labeled GM-1111 demonstrates fluorescence (λ_ex = 633 nm; λ_em = 647 nm; false colored green in the image) consistent with SAGE penetration into LL-37-inflamed skin.

GM-1111 was studied to determine binding affinity for P-selectin (A), Mac-1 (B), and RAGE (C). Binding affinity (K_D) values were 0.0036 nM for GM-1111 binding to P-selectin, 0.175 nM for GM-1111 binding to Mac-1 and 1.69 nM for GM-1111 binding to RAGE.

A. SAGE binds LL-37. Microwell plates coated with GM-111 were incubated with LL-37 at 37°C for 2 h. Plates were washed, incubated with anti-LL-37 antibody, incubated for 1 h, washed again four times and incubated with peroxidase-conjugated secondary antibody for 1 h. A colorimetric reaction was produced by addition of TMB and quantified by absorbance at 450 nm. LL-37 binds to GM-1111 with a K_D of 0.225 nM. B. SAGE inhibits IL-8 production LL-37 stimulated keratinocytes. Human keratinocytes were grown to confluence and treated with 3.2 µM LL-37 or LL-37 and a 4x molar excess of GM-111101 for 6 h. Supernatants were collected and placed in a sterile 96-well plate. Production of IL-8 was determined by ELISA (R&D Systems, Minneapolis, MN) in accordance with manufacturer's instructions. Co-addition of GM-1111 significantly inhibited IL-8 release into medium. *P<0.01 as compared with negative control group without LL37; **P<0.05 as compared with positive control group without GM-111101 treatment.

LL-37, PBS and LL-37 and SAGE-injected mouse skin was stained with antibody to mouse Gr-1 to examine neutrophil infiltration, and also for S100A8 calgranulin, the major RAGE-binding calgranulin present in murine PMNs [17] as described in Methods. A. Staining of normal control skin for the PMN antigen Gr-1. B. Staining of LL-37 injected skin for the PMN antigen Gr-1. C. Staining of skin injected with LL-37 plus GM-1111 (applied at t = 0) for the PMN antigen Gr-1. D. Staining of skin injected with LL-37 plus GM-1111 (applied at t = 12 h) for the PMN antigen Gr-1. E. Staining of normal control skin for the RAGE ligand S100A8. F. Staining of LL-37 injected skin for the RAGE ligand S100A8. G. Staining of skin injected with LL-37 plus GM-1111 (applied at t = 0) for the RAGE ligand S100A8. H. Staining of skin injected with LL-37 plus GM-1111 (applied at t = 12 h) for the RAGE ligand S100A8. Figures and insets are shown at 20x and 40x magnification, respectively.

Experiments were performed as in Figure 7, but with immediate (t = 0) application of 1% w/w HA-containing emollient (53 kDa HA) or 1% w/w GM-1111 emollient. *P<0.05 vs LL-37 alone. A. H&E-stained cross-sectional view of LL-37 injected skin. B. H&E-stained section of LL-37 injected skin treated with topical HA emollient alone. C. H&E-stained section of LL-37 injected skin treated with topical emollient containing 1% SAGE GM-1111. D. MPO activity measurement of LL-37 injection with different treatments. E. Area of erythema from LL-37 injection. Erythema scores after LL-37 injection were: 5.1±0.1 after LL-37 alone; 4.9±0.44 after LL-37+ HA; 2.3±0.7 after LL-37+ SAGE (P<0.05 LL-37 alone vs LL-37+ SAGE). *P<0.05 vs intradermal LL-37 alone; n = 6 per group.

GM-1111 (1%) was applied topically in the croton oil model. A. Normal control right ear (top) and croton oil painted left ear (bottom). B. H&E section of control normal ear. C. H&E-stained cross-sectional view of a croton oil painted ear with vehicle treatment. D. H&E-stained cross-sectional view of croton oil painted ear with SAGE treatment. E. MPO activity measurement in ears. F. Ear thickness differences between left (croton oil) and right (normal) ears. G. Redness difference between right and left ears. *P<0.05 vs croton oil with vehicle emollient alone (negative control).