A novel approach for emerging and antibiotic resistant infections: Innate defense regulators as an agnostic therapy

Abstract

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work, further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR, SGX94, has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies, this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells, resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels, thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation, IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections.

Keywords: Anti-infective; Anti-inflammatory; Antibacterial; Broad spectrum; Cefepime (PubChem CID: 9571075); Doxycycline (PubChem CID: 54671203); Dusquetide (PubChem CID: 71722017); Immune; Innate; Vancomycin (PubChem CID: 14969).

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Figure 1. Broad-spectrum activity of SGX94 against bacterial infections

a) Peritonitis: S. aureus infection in CD-1 mice (n=8/group). Control and SGX94 (9.5 mg/kg) were injected IP 4 h later. Bacterial counts were assessed 24 h after infection. Open symbols indicate dead mice. b) Thigh abscess: SGX94 (50 mg/kg) was administered IV at 24 h or 4 h before infection of neutropenic male CD-1 mice with MRSA (right thigh). Vancomycin (30 or 100 mg/kg) was given SC at 2 h and 14 h after infection. CFU counts were determined 26 h post-infection. c) Skin infection: SGX94 (n=6/gp) was administered IV or SC. Oral linezolid (12.5 mg/kg) was administered daily after infection with MRSA in CD-1 mice. A blinded board-certified pathologist scored the digital images. d) Bacteremia: SGX94 (5 mg/kg) or saline was administered IV 4 h or 24 h after infection of BALB/c mice with MRSA via the tail vein (n=10/group). A single dose of Linezolid (6.25 mg/kg) was administered orally after infection (n=10). e) Peritonitis: SGX94 (50 mg/kg) or saline treatment was administered IV 48 h before MRSA infection of female CF-1 mice (N=10/group), followed by vancomycin (3 mg/kg) was administered to all animals SC, 1 h and 5 h after infection. f) Melioidosis: Female BALB/c mice (N=12/group) were infected intranasally with B. pseudomallei. SGX94 (50 mg/kg) or saline was administered IV 4 h prior to infection and every second day to Day 8. Doxycycline (20 mg/kg) was administered orally upon infection and daily through Day 10. MST = median survival time. N=12/group. The combination of SGX94 + doxycycline was found to be more effective than the additive combination of each treatment alone (p<0.0001). g) Sepsis: Female Sprague-Dawley rats were infected with oral P. aeruginosa on Days 0, 2 and 4 (n=8 SGX94, Saline, n=4 Cefepime). With the onset of fever (Day 5), animals received SGX94 (10 mg/kg IV), Cefepime (40 mg/kg IM on Days 6-8) or saline (IM on Days 6-8). h) Bacteremia: SGX94 (50 mg/kg) or saline was administered IV 4 h prior to infection of BALB/c mice with MRSA via the tail vein (n=10/group). The animals were monitored for 21 days post-infection.

Figure 2. Prophylactic and persistent impacts of SGX94 on MRSA bacteremia

a) SGX94 (50 mg/kg) or saline (n=10/group) was administered IV to female BALB/c mice 5 days prior to infection via the tail vein with MRSA (USA300, 1.9 × 107 CFU). The statistical significance of differences in survival were assessed using Kaplan Meier analysis. b) SGX94 (5 mg/kg) or saline (n=10/group) was administered IV at the indicated times to female BALB/c mice prior to or after infection via the tail vein with MRSA (USA300, 2 × 107 CFU). Sub-optimal antibiotic treatment (linezolid, 6.25 mg/kg) was administered orally immediately after infection. c) SGX94 (IV) or saline (IV) was administered once 4h prior to infection with MRSA (strain USA300, 1.1 × 107 CFU) via the tail vein into female nu/nu mice. Statistically significant (p≤0.05) increases in survival were found with the 50 mg/kg dose level relative to the saline control, as assessed using Kaplan Meier analysis.

Figure 3. Cytokines and cell migration during S. aureus peritoneal infection

a) SGX94 (30 mg/kg) was administered IP to female CD-1 mice (n=8/group). 24 h later an inoculum of S. aureus (Catalog No. 25923, ATCC, ~0.5-1 × 108 CFU) with 5% mucin was injected IP. Mice were sacrificed 3 h after infection and peritoneal lavage fluid was assessed for cytokine levels. b) Female CD-1 mice were injected IP with S. aureus (Catalog No. 25923, ATCC, 8.4 × 107 CFU) with 5% mucin. Saline (n=8/group) or SGX94 (15 mg/kg; n=12/group) was administered IP or IV 4h after infection. Mice were sacrificed 24 h after infection and peritoneal lavage fluid was assessed for macrophage and neutrophil numbers. Only 1/8 animals survived in the control saline IV group, whereas 5/12 and 9/12 animals survived in the SGX94-treated IV and IP groups, respectively. Each data point represents a single animal and the bars represent average cell counts for each treatment group. c) SGX94 (30 mg/kg) was administered IP to female CD-1 mice (n=8/group) 24 h prior to an IP inoculation with a low dose of S. aureus (Catalog No. 25923, ATCC, 1.8 × 106 CFU) with 5% mucin. Mice were sacrificed 24 h after infection and peritoneal lavage fluid was assessed for TNFα levels. A t-test was performed comparing the SGX94 and saline groups, assuming that any data point with a value less than the detection limit of the assay (dotted line) was 0.

Figure 4. Splenocyte supernatant transfer study

a) Schematic of study design b and c) BALB/c (b) and C57BL/6 (c) splenocytes were pre-incubated with control (black circles) or SGX94 (blue square), 200 μM, as described in the methods and the supernatant medium was applied to the RAW264.7 cells. M1 through M5 indicate splenocyte cultures from individual mice. Purple symbols and lines on the graphs indicate the “input” levels of the cytokines from the splenocyte conditioned media that was applied to the RAW cells. Each symbol represents one well of the RAW cell culture. d) C57BL/6 splenocytes were pre-incubated with each IDR, 200μM, as described in the methods and the pooled supernatant medium was applied to the RAW264.7 cells.

Figure 5. Consistent cytokine profiles across species

a) In a Phase 1 study in healthy human volunteers blood samples were obtained 1 h post-treatment from the placebo (n=22) and SGX94-treated populations (Low dose: 0.15-2.0 mg/kg, n=26; High dose: 3.0-8 mg/kg, n=30). Blood samples were immediately incubated with LPS (E. coli) for 4 h and analyzed for cytokine/chemokine responses. Placebo group data and combined data from low dose groups of the SAD and MAD data sets are represented in Figure 5a as the ratios of IL-1ra/IL-1β and of TNF RII / TNFα responses, respectively. Solid lines with symbols in Figure 5a indicate the mean responses of the placebo (black triangles) and low dose (blue circles) populations, dashed black lines and solid blue lines without symbols indicate the respective 95% Confidence Intervals (CI) for the placebo and low dose populations, respectively. The 95% CI for the SGX94 treated population is also shaded to emphasize areas of minimal overlap between the 2 populations. Boxes indicate non- or minimally-overlapping 95% CI between the 2 populations. b) SGX94 was administered to one male (1001A) and one female (1501A) cynomolgus monkey at the indicated doses and blood samples were collected up to 24 h post-dose. The levels of IL-1ra in the plasma are shown. c) 50 mg/kg SGX94 or saline was administered IV to female BALB/c mice (n=10/group). One hour later, 500 ng of LPS was administered intranasally in a volume of 30 μL. Four hours after LPS administration, bronchoalveolar lavage was collected. Statistical significance was assessed using a one-sided t-test.