Beneficial effects of recombinant CER-001 high-density lipoprotein infusion in sepsis: results from a bench to bedside translational research project

Abstract

Background: Sepsis is characterized by a dysregulated immune response and metabolic alterations, including decreased high-density lipoprotein cholesterol (HDL-C) levels. HDL exhibits beneficial properties, such as lipopolysaccharides (LPS) scavenging, exerting anti-inflammatory effects and providing endothelial protection. We investigated the effects of CER-001, an engineered HDL-mimetic, in a swine model of LPS-induced acute kidney injury (AKI) and a Phase 2a clinical trial, aiming to better understand its molecular basis in systemic inflammation and renal function.

Methods: We carried out a translational approach to study the effects of HDL administration on sepsis. Sterile systemic inflammation was induced in pigs by LPS infusion. Animals were randomized into LPS (n = 6), CER20 (single dose of CER-001 20 mg/kg; n = 6), and CER20 × 2 (two doses of CER-001 20 mg/kg; n = 6) groups. Survival rate, endothelial dysfunction biomarkers, pro-inflammatory mediators, LPS, and apolipoprotein A-I (ApoA-I) levels were assessed. Renal and liver histology and biochemistry were analyzed. Subsequently, we performed an open-label, randomized, dose-ranging (Phase 2a) study included 20 patients with sepsis due to intra-abdominal infection or urosepsis, randomized into Group A (conventional treatment, n = 5), Group B (CER-001 5 mg/kg BID, n = 5), Group C (CER-001 10 mg/kg BID, n = 5), and Group D (CER-001 20 mg/kg BID, n = 5). Primary outcomes were safety and efficacy in preventing AKI onset and severity; secondary outcomes include changes in inflammatory and endothelial dysfunction markers.

Results: CER-001 increased median survival, reduced inflammatory mediators, complement activation, and endothelial dysfunction in endotoxemic pigs. It enhanced LPS elimination through the bile and preserved liver and renal parenchyma. In the clinical study, CER-001 was well-tolerated with no serious adverse events related to study treatment. Rapid ApoA-I normalization was associated with enhanced LPS removal and immunomodulation with improvement of clinical outcomes, independently of the type and gravity of the sepsis. CER-001-treated patients had reduced risk for the onset and progression to severe AKI (stage 2 or 3) and, in a subset of critically ill patients, a reduced need for organ support and shorter ICU length of stay.

Conclusions: CER-001 shows promise as a therapeutic strategy for sepsis management, improving outcomes and mitigating inflammation and organ damage.

Trial registration: The study was approved by the Agenzia Italiana del Farmaco (AIFA) and by the Local Ethic Committee (N° EUDRACT 2020-004202-60, Protocol CER-001- SEP_AKI_01) and was added to the EU Clinical Trials Register on January 13, 2021.

Keywords: ApoA-I complexes; Cytokine storm; Multi-organ dysfunction; Sepsis.

Fig. 1

Improvement of survival rate and decrease of systemic pro-inflammatory response and endothelial dysfunction in a swine model of LPS-induced AKI. All animals received an infusion of LPS 300 µg/kg (T0) without (control group) or with infusion of CER-001 at 20 mg/kg (CER20 group) followed for half of this group by a second infusion at 3 h of 20 mg/kg of CER-001 (CER20 × 2 group). At T24, surviving animals were sacrificed (gray band). A Survival curve of pigs upon challenge with LPS and CER-001 infusions. A significant statistical treatment trend was observed by log-rank, in the three groups (p = 0.0265 log-rank trend test, n = 6). B–D Serum levels of TNF-α, MCP-1, and IL-6 were measured by ELISA assay (n = 6 independent samples per time point and group). E–G Systemic complement activation was measured by Wieslab assay (n = 6 independent samples per time point and group). H, I Serum levels of VCAM-1 and ICAM-1 were measured by ELISA assay (n = 6 independent samples per time point and group). In each graph, the gray bands show two infusions (0–1 h and 3–4 h) of saline or CER-001 with flow rates of 250 ml/h. Results are presented as mean ± SEM. Significant differences were assessed using a two-way ANOVA for repeated measures with Tukey correction for multiple comparisons (*p < 0.05 **p < 0.005, ***p < 0.0005 vs the LPS group; §p < 0.05; §§p < 0.005, §§§p < 0.0005 vs the CER20 group). Details of differences between Tukey correction and uncorrected Fisher’s LSD for significant comparisons are listed in Additional File 1: Table S1

Fig. 2

Improvement of liver dysfunction following ApoA-I-complexes (CER-001) infusions. A Top panel shows representative H&E staining of hepatic tissue of LPS and treated groups (n = 6 independent pigs per group). Liver sections (upper panel, scale bar = 400 μm; middle panel, scale bar = 200 μm; lower panel, scale bar = 80 μm). Hepatic injury was defined as the amount of destruction of hepatic lobules (black arrow) and infiltration of inflammatory cells (black dashed arrow). B Liver injury was calculated from whole images of stained liver, as calculated from five randomly selected fields per sample (n = 6 independent pigs per group) and scored from 1 through 4 according to % area of involvement per HPF. Results are presented as mean ± SEM. Significant differences were assessed by one-way ANOVA with Tukey correction (*p < 0.05, **p < 0.005, ***p < 0.0005). C Serum levels of ALT enzyme were measured by ELISA assay (n = 6 independent samples per time point and group). The gray bands show two infusions (0–1 h and 3–4 h) of saline or CER-001 with flow rates 250 ml/h. Results are presented as mean ± SEM. Significant differences were assessed using a two-way ANOVA for repeated measures with Tukey correction (n.s.: p > 0.05, *p < 0.05, **p < 0.005, ***p < 0.0005 vs the LPS group; §p < 0.05; §§p < 0.005, §§§p < 0.0005 vs the CER20 group). Details of differences between corrected and uncorrected comparisons are listed in Additional File 1: Table S1

Fig. 3

Improvement of renal dysfunction following ApoA-I-complexes (CER-001) infusions. A–F sCr, sCystatin C, sKIM-1, UCr, U-Cys, and U-KIM-1 levels were measured by ELISA assay (n = 6 independent samples per time point and group); urinary output (ml/kg/h) was recorded for each animal. The gray bands show two infusions (0–1 h and 3–4 h) of saline or CER-001 with flow rates 250 ml/h. Results are presented as mean ± SEM. Significant differences were assessed using a two-way ANOVA for repeated measures with Tukey correction (n.s.: p > 0.05, *p < 0.05, **p < 0.005, ***p < 0.0005 vs the LPS group; §p < 0.05; §§p < 0.005, §§§p < 0.0005 vs the CER20 group). G, H & E staining was performed on paraffin kidney sections. Renal sections (upper panel, scale bar = 400 μm; middle panel, scale bar = 200 μm; lower panel, scale bar = 80 μm). The kidney desquamation and vacuolization of proximal tubular epithelial cells (zoomed image, 80 µm, black dashed arrow), fibrin deposition and loss of capillaries in numerous glomeruli, Bowman’s capsule expansion (zoomed image, 80 μm, black arrow), and interstitial inflammatory infiltrate (zoomed image, 80 μm, blue arrow) were observed by H&E staining. H, I Tubular and glomerular pathological score was obtained as described in the “Methods” section (n = 6 for each group). Results are presented as mean ± SEM. Significant differences were assessed by one-way ANOVA with Tukey correction (*p < 0.05 **p < 0.005, ***p < 0.0005). Details of differences between corrected and uncorrected comparisons are listed in Additional File 1: Table S1

Fig. 4

Excretion of LPS into the bile via CER-001 transport. A Serum levels of LPS were measured by ELISA assay (n = 6 independent samples per time point and group). B Representative western blot and densitometric analysis of LPS and β-actin protein expression (n = 6 per group). C Endotoxin levels in bile samples were dosed by ELISA assay. D Human ApoA-I levels in bile samples were evaluated by ELISA assay. E The serum human ApoA-I for CER-001 was determined at different time points by ELISA assay. The gray bands show two infusions (0–1 h and 3–4 h) of saline or CER-001 with flow rates 250 ml/h. The data are presented as the mean ± SEM. Statistically significant differences were assessed by one-way ANOVA with Tukey correction (*p < 0.05, **p < 0.005, ***p < 0.0005 vs the LPS group; §p < 0.05; §§p < 0.005, §§§p < 0.0005 vs the CER20 group)

Fig. 5

Effect of CER-001 on ApoA-I level, LPS removal, and inflammatory response in the pilot study RACERS. A–I Serum levels of ApoA-I, LPS, IL-6, IL-8, MCP1, TNF-α, sTREM-1, VCAM, and ICAM were measured using ELISA kits. Data for panels A2–I are represented as changes from baseline (day 1 pre-dose) values. Treatment differences were assessed by using a mixed model ANOVA for values on days 3, 6, and 9 with a significance level of α = 0.05; significant pairwise comparisons are shown. No adjustments were made for multiple comparisons. Abbreviations: ApoA-I apolipoprotein A-I; ICAM endothelial intercellular adhesion molecule; IL-6 interleukin 6; IL-8 interleukin 8; IL-10 interleukin 10; LPS lipopolysaccharides; MCP-1 monocyte chemoattractant protein-1; TNF-α tumor necrosis factor α; sTREM-1 soluble triggering receptor expressed on myeloid cells 1; VCAM vascular cell adhesion molecule

Fig. 6

Effect of CER-001 on organ systems and survival. A Evolution of AKI among patients randomized to CER-001, according to KDIGO criteria from baseline (day 1 pre-dose) to day 6. B AKI stages among patients randomized to the Standard of Care group, according to KDIGO criteria from baseline (day 1 pre-dose) to day 6. C–F Serum levels of AST, ALT and albumin measured by the local hospital lab. Data are represented as changes from baseline (day 1 pre-dose) values. Treatment differences were assessed by using a mixed model ANOVA for values on days 3, 6, and 9 with a significance level of α = 0.05; significant pairwise comparisons are shown. No adjustments were made for multiple comparisons. G, H 30-day survival curves for all patients and for ICU patients. Treatment differences were assessed using the log-rank test with a significance level of α = 0.05. Abbreviations: AKI acute kidney injury; ALT Alanine aminotransferase; AST Aspartate aminotransferase