Sepsis is an inflammatory syndrome, in which a dysregulated host response to an infection results in life-threatening organ dysfunction (1). It is a frequent reason for admission to the intensive care unit (2), and despite many advances in medical care, its incidence is increasing (3) and mortality remains high (4). The current treatment of sepsis consists of source control, antimicrobial therapy, and supportive treatment such as fluid resuscitation, vasopressor use, and mechanical ventilation (5). Currently, no adjunctive pharmacological therapies are used in clinical practice (6). Hemodynamic instability plays an important role in the development of septic shock, and arises due to a combination of sepsis-induced vasodilation and vascular leakage, the latter of which is caused by disrupted endothelial integrity. Extensive changes occur in the endothelium as a result of circulating damage-associated molecular patterns and pathogen-associated molecular patterns that activate inflammatory and coagulation pathways during sepsis. In turn, this can result in increased leukocyte adhesion, a procoagulant state, vasodilation and endothelial cell permeability, and ultimately widespread edema, shock and lethal organ dysfunction (5, 7, 8).
Adrenomedullin (ADM) appears to play an important role in the regulation of the endothelial barrier function and modulation of vascular tone. ADM is a free circulating peptide synthesized by several cell-types, including vascular endothelial and vascular smooth muscle cells (9, 10). ADM signals through binding to the AM1- and AM2-receptors, which are composed of a calcitonin-receptor-like receptor (CLR), and receptor activity modifying proteins (RAMP2 or RAMP3 for AM1 and AM2, respectively) (11). Results on the role of ADM in sepsis are ambiguous. During sepsis, ADM levels are correlated with relaxation of vascular tone (12) as well as with disease severity and mortality in septic patients (13–15). In vitro and in vivo data demonstrate that ADM exerts beneficial effects on the endothelial barrier, as it prevents endothelial hyperpermeability and subsequent edema formation by inhibition of actin-myosin-based endothelial cell contraction and junctional disruption (16–19). Furthermore, ADM administration was shown to improve gut microcirculation in models of inflammation (17, 20). However, other work revealed that ADM promotes vasodilation, and infusion of high doses of ADM decreases blood pressure and induces a compensatory increased heart rate in rats, cats, sheep, and humans (21–25). These data suggest that the ADM response needs to be tightly regulated, maintaining adequate, but avoiding excessive signaling.
Recently, a high-affinity antibody targeting the N-terminus of ADM (HAM1101), which only partially inhibits ADM signaling, showed beneficial effects on outcome in two cecal ligation and puncture (CLP) studies (26, 27). The observation that the antibody that only partially inhibits ADM exerts more benefit than the antibody that completely blocks ADM (26), supports the paradigm that a moderate ADM response is needed. Subsequently, a humanized version of this antibody (HAM8101, also known as Adrecizumab) has been developed for clinical use. In the present study, we investigated the effects of HAM8101 on vascular barrier dysfunction in a rat model of systemic inflammation induced by endotoxin administration as well as in the more clinically relevant model of sepsis induced by cecal ligation and puncture in mice (CLP) (28). Furthermore, we compared the effects of HAM8101 and HAM1101 on survival in the murine CLP model.
Animal experiments were performed according to the guidelines of the Federation of Laboratory Animal Science Associations (FELASA) and the society of Laboratory Animal Science (GV-SOLAS). All animals were obtained from Charles River Laboratories, Sulzfeld, Germany. Male Wistar rats were used for the lipopolysaccharide (LPS) vascular permeability study (n = 48, aged 2–4 months) performed by Preclinics (Potsdam, Germany). The CLP kidney barrier dysfunction study was performed by Phenos GmbH (Hannover, Germany), and male mus musculus C57BL/6 mice (n = 24, 12–15 weeks old) were used. For the CLP survival experiments (also performed by Phenos GmbH), 12 to 15-week-old male mus musculus C57BL/6 mice were used (n = 30 for single and n = 30 for repeated dose experiments respectively. These were separate experiments, using different batches per survival experiment). To determine the robustness of HAM8101 during sepsis/inflammation, its efficacy was investigated in different animal species, using different sepsis models and end-points. Animals were housed under routine laboratory conditions, kept under 12 h/12 h light–dark cycle conditions and fed ad libitum with standard chow (Ssniff R/M-H diet) with unlimited access to water. Substance administration, surgery, and blood withdrawal were performed under isoflurane anesthesia to avoid stress and pain in animals. This study is reported according to the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines.
Generation of the murine monoclonal anti-Adrenomedullin antibody HAM1101 has been described previously (26, 27). From this murine monoclonal antibody a humanized recombinant monoclonal antibody (HAM8101;IgG1) was generated by CDR grafting, and it was produced in Chinese hamster ovary cells. HAM8101 was produced by Glycotope Biotechnology GmbH (Heidelberg, Germany) under Good Manufacturing Practice (GMP) conditions, and this antibody is also known as Adrecizumab; HAM1101 was produced by InVivo GmbH (Hennigsdorf, Germany). The antibodies are directed against the N-terminus of ADM. The antibodies were kindly provided by Adrenomed AG (Hennigsdorf, Germany) in stock solutions in PBS and were stored at 2°C to 8°C under temperature controlled and restricted access conditions.
LPS-induced vascular permeability study procedures
Rats (n = 48 total) were randomly divided into six groups (n = 8 each), of which one group received placebo twice (saline and PBS [control]), whereas the other five groups received 5 mg/kg bodyweight LPS (E coli 055:B5; Sigma-Aldrich, Taufkirchen, Germany) in combination with different dosages of HAM8101 (Adrecizumab, 0.02 mg/kg, 0.1 mg/kg, 0.5 mg/kg, or 2.5 mg/kg) or PBS. HAM8101/PBS was administered 5 min before LPS/saline. All substances were administered as a bolus intravenously through a 24G indwelling catheter placed in the tail vein. 300 μL blood from each animal was collected in Multivette K-EDTA-tubes (Sarstedt, Nümbrecht, Germany) through the tail vein at baseline as well as 3, 6, and 24 h after injection of LPS/saline. Tubes were stored on ice until centrifugation (3,220 × g at 6°C for 10 min). Plasma was then transferred to a micro tube and stored at −80°C until analysis of plasma ADM concentrations. Twenty-four hours after LPS/saline administration, deep isoflurane anesthesia was induced and Evans Blue (40 mg/kg, dissolved in saline, [Sigma-Aldrich, Taufkirchen, Germany]) was administered. Approximately 15 min after Evans Blue administration, the aorta was cannulated and whole body perfusion was started (15 min, saline + 50 IU/mL heparin [Heparin-Sodium 25000 I.E./5 mL; B. Braun Melsungen AG, Melsungen, Germany]). Following perfusion, kidney and liver was harvested and directly processed for Evans Blue absorption (see further below).
CLP-induced kidney barrier dysfunction study procedures
Mice were randomly divided into four groups (n = 6 each, n = 24 total) and received a single i.v. injection of saline (control group) or HAM8101 (0.1 mg/kg, 2 mg/kg, or 20 mg/kg, respectively), 5 min prior to CLP surgery by tail vein injection. Peritonitis was surgically induced under isoflurane anesthesia. Incisions were made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum (at 70–75% cecum length). One puncture wound was made with a 24G needle into the cecum and small amounts of cecal contents were pressed through the wound. The cecum was returned into the peritoneal cavity and the laparotomy site was closed. Animals were returned to their cages with free access to food and water. To prevent dehydration, 500 μL saline was administered subcutaneously. For analgesic treatment, carprofen (Rimadyl, Zoetis, Berlin, Germany) 5 mg/kg was administered subcutaneously directly after surgery. Eighteen hours following surgery, mice were sacrificed by retrobulbar exsanguination, whereafter the left kidney was removed immediately and cut in two parts, which were processed for immunohistochemistry (see further below).
CLP survival study procedures
In the single-dose administration study, mice (n = 30 total) were randomly divided into three groups (n = 10 each) to receive either a single dose of PBS, 2 mg/kg HAM1101, or 2 mg/kg HAM8101, administered by intravenous tail vein injection, 5 min prior to CLP surgery (performed as described in the previous section “CLP-induced kidney barrier dysfunction study procedures”). For analgetic treatment, 5 mg/kg carprofen (Rimadyl, Zoetis, Berlin, Germany) was injected subcutaneously after surgery, and metamizole (Novalgin, Sanofi-Aventis, Frankfurt, Germany) was added to the drinking water for 3 days after CLP surgery (0.8 mL/500 mL water). To prevent dehydration, 500 μL saline was administered subcutaneously. Mice were followed up for 7 days and physical condition was monitored twice daily. If still alive on day 7, animals were sacrificed by retrobulbar exsanguination. In the repeated dose administration study, mice (n = 30 total) were randomly divided into three groups (n = 10 each) to receive doses of either PBS, 4 mg/kg HAM1101 or 4 mg/kg HAM8101 5 min prior to CLP surgery (performed as described in the previous section “CLP-induced kidney barrier dysfunction study procedures”). To prevent dehydration, 500 μL saline was administered subcutaneously. Additional dosages of PBS, 2 mg/kg HAM1101, or 2 mg/kg HAM8101 were administered 24 and 48 h after CLP surgery by retrobulbar injection. Mice were followed up for 14 days, in which physical conditions were monitored twice daily, and if still alive on day 14, they were sacrificed by retrobulbar exsanguination.
Evans Blue analysis
Organs were homogenized and incubated in 1 mL formamide per gram organ sample (Sigma-Aldrich, Taufkirchen, Germany) at 55°C for 3 h to extract Evans Blue. Subsequently, the solution was transferred to fresh centrifugation tubes, of which 500 μL was transferred onto a centrifugal filter (3K, VWR, Radnor, Pa) unit and centrifuged at 25,000 × g at room temperature for 30 min. The filtrate was transferred to a 96-well-plate (non-treated; Costar) and absorption of samples and reference standards was measured at 620 nm using a plate reader (Mithras LB940; Berthold Technologies, Wildbad, Germany).
The left kidney tissues were formalin-fixed, dehydrated, and embedded in paraffin. Study procedures were done as described previously (29). In short, 5-μm-thick kidney sections were cut and deparaffinized with xylene, graded ethanol, and deionized water, followed by heat-induced antigen retrieval performed by microwaving with citrate buffer. Depending on the source of the secondary antibody, blocking was performed with normal (goat or donkey) serum. Next, primary antibodies were used for immunohistochemical localization of VEGF (rabbit polyclonal, Abcam, Cambridge, UK), angiopoietin-1 (goat polyclonal, R&D Systems, Minneapolis, Minn), and extravasation of albumin (goat polyclonal M-13, Santa Cruz Biotech, Santa Cruz, Calif). The primary antibodies were detected with secondary antibodies (goat anti-rabbit or donkey anti-goat IgG alkaline phosphatase), followed by Dako REAL Detection System Chromogen Red, and counterstained with Mayer's hematoxylin. Slides were analyzed using the Axio Vision software (release 4.8), multiple 800,000 μm2 fields were evaluated, and data are represented as mean densitometric sums red.
The ADM assay was performed as described previously (13). Briefly, plasma ADM (both free and antibody-bound) concentrations were measured using a sandwich-coated luminescence immunoassay, based on acridinium NHS-ester labeling and anti-ADM antibodies (a solid phase antibody targeted against the mid region of ADM and a labeled antibody against the amidated C-terminal moiety of ADM). Dilutions of rat ADM and labeled tracer were subsequently added to antibody-coated wells. After washing of unbound tracer, chemiluminescence was measured and evaluated against a standard curve from rat ADM standards.
Data were non-normally distributed, and therefore expressed as median and interquartile range. Differences across three or more groups were analyzed using Kruskal–Wallis tests. In case the two-sided P value of the Kruskal–Wallis test was < 0.05, pairwise comparisons were made using Mann–Whitney U tests. P values of the Mann–Whitney U tests were not adjusted for multiple testing in view of the exploratory nature of the experiments. For survival analysis, Kaplan–Meier curves were generated and log-rank tests were performed to test statistical significance of observed differences. A two-sided P value <0.05 was considered statistically significant. Calculations and statistical analyses were performed using GraphPad Prism version 6 for Windows (Graphpad Software Inc, La Jolla, Calif).
HAM8101 prevents LPS-induced vascular hyperpermeability in the kidney
The effect of HAM8101 on LPS-induced vascular hyperpermeability was studied in kidney and liver tissue using Evans Blue dye to detect albumin leakage. LPS administration resulted in a 3.5-fold increase in renal albumin leakage compared with saline-treated controls, which was significantly attenuated by HAM8101 at dosages of 0.1 mg/kg and 2.5 mg/kg (71% and 40% attenuation, respectively), whereas statistical significance was not reached for the 0.5 mg/kg dose (33% attenuation, P = 0.083, Fig. 1A). LPS administration also resulted in a 2.3-fold increase in hepatic albumin leakage (Fig. 1B). Only the highest dose of HAM8101 tended to attenuate albumin leakage in the liver, approaching but not reaching statistical significance (36% attenuation, P = 0.065, Fig. 1B). LPS administration resulted in increased ADM plasma levels (Fig. 2). HAM8101 administration in dosages of 0.5 mg/kg or higher caused a dose-dependent increase in plasma ADM concentrations (Fig. 2).
HAM8101 improves kidney barrier function during murine sepsis
The effects of HAM8101 on albumin accumulation, VEGF, and angiopoietin-1 expression were investigated in murine CLP-induced sepsis. Representative images of immunohistological stainings are provided in Figure 3A. Densitometric evaluation of immunohistological stained slides of renal tissue revealed significantly lower extravascular albumin accumulation with all three dosages of HAM8101 compared with saline-treated CLP animals (78%, 77%, and 77% attenuation for 0.1, 2.0, and 20 mg/kg Adrecizumab, respectively), without HAM8101 dose dependency (Fig. 3B). Likewise, significantly lower VEGF expression was observed in all HAM8101-treated CLP mice (55%, 45%, 59% attenuation, respectively, Fig. 3C). Expression of the protective protein angiopoietin-1 was significantly augmented in CLP mice treated with dosages of 0.1 and 2.0 mg/kg HAM8101 (387% and 474% augmentation, respectively, Fig. 3D), whereas this effect did not reach statistical significance in the 20 mg/kg group (379% augmentation, P = 0.065).
Both single- and repeated-dose administration of HAM8101 improve survival during murine sepsis
Finally, the effects of single- and repeated-dose administration of HAM8101 on survival in CLP-induced sepsis were studied in mice, and compared to the effects of the murine analogue HAM1101, which was previously shown to increase survival in the same model (26). In single-dose administration experiments, CLP caused 90% 7-day mortality, which was significantly improved by both HAM8101 and HAM1101 (50% and 30% mortality, respectively, difference between both antibodies was not statistically significant, Fig. 4A). In the repeated-dose administration experiments, CLP caused 100% 14-day mortality, which was also significantly improved by the administration of both HAM8101 and HAM1101 (60% and 80% mortality, respectively, no significant difference between both antibodies, Fig. 4B).
In the present study, we investigated the effects of the humanized recombinant monoclonal anti-Adrenomedullin antibody HAM8101 (Adrecizumab) on vascular barrier dysfunction and survival in rodent models of systemic inflammation and sepsis. We demonstrate that treatment with HAM8101 prevents LPS-induced vascular hyperpermeability in the kidney. Second, we reveal that HAM8101 improves kidney barrier function during murine sepsis, exemplified by significantly reduced extravascular albumin and VEGF expression, as well as increased expression of the protective protein Ang-1. Finally, the humanized recombinant monoclonal antibody HAM8101 enhanced survival during murine sepsis to a similar extent as the murine anti-ADM antibody HAM1101, which was previously shown to be efficacious (26, 27).
Our findings with HAM8101 support previous studies in which beneficial effects of ADM-binding with HAM1101 were demonstrated during murine sepsis, including reduced catecholamine infusion rates, attenuated kidney dysfunction, and improved survival (26, 27). Our finding of reduced renal interstitial edema formation in endotoxemic rats underscores findings from a previous study in which indirect evidence also pointed toward reduced vascular leakage: urine output was shown to be significantly higher in the HAM1101-treated mice, while fluid administration rates were similar (27). This was accompanied by improved creatinine clearance, urea levels, and less urinary excretion of tubular damage marker neutrophil gelatinase-associated lipocalin (NGAL), which were not measured in the present study (27).
HAM8101 pretreatment attenuated sepsis-induced VEGF expression in the kidney. VEGF is a potent inducer of vascular permeability (30), cell adhesion molecule expression (31), as well as cyto- and chemokine release (32). Interestingly, increased VEGF concentrations are observed in septic patients, and associated with their fluid balance (33, 34), and anti-VEGF treatment improved outcome during models inflammation and sepsis in preclinical studies (35). HAM8101 enhanced renal expression of angiopoietin-1, a vascular specific growth factor, also involved in the regulation of vascular permeability during sepsis and septic shock (36) that appears to counteract the observed VEGF effects. Angiopoietin-1 levels are also increased in sepsis and septic shock patients, and lower values are associated with increased mortality (37). Preclinical studies have demonstrated beneficial effects of Angiopoietin-1 on microvascular dysfunction and endothelial barrier function (38, 39). Collectively, our immunohistological findings of decreased VEGF expression and enhanced angiopoietin-1 expression provide mechanistic support for HAM8101's protective effects on vascular permeability that we observed in rats. As VEGF is also implicated in endothelial dysfunction, it appears plausible that HAM8101 may improve endothelial dysfunction as well, although this remains to be determined.
We show that the survival benefit in murine sepsis is comparable for HAM1101 and HAM8101 for both single and repeated dose administration. This is not unexpected, because HAM8101 is derived from HAM1101, and therefore both antibodies target the exact same epitope. Furthermore, both antibodies were shown to be ideally cross-reactive among all mammalian species tested (mice, rats, dogs, pigs, and humans; unpublished data). Also, as mentioned previously, both antibodies, despite their high affinity for ADM, only partially and to a similar extent inhibit the ADM-induced cAMP response in CHO cells overexpressing the ADM (CRLR/RAMP3) receptor (26).
Of particular interest is the fact that HAM8101 administration led to a dose-dependent increase in total ADM levels in our study. This is in line with the results from our recently performed phase I study in healthy volunteers (40), and we have formulated a hypothesis on this aspect of HAM8101 therapy (41). Briefly, we believe that it involves elongation of ADMs half-life by binding with the non-neutralizing antibody HAM8101, providing protection from N-terminal proteolytic degradation. Second, we hypothesize that ADM distribution is shifted from another compartment (likely the interstitium) toward the circulation, based upon the fact that ADM can normally diffuse freely over the endothelial barrier (6 kD peptide), whereas it likely cannot after binding to HAM8101. This is supported by data from our phase I study, showing a low volume of distribution (∼100 mL/kg) (40). Nevertheless, more research is required before definite conclusions can be drawn on the purported mechanism of action of HAM8101.
It could be argued that the HAM8101-induced increase in circulating ADM levels negatively influences blood pressure, as high dosages of ADM have been reported to exert vasodilatory effects (21–25). We cannot draw direct conclusions on this possible untoward effect, because we did not measure blood pressure in our experiments. It has to be kept in mind, though, that the increase of circulating ADM does not represent free ADM, but ADM complexed with Adrecizumab. As alluded to in the section above, this complex is too large to cross the endothelial barrier, and therefore cannot exert vasodilation through direct effects on vascular smooth muscle cells. Furthermore, data from previous studies do not support detrimental effects on blood pressure either. For instance, pretreatment with the murine form of the antibody (HAM1101) resulted in reduced vasopressor infusion requirements, which argues against hypotensive effects of the antibody, at least under inflammatory conditions (27). Finally, no effects on blood pressure or heart rate were observed in our phase I study (40).
A strength of the current work is the use of different animal species, sepsis(-like) models, and end-points. Several limitations also need to be addressed. Although our results are encouraging, it needs to be acknowledged that HAM8101 and HAM1101 were administered 5 min prior to LPS-administration/CLP-surgery in our experiments, to demonstrate proof of principle. As time dependency of the effects was not investigated, it remains to be determined whether HAM8101 also exerts beneficial effects when treatment is delayed, which is more representative of the clinical setting in sepsis patients. Moreover, measurements of vascular permeability were only performed at one time-point in each study and were restricted to the kidney and liver. It would also be of interest to evaluate whether HAM8101 exerts similar effects in the lung, as vascular leak in this organ is the major cause of acute respiratory distress syndrome in sepsis patients. Another limitation is the fact that the CLP-induced kidney barrier dysfunction experiment did not include a sham control group.
Pretreatment with the humanized anti-ADM antibody HAM8101 improves vascular barrier function and survival in rodent models of systemic inflammation and sepsis. The promising findings from the current animal experiments warrant further research in humans. Currently, a phase I study (ClinicalTrials.gov identifier NCT02991508) is in its final stages and a phase II study in septic patients is planned.
The authors thank Oliver Hartmann for his help with statistical analysis.
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