A non-interventional comparative study of the 20:1 combination of cafedrine/theodrenaline versus ephedrine for the treatment of intra-operative arterial hypotension: the ‘HYPOTENS’ study design and rationale

<p><b>Objective:</b> To compare the effectiveness of 20:1 cafedrine/theodrenaline approved for use in Germany to ephedrine in the restoration of arterial blood pressure and on post-operative outcomes in patients with intra-operative arterial hypotension of any origin under standard clinical practice conditions.</p> <p><b>Methods and results:</b> ‘HYPOTENS’ is a national, multi-center, prospective, open-label, two-armed, non-interventional study. Effectiveness and post-operative outcome following cafedrine/theodrenaline or ephedrine therapy will be evaluated in two cohorts of hypotensive patients. Cohort A includes patients aged ≥50 years with ASA-classification 2–4 undergoing non-emergency surgical procedures under general anesthesia. Cohort B comprises patients undergoing Cesarean section under spinal anesthesia. Participating surgical departments will be assigned to a treatment arm by routinely used anti-hypotensive agent. To minimize bias, matched department pairs will be compared in a stratified selection process. The composite primary end-point is the lower absolute deviation from individually determined target blood pressure (IDTBP) and the incidence of heart rate ≥100 beats/min in the first 15 min. Secondary end-points include incidence and degree of early post-operative delirium (cohort A), severity of fetal acidosis in the newborn (cohort B), upper absolute deviation from IDTBP, percentage increase in systolic blood pressure, and time to IDTBP.</p> <p><b>Conclusion:</b> This open-label, non-interventional study design mirrors daily practice in the treatment of patients with intra-operative hypotension and ensures full treatment decision autonomy with respect to each patient’s individual condition. Selection of participating sites by a randomization process addresses bias without interfering with the non-interventional nature of the study. First results are expected in 2018. ClinicalTrials.gov identifier: NCT02893241; DRKS identifier: DRKS00010740.</p

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

<div><p>The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces <i>in situ</i>, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.</p></div

Study flow diagram.

<p>The most common major protocol deviations were ‘disallowed concurrent medications’ (15 patients in the placebo and 19 patients in the FCM group) and ‘selection criteria not met’ (5 patients in the placebo and 8 patients in the FCM group).</p

Mean changes (Δ) in reticulocyte counts, Hb levels, MCV and TSAT (error bars SD).

<p>The rapid increase of reticulocyte counts in the FCM group is in line with early FCM-associated improvement of the fatigue total score (fig. 2B). The significantly higher increase in reticulocyte counts in the FCM group at Days 7 and 28 (A) translated into higher increases of Hb and MCV at Days 28 and 56 (B,C). The early FCM-associated increase in reticulocyte counts remained significantly higher compared to placebo when analysing the subgroup of patients with Hb≥120 g/L (P<0.001 for Days 7 and 28). This suggests that iron deficiency without anaemia affected erythropoiesis in these apparently healthy women and raises doubts whether the currently recommended Hb cut-off level of 120 g/L to diagnose anaemia in premenopausal, menstruating women is appropriate. TSAT remained significantly improved until the end of the 56-day study period (D). *P<0.0001 for difference between FCM and placebo group in reticulocyte counts and Hb levels, respectively.</p

Patient baseline characteristics, SAF.

<p>*Weight, PFS, Hb, MCV, serum ferritin, TSAT and CRP shown as median (Q1, Q3).</p><p>CRP C-reactive protein; Hb hemoglobin; MCV mean corpuscular volume; PFS piper fatigue scale; TSAT transferrin saturation.</p

Secondary endpoints – Hematologic parameters (% [n] or mean [SD]) (ITT).

‡<p>A positive value of the difference FCM–placebo is in favor of FCM;</p>§<p>Mean Hb levels at Day 56 were 134±7.7 g/L (FCM) vs. 127±10.1 g/L (placebo), median serum ferritin levels at Day 56 were 169 µg/L (FCM) vs. 16 µg/L (placebo) and median TSAT was 27% (FCM) vs. 15% (placebo).</p><p>Hb hemoglobin; TSAT transferrin saturation; MCV mean corpuscular volume.</p

Secondary endpoints – Fatigue, QoL and cognitive function tests (mean [SD]) (ITT).

†<p>A negative value of the difference FCM–placebo is in favor of FCM;</p>‡<p>A positive value of the difference FCM–placebo is in favor of FCM.</p><p>PFS Piper fatigue scale; QoL quality-of-life; SF short form; VAS visual analogue scale (results in mm); CFT cognitive function test (results in msec).</p

scFv-Fc B6-11 fusion antibody inhibits motility of BECs in a scratch wound assay.

<p>A: Migration analysis of BECs by scratch wound assay in the presence of scFv-Fc B6-11, Fc fragment or PBS as controls. Shown are representative images of wounds created in BEC monolayers (n = 3 in each group) at indicated time points. Size bars: 500μm. B: Reoccupation of the gap by BECs after t = 24 hrs and t = 48 hrs versus t = 0 hrs was measured using AxioVision 4.7 software. Areas repopulated by BECs and % of gap area newly covered by BECs were calculated at each timepoint. Assay was performed twice and values are means ± SD. Gap closure was analysed by one-way Anova (<i>P</i> < 0.05) followed by pairwise Student´s t-testing. Shown are significant differences between scFv-Fc versus Fc control, and scFv-Fc versus PBS control, respectively (<i>P</i>-values: * < 0.05, ** < 0.005, ***< 0.0005).</p

Treatment effects of FCM vs. placebo in fatigued, iron-deficient, non-anemic women (ITT; error bars SD).

<p>(A) Proportion of patients with ≥1 point reduction in PFS (primary endpoint). (B) Improvement of mean PFS total score in FCM- vs. placebo-treated patients throughout the study (Mean differences ± SD vs. baseline: Day 7: −1.2±1.8 vs. −0.8±1.5 points; Day 28: −1.8±2.1 vs. 1.2±2.0 points; Day 56: −2.2±2.1 vs. −1.4±2.0 points). P-values given for intergroup differences. (C) Mean change (Δ) in PFS total score and subscale scores from baseline to Day 56. *P≤0.01 for all scales. (D) Mean change (Δ) in self-rated alertness, contentment and calmness (computer-based VAS scales). *P<0.05; †P = 0.055 vs. placebo.</p

Enrichment of phage antibodies that bind to BECs and LECs with successive biopanning rounds.

<p>A: Numbers of recovered phage particles after each panning round on BECs and LECs. For each selection round, the phage output/input titer ratios are given on the y-axis, showing an increase on BECs (red line) and LECs (green line) when adding polyclonal ETH-2 phage. In contrast, control wild-type VCS-M13 (grey and black lines) phage did not shift significantly on either cell population. B: Enrichment of BEC- and LEC-binding phage antibodies as seen by FACS-analysis of phage antibody pools. Shown are representative histograms of BECs and LECs stained with phage antibodies after respective panning rounds (black line) and WT phage as negative control (grey-filled graphs). The number of cells (counts: Y-axis) is given as function of the fluorescence intensity of phage antibody staining of the cells (FL1-H: X-axis). Percentage of BECs and LECs shifted by phage binding is depicted in the graphics. In all experiments, cells were incubated with phage antibody pools, and cell-binding was detected by anti-M13 antibody and FITC-conjugated anti-rabbit antibody. Grey-filled graphs show WT phage binding as negative control. C: <i>BstN</i>I DNA fingerprint analysis of single scFv clones. Representative gel images of respective panning rounds (#1 - #6) performed on BECs and LECs are shown. Over the course of panning rounds, the number of intact clones is increasing. Phagemid DNA isolated from single clones was digested with <i>BstN</i>I and analyzed in agarose gel electrophoresis. M: 1 kbp molecular weight marker.</p